Comparison of the binding affinities of rabbit IgG fractions to the rabbit fetal yolk sac membrane: use of 22Na to facilitate quantitation of 125I-IgG binding. Rabbit IgG was purified, Fr-1-(G-200)2, and then separated by DEAE-cellulose column chromatography into three major fractions, Fr-I, -II, and -III. Binding affinities of the 125I-labeled IgG and its fractions to the rabbit fetal yolk sac membrane were studied. At a concentration of 2 mg/ml, rabbit IgG is bound to the extent of 9 mug/cm2 membrane, whereas the values for fractions Fr-I, -II, and -III are 13, 7, and 5 mug/cm2, respectively. The maximal amount of IgG bound appears to be approximately the same, i.e., 23 mug of IgG/cm2 membrane, for Fr-1-(G-200)2 and its three fractions. In contrast, bovine IgG does not bind to the membrane over the range of concentration tested. Binding constants for Fr-1-(G-200)2, fraction Fr-I, -II, and -III are estimated to be 5.4, 8.6, 4.0 and 2.0 times 10(4) M-1, respectively. The binding affinities of IgG fractions to the yolk sac membrane correlate with the chemical and physicochemical properties of the fractions. Also detailed in the text is the use of 22Na to facilitate quantitation of specific binding of the 125I-IgG to the membrane under equilibrium conditions.


Reactivity of rabbit antiserum to guinea pig eosinophils. Although the eosinophil has been recognized as a distinctive cell type for almost 100 years, the major functions of these cells remain unknown. As an approach to defining these functions we have treated guinea pigs with rabbit antiserum to eosinophils (AES) in an attempt to ablate these cells from tissues. Rabbits were immunized thrice with purified eosinophils and the antisera were absorbed with peripheral blood cells from guinea pigs made eosinopenic with methyprednisolone to remove antibodies reactive with serum proteins and erythrocytes. The resulting sera reacted strongly with eosinophils in cytotoxicity tests and had weak or no reactivity with neutrophils. However, absorption of AES with purified neutrophils removed antieosinophil activity. Intraperitoneal injection of potent AES into guinea pigs resulted in complete absence of eosinophils from the peripheral blood and from the peritoneal cavity with only transient or no reduction in circulating neutrophils. Eosinophils were also reduced in bone marrow, spleen, and intestine. The ability of neutrophils to absorb AES activity in spite of weak reactivity in cytotoxicity tests may reflect a quantitative difference in antigenic determinants between eosinophils and neutrophils.


Peculiar secretory IgA system identified in chickens. II. Identification and distribution of free secretory component and immunoglobulins of IgA, IgM, and IgG in chicken external secretions. A homologue of a free secretory component (SC) was identified in chicken intestinal secretion by criteria based on its antigenic relationship with intestinal secretory IgA (SIgA), molecular size, sugar content, and electrophoretic mobility, as well as its elution characteristic from ion-exchange chromatography. SC was obtained in a form free from IgA from the intestinal secretion by salting out and DEAE chromatography, followed by density ultracentrifuguation or Sephadex G-200 gel-filtration. However, the free SC revealed some antigenic deficiency when compared to bound SC of intestinal SIgA and showed a failure of binding to serum-type-polymeric IgA of biliary IgA in vitro. Several kinds of chicken external secretions were examined for detection of SC and immunoglobulin classes of IgG, IgA, and IgM. In spite of the wide distribution of immunoglobulins in the external secretions, SC antigen could be detected only in intestinal secretion. Most IgA in the secretions had a molecular structure of a tetramer of serum-type IgA, lacking in SC and having 17S to 18.5S and 600,000 to 700,000 daltons. On the other hand, IgA in the intestinal secretion showed close similarity to the mammalian SIgA, associated with SC and having 11.2S and 350,000 daltons. Presence of antibody activity in the intestinal IgA to avian reovirus was confirmed by plaque reduction tests.


Alloantiserum-mediated supperssion of Ir gene controlled antigen-induced lymphocyte activation: effect on uridine and leucine incorporation. Antisera to guinea pig histocompatibility antigens specifically suppress Ir gene-controlled antigen-stimulated, DNA-synthetic responses in vitro. To define further the mechanisms of alloantiserum-mediated suppression and to utilize this suppression as a probe of the cellular events occurring during lymphocyte activation, we have examined the effects of alloantisera on 14C-leucine and 3H-uridine incorporation, earlier events after antigen stimulation. The RNA and protein synthetic responses of peritoneal exudate lymphocytes from immunized guinea pigs to DNP-GL (controlled by a strain 2-linked Ir gene) are 40 to 50% of maximum by 24 hr in culture and at or near maximum by 48 hr. DNA synthesis was only 2% of maximum at 24 hr and maximum at 72 hr. Comparisons of the degree of stimulation of the incorporation of all three precursors reveals an excellent correlation between leucine and uridine but poor correlation between leucine and thymidine. Despite the observed differences in time course of incorporation and magnitude of response, the susceptibility to suppression by alloantisera of all three responses was virtually identical. Anti-2 sera, added at the initiation of culture, completely suppress all three responses. If addition was delayed 8 hr, 50 to 60% suppression was still demonstrated, whereas only minimal suppression was evidenced if addition was delayed 18 hr. These results suggest that by 8 to 10 hr of incubation with antigen, the responding cells have undergone the necessary biochemical and structural changes to eventuate in an immune response; and, furthermore, that alloantisera do not suppress by blocking antigen access to, or release from, its lymphocyte membrane receptor, but rather by a dynamic alteration of the lymphocyte membrane surface which interferes with the stimulus being generated by the antigen-receptor complex.


Splenic suppressor macrophages induced in mice by injection of Corynebacterium parvum. Spleen cells from C57BL/6N mice injected with killed Corynebacterium parvum (CP) had a marked growth inhibitory effect on the in vitro proliferation of RBL-5 murine lymphoma cells. It was most marked 12 to 14 days after injection and was usually no longer detectable later than 21 days. It could be demonstrated at effector cell to target ratios between 20:1 and 5:1 at which normal spleen cells had a growth-promoting effect. Addition of CP to an in vitro mixture of spleen cells and tumor cells augmented the inhibitory effect of spleen cells from CP-injected mice although it conferred no inhibitory potential on normal spleen cells. Growth inhibiton by CP spleen cells was not mediated by T cells and various depletion experiments suggested that the effector cells of the phenomenon were macrophages. Spleen cells of CP-injected mice also showed strongly depressed responses to the T cell mitogens PHA and Con A and suppressed the mitogen responses of syngeneic normal spleen cells. The characteristics of the suppressor cells mediating this effect appeared to be very similar to those inhibiting lymphoma cell growth. The responses to LPS were also strongly suppressed in mice injected with 2.1 mg of CP. However, after injection of one-tenth of the dose a relative sparing of the LPS response was noted, whereas the PHA response was still suppressed.


Receptors for IgG and complement in human spleen lymphoid cells. Preferential binding of particulate immune complexes through complement receptors. Human lymphoid spleen cells attached to Petri dishes by poly-L-lysine bind 51Cr-labeled erythrocytes coated with IgG antibodies or complement but not uncoated erythrocytes or those coated with IgM antibodies. The number of erythrocytes bound through complement receptors is several times larger than that bound through IgG receptors. Increasing up to five times the number of IgG molecules on the red blood cells only leads to a slight increase of binding. However, the addition of complement to the IgG-coated erthrocytes increases 10 times the binding to spleen cells, even in the presence of an excess of normal IgG. These results can be explained by postulating that there is a larger number (or greater affinity) of spleen cell receptors for complement than that of spleen cell receptors for IgG.


Peanut agglutinin, a new mitogen that binds to galactosyl sites exposed after neuraminidase treatment. Peanut agglutinin, purified by affinity chromatography, agglutinates lymphocytes from mouse, rat, guinea pig, and man only after their treatment with neuraminidase. However, it stimulates only neuraminidase-treated rat and human cells. A similar number cell surface receptors for peanut agglutinin was found on neuraminidase-treated rat and mouse lymphocytes although the latter cells were not stimulated by the lectin. Galactose specifically inhibited the agglutination and stimulation of lymphocytes by peanut agglutinin. Sequential treatment of lymphocytes with neuraminidase and beta-galactosidase markedly reduced the response of the cells to stimulation by peanut agglutinin, soybean agglutinin, and galactose oxidase. It is suggested that the same galactosyl residue may be the target for the initial step in triggering lymphocytes by the above mentioned mitogens.


Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity. Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3  to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.


The humoral response of mouse spleen cells to two types of sheep erythrocytes. III. A new VH region marker. It was previously shown that there is multigene control of the response of mouse spleen cells to two types of sheep erythrocytes (H and L). Discriminator strains of mice make a much higher response to extra antigens found only on H SRBC than to the shared antigens found on both types of erythrocyte. Non-discriminator strains respond only to the shared antigens, making a response as great as the discriminator response to the extra antigens. In this study we have shown that the response to the extra antigen(s) on H SRBC is under the genetic control of a gene(s) located in the VH region near or to the left of the A5a, alpha 1, 3-dextran and NP makers.


Isolation of the most immunoreactive antigenes of echinococcus granulosus from sheep hydatid fluid. This paper describes a simplified procedure for obtaining purified Echinococcus granulosus antigens from sheep hydatid fluid by using affinity chromatography on concanavalin A-Sepharose. The presence of two "major" antigens (4 and 5) was confirmed. Antigen 5 was isolated by preparative polyacrylamide gel electrophoresis. Antigen 4, eluted by diffusion from the gel, was seen to be "contaminated" by antigen 5 and was isolated by using anti-5 Sepharose-linked serum. These two major antigens were then tested separately against the sera of hydatidosis patients by using very simple immunolgic tests. The best results were obtained in passive hemagglutination with antigen 4. Antigen 4 is the most immunoreactive parasitic antigen; antibodies against it were found in the sera of all hydatidosis patients showing positive reaction. Apart from the direct use of this antigen in serologic tests, it appears possible to standarize the most frequently used and commerically available antigenic materials by titrating this component.


Effects of retinoic acid on prostaglandin biosynthesis in guinea-pig skin. Topical application of retinoic acid on guinea-pig skin resulted within 70 hours in erythema with a concomitant elevation of endogenous prostaglandin E2 (PGE2) in the treated areas of the skin. Prolonged daily treatment resulted in the development of severe scaly dermatoses and a corresponding decrease in the level of PGE2 in the skin. Examination of retinoic acid effects on the in vitro biosynthesis of PGE2 from arachidonic acid by extracts from guinea-pig skin and sheep vesicular gland demonstrated that retinoic acid inhibits prostaglandin synthesis in a concentration and time-dependent manner. These results indicate that retinoic acid may exert a regulatory role on prostaglandin biosynthesis in the skin.


Age-related changes in the resident bacterial flora of the human face. Quantitative levels of resident aerobic and anaerobic bacteria of the face, show a characteristic age-related pattern. The density of anaerobic diptheroids and surface aerobic micrococci is higher in infancy than in early childhood. At puberty the quantity of organisms increases, with significantly higher levels achieved in late adolescence. Maximum counts are attained in early adulthood and remain constant until old age when a trend toward lower numbers occurs. These changes seem to correlate with the production of sebum.


The mode of growth of eccrine duct milia. Light and electron microscopic studies were performed utilizing serial sections in 10 patients with secondary milia to clarify the incidence of eccrine duct milia and to demonstrate the life history of the milia. Out of 69 milia after blistering taken from 8 patients, 52 were found to connect with the eccrine sweat duct (75.4%) while only 1 connected with the hair follicle (1.5%). In the control group, of 4 milia taken from 2 patients, 2 were found to connect with the eccrine sweat duct and 1 with the hair follicle. Various degrees of milium formation were observed ranging from incomplete and having either concave-shape, U-shape, capped U-shape, or O-shape. The eccrine duct milia in all types possess structures resembling the epidermal sweat duct unit. The epithelial cells constituting the recess or cyst differ from epidermal cells.


Mouse vaginal assay for topically effective chemotherapeutic agents. A number of chemotherapeutic drugs have been subjected to intravaginal testing in mice to measure their local inhibitory activity on DNA, RNA, and protein synthesis in vaginal epithelium. The drugs have been tested at various concentrations and in different vehicles and evaluated by autoradiographic techniques. Systemic absorption of the drugs was monitored by simultaneous study of the gastrointestinal mucosa of the rectum. Methotrexate inhibited deoxyuridine incorporation into DNA in both vaginal and rectal epithelium. Several lipid-soluble analogues of methotrexate were found to have no effect on deoxyuridine incorporation. Nitrogen mustard and emetine have been shown to selectively inhibit DNA and protein synthesis, respectively, without systemic effects. This animal assay system may be useful for predicting the effectiveness of potential drugs for the topical treatment of psoriasis.


Effect of pharmacologic agents on human keratinocyte mitosis in vitro. III. Inhibition by histamine and methylated analogs. Keratinocytes from normal human skin were propagated in vitro. Histamine at 2 times 10(-6) M inhibited mitosis 68%. The side-chain methylated analogs, n-methylhistamine and n,n-dimethylhistamine, produced an inhibitory response of 53 and 60%, respectively, at 1 times 10(-6) M. The side-chain acetylated analog, n-acetylhistamine, did not elicit an inhibitory response in concentrations as high as 1 times 10(-4) M. The ring-methylated analogs, 1-methylhistamine and 4-methylhistamine, produced some degree of mitotic inhibition at 1 times 10(-4) M. When the H1-blocking agent, pyrilamine, or the H2-blocking agent, metiamide, was added in conjunction with histamine, the histamine-induced mitotic inhibition was abolished. These data indicate that human keratinocytes may possess receptors for histamine which could play a role in the regulation of human keratinocyte proliferation in vivo.


Thermal analysis of human stratum corneum. A thermal analysis technique has been developed and used to detect the melting of lipids and denaturation of proteins in stratum corneum. Trasitions were observed at 40 degrees C, 75 degrees C, 85 degrees C, and 107 degrees C. The transitions at 40 degrees C were attributed to the melting of lipids. The transition at 85 degrees C was identified as due to the denaturation of alpha-keratin, and the transition at 107 degrees C to the denaturation of a nonfibrous protein. It was found that alteration of the conformation of the nonfibrous protein changed the state of water absorbed, and that water contributed to the ordering of the lipid and protein. The thermal analysis technique may prove to be of value in studying the interaction of materials with skin.


Elevated spermidine and spermine levels in the blood of psoriasis patients. Samples of blood, anticoagulated with EDTA, from 11 patients with psoriasis and 11 individuals without psoriasis were analyzed for their polyamine content. The average spermidine level in patients with psoriasis was approximately twice that of the controls and the average spermine level was three times that of the controls. The level of spermidine and spermine in the skin of two patients with psoriasis were significantly depressed as compared to those of controls.


Suppression of ultraviolet light-induced tumor formation by dietary antioxidants. Dietary antioxidants were effective in reducing the number and severity of ultraviolet light-induced squamous cell carcinomas in skin of hairless mice. This and previous studies suggest that antioxidants may play a role in both inhibition of the primary event(s) involved in ultraviolet light-mediated tumor induction and the subsequent development of precancerous lesions into tumors.


Inhibition of platelet adherence to damaged surface of rabbit aorta. A method has been developed for quantitative measurement of adherence of rabbit platelets to the damaged intimal surface of everted segments of rabbit thoracic aorta. Platelets were labeled with 51Cr, washed, and resuspended in Tyrode solution containing 0.35 per cent albumin and apyrase. This suspending medium contains physiologic concentrations of calcium and magnesium; apyrase degrades any ADP lost from the platelets or from the damaged wall. Everted aorta segments were rotated in the platelet suspensions. Neither platelet aggregation nor lysis occurred and the platelets adhered to the subendothelium either as individual platelets or as a single layer. Damage caused by scraping the everted segments with a scalpel blade increased adherence 50-fold. Acetylsalicylic acid (ASA) in vitro, or administered orally to the rabbits from which platelet suspensions were prepared, significantly reduced the number of platelets adherent to the damaged aorta wall. ASA affected only the platelets, and did not affect the damaged wall. Platelet adherence to the damaged wall was also reduced by the use of 4 per cent albumin in the suspending medium, or by the addition of citrate. Adherence of platelets resuspended in citrated plasma was low and further inhibition by ASA was not demonstrable. ASA may affect two aspects of thrombus formation: platelet adherence to subendothelial structures and the platelet release reaction induced by collagen (and possibly by other subendothelial structures). These studies show that ASA has a marked effect on adherence of platelets to subendothelium under conditions in which aggregation and thrombus formation are prevented.


The kinetics of the sol-gel transformation of deoxyhemoglobin S by continuous monitoring of viscosity. By continuous monitoring of viscosity during the sol-gel transformation of deoxygenated sickle hemoglobin a time: viscosity profile has been demonstrated that can be subdivided into: (1) initial lag phase, (2) gradual and minor increase in viscosity, (3) rapid and major (180 times initial value) increase in viscosity, (4) moderately rapid decrease in viscosity, and (5) achievement of equilibrium at approximately 50 per cent of maximum viscosity increase. The duration of the lag phase, rate of increase in viscosity, and maximum change are greatly influenced by hemoglobin concentration and markedly altered by temperature. Admixtures of hemoglobins A and F lengthen the lag phase and attenuate the rate of increase and magnitude of viscosity change according to proportions added and capacity to interact with deoxyhemoglobin S, but the general configuration of the curve is maintained. A different time: viscosity profile is obtained for mixtures of S and C hemoglobin that is lacking the phase with decreasing viscosity. Relevance to the pathophysiology of the sickling phenomenon is evidence because the quantitative and qualitative changes induced by variations in concentration of deoxygenated hemoglobin S, temperature, amount and type of admixed hemoglobin (A, C, and F), ionic strength, and 2, 3-DPG are in agreement with their known effects upon the sickling of intact cells and upon the minimum gelling point of deoxyhemoglobin S. No final conclusions can be drawn concerning the extent or form of hemoglobin aggregation present in the various phases of the time: viscosity profile; however, the technique lends itself readily to obtaining samples at various points along the curve for additional studies such as electron microscopy and light scattering.


In vivo evidence for the functional heterogeneity of transferrin-bound iron. III. Studies of transferrin at high and low iron saturation. The functional heterogeneity of the transferrin iron pool of rats was studied by means of selective radioiron labeling of transferrin at high and low iron saturations. A sample of iron-poor plasma transferrin brought to 90 per cent iron saturation by the addition of 59Fe-nitrilotriacetate was mixed with a similarly labeled plasma sample of 55Fe-transferrin at 10 per cent iron saturation. The mixture was injected intravenously into groups of normal rats which were killed after 30 minutes, 3, and 24 hours for measurement of the distribution of the 59Fe and 55Fe in various tissues. 59Fe from diferric transferrin disappeared more rapidly from plasma and was preferentially removed by red blood cells, bone marrow, liver, and spleen. This phenomenon was most apparent at 30 minutes and 3 hours with little difference in the distribution of 59Fe and 55Fe at 24 hours. These studies add further support for the Fletcher-Huehns hypothesis of the functional heterogeneity of the transferrin iron pool in the rat.


The determination of leukocyte phagocytic oxidase activity by measurement of the initial rate of stimulated oxygen consumption. A method for the estimation of leukocyte phagocytic oxidase activity is described, which is based upon the polarographic measurement of the initial rate of oxygen consumption of peripheral blood leukocyte suspensions during maximally active phagocytosis. Mean values are presented for 22 normal donors, and the effects of centrifugal force, serial washing, temperature of storage, and the presence of platelets in the incubation mixture are described and discussed.


Degradation of insulin in serum by insulin-specific protease. The effect of insulin specific protease (ISP) in degrading plasma insulin over a wide concentration range was studied. In sera containing more than 50 muU per milliliter, more than 85 per cent of the insulin was destroyed. However, a smaller percentage of the hormone was degraded in sera containing less than 20 muU per milliliter and the results were highly variable. The residual insulin-like immunoreactivity in plasma after exposure to ISP thus represents both undegraded insulin as well as proinsulin. These findings explain the higher plasma proinsulin values obtained by the enzyme method when compared to gel filtration.


In vitro uptake of calcium by dermis of patients with pseudoxanthoma elasticum. The calcification of dermal elastic fibers is a characteristic feature of affected skin from patients with pseudoxanthoma elasticum (PXE). Punch biopsies of normal skin and of affected and unaffected skin from patients with PXE were obtained to study dermal calcification in vitro. Before incubation, the calcium, magnesium, and phosphorus content of normal and PXE unaffected dermis were similar and that of PXE affected dermis was significantly higher. Dermal samples were incubated for 2 and 4 days at 37 degrees C. in a calcium phosphate buffer and the Ca and P content were measured. After 4 days of incubation, both PXE affected and unaffected dermis took up significantly more Ca and PO4 than normal dermis. Thus in vitro uptake study could clearly distinguish between normal and PXE dermis. PXE is a disease of variable expression and mild or subclinical forms are difficult to diagnose. Dermal biopsies from clinicallu normal relatives of patients with PXE were obtained and the in vitro uptake of Ca and PO4 was measured. The results show that several of these individuals had elevated uptakes of Ca and PO4 similar to the unaffected dermis of the PXE patient. The results suggest that this test may be used as a diagnostic aid in the detection of individuals with mild forms of PXE and may be a genetic marker for PXE.


Rheological properties of fibrin clots. Effects of fibrinogen concentration, Factor XIII deficiency, and Factor XIII inhibition. The effect of fibrinogen concentration, Factor XIII deficiency, and Factor XIII inhibition, utilizing hydroxylamine, on the formation of clot structure in vitro was studied in human platelet-free plasma systems. Rheological and biochemical techniques were employed to relate changes in clot elasticity and viscosity to clot structure formation following recalcification of citrate anticoagulated samples. Classical theories of linear viscoelasticity for swollen crosslinked materials were shown to give an excellent estimate of the number of covalent crosslinks per fibrin monomer, which is directly attainable from rheological data. SDS gel electrophoresis was utilized to show qualitatively that decreases in maximum clot elasticity, at constant fibrinogen concentration, are directly related to a decrease in Factor XIII-mediated intermolecular crosslinking. The use of the technique to investigate both kinetic and equilibrium crosslinking (or structure formation) abnormalities in plasma systems is discussed.


Normal vitamin B12 turnover in subacute combined degeneration of the spinal cord. Vitamin B12 turnover studies were carried out in 2 patients with pernicious anemia and subacute combined degeneration of the spinal cord. The retention of a tracer dose (0.5 mug) of intravenously administered 58Co-labeled vitamin B12 in these 2 patients was measured by whole-body monitoring over periods of 94 and 134 days. Vitamin B12 turnover (per cent per day) was calculated and the results compared with normal subjects and with pernicious anemia patients having no neurologic dysfunction. The loss of radioactive-B12 from the body was described by a single exponential model, confirming that the loss of vitamin B12 takes place as though from a single pool. There was no difference in vitamin B12 turnover between the patients with subacute combined degeneration of the spinal cord and normal subjects or other pernicious anemia patients without neurologic involvement. These findings contradict the suggestion that the development of neuropathy in vitamin B12 deficiency might be related to an increased requirement for vitamin B12.


Microchromatography of hemoglobins. II. A simplified procedure for the determination of hemoglobin A2. A modification of the microchromatographic procedure for Hb-A2 which utilizes DEAE-cellulose (Reference 1) allows the quantitation of Hb-A2 without interference from any Hb-S in the sample. Elution of Hb-A2 with a glycine-KCN developer is much less sensitive to minor change in pH of the developer, but is greatly dependent on the pH of the ion exchanger.


Evaluation of a cytocentrifuge method for measuring neutrophil granulocyte chemotaxis. An in vitro method for the assessment of neutrophil granulocyte chemotaxis employing a cytocentrifuge has been evaluated. The cytocentrifuge is used to directly sediment leukocytes on a Millipore filter, and thus (1) eliminates errors due to abnormal or unequal settling of the cells and (2) eliminates the need for serum, plasma, or albumin on the cell side of the chemotactic chamber to obtain optimal chemotaxis. In addition, other advantages of the method are that it requires smaller volumes of blood, less handling of the cells, and less time is required for visual counting of the neutrophils. The method has been utilized to assess chemotactic activity of neutrophils from 80 control subjects and over 100 patients with various diseases and to study the effects of various pharmacologic agents on neutrophil chemotactic responsiveness.


Congenital achalasia of the oesophagus in children. Nine cases of congenital achalasia of the cardia in children are reported, of which four were below 1 year old and recording the youngest age of 3 months. A hereditary element is present in four children. Association with Hirschsprung's disease of the colon in one case and with hiatal hernia in another case, favours a more generalized neuromuscular pathology than a simple spasm of the cardia.


Rupture of the round window membrane.

Rupture of the round window membrane. A perilymph leak into the middle ear through a ruptured round window membrane results in the symptoms of hearing loss, tinnitus and vertigo, either singly or in combination. The case histories of thirteen patients with such a fistula are described, these patients having in common a predisposing incident which had led to a rise of C.S.F. pressure. Symptomatology and the results of investigation are analysed and operative technique and results discussed. While it appears that vertigo uniformly responds very satisfactorily to operative treatment the improvement in hearing loss and tinnitus is more difficult to predict.


Septoplasty. Rational surgery of the nasal septum. The Submucous Resection (S.M.R.) operation ascribed to Killian, still widely taught as the standard surgical treatment of obstructive septal deformities, has serious deficiencies in many common clinical situations. The reasons for this are critically discussed in the light of the surgical anatomy and physiology of the nose as a whole. The rational, conservative and versatile principle of septoplasty has evolved over half a century, until today the author considers that it should largely supersed the old S.M.R. The principles and the indications for septoplasty are discussed. The basic septoplasty procedure used by the author is described with practical details, together with all the variations and extensions possible as far as a complete septo-rhinoplasty. The importance is stressed of accurate assessment of the whole nasal problem and precise correction of all primary and secondary obstructive factors.


Laryngocoele.

Laryngocoele. All cases of laryngocoele in the English literature have been reviewed and a further nine added, making a total of 139. The sex incidence is 5:I in favour of men, and the maximum age incidence is in the sixth decade. Approximately two out of three laryngocoeles are unilateral and they may be combined, external or internal, with roughly equal frequency; about 8 per cent become infected and present as pyocoele. The commonest presenting symptoms are hoarseness and a lump in the neck, with almost equal frequency. There is only one case on record where the laryngocoele could have been caused by prolonged and repeated blowing against resistance. This factor appears to play no part in the cause of laryngocoele though increased air pressure in the larynx may make an already existing laryngocoele manifest; this increased air pressure may be caused by a co-existing carcinoma. Laryngocoeles appear to be an atavistic remnant from the higher apes, particularly those who use their arms with the thoracic cage fixed whilst swinging through the trees.


Rhabdomyosarcoma of the larynx.

Rhabdomyosarcoma of the larynx. A case of rhabdomyosarcoma of the larynx in a 54-year-old man is reported. Patient presented dramatically with a large tumour mass obstructing his laryngeal inlet. The patient remains alive and well sixteen months after a total laryngectomy. Only seven previous reports of a rhabdomyosarcoma of the larynx could be found in the literature and these are compared with the case reported. It would seem that a rhabdomyosarcoma of the larynx has a better prognosis than elsewhere in the body, probably because of its earlier recognition and accessibility to radical surgery.


Cryo-labyrinthectomy for post-mastoidectomy vertigo. A case of severe vertigo, following multiple operations for chronic suppurative otitis media is described. Sensitive nystagmus-producing spots in the mastoid cavity were treated by cryosurgery. Despite the absence of histological evidence of sensory epithelium or nerve endings, a good result was obtained.


Tissue distribution of cholesterol feedback control in the guinea pig. The level of cholesterol synthesis and the activity of the cholesterol feedback system were studied in tissue slices from a number of organs of the guinea pig. In contrast to the tissue distribution of sterol synthesis in the rat, liver slices of the guinea pig have a low rate of sterologenesis, with ileum and lung being the most active sterologenic tissues. More surprising, all tissues studied in the guinea pig, including lung, ileum, and brain, were shown to possess an active cholesterol feedback system. The basis for the widespread organ distribution of cholesterol feedback control in the guinea pig is probably the ability of the various tissues of the guinea pig to take up and concentrate exogenous cholesterol and is not the result of any inherent differences in the lipoprotein composition in this species.


Interactions of cationic bile salt derivatives with the ileal bile salt transport system. Previous structure-activity studies of the active ileal bile salt transport system have demonstrated that a single negative charge on the side chain is essential for active transport. Furthermore, mutual inhibition studies between different pairs of bile salt substrates indicated that dihydroxy bile salts had a greater apparent affinity for the transport system than the trihydroxylated compounds and triketo bile salts had the least such affinity. In this study, a series of cationic bile salt derivatives (cholamine conjugates) were prepared with one, two, and three alpha-hydroxyl groups on the steroid moiety. Based on the previous observations one would expect (1) no active transport of any of the cholamine conjugates by the ileal transport system; (2) interaction of these compounds with the transport system in such a way as to inhibit the transport of bile salts, with inhibition potency of the transport of any single bile salt inversely related to the number of hydroxyl groups present on the cholamine conjugate; and (3) transport of triketo anionic bile salts to be most readily inhibited, trihydroxy compounds less readily inhibited, and dihydroxy bile salts least inhibited. Using everted gut sac preparations it was demonstrated that all three aforementioned expectations did occur. Furthermore, reversible inhibition of ileal absorption of taurocholate and the bile salt derivative taurodehydrocholate could be demonstrated in vivo. The dihydroxy cholamine conjugates were better inhibitors than the trihydroxy compound. Relative specificity for the bile salt system of these cationic bile salt derivatives was demonstrated in the in vivo preparation by comparing its inhibition of taurodehydrocholate absorption with their lesser capacity to inhibit glucose transport.


Tocopherol in human platelets.

Tocopherol in human platelets. A spectrophotometric method was used to determine the total tocopherol levels in platelets, plasma, and erythrocytes from human subjects. The platelets contained about three times as much total tocopherol per cell as erythrocytes. This difference was not related to the content of polyunsaturated fatty acids in platelets and erythrocytes. In vitro incubation resulted in significant uptake of tocopherol by plasma and RBC, whereas no uptake was observed into platelets. A 3-month period of tocopherol treatment increased the level of tocopherol in plasma and erythrocytes, whereas the platelet level was unchanged. Tocopherol treatment did not interfere with platelet function or platelet lipid metabolism. The tocopherol fractions of platelets, red cells, and plasma were similar, and alpha-tocopherol was the main fraction.


A simplified procedure for synthesis of di-[14C]acyl-labeled lecithins. A simplified procedure for synthesis of 1,2-di-[1'-14C]oleoyl-, 1,2-di-[1'-14C]linoleoyl-, and 1,2-di-[1'-14C]eicosatrienoyl-sn-glycero-3-phosphorylcholine is described. The method involves acylation of the CdCl2 complex of glycerophosphorylcholine with a 14C-labeled fatty acid in the presence of trifluoracetic anhydride and pyridine. The 14C-labeled lecithin is isolated in pure form by preparative thin-layer chromatography and alumina column chromatography in an overall yield of 12-24%. No isomerization or peroxidation of the unsaturated acids was detected.


A simple, rapid method for measurement of acetate in tissue and serum. A simple and rapid method is described for determining the free acetate concentration in liver and serum. After extraction, the acetate is converted to its benzyl ester by thermal degradation of its benzyldimethylphenylammonium salt in the vaporizer of a gas chromatography. Good quantitation is achieved in the range of 0.033-2.5 mumoles of acetate per gram of liver or per milliliter of serum.


Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A. A novel radiochemical assay for long-chain fatty acid:CoASH ligase activity (AMP) (EC 6.2.1.3) has been developed based on the conversion of [3H]CoASH to long-chain fatty acyl CoA. Fatty acyl [3H]CoA was quantitatively retained on Millipore filters upon filtration of the acidified reaction mixture under conditions where the [3H]CoASH was not retained. The assay was developed using microsomes derived from isolated fat cells as the source of fatty acid:CoASH ligase activity. The assay performed at 25 degrees C for 10 min was linear with added microsomal protein up to 7 mug. The assay was linear with time up to 24 min when 1 mug of protein was employed. Fatty acid:CoASH ligase activity was strongly dependent on ATP and magnesium, was stimulated by Triton WR-1339, and was two- to fivefold dependent on added fatty acid. The filter assay is easier than existing assays based on incorporation of labeled fatty acid and is equally sensitive.


Automated modification of Duncombe's method for the ultramicro determination of serum free fatty acids. An automated continuous-flow method is described for estimating free fatty acids in serum using 25-mul samples. The procedure depends on the formation of copper soaps on the surface of a semipermeable membrane, their transfer into chloroform, and subsequent determination of dissolved copper. The membrane separating copper reagent and chloroform is supported between thin-channel dialysis plates. Chloroform extracts of serum free fatty acids are passed through the dialysis unit, and dissolved copper in the outflow is estimated colorimetrically. The procedure gives values that agree with a standard titrimetric method.


Microelectrode study of high grade block in canine Purkinje fibers. Previous reports from this laboratory described an electrotonic mechanism for simple impulse transmission through blocked segments of canine Purkinje tissue with slow diastolic depolarization assuming a vital role in second degree block. Utilizing an electrical blocking current, a blocked segment of canine Purkinje tissue was produced. Transmembrane events were recorded from  blocked segments during higher grades of block (3:1 to complete) to delineate further mechanisms responsible for a periodic distal boundary response. Our results confirm that slow diastolic depolarization is an important determinant in sustaining periodic impulse conduction. Its importance is related to (1) progressive decrease of the resting membrane potential toward threshold at the distal block boundary, and (2) augmentation of the transmitted electrotonic potential in accordance with voltage dependent changes in membrane resistance. These data further lend definition to the distinction between electrotonic, partially active, and active transmembrane potentials. Impulse transmission through a segment of inactivated tissue is electrotonic and slow diastolic depolarization plays an important role in the maintenance of periodic impulse transmission.


An analysis of Wenckebach periodicity.

An analysis of Wenckebach periodicity. His bundle electrograms of 40 patients developing Wenckebach block during atrial pacing and four with spontaneous Wenckebach block above the His were reviewed to determine the frequency of classical Wenckebach periodicity. Thirty patients had 143 Wenckebach cycles that were suitable for analysis. Cycles were evaluated for the following features: 1) the first A-H interval as the shortest, 2) the first R-R interval as the longest, 3) the last R-R interval as the shortest, 4) a progressive diminution of the increment of A-H interval prolongation, 5) a progressive diminution of the R-R interval and 6) the R-R interval containing the nonconducted A wave being equal to twice the A-A interval less the sum of the increments of A-H prolongation. Wenckebach cycles that occurred during atrial pacing were not significantly different from those that occurred spontaneously. Fifteen per cent of all cycles met all six criteria- 14% had five, 6% had four; 17% had three; 20% had two; 27% had one; and 1% had none. Short cycles were the most likely to show typical Wenckebach periodicity: 56% of the cycles with conduction ratios of 4:3, 28% with 5:4 and 4% with 6:5 met at least five criteria, whereas none of the 22 cycles having ratios 7:6 or greater had more than three features. The first A-H interval as the shortest was the most common feature occurring in 98% of cycles, whereas the features of a progressive diminution of the increments of the A-H interval prolongation or the progressive diminution of the R-R interval were the least common, occurring in 35% of cycles. These findings indicate, therefore, that classical Wenckebach periodicity is uncommon, especially when conduction ratios are 5:4 or greater. The implications of these observations and the suggested mechanisms are discussed and literature reviewed.


Left bundle branch block and coronary artery disease. This study tries the concept that left bundle-branch block (LBBB) connotes coronary artery disease (CAD). The findings indicate that prior studies both supporting of and in contradiction to the premise of a positive correlation have been biased by pre-selection of the patients reviewed. The data indicate, therefore, that LBBB is related to multiple entities. The major categories are CAD and/or hypertension myocardiopathy and aortic valvular disease. In addition, LBBB may develop during the acute phase of myocardial infarction. Its existence as a wholly benign entity has been documented as well. Further, this study adds still another group with LBBB. Six of the nine LBBB patients were female. Five of these, in spite of typical anginal histories, had no arteriographically demonstrable CAD. The absence of disease was surprising and the incidence of women with LBBB was greater than anticipated, thus providing some basis for suggesting that these women may be representative of still another group with LBBB. Further, this study supports the findings of Lewis et al by confirming an association between LBBB and a statistically shorter LCA mainstem (p less than 0.001).


Prognosis of left anterior hemiblock in acute myocardial infarction. Four of 32 patients with left anterior hemiblock and an acute anterior wall myocardial infarction died. Left anterior hemiblock was present on admission in 24 patients, and subsequently appeared in 8. Of the 28 survivors, 21 are still alive an average of 2.8 years after the acute myocardial infarction.


Relation of left ventricular contractile patterns in coronary artery disease to the electrocardiogram and vectorcardiogram. Two hundred thirty patients with coronary artery disease (CAD) were studied with left ventriculography, coronary arteriography, electrocardiography (ECG) and vectorcardiography (VCG) to determine how well left ventricular (LV) contractile defects could be predicted from the ECG-VCG patterns and how this was related to the coronary disease location and severity. Of 124 patients with infarction patterns on ECG-VCG about 50% had LV contractile defects localized to the corresponding ECG-VCG abnormalities, i.e., antero-apical asynergy with anterior infarction patterns, inferior asynergy with inferior infarction patterns, or antero-apical plus inferior asynergy with anterior plus inferior patterns. About 20% in each infarction group had unexpected synergy on ventriculography except for patients with dorsal infarction patterns (synergy in 68%) who are discussed as a special problem. Another 25-30% of patients had more extensive contractile abnormality than indicated by the ECG-VCG patterns. In 106 patients with left ventricular hypertrophy, normal QRS-abnormal T and normal QRS-T on ECG-VCG, 65-70% had synergy. However, 30-35% had asynergy in various combinations not suspected from the ECG or VCG. Coronary artery disease severity was less pronounced in patients with synergy than with asynergy and single vessel disease was more common in the former, 47% versus 18-30% in the latter. However, coronary artery disease severity was the same for all ECG-VCG groups except for anterior plus inferior infarction patterns where it was most severe.


Refractory paroxysmal supraventricular tachycardia incited by multiple mechanisms. A patient with refractory paroxysmal supraventricular tachycardia post acute myocardial infarction is presented. His bundle recordings and atrial stimulation studies suggest atrioventricular nodal reentry precipitated by three different mechanisms. Therapy required permanent coronary vein pacing and drugs.


Chronic binodal block with Wenckebach phenomenon. Coexistent sino-atrial and atrioventricular block with Wenckebach phenomenon is an extremely unusual cardiac arrhythmia. Observations on the natural history of sinus node dysfunction are rare. For over six years our elderly patient has been followed without symptoms but with increasing severity of atrio-ventricular block.


A simple method of checking the ECG paper speed. A simple method of determining accurate rates on ECG machines has been described. A short-wave radio, capable of tuning station WWV (5, 10 or 15 MHz) and a telephone amplifier may be used to place the precise 1 second intervals pulses on the ECG, from which exact paper speed is determined. This information may then be used to calculate the precise rate of an implanted pacemaker stimulus for evaluating pacemaker battery life.


The triple pulse generator: Clinical evaluation of a new multipurpose external pulse generator. This report describes our experience with a new, versatile battery operated, multi-purpose pulse generator especially designed for the electrophysiological investigation and treatment of cardiac arrhythmias in man. The unit was constructed according to our specifications and has been clinically in 75 patients over a period of 18 months. We have found the triple pulse pacemaker safe, reliable and functioning precisely according to specifications both clinically and when repeatedly checked with a storage oscilloscope.


[Role of the somitic mesoderm in the development of the rib cage of bird embryos. I. Origin of the sternal component and conditions for the development of the ribs (author's transl)]. The developmental origin of the sternal component of the ribs and the conditions for the development of the rib cage have been elucidated in bird embryos. Experiments consisted in homo- or heterospecific transplantations of 2-day-old quail or chick embryo somitic mesoderm into chick hosts of the same age in ortho- or heterotopic position along the cephalo-caudal axis. Results show that vertebral halves, ribs (not only their vertebral segment, but also their sternal component when they possess one), the trunk and intercostal muscles, as well as at least part of the scapula originate exclusively from the somitic material, while the sternum, ventral muscles and the other parts of the pectoral girdle are derived from the somatopleural mesoderm. The development of the rib basket is subjected to following rules: -only the somitic mesoderm of the prospective thoracic region (somites 19-26) is able to give rise to ribs. -only the somitic mesoderm of the posterior thoacic region (somites 22-26) is able to develop ribs with a sternal component. -the vertebral component of ribs can develop outside the thoracic region. -contrariwise, the sternal component can form only in the vicinity of the sternal anlage, i.e. within about three somites in front and rear of the normal limits of the thoracic region. It is concluded that the somitic mesoderm is already regionalized at a stage slightly preceding its metamerization and that the somatopleural territory of the sternum plays a morphogenetic role in the development of the sternal component of ribs, although it does not make a cellular contribution to their construction.


[Development of the thalamus in anurans during metamorphosis (author's transl)]. A developmental study of the anuran diencephalon from stage 30 to 2 months after metamorphosis has been done. At stage 30 in Discoglossus pictus, the diencephalic wall is divided into a superior area and an inferior area, where two kinds of cellular migrations are described. Comparison with the urodele diencephalon suggests that the building of a new area (area dorsolateralis) in anurans, by means of an 'external migration', characterizes a level of organization which is related to the optic system in Anura. During metamorphosis, opitcal morphogenetic influences would come from the tectum opticum to the dorsal thalamus. It is possible that the dorsal thalamus could be involved in binocular vision by way of tectofugal fibres. Some typical anuran diencephalic nuclei (i.e. nucleus corpus geniculatum laterale) are missing or are not well differentiated (i.e. nucleus rotondus) in Discoglossus. This species gives a simplified pattern of the anuran diencephalon.


Zona pellucida denudation, blastocyst proliferation and attachment in the rat. The mode of zona pellucida denudation and blastocyst proliferation and attachment was investigated in Wistar rats during day 5 of pregnancy and in ovariectomized pregnant animals given 5-0 mg progesterone daily until the 10th day, when 0-2 mug oestradiol was injected together with progesterone. During day 5 of pregnancy, when only 5 empty zonae were detected from 32 animals, zona denudation occurred by lysis, which took 6 h. Zona denudation occurred by shedding in the ovariectomized pregnant animals; 190 empty zonae were detected from 40 animals. Oestradiol administration to the experimental animals caused lysis of the shed zonae 24-30 h after oestradiol treatment. Air-dried preparations of the embryos showed that the blastomeres proliferated exponentially from a mean of 27-52 +/- 4-0 cells per embryo at 12.00 h on day 5 of pregnancy to 53-96 %/- 2-58 cells per embryo at 22.00 h, with a cell doubling time of 10 h in normal animals. In the ovariectomized animals, blastocysts were arrested at about the 100-cell stage on the 10th day. Oestradiol administration to the experimental animals did not induce mitosis; the embryos implanted without further cell division. The number of free embryos recovered declined from a mean of 12-0 per animal at 12.00 h to 4-25 per animal at 22.00 h on day 5 of pregnancy. The number of embryos recovered from the ovariectomized animals varied between one and 11 per animal. The results show that cell division continued in embryos within ovariectomized pregnant animals in the absence of oestrogen, until about the 100-cell stage. There was no further cell division before nidation when oestradiol was administered. The empty zonae were, however, lysed between 24 and 30 h after oestradiol treatment. The zona lytic factor appears to be oestradiol dependent and of maternal origin. A causal relationship exists between zona lysis and embryo attachment, which is independent of the number of cells per blastocyst.


Limb development in mouse embryos: protection against teratogenic effects of 6-diazo-5-ox-L-norleucine (DON) in vivo and in vitro. The glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), has been shown to inhibit biosynthesis of purines and glycosaminoglycans, presumably by blocking the glutamine-dependent steps in the biosynthetic pathways. The teratogenic potential of DON on the developing mouse limb-bud in vivo and in vitro was studies in an attempt to discriminate whether DON is exerting its teratogenic effect by interfering with glycosaminoglycan or purine metabolism. A single intramuscular injection of DON (0-5 mg/kg) to ICR/DUB mice on day 10 of gestation resulted in 76% resorption, while fetuses surviving to day 17 exhibited growth retardation, median cleft lip, and limb malformation. Concurrent administration of L-glutamine (250 mg/kg) provided no protection against resorption of malformations, while 5-aminoimidazolecarboxamide (AIC, 250 mg/kg) decreased the resorption rate to 34% without significantly altering the incidence of malformations. Injection of DON alone on day 11 resulted in 87% of fetuses exhibiting limb formations, with only 2% resorption. Concurrent injection of AIC decreased the frequency of limb malformations to 32% L-Glutamine, D-glucosamine, or inosinic acid were without any protective effect in vivo. DON (5mug/ml medium) added in vitro to organ cultures of day 11 mouse limb-buds caused all limbs to evidence cartilage abnormalities. In this system, either L-glutamine ro D-glucosamine (0-5 mg/ml medium) provided protection against DON effects while AIC (0-5 mg/ml medium) offered no protection in vitro. These data suggest that DON exerts its effects in vivo by interfering with purine metabolism while in vitro its teratogenic action may be interruption of glycosaminoglycan biosynthesis. This may reflect upon the relative importance of growth and differentiation to limb development in vivo and in vitro. These data infer that limb development in vitro relies more on the differentiative process (differentiation of cartilage) than on growth, whereas limb development in vivo is dependent, at this stage, to a greater extent on growth for normal phenotypic expression.


In vitro chondrogenesis of limb mesoderm from normal and brachypod mouse embryos. This paper describes the cytodifferentiation of hind limb mesodermal cells from 12-day-old normal and brachypod (pbH) mouse embryos grown in vitro at high densities. Over a 3-day culture period normal cells underwent aggregation, nodule formation, and coalescence of nodules into large masses of cartilage. This was associated at the biochemical level with a cessation of cell division, with a concomitant increase in metachromatic matrix, and synthesis of collagen. Under the described culture conditions the collagen synthesized by 48 h cultures was predominantly of cartilage type with an alpha1:alpha2 ratio of 9:1. A change in the collagen synthetic program was observed when the entire medium was replaced after 48 h incubation with fresh, serum-free medium. Under these conditions the alpha1:alpha2 ratio was 4:1. In contrast, brachypod cells plated at the same density appeared large, flattened, and stellate. Upon aggregation, normal nodule morphology was only rarely observed. More often large, irregular clusters formed from suspended cells loosely attaching to the surface aggregates. Concomitant with the marked changes in the morphology of the mutant cells was a linear increase in DNA synthesis and the appearance of many mitotic figures. A biochemical transformation in matrix synthesis was not observed, however. After a 24 h delay, mutant matrix accumulated and stained intensely with toluidine blue. Collagen was synthesized at approximately the normal rate and was of the cartilage type with an alpha1:alpha2 ratio of 9:1. When incubated in fresh, serum-free medium, the response of collagen subunit synthesis was identical to the normal cultures. In view of these results the possible manner in which brachypodism causes developmental anomalies of the limb skeleton is suggested.


The distribution of non-synaptic intercellular junctions during neurone differentiation in the developing spinal cord of the clawed toad. The distribution of intercellular junctions, other than synapses and their precursors, has been described in the developing spinal cord of Xenopus laevis between the neurula and free swimming tadpole stages. At the neurocoel, ventricular cells are joined in the apical contact zone by a sequence of junctions which usually has one or more intermediate junctions but also includes close appositions, gap junctions and desmosomes. This apical complex is more diverse than that reported in other vertebrate embryos and between ependymal cells in the adult central nervous system. Gap junctions are also found between ventricular cells and their processes near the external cord surface. However, no other special junctions occur in this location under the basement lamella which surrounds the cord. Punctate intermediate junctions are generally distributed between undifferentiated and differentiating cells and their processes but were not found in neuropil after stage 28. These results are discussed in relation to cell movements during neural differentiation, possible effects on the freedom of movement of ions and molecules through extracellular pathways in the emryo, and possible intercytoplasmic pathways via gap junctions which may be responsible for the physiologically observed electrical coupling between neural tube cells.


Fate maps and the pattern of cell division: a calculation for the chick wing-bud. Given the growth rate throughout an embryonic rudiment, one can calculate how its parts must shift as it gets bigger, and so plot fate maps showing their future positions. The calculation is done here for the chick wing-bud from stage 18 to 25, using published data for mitotic index and cell packing density, and new measurements of size and shape. Two main findings are: (1) the wing-bud in this period elongates almost uniformly along its proximo-distal axis. (2) Each limb segment derives from the tissue generated by one cell division cycle in the 'progress zone' at the tip.


The determination of polarity in the developing insect retina. The retina in Oncopeltus fasciatus is a polarized structure in which all ommatidia are orientated the same way. By a series of grafting experiments we have shown that the orientation of ommatidia is partly dependent upon the orientation of the epidermis from which they develop and partly on the orientation of the host eye. Grafts which invert the forsoventral axis of the presumptive eye epidermis do not disrupt the orientation of the nascent ommatidia. Grafts which are rotated by 90 degrees or 180 degrees produce altered patterns of orientation. The polarity of the epidermis is to some extent conserved in these cases. Significantly ommatidia at the graft/host border take up orientations intermediate between the extremes found in the graft and host. Small rotated grafts assume the polarity of the host retina. These results are compared with the effects of similar grafts on developing insect bristle patterns.


Positional information and pattern regulation in Hydra. Dynamics of regions at the boundary. The dynamics of boundary regions have been investigated mainly by axial grafting and with the emphasis on the head end. The time to resist inhibition of head-end formation and the ability to inhibit head-end formation have been assayed under a variety of conditions. The times increase with distance from the head end. The times required by a boundary region to aquire the inhibitory properties of a head end are longer than those required to aquire resistance to inhibition. Determination of a head end is faster at a cut surbface and at higher temperatures. The results are discussed in terms of a model involving two gradients. Some anomalous results are reported.


Positional information and pattern formation in Hydra. Dynamics of regions away from the boundary. The dynamics of regions away from the head-end boundary have been investigated by a variety of assays, and the changes appear to be slow. If, for example, a head end is grafted onto a peduncle to give H/56F, region 5 does not become a region 1 with 72 h. Such results support a gradient type model, and exclude a head end inducing a region 1 in the tissue adjacent to it. The changes can be interpreted if the positional value were effectively slowly diffusible: about 10 times more slowly than the positional signal. The results also have implications for studies on polarity reversal.


Cell death during the formation of tubular heart of the chick embryo. Light and electron microscopical examination of the heart of the chick embryo between stages 9 and 11 (Hamburger & Hamilton) revealed degenerating and dead cells specifically localized in the midline of endocardial tubes. The morphology of cell death in this system does not differ from that described in other embryonic tissues and organs. Phagocytosing cells are rarely seen. The results of this study show that a destruction of midline cells of both endocardial tubes takes place during the fusion of heart anlages. The possible roles of this cell death in the morphogenesis of the tubular chick heart are discussed.


[Role of the nervous system during the regeneration of an arm in a starfish: Asterina gibbosa Penn. (Echinodermata, Asteriidae)]. In Asterina gibbosa regeneration of a cut-off arm tip cannot occur if the radial nerve has been removed. This regeneration needs the presence of the oral nerve ring connected with the radial nerve intact up to the level of the wound tissues. The oral nerve ring can be considered as a central nervous system. Exposure of the radial nerve to irradiation induces a delay in regeneration. These data suggest there is a stimulation from the oral nerve ring up to the wound tissues. When the tissues involved in regeneration, except for the radial nerve, have been destroyed by irradiation, regeneration is inhibited. Stimulation by a healthy radial nerve is necessary to maintain differentiation and growth of the blasternal cells. The amount of blasternal growth and differentiation depends on the time when the radial nerve ceases to stimulate regeneration. The action of the nervous system is needed throughout the whole course of regeneration.


An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb. An analysis has been made of the pre-cartilaginous condensation stage in the development of the femur and tibia/fibula skeletal blastemata of the embryonic chick hind limb. Light microscopy serial sections were used to 'map' the mesenchymal cell condensations of both myogenic and chondrogenic anlagen in the limb-bud from stages 22 to 26 (Hamburger & Hamilton, 1951). Cell counts reveal that an increase in mesenchymal cell number per unit area occurs in the central chondrogenic locus at stage 24 (4 1/2 days) prior to matrix formation. Electron microscopy, using a simultaneous double fixation with osmium and glutaraldehyde, reveals that the pre-chondrogenic cells are characterized by large areas of close surface contact between adjacent cells, as compared with the extensive intercellular spaces associated with undifferentiated mesenchymal cells. The results are discussed and related to other investigations of in vivo chondrogenesis and to analyses of cellular events during in vitro chondrogenesis. These observations are consistent with the theory that condensations are formed by a process of aggregation rather than by localized increased mitosis.


On the origin of haemopoietic stem cells in the avian embryo: an experimental approach. It is currently accepted that stem cells of the definitive blood cell lines originate from the yolk-sac blood islands. Experiments were devised to examine the validity of this theory in the avian embryo. These involved grafting two-day-old quail embryos on to chick yolk-sacs of comparable developmental stages, i.e. before or shortly after the establishment of vascularization. The conclusions of the experiments are based on the possibility of distinguishing chick cell nuclei from those of the quail. In the developing haemopoietic organs (spleen and thymus) of quail embryos grafted on to the chick and subsequently incubated for 6-11 days, all cells, whether belonging to the granulopoietic, erythropoietic or lymphopoietic series, are of quail type. Thus these organs have not been colonized by chick stem cells. On the other hand, coelomic graft experiments show that the development of these organs is indeed dependent on an extrinsic colonization by haemopoietic cells; quail spleen or thymus rudiment, developing in the coelom of a chick is populated by chick cells. Thus no incompatibility which would prevent heterospecific colonization exists in this system. It is concluded that haemopoietic stem cells of the definitive blood cell series originate from some source other than the yolk-sac, and that this source must be intra-embryonic.


Time, place and positional value in the chick limb-bud. Quantitative experimental evidence is presented for the progress zone theory of limb development. The theory, here formulated mathematically, states that the parts of the limb are specified in proximo-distal succession by an autonomous timing mechanism operating in a 'progress zone' of undifferentiated growing mesenchyme under the influence of the apical ectodermal ridge. By the exchange of distal tips between young and old wing-buds, it is shown that there are no long-range morphogenetic signals from proximal to distal tissue. The width of the progress zone is calculated, and it is found autoradiographically that practically all its cells are dividing.


Ultrastructural studies of tw32/tw32 mouse embryos. Homozygous tw32/tw32 mouse embryos, obtained from both spontaneously ovulated and superovulated T+/tw32 females mated to T+/tw32 males, have a lethal period which extends from the 8-12 cell stage to the late morula stage. Most of the homozygous mutant embryos die at the early morula stage and are characterized by excessive amounts of cytoplasmic lipid, mitochondrial abnormalities, binucleated cells and nuclear lipid droplets. The excessive cytoplasmic lipid and nuclear lipid droplets distinguish 35-50% of the embryos (presumably tw32 homozygotes) from their litter-mates prior to the lethal period. The remainder of the distinguishing characteristics appear in the later (8-cell to late morula) tw32/tw32 embryos in frequencies high enough to be considered phenotypic expressions of the mutant genome. The present study indicates that the non-complementary tw32 and t12 alleles are in fact separate T locus recessive alleles.


Studies of t6/t6 mouse embryos.

Studies of t6/t6 mouse embryos. The phenocritical period of the t6/t6 genome extends from the late blastocyst substages through the elongated egg-cylinder stage. Most of the homozygous mouse embryos die during the short egg-cylinder stage. The viable egg-cylinder staged t6/t6 embryos can be distinguished from their phenotypically wild-type litter-mates at both the light and electron microscopic levels. The distinguishing characteristics of these embryos are aberrantly arranged entodermal cells, excessive cytoplasmic lipid and crystal-containing mitochondria. These same features are also characteristic of those mutant embryos which are developmentally arrested at both the short and elongated egg-cylinder stages. Over 50% of the t6/t6 embryos can be identified as early as the late blastocyst substages by the presence of large, electron-dense cytoplasmic lipid droplets.


ATP metabolism in tn/tn mouse embryos.

ATP metabolism in tn/tn mouse embryos. Total ATP, ATP/ADP ratios, and the rates of synthesis and turnover of ATP were determined for t12-, tw32- and T+/T+-litters at the 2-cell, 4-cell, 8-cell, early morula, late morula and late blastocyst stages. The results show that tn-litters have excessive rates of ATP metabolism up to the cleavage stage immediately before the stage of lethality for most tw32/tw32 embryos (early morula) and most t12/t12 embryos (late morula); mutant ATP metabolism then falls below control levels. After the death of the tn homozygotes, ATP metabolism in the tn-litters (composed of only T+/T+ and T+/tn embryos) returns to control levels. These data are discussed in relation to the phenotypes of the homozygous tn embryos.


Autoradiographic studies of tn/tn mouse embryos. High-resolution autoradiographic studies were used to determine whether t12/t12 and tw32/tw32 mouse embryos synthesize the excessive lipid which distinguishes these embryos prior to their death. The studies show that the tn homozygotes synthesize neutral lipid which is stored in intracellular lipid droplets. Cholesterol and phospholipid precursors are not incorporated into these droplets.


Pathogenesis of preaxial polydactyly of the hand in human embryos. Hand plates with preaxial polydactyly from 13 human embryos of the Carnegie stages 17-23 were examined macro- and microscopically. Morphological features in early pathogenesis of preaxial polydactyly are (1) an abnormal extension and a delayed involution of the apical ectodermal ridge on the preaxial border of the hand plate in stages 17 and 18, (2) a precocious development of an interdigital notch between the duplicated thumbs in stages 17 and 18, and (3) bifurcation of the distal part of the first digital ray in stage 19. A disorder of the interaction between limb ectoderm and mesoderm is considered to be the pathogenetic event.


Myogenesis in the trunk and leg during development of the tadpole of Xenopus laevis (Daudin 1802). Xenopus embryos pass through five behavioural stages between the end of neurulation (stage 20) and the accomplishment of free swimming (stage 33). These are (I) non-motile (stage 20-22) when the myotome muscle begins to differentiate; (II) pre-motile (stage 22-24) when the first striated fibrils are visible and contractions are possible; (III) early flexure (stage 24-27) when reflex responses are given and peripheral nerves are present; (IV) early swimming (stage 28-33); and (V) free swimming (stage 32-46) when co-ordinated swimming is possible but the myotome muscles are still uninucleate. At the onset of metamorphosis (stage 48-50) the myotome muscle becomes multinucleate, possibly by fusion with satellite cells at the ends of the fibres, and has the appearance of adult skeletal muscle. The hind limb of Xenopus passes through similar behavioural stages but at a later stage in development: (i) non-motile (stage 48-52) when little differentiation of the limb-bud has occurred but nerves are present; (ii) pre-motile (stage 53-54) when the limb trembles and muscles are just beginning to acquire striated fibrils; (iii) motile (stage 55-58) when the limb can make stepping movements and the muscles are striated and multinucleate; and (iv) fully functional (stage 60-63) when the limbs are fully differentiated. Unlike the myotome muscle the limb muscle becomes multinucleate before striated myofibrils are assembled. By stage 60 myotome and limb muscle are similar in appearance except that the myotome muscle has larger fibres with fewer nuclei than the limb muscle. In Xenopus, myotome and limb muscle become multinucleate at more or less the same time in the development of the tadpole. In the myotome this is long after contractility and nervous control have appeared, in the limb it precedes the formation of striated fibrils and the ability to contract. It is suggested that the difference in development of the myotome and limb muscles with respect to the stage at which they become multinucleate may be due to some substance produced just before or during metamorphosis.


The development of the retinotectal projection in Xenopus with one compound eye. Double-nasal and double-temporal compound eyes were constructed in Xenopus embryos at stages 32 and 37/38. A particular half was removed from the host eye anlage and replaced with the opposite half-eye from the contralateral side of a donor embryo. Control operations consisted of removing a half-eye and replacing it with a similar half from the ipsilateral side of the donor embryo. Whereas in control animals, each half-eye projects its fibres to the appropriate half-tectum, in operated animals each half of the compound eye spread its optic terminals across the entire rostrocaudal extent of the dorsal tectal surface. The area of tectal surface covered by ganglion fibre terminals was similar in operated animals mapped at successive stages of development to that previously observed in normal animals at equivalent stages. Therefore the factors responsible for the extended distribution of fibre terminals from each half of a compound eye must exist at least from mid-tadpole life, and thereafter be continuously present throughout development.


Skull growth in achondroplasic (cn) mice; a craniometric study. Skull morphology in achondroplasic (cn/cn) mice was compared with that of normal siblings in order to determine the effects of this chondrodystrophy on skull growth, particular attention being given to dimensions reflecting growth at the synchondroses of the cranial base, the nasal septal cartilage and the condylar cartilage of the mandible. The central section of the cranial base (basicranial axis) was reduced by 25 percent, the length of the viscerocranium by 18 percent and the length of the condylar process by 11 percent. The evidence indicates that these reductions are due to diminished growth at respectively the spheno-occipital and missphenoidal synchondroses, the nasal septal cartilage and the condylar cartilage. The relative sizes of the reductions in cranial base, vicerocranium and condylar process suggest that the growth of synchondrotic and septal cartilages is diminished to a greater extent than that of condylar cartilage. This finding is in agreement with the observations that condylar cartilage, unlike synchondrotic and septal cartilage, grows by surface apposition and that the principal defect in cn/cn mice is a disturbance of interstitial cartilaginous growth. The posterior extension of the basicranial axis of the cn/cn mice was reduced by 14 percent and the anterior extension by 2 percent. The width of the cranial base was decreased by 9 percent and the angle between the basicranial axis and its anterior extension was decreased by 3 percent. The length of the neurocranium was reduced by 19 percent in the cn/cn animals while the volume of the endocranial cavity was diminished by only 18 percent. The latter reduction is less than would be expected from the cube relationship between volume and linear dimensions but is readily accounted for by the lack of reduction in the height or width of the neurocranium, the slight flattening of the cranial base and the doming of the neurocranial vault.


The matrix of the optic vesicle-presumptive lens interface during induction of the lens in the chicken embryo. The cell coats of the presumptive lens cells and the extracellular interface between the lens rudiment and optic vesicle were investigated in the chicken embryo throughout the period during which lens induction is presumed to take place. Histochemical methods showed that the cell coats contained both glycoproteins and glycosaminoglycans. Autoradiography after [3H]glucosamine injection indicated incorporation of the precursor with subsequent localization primarily at the cell surface. No obvious changes in the properties of the coat were noted with the progression of early lens morphogenesis. The extracellular matrix at the interface between ectoderm and optic vesicle also contained glycoprotein and glycosaminoglycan. There was a heavy concentration of [3H]glucosamine-containing macromolecules in the area. Electron microscopy revealed that the interface consisted of the basement membrane systems of lens and optic vesicle, fused with their external fibrillar layers. In contrast to the findings on cell coats the density of the interfacial matrix increases appreciably during the lens induction period. Evidence suggests that the cells of the two ocular epithelia are themselves the source of the matrix materials. It is proposed that the macromolecules excreted by the epithelial cells into the interface interact at different concentrations to form aggregates of various structure by a process of self-assembly. This may be reflected in the different ultrastructure of the layers of the interfacial matrix. Quantitative changes in the density of the matrix, leading to increased adhesion between lens rudiment and optic vesicle, may restrict the lateral spreading of the lens cells and so fix the basal area of the lens rudiment. This, together with continued cell replication, may produce the cell crowding, placode formation and invagination characteristic of lens morphogenesis.


[Biological effects of solutions of non-histone chromatin-associated proteins on cell differentiation in vitro (author's transl)]. Nuclear non-histone (NNH) proteins, both chromatin-associated and otherwise, have been prepared in defined conditions, either from nuclei or from washed chromatin. When these NNH proteins are homospecific, they produce inhibitory effects on morphological differentiation of embryonic Urodelan Amphibian cells cultivated in vitro. When they are heterospecific, on the contrary, they have no action on the differentiation of those cells. It is concluded that the effects of the non-histone proteins on cell differentiation depend upon the relationship between the animal species which is used to provide the proteins and that used to provide the reactive material.


Epithelial autolysis during implantation of the mouse blastocyst: an ultrastructural study. An ultrastructural investigation was made of cell death in the uterine epithelium during the implantation of the mouse blastocyst. Cell death occurs in two phases: (i) early individual cell death, and (ii) later general cell death. Individual dead epithelial cells are phagocytosed and digested by trophoblast cells. At 95 h post coitum (p.c.) there are 1-4 dead cells; at 105 h and 113 h, 6-8 dead cells, and at 116 h, 2-3 dead cells. No lysosomal involvement could be identified in the death of these cells. The general breakdown of the uterine epithelium around the implanting blastocyst is first recognizable at the ultrastructural level at 113 h p.c., and continues until the adjacent deteriorated epithelium has been phagocytosed by the trophoblast cells at 119 h p.c. Ultrastructurally, from 113 h p.c. cytoplasmic portions of epithelial cells are trapped within cytosegrosomes, and there is an increase in size of the dense lysosomal bodies found in viable epithelial cells at 105 h p.c. The dense bodies, which are positively stained for acid phosphatase enzyme at 105 h p.c., increase in diameter approximately 3-5 times between 105 h p.c. and 119 h p.c. These results provide evidence of intracellular digestion of small portions of the cytoplasm possibly through the formation of cytosegrosomes which then fuse with residual bodies already present. Thus the evidence favours a process of autolytic breakdown of the uterine epithelium around the implanting blastocyst.


Differentiation in vitro of innervated tail regenerates in larval Ambystoma. Larval Ambystoma tail regenerates, innervated by the intact spinal cord, were cultured with their epidermal covering in modified Parker's medium (CMRL-1415) and the morphological integrity of the explanted regenerates was maintained with consistent success. The results show that the larval urodele tail blastema is capable of undergoing growth and differentiation when isolated in vitro and regeneration follows the normal in vivo pattern. A diffuse aggregate of blastema cells, which extended caudally from the cut end of the notochord at the time of explantation, underwent differentiation in vitro in the presence of the regenerating spinal cord. The importance of the nerve in regeneration is discussed in relation to the in vitro system.


In vitro studies of limb regeneration in adult Diemictylus viridescens: neural dependence of blastema cells for growth and differentiation. Explants of 99 adult newt forelimb blastemata (21- to 24-day regenerates) were cultured, with and without implanted dorsal root ganglia, in modified Parker's medium (CMRL-1415) for periods of 72-144 h. Growth and differentiation of the cultured blastemata were compared with ganglionated and non-ganglionated controls fixed at the start of the culture period. The results of these experiments establish that implanted spinal ganglia are able to sustain growth and differentiation of forelimb blastemata in vitro: active proliferation amongst the blastema cells was found to be correlated with the presence of an implanted ganglion. In addition, the blastema cells exhibited a differential growth response which was most pronounced when the ganglion was eccentrically implanted 2-3 days before explantation of the limb regenerate. These results suggest that a causal relationship exists between the position of the implanted ganglion and the localization of growth within the blastema. The nerve influence, believed to be mediated by a chemical factor(s), was localized in the region of the implanted neurons, indicating that a close association between the nerves and the responding blastema cells is essential for normal growth. The importance of the physical presence of nerves for the cultivation of blastemata in vitro is emphasized.


The growth of rats and mice vibrissae under normal and some abnormal conditions. The growth of hair from the mystacial vibrissal follicles of C3H mice and Wistar rats has been measured for up to seven cycles. Normally growth in length and thickness was regular and little affected by age or sex. Plucking vibrissae at any stage during the growing period was followed by the appearance of a new vibrissa 8-11 days later. Plucking when growth had ceased had no effect on the time of appearance of the subsequent cycle. New whiskers emerging after plucking grew at the normal rate. Withholding food slowed the growth of vibrissae within 1 day. Normal growth was re-established 3 days after return to full diet.


Polarity reversal in hydra by oligomycin. Intact hydra treated for 24 h with oligomycin gradually lose their head structures and the distal ends form feet. Grafting experiments show that the distal ends of treated animals induce proximal structures.


On the determination of the dorso-ventral polarity in Xenopus laevis embryos. When embryos, or dorsal or ventral half-embryos, of Xenopus laevis are subjected to unilateral restriction of oxygen supply, the posterior end will always appear at the aerobic side, while the development of the anterior end, oriented towards the anaerobic side, will be partly suppressed. The shorter the time treatment lasts, the more normal the development will be. When the restriction of oxygen effects an inversion of the dorso-ventral polarity, development is retarded, otherwise not. Measurements of oxygen consumption show a substantial reduction in the experimental embryos, as compared with normal ones. The change in oxygen consumption in inverted embryos is delayed relative to non-inverted ones, but there is no significant difference in the total consumption of oxygen. Our results support the idea that the dorso-ventral polarity is associated with a gradient in oxygen consumption, and various kinds of evidence suggest that oxygen consumption is, in part, required for the formation of Ruffini's flask-cells, responsible for the initiation of invagination. It is suggested that the basic mechanisms involved in the determination of the normal, and the inverted, dorso-ventral polarity are fundamentally different, the latter being in fact an induction of a new polarity.


Haploid mouse embryos obtained by microsurgical removal of one pronucleus. A pronucleus can be microsurgically removed from the fertilized mouse egg. Out of 145 haploid eggs obtained by this method and transplanted into the oviduct of pseudopregnant recipients, 36 multicellular embryos were recovered on the 4th or 5th day. On the 4th day all embryos were morulae composed of 8-50 cells, with the majority containing 8-16 cells. After an additional 24 h in vivo or in vitro the cell number increased considerably, sometimes up to as many as 80. Out of 36 multicellular embryos only one developed into a blastocyst while the others remained at the morula stage. Karyological investigations confirmed that the embryos were haploid and revealed that all were gynogenetic. Possible reasons for the absence of the androgenones and for the scarcity of blastocysts are discussed.


Developmental precocity in transferred mouse embryos influencing the teratogen response to salicylate. Asynchronous blastocyst transfer, supposed to equalize the developmental stage of native and alien embryos during the organogenic period, was used as a tool in a teratological investigation. A spurious protection by the transfer as such was shown to depend on a persisting asynchrony between native and alien foetuses. The initial difference of 24 h was not nullified, but decreased to 8 h. This difference allowed transferred foetuses to pass the period of maximum sensitivity before salicylate treatment.


The structural and functional development of the retina in larval Xenopus. The structural transformations of the larval Xenopus retina at successive stages of development, and concomitant changes in response characteristics of retinal ganglion cells, were studied using histological and electrophysiological techniques. The first sign of visually evoked electrical responses appears at about the time when the ganglion cells spread out into a single layer and shortly after the inner and outer plexiform layers become discernible. Initially giving simple "on" responses, the cells progressively change their response characteristics and become "event" units. Subsequently, "dimming" units can be identified. Throughout larval life, response properties of these two types become more distinct from one another and approximate to those found in the adult. So do the arborization patterns of the dendritic trees of the ganglion cells. Two types of branching patterns are identifiable in Golgi preparations. Around metamorphic climax, a new type of ganglion cell appears, coinciding with the emergence of "sustained" units electrophysiologically. After metamorphosis, the retina still grows both in thickness (mainly in the inner plexiform layer) and diameter. The three unit types change such they they come to show pronounced inhibitory effects from the peripheral visual field on the receptive field and each unit type acquires a distinct pattern of endogenous discharge.


Xenopus laevis cement gland as an experimental model for embryonic differentiation. II. The competence of embryonic cells. Explants of the blastocoelic roof of Xenopus laevis embryos were incubated for 6 h in Holtfreter solution containing 10 mM ammonium chloride and 10 mM sodium bicarbonate. After this incubation they were transferred for 5 days to Barth's physiological salt solution. Under these conditions the explants underwent differentiation into cement gland tissue. The ability to produce cement gland was highest when explants were dissected from young gastrulae (stage 10). Explants from younger and older embryos displayed much smaller cement glands. There is no difference in the response of ventral or dorsal portions of the blastocoelic roof of stage-10 embryos under these in vitro conditions involving ammonium chloride stimulation. Although the treatment changed the fate of some ectoblastic tissues, it was unable to force endodermal or mesodermal tissues of the blastoporal lip to differentiate into cement gland. The amount of cement gland in the ectoblastic explants varied according to the egg-batch and on the average accounted for 55 percent of the total tissue. The remaining tissue was undifferentiated. Most of this undifferentiated tissue originates from the adjacent layer of the ectoblast. Therefore it may be estimated that the cement gland accounts for 80 to 100 percent of the volume of the competent superficial layer.


Investigation of the determinative state of the mouse inner cell mass. I. Aggregation of isolated inner cell masses with morulae. Inner cell masses (ICMs) were dissected from 3 1/2- and 4 1/2-day blastocysts and cultured in contact with 2 1/2-day morulae. Blastocysts and morulae were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed that most were chimaeric. However, donor ICM-type isozyme was only detected in the embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic fraction. Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to 'outside' conditions as experienced by developing trophoblast cells. This is evidence that ICM cells, although not overtly differentiated, are determined by 3 1/2 days.


Investigation of the determinative state of the mouse inner cell mass. II. The fate of isolated inner cell masses transferred to the oviduct. Inner cell masses (ICMs) were dissected from 3 1/2- and 4 1/2-day mouse blastocysts and inserted into empty zonae before transfer to the oviducts of pseudopregnant mice. The ICMs survived in the oviduct for at least 2 days with little evidence of reduction in cell number. After 1 day, isolated 3 1/2-day ICMs were still viable, since they could form chimaeras when injected into 3 1/2-day blastocysts. However, no evidence of trophoblast formation could be detected in any of the isolated ICMs, despite exposure of some of their cells to 'outside' conditions. This is further evidence that ICM cells, although not overtly differentiated, are determined by 3 1/2 days. Although no trophoblast was formed, 3 1/2-day ICMs formed an outer endoderm layer after 1 day in the oviduct, as judged by light and electron microscopical evidence. It is suggested that cell position may be important in endoderm differentiation.


A receptor for the third component of complement in the human renal glomerulus. In the course of studying the nature of mononuclear cellular infiltrates in tissue sections of human kidney it was noted that indicator sheep erythrocytes densely coated with the third component of complement (C3) specifically adhered to all of the glomeruli in the tissue sections. The deposition of complement (C) within the glomerulus is a feature of many immunologically related renal diseases (1,2), yet the precise mechanism by which C is deposited remains unexplained. We feel that this observation, suggesting the presence of a receptor for C, is, therefore, of particular interest.


Immunologic injury in measles virus infection. II. Suppression of immune injury through antigenic modulation. Upon the addition of antibody to measles virus, measles virus antigens expressed on the surface of infected cells can be modulated from the cell's membrane in vitro. Removal of measles virus antigens from the surface of cells occurs relatively rapidly and is accompanied by a parallel reduction in the ability of antibody and complement to lyse these cells. Modulation of surface viral antigens can occur in the absence of cap formation and is fully reversible once measles virus antibodies are removed from culture medium. Protracted exposure of acutely infected cells to measles virus antibodies results in a population of cells that exhibit normal cytomorphology and growth behavior. These cells continue to express measles virus antigens internally, but not at the cell surface, and are refractory to immune lysis. Once antiviral antibody is removed, measles virus antigens again appear on the cell surface, giant cell and syncytial formation occur, and cell death follows. These observations may explain the persistence of virus in spite of a vigorous host antiviral immune response in certain chronic infections of man.


Phagolysosome formation in normal and colchicine-treated macrophages. Intracellular lysosomal fusion has been evaluated in cultivated mouse peritoneal macrophages by measurement of transfer of acid phosphatase to polyvinyltoluene (PVT)-containing phagolysosomes. Enzyme transfer was found to be directly and significantly related to the uptake of PVT and to be independent of time allowed for phagolysosome formation over time periods of 15 min to 18 h. In addition, the extent of transfer of lysosomal enzyme to phagolysosomes was unaffected by treatment of the cells with 10(-6) M colchicine, a dose which eradicates morphologically identifiable microtubules in this cell type within 2 h. The data indicate that intracellular fusion of lysosomes with phagosomes in the macrophage does not require formed microtubules and suggest that fusion occurs promptly after interiorization of inert particles.


Continuous replication of Friend virus complex (spleen focus-forming virus-lymphatic leukemia-inducing virus) in mouse embryo fibroblasts. Retention of leukemogenicity and loss of immunosuppressive properties. Exposure of NIH Swiss mouse embryo fibroblasts (MEF) to infectious Friend virus (FV) complex [containing defective spleen focus-forming virus (SFFV) and endogenous NB-tropic leukemia-inducing helper virus (LLV-F)] resulted in the productive infection of these cells by both SFFV and LLV-F. Stocks of SFFV derived after extensive growth in this Swiss MEF cell culture system are fully leukemogenic in adult mice for the induction of erythroleukemia and spleen foci. In addition, in vitro-derived LLV-F, when isolated free of SFFV, is fully leukemogenic for the induction of lymphatic leukemia when inoculated into susceptible newborn BALB/c mice. The host range of in vitro-derived FV complex (i.e., FV-TC) for focus formation in vivo is NB-tropic. Unlike in vivo-derived FV complex, FV-TC does not suppress the responsiveness of murine thymocytes to concanavalin A (Con A) in vitro. Rather, FV-TC acts as a mitogen to nonspecifically stimulate the proliferation of BALB/c thymocytes. The mitogenicity of in vitro-derived FV complex is directly associated with the presence of type-C virus particles, is a heat-labile and UV-sensitive property of the virus, and may be primarily due to LLV since equivalent amounts of LLV with or without SFFV present are equally mitogenic. One in vivo passage of FV-TC resulted in the total loss of this mitogenic property with the reappearance of full immunosuppressive properties. This result demonstrates a clear association between in vivo growth of FV and its ability to suppress mouse thymocytes, and suggests that FV complex (SFFV-LLV) is not inherently immunosuppressive for these cells. While the mechanism of this interconversion between immunostimulatory and fully suppressive virus is unknown, both virus markers appear to be dependent upon the presence of infectious FV.


Serum albumin is essential for in vitro growth of activated human lymphocytes. The effect of human plasma, the plasma protein fractions of Cohn, and crystallized serum albumin on the in vitro growth of human lymphocytes activated by concanavalin A (Con A) or bacterial lipopolysaccharide was compared. It was found that fraction V or serum albumin (SA) is essential for growth of activated T and B lymphocytes. The other plasma proteins have no effect. The growth response of Con A-activated T lymphocytes to increasing concentrations of SA is similar to the response to increasing equivalent concentrations of plasma suggesting but not proving that SA is the only growth-stimulating factor in plasma when added to a protein-free culture medium. The growth-promoting effect of SA is not due to the fatty acids or hormones bound to SA but is attributed to the albumin molecule itself or to a factor tightly bound to it. SA can also effectively replace plasma to stimulate proliferation of lymphocytes activated by allogeneic lymphocytes or purified protein derivative of tuberculin.