Diseases of the breast.

Diseases of the breast. As a result of health education programs that have stressed self-examination and the importance of early diagnosis and treatment, less than 5 percent of breast lesions seen in clinical practice today are malignant. Nevertheless, breast cancer is the leading cause of deaths due to cancer in women and the second most common breast lesion. Chronic cystic mastitis, the most common breast lesion seen in women, can often be treated conservatively by aspirating the cystic fluid. Close follow-up is necessary so that if carcinoma develops, it can be treated early. Carcinoma is unpredictable but not hopeless. Although controversy exists regarding treatment, radical mastectomy is still the preferred treatment in most cases. Other breast diseases include adenofibroma, a common lesion requiring excisional biopsy for positive diagnosis; nipple discharge, which should be carefully examined cytologically; and relatively rare breast lesions, including intraductal papilloma, adenosis, traumatic fat necrosis, and cystosarcoma phylloides.


The radiosensitization of hypoxic tumor cells by hyperthermia. The influence of hyperthermia on the survival of irradiated oxic and hypoxic HeLa cells was determined. A significant reduction in OER was obtained for irradiated cells subsequently cultivated at 42-degrees C for two hours compared to irradiated cells cultivated at 37-degrees C. These results suggest that heating irradiated hypoxic cells is an effective means of reducing the oxygen dependence factor in radiation effects.


Pneumomediastinum complicating pregnancy. Pneumomediastinum and subcutaneous emphysema is a rare complication of the second stage of labor. Although this entity runs a benign course, fatalities have been reported. Clinical findings of subcutaneous emphysema necessitate a chest X-ray to document pneumomediastinum. If pneumomediastinum is present, rapid delivery via elective forceps or even surgery may be considered to prevent serious complications.


Concanavalin A: its action in inducing Oocyte maturation-inducing substance in starfish follicle cells. Maturation of starfish oocytes is induced by 1-methyladenine produced in follicle cells under the influence of a gonad-stimulating hormonal peptide released from the nervous system. Concanavalin A stimulates isolated follicle cells to produce 1-methyladenine, which suggests that it has the same capacity as the peptide hormone to indirectly induce oocyte maturation.


Anomeric specificty of 3-0-methyl-D-glycopyranose against alloxan diabetes. The individual alpha and beta anomers of the nonmetabolized glucose analog 3-O-methyl-D-glucopyranose (3MG) were studied as protective agents against the alloxan toxicity to pancreatic beta cells in an in vivo rat model. The alpha 3MG provides greater protection than either the beta or the equilibrated compound, as indicated by plasma glucose concentrations 24 hours after the experiment. This specificity suggests that the beta cell membrane is extremely stereospecific, and that glucose or 3MG provide protection against alloxan injury directly by an interaction with the cell membrane and not by subsequent metabolism of the protecting compound.


Lactate dehydrogenase X: effects of antibody on mouse gametes but not on early development. A rabbit antiserum specific for LDH-X, the spermatozoal form of mouse lactate dehydrogenase, was prepared. This antiserum had no effect on fertility of female mice when injected before or after insemination. Similarly, there was no toxicity to the embryo when high concentrations of the antiserum were added to cultures of 2-cell and 8- to 16-cell embryos. There was, however, a moderate inhibitory effect on fertilization in vitro, which may be attributable to a direct action of antiserum to LDH-X on sperm.


Effect of antimycin A on sensivitivy of fertilized sea urchin egg cells to embryotoxic analogs of serotonin. Antimycin A blocks cleavage divisions in concentrations 1-10-15-1-10-14 g/ml (experiments on Arbacia lixula) or 1-10-11-1-10-9 g/ml (experiments on sea urchins of the genus Strongylocentrotus). Subthreshold and threshold concentrations of this antibiotic do not affect the sensitivity of fertilized Strongylocentrotus intermedius to 1-benzyl-2, 5-dimethylserotonin, but increase their sensitivity to 5-butyltryptamine by a factor of 3-5. These data provide the basis for a discussion of the possible nature of the reception of serotonin and other low-molecular-weight regulators of early embryogenesis.


The ultrastructure of mouse neuroblastoma cells in tissue culture. Neuroblastoma cells grown in suspension culture are round and have no distinctive structural characteristics. However, cells transferred to substrates flatten, develop long neurites, and assume the morphology of normal neurons. The resemblance of monolayered neuroblastoma cells to normal neurons is amplified by treatment with hypertonic medium; under these conditions, cell division is inhibited and the neurites become long and differentiated. The treated cells contain clusters of clear vesicles, 400-600 A in diameter, which are morphologically indistinguishable from the synaptic vesicles of normal neurons. Specialized cell contacts are observed between the treated cells as well as between confluent cells grown in normal medium.


[Primary pathological changes in cells infected with tick-borne encephalitis virus]. Synthesis and the content of RNA and protein in cells of the pig embryo kidneys infected with tick--borne encephalitis virus were studied by means of autoradiography and cytophotometry. Acceleration of synthesis and accumulation of RNA (in cellular structures) were estimated as over 125--200% of the initial level followed by a decrease in 12 hr when the virus release from the cells occurred. By the 2nd and 6--8th hr of infection (in the period of synthesis of early and late virus material), two peaks of protein contents over 123--131% were observed. The RNA and protein shifts are well correlated with the virus reproduction stages.


Urodynamic evaluation of female incontinence. Several diseases of the female bladder are accompanied by stress incontinence or some other form of incontinence. By using micturition cystourethrography, visualizing the bladder in lateral views, with simultaneous recording of micturition pressure and flow, a distinction may be made between the individual diseases, as they often exhibit characteristic radiological configurations and urodynamic patterns. It is also possible, by comparing with measurements in control material, to decide the best physiological method of abolishing the incontinence. This method of examination has proved particularly suitable in cases with a history of unsuccessful operation for incontinence. Stress incontinence has been found to be associated mainly with descent of the bladder, a reduced urethral resistance, and cystoceles; it also occurs as a sequel to radical operations on the true pelvis and in a number of neurological diseases. In practically all cases it has been possible to abolish incontinence by the following surgical plan: descent of the bladder to be treated by colpoperineoplasty, reduced urethral resistance and incontinence following radical hysterectomy or extirpation of the rectum by colposuspension, and cystoceles by reduction of the cystocele and colpoperineoplasty.


Urodynamic analysis of differential sacral nerve blocks and sacral neurectomy. Urodynamic analysis in 17 patients after long-acting anaesthetic root blocks of S2-S4 has shown that it is possible to increase bladder capacity and reduce uninhibited contractions without significant adverse effects on sphincter function. Satisfactory voiding dynamics are observed after blocks. This has provided a physiological rationale for selective sacral neurectomy in eight patients. Initial improvement after operation has been noted in seven cases.


Effect of posture on bladder and urethral function in normal pregnancy. A preliminary report. 25 pregnant women, 15 of whom developed stress incontinence in pregnancy, have been investigated by cystometry and urethrometry in both erect and supine posture. The resting bladder pressure rises throughout pregnancy in both erect and supine positions. Those who are not incontinent have normal bladder and urethral function. Incontinent patients show no residual urine, normal bladder capacity, and either detrusor instability and/or sphincter weakness. The stress of change of posture makes both bladder dysfunction and sphincter weakness more evident. Although the results of this investigation are not yet complete, there is a suggestion that detrusor instability may be produced by pregnancy in some patients and that in some instances recovery to normal bladder function occurs after delivery. It is postulated that these patients may be the ones who, in later life, develop stress incontinence due to bladder dysfunction.


Glanzmann's thrombasthenia; assessment of the response to platelet transfusions. The response to platelet transfusions was studies in two patients with Glanzmann's thrombasthenia. Antibodies to platelets were detected in one patient who had failed to respond to platelet transfusions, and bled during and after surgery despite fresh whole blood transfusions. The other patient had no detectable antiplatelet antibodies, exhibited improved platelet function when normal platelets were added to her platelet-rich plasma and experienced a favorable response to platelet transfusions during major surgery. These cases demonstrate the importance of antiplatelet antibodies as a determinant of a patient's response to platelet transfusion, the value of this therapy in qualitative platelet disorders, and the potential usefulness of in vitro studies to predict the outcome of platelet transfusions in such patients.


Proliferative and functional impairment of pancreatic epithelial cells maintained in vitro. Studies on the proliferative and functional properties of epithelial cells from human and guinea pig pancreatic tissue were presented and discussed. A novel technique for the isolation of epithelial cell groupings as colonial aggregates in two dimensional culture was utilized. More conventio;al clonal analyses were also performed. Irrespective of the methodology employed, epithelial cells from both species exhibited reduced proliferative activity with time in vitro. Total degeneration occurred in less than five months in every case studied. Aggregates and colonial aggregates, formed using fractionated suspensions containing up to 95% guinea pig acinar cells, were found to release amylase only during the first few days in vitro. Positive response to secretogogue stimulation was interpreted as evidence favoring the thesis that permanent cellular damage had not been sustained during tissue dissociation. These findings were discussed with reference to the phenomenon of human fibroblast degeneration with time in culture. Differences between fibroblast and epithelial cell behavior in vitro and in vivo were emphasized.


Observations of age and environmental influences on the thymus kept in tissue culture. In tissue cultures of thymus of rats of various ages only the cells of Gomori- and PAS-positivity showed iodine incorporation as demonstrated by radioautography. Above other cells of the migration zone no grains indicating iodine accumulation were observable. The capacity of iodine accumulation depends on the age of the animal, namely it decreases with the ageing.


Relationship between cell kinetic changes and metabolic events during cell senescence in vitro. Cell kinetic studies performed throughout the lifespan of fibroblasts with a limited lifespan in vitro have led to the conclusion that although division slows down, almost all cells are able to divide until the last subcultivation. The prolongation of the division cycle is primarily due to the impariment of mechanisms preceding DNA synthesis and mitosis. An attempt was made to distinguish between primary and secondary changes and to correlate the findings concerning cell kinetics with alterations observed at the molecular level. A decline in protein synthesis was the first modification detected. The two parameters that are always present during cell senescence in vitro, i.e., and increase in cell volume and a decrease in saturation density could be due respectively to a change in cell permeability and a decline in ribosome synthesis. The latter could also be the step responsible for the limited potential of division.


The association of fixed and dynamic left ventricular outflow obstruction. Twelve patients were investigated echocardiographically and angiographically and were shown to have severe fixed obstruction to the left ventricular outflow tract.  Eight had valvular stenosis, and four had discrete subvalvular membranes.  Two of the patients had additional dynamic obstruction of the left ventricular outflow tract.  This was recognized preoperatively by echocardiography because of abnormal systolic motion of the mitral leaflet. At the time of definitive surgery for relief of the fixed obstruction, the additional dynamic obstruction was identified and treated, since persistent residual obstruction may lead to death in the immediate postoperative period or to long-term symptoms.  The dynamic left ventricular outflow obstruction is probably a result of the hypertrophy produced by the fixed obstruction.


Coronary arteriographic findings in 1,000 women under age 50. In a consecutive series of 1,000 women under age 50 examined by coronary cinearteriography principally for evaluation of chest pain, 236 had at least 50 percent narrowing of one or more coronary arteries. The youngest, and the only patient who died, was 26 years old. The anterior descending coronary artery was the vessel most frequently involved; the right coronary artery was most often totally occluded. The amount of arterial involvement seemed to be related to the duration of symptoms. Only 10 percent of women with serum cholesterol levels under 200 mg/100 ml had significant coronary artery disease, whereas 44 percent of those with levels over 275 mg/100 ml had significant disease. Electrocardiographic evidence of myocardial infarction was uncommon in women with single vessel disease, but left ventriculograms disclosed that additional patients had areas of decreased contractility. Eleven of 46 women with electrocardiographically diagnosed myocardial infarction had no significant coronary artery disease. The cause of the electrocardiographic finding remained obscure in some. Although there was an excellent correlation of clinical diagnoses with arteriographic findings in women thought not to have angina pectoris, only half of those thought to have angina pectoris had significant coronary artery disease. This finding may be a major reason for recommending coronary arteriography for the evaluation of angina-like pain in women under age 50.


Studies on the progestational endometrium of the rabbit. II. Electron microscopy, day 0 to day 13 of gonadotrophin-induced pseudopregnancy. The fine structure of the endometrial epithelium of the pseudo-pregnant rabbit from the day of induced ovulation (day 0) to the 13th day is here correlated with previously defined light microscopic phases. In Phase 1 (0-1 day), in which there is a presumed "priming" of the endometrium by ovarian steroidal hormones, no changes were observed. In Phase 2 (1-3 days), in addition to mitotic activity, the epithelium showed a disappearance of the mucification and lymphocytic migration typical of Phase 1 and also of the non-pregnant or "estrous" phase, and showed other nuclear and cytoplasmic changes which probably reflect endogenous growth and protein synthesis. In Phase 3 (4-6 days), two distinct populations of reacting cells were present: (1) surface and cryptal cells investing the now folded mucosal surface, and (2) glandular cells. The first group showed characteristic dome-like protrusions of the cytoplasm into the lumen, and also showed distinct cytoplasmic and nuclear changes which appear to be a prelude to the succeeding phase of fusion but are not necessarily secretory in type. The glandular cells, in contrast, showed cytoplasmic changes which appear to reflect active secretory activity (hypertrophy of the Golgi area, cytoplasmic vacuoles containing electron-opaque material, etc.). This phase coincides with the maximal secretion of uterine-specific proteins, and electron-opaque material is abundant within the endometrial lumen. In Phase 4 (6-8 days), the surface and cryptal epithelium undergoes a transformation into multinucleated cells, the result of a process of lysis of intervening plasma membranes, the precise mechanism of which (i.e., with or without initial membrane fusion) was not determined. Cell fusion proceeded earlier and more actively mesometrially than antimesometrially. The glandular cells showed evidence of reduced secretory activity, but did not at any stage undergo multinucleate-cell transformation. In Phase 5 (8-13 days) there was progressive fusion, and the number of nuclei per cytoplasmic sac appeared increased, presumably due to the continued action of progesterone which is maximal during this phase. Glandular cells showed further reduced secretory activity but remained columnar. Ciliation of the epithelium was sporadic in the pre-secretory phases and rare or absent in the secretory and fusion phases; it became widespread during the phase of decline after day 14, a period which will not be included in this study. The fine structure of the ciliated cells was the same at all stages; there was no evidence for their origin from a reserve population; it is possible that they arise by modification of the multinucleated cells. Cytoplasmic crystals and intramitochondrial densities or lamellae were observed during the secretory and fusion stages, the former only in the glands, the latter in the surface and cryptal epithelium. They appear to be associated with rising or maximal progesterone secretion.


Variability of creatinine excretion of normal, phenylketonuric and galactosemic children, and children treated with anticonvulsant drugs. Creatinine per minute excretion rates in individual voidings varied as much as 300-500 percent of the 24-hour value for several children. Creatinine excretion rates were significantly more variable for children treated with anticonvulsant drugs than for normal children. Riboflavin-creatinine ratios determined on individual voidings were variable but adequate although total riboflavin was low. No time of day was found when creatinine excretion was representative of the 24-hour value. The creatinine height index when calculated from timed individual voidings during a 24-hour period varied for some children from 0.5 to more than 1.5 times published norms.


Effect of oral contraceptive agents on nutrients: II. Vitamins. Clinical, biochemical and nutritional data were collected from a large population of women using oral contraceptive agents. Higher incidence of abnormal clinical signs related to malnutrition were observed in the lower (B) as compared to the higher (A) socioeconomic groups, and also in the nonsupplemented groups as compared to the supplemented groups in the B subjects. As a rule the intake of oral contraceptive agent subjects of vitamin A, C, B6 and folic acid did not differ from that of the controls As expected, subjects from the supplemented groups had higher intake of vitamin A, C, B6, thiamin, riboflavin and folic acid, and A groups had higher intake of vitamin C, B6, riboflavin and folic acid. Increased plasma vitamin A and decreased carotene levels were observed in oral contraceptive agent users. In general oral contraceptive agents had little or no effect on plasma ascorbic acid. Urinary excretion of both thiamin and riboflavin in subjects using oral contraceptive agents were lower in A groups. Erythrocyte folate and plasma pyridoxal phosphate was decreased in A groups due to oral contraceptive agents. Subjects who took supplements had higher levels of plasma vitamin A, ascorbic acid and folate. But urinary thiamin and riboflavin were higher only in group A subjects who took supplements.


Chronic alcoholism and endogenous gastrin. Gastirn blood levels after the ingestion of a meat meal, either alone or associated with ethanol (1.0 gm./kg.), are higher and better sustained in dogs treated chronically with alcohol than in control animals. This greater gastrin-releasing capacity of the dog gastric antrum would be responsible for the increased parietal cell mass and gastric acid secretion shown by animals subjected to chronic alcohol intoxication.


A structural analysis of the myogenic control systems of the human Fallopian tube. The structural basis for myogenic and neurogenic control of motility of human Fallopian tubes was studied. In the ampulla distinct muscle layers were absent, and bundles of cells oriented in different axes were in contact with one another by means of randomly located simple apposition contacts; no nexuses were present. The organization was consistent with a spread of electrical activity in either direction throughout all muscle bundles. Nerves were not found in close proximity to muscle and were mostly peripheral, surrounded by connective tissue sheaths and by Schwann cells within the sheath. They could play little role in control of ampullar motility. In the isthmus muscle bundles were grouped into innermost longitudinal layer, circular layer, and outermost layer of mixed orientation. Cell-to-cell connections were mainly by simple apposition contacts; no nexuses were found. There were such connections between cells of bundles oriented in different axes. The organization of muscles suggested that electrical activity could spread in either direction and throughout all layers. Nerves like those in the ampulla were located peripherally, and smaller bundles without connective tissue sheaths were in circular muscle. Few close contacts between nerve and muscle were observed.


The effects of long-term administration of 3H-digoxin to the pregnant ewe upon the cardiovascular hemodynamics of the fetal lamb. Placental transfer of tritiated digoxin in the ewe and its effect upon the left ventricular output (LVO) of the exteriorized fetal lamb were studied. Seven ewes were given 6 mug per kilogram of body weight of tritiated digoxin daily (20 per cent of total digitalizing dose 0.03 mug per kilogram of body weight) during their entire pregnancies. The fetuses were exteriorized between 130 and 140 days of gestation, and the LVO was determined serially by the dye-dilution technique for one hour. The mean tissue concentrations in millimicrograms per gram for the ewes were: heart, 5.7 plus or minus 1.76; liver, 3.38 plus or minus 1.38, kidney, 01.9 plus or minus 12.3. Corresponding values for the fetuses were: 1.55 plus or minus 1.01, 0.78 plus or minus 0.35, and 2.48 plus or minus 1.18. The mean serum level for the ewe at the end of one hour was 1.65 ng. per milliliter plus or minus 0.48, and that for the fetus was 1.22 plus or minus 0.20. The amniotic fluid contained 1.45 ng. per milliliter plus or minus 0.27. At the end of one hour, the mean LVO of the fetus had decreased by 39 plus or minus 15 per cent of its control value. Although the fetal serum level of tritiated digoxin was similar to that found in the mother and the amniotic fluid contained a significant amount of tritiated digoxin, the fetal tissue concentration of tritiated digoxin was not sufficient to produce any demonstrable inotropic effect.


Diurnal variation of intraocular pressure in primary open-angle glaucoma. Intraocular pressure was measured with a Goldmann applanation tonometer in 12 normal individuals (24 eyes), 14 ocular hypertensives (28 eyes), and 14 patients (27 eyes) with primary open-angle glaucoma every hour for 24 hours. In most subjects, pressure was highest sometime during the day and pressure elevation before rising was not demonstrated. The lowest intraocular pressure was most frequently observed early in the morning, whether the patient was normotensive or hypertensive. Fourteen of 27 glaucomatous eyes had intraocular pressure below 20 mm Hg early in the morning.


Photocoagulation in venous occlusive disease. Fifty-one eyes with branch or tributary retinal venous occlusive disease underwent photocoagulation (1970-1973) after certain criteria were met: reduced visual acutity, fluorescein angiographic documentation of vessel permeability changes, and either dye leakage into the macula or neovascularization of the retina, or both. Eighty-four percent of the eyes responded to treatment with an improvement or stabilization in visual acuity, a reduction in collateral vessel caliber and permeability, and reduced dye accumlation in the macular area and increased fluorescein transit time. Photocoagulation is an affective means of reducing macular edema resulting from vascular compensatory changes following venous occlusion and decreases the risk of vitreous hemorrhage from sites of retinal neovascularization.


Tissue culture methods for testing the toxicity of ocular plastic materials. A screening method using tissue culture techniques determined inertness of ocular alloplastics. Secondary rabbit kidney cells revealed the presence of toxic constituents by absence of growth or cytopathic changes, or both. The optical portions of the lenses studied were nontoxic, whereas the polyamide loops of one lens were toxic. A keratoprosthesis that had been in situ in a patient for two years was highly toxic in culture. A neutralized agar overlay technique was not sufficiently sensitive to screen anterior chamber implants. The tissue culture technique, in combination with chemical, optical, and animal experimentation, may be helpful in determining which implant systems are most suitable for in vivo use.


Twins in a child psychiatry clinic.

Twins in a child psychiatry clinic. The authors found that 22 of the 2,015 referrals to a children's psychiatric clinic during a six-year period were twins--half the number that would be expected on the basis of the national incidence of twins. Evidence of minimal brain dysfunction was found in a higher percentage of the twins than in a matched nontwin group. The authors suggest that factors often inherent in twins (e.g., prematurity and birth difficulties) may be related to this finding.


A comparison of traditional and symptom-checklist-based histories. The authors compared the results of independent evaluations of 96 patients, each of whom was given a traditional psychiatric interview, resulting in a narrative report, and a structured interview based on a self-report questionnaire, resulting in a standardized report. Although the latter method produced a higher frequency of report of all symptoms, the overall accuracy of diagnoses was similar for the two methods. The authors enumerate the potential benefits of the use of standardized histories.


The selection of a psychiatric curriculum for medical students: results of a survey. A survey of 131 psychologists, psychiatrists, and nonpsychiatrist physicians taken to determine what doctors of medicine should know about psychiatry revealed that of the 21 topics assessed, there was a significant intergroup agreement on the 10 most important and the 5 least important topics. Interviewing, suicide evaluation, the chronically ill or dying patient, and psychiatric referral received high ratings by each group, and psychoanalytic theory, psychodynamics, and mental retardation received uniformly low ratings. The authors believe that these findings provide useful information for planning an undergraduate curriculum in psychiatry.


Dipetalonema streptocerca (Macfie and Corson, 1922): description of the adult female. Morphologic features of the adult female Dipetalonema streptocerca and microfilariae are described and illustrated from serial paraffin sections of biopsied skin from eight Africans. Characteristic features include a maximum diameter of 85 microns, a thin, smooth cuticle, and uteri that fill the body cavity. D. streptocerca is briefly compared to Onchocerca volvulus.


Evaluation of whole and purified hydatid fluid antigens in the diagnosis of human hydatidosis by the immunoelectrophoresis test. Whole sheep hydatid cyst fluid (WHF) and purified hydatid fluid antigens (PHF) were evaluated in the immunoelectrophoresis (IEP) test for the diagnosis of human hydatid disease. A higher sensitivity was obtained using the PHF than with the WHF antigen but false positive results were observed with the former in 22.4% of the 102 non-hydatid sera examined. Using the presence of arc 5 as the criterion of positivity however, no false positives were observed in non-hydatid sera with the WHF antigen. It is postulated that the number of bands other than arc 5 may be of value in the diagnosis of hydatid disease by the IEP test, in cases where this diagnostic arc is absent.


Management of cystic disease of the breast. The sudden appearance of a large cyst in the breast is a frightening experience to the average female patient. Needle aspiration of the mass not only alleviates this fear quickly but also is a safe and economical method of management. Yet, surveys show that the majority of surgeons still do not routinely aspirate breast cysts. The concomitant use of progesterone apparently reduces the frequency of recurrence of these cysts. In addition, follow-up studies indicate no increased incidence of carcinoma after aspiration.


Meningeal mycosis fungoides: clinical and cellular characteristics. A patient with mycosis fungoides developed meningeal disease while his skin disease was in remission with systemic chemotherapy. His central nervous system involvement with mycosis fungoides was controlled with intrathecal methotrexate for 7 months. The proliferating cells recovered from the spinal fluid showed similarities to the Sézary cell by light and electron microscopy. Surface receptor sutudies suggested that these cells were lymphoid cells of thymic derivation. Although mycosis fungoides has been shown to spread to the central nervous system in autopsied cases, reports of clinical neurologic disease are rare, and in only one earlier report have malignant cells have been found in the spinal fluid. Thus, as in other lymphoproliferative disorders, prompt consideration of meningeal involvement in a patient exhibiting neurologic symptoms while in peripheral remission may allow earlier treatment of this complication.


Brain abscess: a review of recent experience. From 1961 through 1973, 60 patients with brain abscess were treated at the Mayo Clinic by surgical excision and antimicrobial therapy. The operative mortality was 17%, which was identical to a similar series reported in 1959. The patients who had the diagnosis of brain abscess confirmed at autopsy and who never underwent operation (24 patients) were more elderly than the group operated on, and a higher percentage had multiple brain abscess. Severe systemic disease or multisystem disease, or both, usually precluded a vigorous surgical approach among the nonsurgical group. Etiologic, diagnostic, and microbiologic factors were examined to develop an approach to antimicrobial therapy.


Hemangioma-thrombocytopenia syndrome; a disseminated intravascular coagulopathy. A patient who had multiple hemangiomas and for whom results of coagulation studies were consistent with hemangioma-thrombocytopenia (Kasabach-Merritt) syndrome and a microangiopathic hemolytic process was asymptomatic prior to surgical removal of a lipoma. Postoperatively, she developed bleeding secondary to a disseminated intravascular coagulopathy. Her bleeding was controlled by anticoagulation therapy. A review of the literature provides evidence suggesting that the hemangioma-thrombocytopenia syndrome is due to a disseminated intravascular coagulopathy initiated within the hemangioma. Surgical trauma may precipitate exacerbations of the coagulopathy in these patients and all unnecessary surgical procedures should be avoided.


Strain-specific degradation of a viral glycoprotein in Newcastle disease virus-infected cells. In cells infected with mesogenic or lentogenic strain of Newcastle disease virus the level of neuraminidase and hemagglutinin activities sharply decreased after the addition of cycloheximide. With two velogenic strains such decreases did not occur. The infected cells were labelled with 14C-amino acids (leucine or valine) and further incubated with an excess of unlabelled precursor. Polyacrylamide gel analysis revealed a decrease of the peak correspondig to the "large" glycoprotein after the chase in cells infected with meso- or lentogenic strain (Beaudette, B1). In the cells infected with velogenic strains (Italia, Herts) no such decrease was observed. The degradation of the "large" glycoprotein as the cause of the decrease of hemagglutinin and neuraminidase activities in cycloheximide-treated cells and its possible relation to virulence is discussed.


Characterization of inactivation of myxoviruses and paramyxoviruses by hydroxylamine, N-methylhydroxylamine and O-methylhydroxylamine. A study of the mechanism of myxovirus and paramyxovirus inactivattion by hydroxylamine, O-methylhydroxylamine and N-methylhydroxylamine was conducted. Influenza A (WSN) was used as the sensitive myxovirus and Newcastle disease virus (NDV-L) was used as the relatively resistant paramyxovirus in certain experiments. Inactivation was found to be rapid (15 minutes) and mose effective at high concentrations (2M). All three compounds significantly decreased the hemagglutination titer of WSN treated at pH smaller than or equal to 5.0. There was no detectable change in NDV hemagglutination titer. Adsorption of hydroxylamine (pH 7.0) inactivated WSN appeared normal; however, the rate of adsorption was decreased when virus was inactivated by (pH 5.0) O-methylhydroxylamine. Equilibrium density gradient centrifugation in potassium tartrate showed no density changes in inactivated virus. WSN inactivated virus. WSN inactivated with 14C-O-methylhydroxylamine and subjected to RNA extraction showed greater than or equal to 35 percent of the 14C in the phenol phases and 21 per cent in the RNA. The 14C-O-methylhydroxylamine associated with the RNA of insensitive NDV was about 3 per cent of that associated with sensitive WSN-RNA. Hydroxylamine has no apparent effect on paramyxovirus (NDV) hemagglutination titer and less 14C-O-methylhydroxylamine is associated with the RNA of this virus. The results suggest these compounds may affect both the RNA and the envelope portion of myxoviruses (WSN) to produce inactivation.


Viral polypeptide composition of Japanese encephalitis virus-infected cell membranes. Chick embryo cells infected with Japanese encephalitis virus were separated into smooth and rough membrane fractions, as judged by electron microscopy.  Polyacrylamide gel electrophoretic analysis of the membrane fractions showed them to contain all of the virus specific polypeptides; however, the relative proportions of the polypeptides varied among smooth and rough membranes. The relationship of these observations to current concepts of group B arbovirus morphogenesis is discussed.


Thermostability of Newcastle disease virus strains of different virulence. The thermal inactivation rate constant for infectivity of a total of 24 Newcastle disease virus (NDV) strains was determined at 50 degrees and 56 degrees C. The greater part of the examined NDV strains were found, irrespective of virulence properties, to be thermostable, since the loss of infectivity titre did not exceed 2 logarithmic orders after exposure at 50 degrees C for 60 minutes. Thermostable (I+) and thermolabile (I minus) strains with respect to infectivity were uniformly encountered among the 12 avirulent (lentogenic) and 10 fully virulent (velogenic) strains studied. Strains with both heat stable (Ha+) and heat labile (Ha minus) haemagglutinin were found in the lentogenic group, whereas haemagglutinins of all examined velogenic strains were heat stable. On the basis of I/Ha character, i.e. combination of thermosensitivity of infectivity and haemagglutinin, all examined strains could be classified into one of three categories: I minus Ha minus (8 lentogenic strains), I+Ha+ (4 lentogenic and 7 velogenic strains) and I minus Ha+ (2 mesogenic and 3 velogenic strains). The possible fourth combination, I+Ha+ minus, was not encountered among the strains studied. Determination of the I/Ha character may be helpful in clarifying the origin of a lentogenic NDV strain.


Relief of sever left ventricular outflow obstruction in a case of hypertrophic obstructive cardiomyopathy treated with practolol. The clinical and haemodynamic findings in a patient with hypertrophic obstructive cardiomyopathy and severe left ventricular outflow obstruction are presented. Treatment with an increasing dose of orally administered practolol up to a maximum of 1200 mg a day, resulted in symptomatic improvement, and abolition of the resting gradient when the patient was recatheterized six months later.


Immobilized carboxypeptidase A as a probe for studying the thermally induced unfolding of bovine pancreatic ribonuclease. A method for the preparation of Sephadex-immobilized carboxypeptidase A is presented. This form of the enzyme has the same specific activity as the soluble enzyme at room temperature, but retains its activity at higher temperatures (60-70 degrees). This preparation of immobilized carboxypeptidase A was used, as a proteolytic probe, to investigate the thermally induced unfolding of the C-terminus of ribonuclease A. This technique indicates that the C-terminal residues of ribonuclease A do not unfold until the high-temperature region of the thermal transition (as determined by ultraviolet difference spectrophotometry and optical rotation).


Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase. A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.


Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting. The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.


The rapid labeling of adenosine triphosphate by 32P-labeled inorganic phosphate and the exchange of phosphate oxygens as related to conformational coupling in oxidative phosphorylation. Evidence is presented that extends and amplifies the concept that in oxidative phosphorylation energy input serves to bring about release of ATP formed at a catalytic site by reversal of hydrolysis. The evidence with beef heart submitochondrial particles includes additional demonstration of uncoupler insensitive Pi leads to HOH exhchange, demonstration that this exchange is sensitive to the specific phosphorylation inhibitor, oligomycin, and demonstration that the small burst of uncoupler-insensitive ATP, rapidly labeled after addition of a tracer of 32Pi, behaves in a manner consistent with its participation as a membrane-bound intermediate in the Pi leads to HOH exchange. In addition, data are presented showing that addition of hexokinase plus glucose to submitochondrial particles in presence of ADP and Pi considerably lowers the Pi leads to HOH exchange but that further addition of cyanide or 2,4-dinitrophenol or both has little additional effect. Such data are compatible with no energy requirement for formation of bound ATP. However, with a large excess of hexokinase, the rate of the Pi leads to HOH exchange is further depressed. This could reflect some use of energy to promote formation of ATP at the catalytic site or to maintain the integrity of the phosphorylation system. Relationships of these findings to related information in the field are discussed.


Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges. The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.


Nonidentical alkylation sites in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. These studies establish the specificity of 3,3,3-trifluorobromoacetone for reaction with the active site cysteines of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and suggest the potential use of trifluoroacetonyl groups as 19F nuclear magnetic resonance probes for study of symmetry relations between the four protomers of the enzyme. The alkylation of the holoenzyme follows biphasic kinetics and indicates either preexistent or induced nonequivalence among the sites; these effects are not predisposed by a low coenzyme/enzyme ratio. Two additional alkylation sites not at the active centers are created by acylation with beta-(2-furyl)acryloyl phosphate: it is concluded that pseudosubstrates cause an intramolecular rearrangement which exposes two sulfhydryl functions besides those of the active site (Cys-149).


Nuclear magnetic resonance studies on the solution conformation of histone IV fragments obtained by cyanogen bromide cleavage. Two histone IV fragments obtained by cleavage at Met-84 by cyanogen bromide have been examined by proton magnetic resonance (PMR) spectroscopy as a function of temperature, peptide concentration, ionic strength, and pD. Sedimentation and gel electrophoresis studies on these peptides have also been carried out. The 220-MHz PMR spectrum of the N-peptide in both the high- and low-field regions was shown to be almost identical with that calculated for an extended coil N-peptide monomer. The calculated random coil and experimental C-peptide spectra, on the other hand, differ in many respects. Evidence was obtained for the presence of rigid secondary structure in the C-peptide. In addition, the Val, Leu, Ile CH3 resonance displays a prominent high-field satellite band which shifts downfield with increasing temperature. Sedimentation studies on the N-peptide reveal the formation of extremely large, remarkably homogeneous aggregates at ionic strengths larger than or equal to 0.01. The C-peptide, on the other hand, does not appear to form aggregates of sufficient size to be detectable in velocity sedimentation studies of a few hours duration. The relative area changes which have previously been noted in the PMR spectrum of histone IV with increasing ionic strength were also observed for the N-peptide but not the C-peptide. Interpretation of these relative area changes has been made in terms of the amino acid sequence of histone IV, and an effort was made to identify that segment of the polypeptide which undergoes secondary structural change with increasing ionic strength.


The two-domain structure of cytochrome b5 in deoxycholate solution. The membrane protein cytochrome b5 and the polar and hydrophobic fragments into which it is cleaved by trypsin have been investigated, with major emphasis on the deoxycholate-solubilized form of the protein. Molecular weight measurements show that both the intact protein and the fragments are in a monomeric state in deoxycholate and that a small peptide of perhaps 15 residues is excised when the fragments are formed. Measurements of Stokes radius show that the major fragments are globular, but that intact cytochrome b5 has an asymmetric shape, consistent with a structure composed of two globular domains joined by a link region that may be as long as 30 to 40 A. Circular dichroism measurements were made in the far-ultraviolet and in the Soret region, and they add to previously existing data to make it virtually certain that the polar heme-containing domain is unaffected by proteolysis or by removal of deoxycholate. A significant change in the ultraviolet circular dichroism is, however, observed when proteolysis occurs and it is likely that it arises from the link between the domains, which appears to be highly structured (perhaps helical) in the intact protein, but randomly coiled after it is excised. The binding studies reported previously from this laboratory suggest that these inferences about the structure of cytochrome b5 in deoxycholate solution apply also to the protein as solubilized by detergent micelles, by phospholipid vesicles, or by the microsomal membrane.


Platelet stimulation by thrombin and other proteases. The mechanism of stimulation of platelets by thrombin and other proteases was studied by following kinetics of secretion of Ca2+ or ATP. The progress-time curves of secretion were analyzed for rate and total amount released. The reaction of thrombin was perturbed by addition of hydroxylamine or a competitive inhibitor and by variation of pH and it was compared with the reactions of other proteases. Trypsin and papain, with specificities for arginyl residues, induced secretion with a time course that was nearly identical with that induced by thrombin when saturating levels of enzyme were used. At low levels of enzyme, trypsin and papain gave extended lags in the progress-time curves. Higher concentrations of trypsin and papain were required for saturation of the measured parameters. Human plasmin (lysly specificity) and bovine chymotrypsin (aromatic amino acid specificity) failed to induce platelet secretion. Active site inhibited thrombin was also ineffective. Both yield and kinetics depended on pH, with the pH profile for each enzyme similar to its profile for hydrolysis of synthetic substrates. Studies at low pH also showed that the early part of the reaction undergoes a change in rate-determining step from enzyme dependent at low enzyme to enzyme indepdenent at high enzyme. Hydroxylamine, a nucleophile that would be expected to accelerate hydrolytic reactions, actually decreased both the rate of initial reactions and yield. A competitive inhibitor of thrombin also decreased both rate and yield; a calculated inhibition constant was in agreement with the value for a synthetic substrate, suggesting that the interaction of thrombin with platelets is analogous to reaction with substrates. A modification of our previous model is proposed in order to accommodate the results described here and to reaoncile the apparent contradictions that enzyme was found not to turn over in the reaction (Detwiler, T. C., and Feinman, R. D. (1973), Biochemistry 12, 282), that catalytic activity is required (Davey, M. G., and Luscher, E. F. (1967), Nature (London) 216, 875; this paper), and that the reaction is characterized by an apparent equilibrium binding (Tollefsen, D. M., Feagler J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). The essential feature is a reversible catalytic step with no dissociation of enzyme from product. This is followed by irreversible, thrombin-independent platelet processes leading to secretion, with yield dependent on the equilibrium concentration of the thrombin product. The model thus has aspects of catalysis, stoichiometry, and an agonist-receptor equilibrium.


Shape and volume of fragments Fab' and (Fab')2 of anti-poly(D-alanyl) antibodies in the presence and absence of tetra-D-alanine as determined by small-angle x-ray scattering. The conformation of two fragments derived from anti-poly(D-alanyl) antibodies, the divalent fragment (Fab')2 and the monovalent fragment Fab', was studied by small-angle X-ray scattering before and after interaction with the tetra-D-alanine amide hapten. More than 90% of the combining sites were occupied by the hapten. No significant changes were observed in the volume or in the radius of gyration, with either of the fragments. This contrasts with the significant decrease in these two parameters found upon reacting the hapten with intact anti-poly(D-alanyl) antibodies (I. Pilz, O. Kratky, A. Licht, and M. Sela (1973), Biochemistry 12, 4998). For Fab', the radius of the whole particle was found to be 3.48 nm in the absence of the hapten and 3.46 nm in its presence, the radius of gyration of the cross-section was 1.37 nm without hapten and 1.38 nm in its presence, and the volume of the particle was 98 nm3 in the absence of the hapten and 91 nm3 in its presence. For (Fab')2 the respective values were 5.06 and 5.05, 1.38 and 1.37, and 182 and 182. These results suggest that a conformational change occurs within the antibody molecule, but not within its Fab fragment, upon reaction with the tetraalanine hapten.


Studies of a serum albumin-liposome complex as a model lipoprotein membrane. Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicu lar, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associates with lipid. Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 A. The same lamellar separation in the positively charged system is 108 A. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 A while that for the positively charged lipoprotein system is 91 A. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer. Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 A spacing, characteristic of hexagonal packing of the hydrocarbon chains. A classical light scattering technique is used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose smaller than arabinose smaller than malonamide smaller than glycerol). K+ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.


Phosvitin phosphate content. Implications for protein kinase assay. The maximal rates of the protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) reaction studied with chicken egg yolk phosvitin as substrate are dependent on the level of dephosphorylation of phosvitin. 30 per cent dephospho-phosvitin gives the optimal initial rates. With varying levels of dephosphorylation, the apparent Km for the substrate also changes in a biphasic manner. If this factor is taken into account, and a suitable adjustment is made for the concentration of dephospho-phosvitin in the reaction it is possible to achieve maximal rates for the kinase reaction with phosvitin preparations of varying levels of dephosphorylation. Such a consideration is important for comparing the results of protein kinase studies using phosvitin as the substrate.


Immobilized electric eel acetylcholinesterase. I. Kinetics of acetylcholinesterase trapped in polyacrylamide membranes. Techniques are described for the trapping of electric eel acetylcholinesterase in polyacrylamide gel. The activity of the trapped enzyme was substantially reduced, the effect being due to inhibition by acrylamide, but the emzyme immobilized in polyacrylamide was considerable more stable than that in free solutionma kinetic study was made of the hydrolysis of acetylthiocholine, covering a range of membrane thicknesses, enzyme concentrations, substrate concentrations and temperatures. The results were interpreted with reference to the theoretical treatment of Sundaram, Tweedale and Laidler, and of Kobayaski and Laidler, and provided support for those treatments; Clear evidence was obtained for diffusion control with the thicker membranes. An activation energy was obtained for the diffusion of the substrate within the membrane, by combining the temperature results for thick and thin membranes at low substrate concentrations. The results lead to the conclusion that the in vivo kinetics of acetylcholinesterase are largely diffusion-free in muscle filaments, but are substantially diffusion-controlled in fibrils and fibers.


Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing. Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.


Poly(A) polymerases in normal and virus-transformed cells. The presence of poly(A) polymerase(s) has been studied in a clone of the established hamster embryo fibroblast line (NIL), and in a subclone of this line transformed by an RNA tumour virus, hamster sarcoma virus, (NIL-HS VIRUS). The results suggest the existence of three distinct poly(A) polymerases, designated I, IIA and IIB, in dense cultures of NIL and NIL-HS virus cells. Forms IIA and IIB have also been found in exponentially growing NIL and NIL-HS virus cells. Poly(A) polymerase I has not been detected in growing NIL cells, while growing and resting NIL-HS virus contain comparable amounts of this enzyme. The poly(A) polymerases differ in chromatographic behaviour and in their requirements for divalent cations. They are highly specific for ATP and require the presence of a primer. Cellular RNA or poly(A), but not the oligoribonucleotide (Ap)4A, can be utilized as primers. The products of the reactions have been identified as poly(A) chains (20-50 nucleotides long) by alkaline degradation and by their resistance to pancreatic RNAase.


Limited proteolysis of the tryptophanyl-tRNA synthetase. Earlier studies have shown that native tryptophanyl-tRNA synthetase from beef pancreas is composed of two apparently identical subunits having a molecular weight of 60000 plus or minus 2000 each. Incubation of the pruified enzyme with trypsin under restrictive conditions results in splitting of each subunit to form an enzymatically inactive polypeptide chain of mol. wt 24500 plus or minus 1500. During proteolysis, two distinct intermediate forms of mol. wt 51000 plus or minus 2000 and 40000 plus or minus 2000 and fragments of mol. wt 14000 plus or minus 2500 are formed. The presence of substrates, viz. ATP, tryptophan or tryptophanyl adenylate, decreases the rate of proteolysis. However, a band pattern monitored by acrylamide gel electrophoresis is qualitatively indistinguishable from that obtained in the absence of substrates. Native and trypsin-modified subunits (the latter having a molecular weight of 24500) have been maleylated, reduced, carbosymethylated and subjected to exhaustive digestion by trypsin followed by peptide mapping. Comparison of the finger prints has shown that the trypsin-modified subunit represents a polypeptide with lowered content of dicarboxylic amino acids. That the number of peptides revealed after complete proteolysis of native and trypsin-modified subunits does not favour the presence of long repetitive sequences in each subunit, is at variance with some bacterial aminoacyl-tRNA synthetases. Study of the fluorescence polarisation of 1-anilino-8-napthalene sulphonate adsorbed on the dimeric tryptophanyl-tRNA synthetase, indicates that the molecule behaves as a complete entity in Brownian rotation. The trypsin-resistant end products, composed of two types of polypeptides (mol. wts 24500 and 14000), remain associated with each other. From the mol. wt of this associate it follows that each fragment is present in the associate in duplicate. When the purification procedure was carried out in the absence of a protease inhibitor, the active modified enzyme form was obtained. As judged from the molecular weight values, it is composed of two equal subunits corresponding to one of the products of limited proteolysis. The data presented are compatible with compact three-dimensional structure of tryptophanyl-tRNA synthetase having very limited regions exposed to exogenous or endogenous proteolysis.


Difference-derivative absorbance spectrophotometry as a technique to measure state changes of phenylalanine residues in proteins. The first derivatives of difference absorbance spectra of several proteins were measured to examine the applicability of this technique as a tool to investigate state changes of phenylalanine residues in proteins. It was found by this technique that phenylalanine residues in insulin and those in lysozyme are exposed to more aqueous environment by denaturation with guanidine hydrochloride. Heat denaturation of collagen caused similar changes of some of its phenylalanine residues. It was thus demonstrated that difference-derivative absorbance spectrophotometry gives the information about state changes of phenylalanine residues in native proteins, which are hardly detected by common difference spectrophotometry.


A high molecular weight RNA fraction in chromatin. Utilizing a new chromatin isolation and fractionation technique we have obtained a high molecular weight RNA fraction from L-929 cell chromatin. The synthesis of this RNA is not greatly inhibited by concentrations of 0.04 mug/ml actinomycin D in the medium. Its synthesis appears to be strongly inhibited by 2 mug/ml of alpha-amanitin. The RNA appears to be quickly degraded (or removed from the chromatin) and does not contain a poly(A) sequence at its 3'-OH terminal end. Our working hypothesis is that this RNA is "nascent" heterogenous nuclear RNA partially transcribed from regions of the chromatin.


Physicochemical properties of chromatin digested with trypsin. Chromatin was prepared from calf thymus and digested with trypsin. Some physicochemical properties of chromatin were examined in connection with the time-course of the tryptic digestion. As the tryptic digestion proceeded, chromatin showed increases in viscosity and susceptibility to DNAase II and exhibited considerable alteration in thermal denaturation. A monophasic melting profile was found in the trypsic (digested) chromatin, but a biphasic one in the native (undigested) chromatin. The melting temperature descended from 78.2 degrees C for the native chromatin to 70.2 degrees C for the chromatin after only 10 min and further to 65.3 degrees C after 180 min tryptic digestions. The molar percent of total basic amino acid or lysine plus arginine in the chromatin increased with the time-course of the tryptic digestion whereas that of the total hydrophobic amino acid decreased. The molar ratio of hydrophobic amino acids to basic amino acids thus descended from 1.46 for the native chromatin to 1.05 for the chromatin after a 180-min tryptic digestion. These findings suggest that the neutral or hydrophobic portions in chromatin protein might play a role in the maintenance of the tertiary structure of chromatin.


Poly(A) polymerase in quail oviduct. Changes during estrogen induction. A nuclear poly(A) polymerase has been isolated from oviducts of immature quails. It could be purified 4300-fold. The enzyme depends specifically on ATP as substrate and requires Mg2+. The most effective primer for the enzyme is a polynucleotide, isolated from oviduct tissue. A poly(A) sequence to a maximum of 60 AMP residues is covalently linked per primer molecule. The poly(A)-rich product of the enzymatic reaction can be annealed to oligo(dT)-cellulose. The purest fraction does not contain any detectable poly(A)-degrading enzyme activity. Only very low activities of RNA polymerase are present. The poly(A polymerase activity in the assay with ATP is reduced by the ATP analogue, beta, lambda-ATP-methylene-diphosphonate. Both K-m and V are lowered. The ATP analogue is incorporated to a smaller extent into the poly(A) sequence, synthesized by the enzyme. Several other analogues of adenine, adenine nucleosides and adenine nucleotides are without effect on the enzymatic reaction. By these properties poly(A) polymerase can be distinguished from RNA polymerases form I and form II, isolated from the same tissue. Actinomycin D and alpha-amanitin failed to inhibit poly(A) polymerase activity. The activity of poly(A) polymerase has been determined during primary stimulation with the estrogen analogue diethylstilbestrol (daily injection for 5 days), after withdrawal of the hormone for 17 days and after secondary stimulation with the hormone analogue. The enzyme activity does not change during primary stimulation, withdrawal of the hormone or secondary stimulation. However the activity of a poly(A) degrading enzyme, localized in the nucleus, is reduced in oviducts from hormone-treated quails.


Relationship between Hb and HbA2 concentrations in healthy and iron deficient subjects. In 23 healthy subjects and in 115 patients with various degrees of chronic iron deficiency anaemia without congenital abnormalities of globin synthesis, Hb ranged from 3.4 to 16.3 g/100 ml. HbA2 ranged from 0.0550 to 0.5250 g/100 ml. Hb and HbA2 were statistically correlated, as shown by linear regression analysis (a equals --0.1387; b equals 0.0372; r equals 0.8198; P smaller than 0.001). The second degree parabola was not statistically different, but it gave a biologically preferable Figure for intercept (a equals --0.0006; b equals 0.0070; c equals 0.0015; r equals 0.8324; P smaller than 0.001). The second degree parabola was to be preferred also on the basis of previous literature results. Shortness of iron seems to reduce more the HbA2 than the Hb levels.


A flow method for determination of the kinetic parameters for immobilized enzymes. A flow method is described for determination of the kinetics parameters (V-m and K-m) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.


Retinal detachment surgery.

Retinal detachment surgery. Retinal surgery commences with careful ophthalmoscopy. The surgeon must be familiar with indirect monocular and binocular ophthalmoscopy, but biomicroscopy is most essential. Buckling procedures can be produced by different techniques and should be adapted to the case. Drainage of subretinal fluid is crucial. One should avoid it if possible, but if it has to be done, care has to be taken to avoid complications. The sealing of retinal holes and tears can be performed by diathermy, cryo- and light coagulation. Each technique has its indication and should be available at the time of surgery. Vitreous surgery is still in the experimental stage, but seems to be very promising, although its main use will most probably lie outside the field of retinal detachment.


Cytotoxicity of chlorhexidine.

Cytotoxicity of chlorhexidine. We evaluated chlorhexidine, a commonly used ophthalmic preservative for sterilization of soft hydrophilic contact lenses. Light microscopy and flat preparations of the endothelium were used. Our studies indicate that chlorhexidine is safe, even when used in higher concentrations than commercially available or required for bactericidal effect. Except for its mucous binding capacity, limiting its use for hydrophilic contact lenses, it is considered to be one of the best preservatives available for ophthalmic solutions.


Hemangiosarcoma of the breast.

Hemangiosarcoma of the breast. This report of the case of a 37-year-old woman with hemangiosarcoma of the breast it is the first such case to be described in the Canadian literature. It is emphasized that lack of familiarity with the lesion is the main reason for its frequent misdiagnosis. This case is unusual in that the patient is alive and well 7 years after her initial operation.


Excretion of inhibitors of calcification in urine Part II. Findings in patients with chronic renal failure. By means of a semiquantitative method incorporating the rachitic rat cartilage technique, the total urinary inhibitory activity with respect to calcification was compared in 11 control subjects and 20 patients with renal failure. The patients had significantly lower mean values of inhibiting units per day than did the control subjects. Both groups showed a significant positive correlation between the number of inhibiting units per day and urine volume. When urine volume was taken into account in the comparison, the numbers of inhibiting units for patients continued to be lower than the numbers for controls. These findings are consistent with the hypothesis that the increase of inhibitory activity observed in uremic serum is secondary to a decrease in excretion of the responsible factor (or factors) in the urine, and that the factor (or factors) in serum responsible for the inhibition are identical to those in the urine.


Isolation of cell subpopulations from in vitro tumor models according to sedimentation velocity. Chinese hamster V79-171 cells grown in vitro as asynchronous single cells, as plateau-phase cultures, and as multicell spheroids contianed subpopulations that differed in cell volume. These subpopulations could be isolated according to their sedimentation velocity at unit gravity. Asynchronous cells were found to sediment at a modal velocity of 17.7 mm/hr, whereas the modal sedimentation velocities of plateau and spheroid cells were found to be 16.0 and 15.1 mm/hr, respectively. When the composition of each subpopulation was determined using either [3H]thymindine incorporation or sensitivity to ionizing radiation as an index of cell cycle position, it was found that G1-like populations developed in both in vitro tumor models. Since the sedimentation velocity technique permits isolation of functionally different cell subpopulations after treatment with cytotoxic agents, it may now be possible to assay differential lethality when such agents are applied to cells growing in situ in a tumor-like situation.


In vitro biological evaluation of the R and S isomers of 1-(Tetrahydrofuran-2-yl)-5-fluorouracil. The S isomer of Ftorafur was synthesized and the ability of the latter to inhibit growth of cultured human fibroblasts was determined relative to both the R isomer and the racemic mixture (Ftorafur) that is presently used clinically. No significant difference in the cytotoxic effects or the relative abilities to prevent an increase in cell numbers was observed with the three forms. Inhibition of DNA synthesis in murine L1210 leukemia cells by either isomer was observed only after prolonged (18-hr) exposure. The data suggest that Ftorafur is a repository form of 5-fluorouracil and that activity is manifested equally by both isomers.


Nuclear magnetic resonance investigations of human neoplastic and abnormal nonneoplastic tissues. A large number of samples of human neoplastic and abnormal nonneoplastic tissues were studied by nuclear magnetic resonance spectrometry in order to evaluate the possible role of this technique in the diagnosis of cancer. The spin-lattice magnetic relaxation times (T1) of abnormal nonneoplastic tissue were longer, in many instances, than those of malignant tumors from similar sites, preventing recognition of the tumors in this manner. The evidence for the nonspecific nature of the prolongation of T1 in abnormal tissue is reviewed, and additional limitations of this technique in the diagnosis of cancer are indicated.


Histochemical studies of the adrenocortical homologue in the kidney of the Australian lungfish, Neoceratodus forsteri. A histochemical study has been made of groups of cells lying adjacent to the blood vessels of the kidney of Neoceratodus forsteri. The cells contain lipid droplets of varying sizes, stain positively for cholesterol and its esters and contain 3betaol-steroid dehydrogenase activity. Similar cells have previously been described in another dipnoan fish, Protopterus, and in a urodele, Pleurodeles waltlii and are considered to be the adrenocortical tissue of these species. Because of the histochemical characteristics of the cells together with their location, we conclude that they represent the adrenocortical homologue of N. forsteri. As is the case in Protopterus, no chromaffin tissue was detected in the kidney of N. forsteri.


Effect of reduced temperatures on protein synthesis in mouse L cells. The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.


The methylated constituents of L cell messenger RNA: evidence for an unusual cluster at the 5' terminus. An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2'-O-methyl nucleosides and N6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2'-O-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5' terminus of mRNA. These sequences seem to contain two 2'-O-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotid kinase reactivity. A strong candidate is a sequence of the type XppY1mpY2mpZp..., where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A)+ mRNA contain all four species of 2'-O-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3' end of the mRNA molecules.


Lipids in plant tissue cultures. V. Effect of environmental conditions on the lipids of Glycine soja and Brassica napus cultures. The environmental conditions prevailing during growth of plant tissue cultures affect the concentration of certain lipid classes and the fatty acid patterns of the total lipids. Irrespective of whether the cultures are grown under continuous illumination or in the dark, aerated cultures contain larger proportions of sterols, steryl esters, steryl glycosides and various sterylglycolipids and lower concentrations of squalene than non-aerated cultures. The lipids of the latter contain larger proportions of saturated very long-chain fatty acids than those of the former cultures.


Right heart volume characteristics in transposition of the great arteries. Right and left heart volume data were obtained during 44 cardiac catheterizations in 24 patients with complete transposition of the great arteries (TGA) prior to "corrective surgery." Patients were divided into three hemodynamic groups: I)TGA with intact ventricular septum, N = 23 studies in 13 patients, ages 1 day-22 months; II)TGA with ventricular septal defect (VSD), N = 12 studies in six patients, ages 7 days-15 months; III)TGA with VSD plus pulmonary stenosis, N = 9 studies in five patients, ages 5 days-5.6 years. In group I, right ventricular end-diastolic volume (RVEDV) averaged 170 plus or minus 52% of normal (P smaller than 0.001), RV ejection fraction (EF) was 0.48 plus or minus 0.09, 74% of normal (P smaller than 0.001), and RV systolic output (SO) was 123 plus or minus 34% (NS). In group II, RVEDV was 163 plus or minus 25% of normal (P smaller than 0.001), RVEF 0.59 plus or minus 0.08, 91% of normal (NS), and RVSO was 158 plus or minus 52% (P smaller than 0.02). In group III, RVEDV averaged 124 plus or minus 26% of normal (P smaller than 0.04), RVEF 0.58 plus or minus 0.15, 89% of normal (NS), and RVSO 125 plus or minus 57% (NS). Right atrial maximal volume was increased in 21 or 22 studies and averaged 185 plus or minus 47% of normal (P smaller than 0.001). RVEDV was greater than left ventricular end-diastolic volume in all but one patient in group I, but RVEDV/LVEDV was smaller than 1.00 in four VSD patients with very large pulmonary flows. A majority of patients with TGA without VSD have abnormal right heart function as indicated by an increased RVEDV and a low ejection fraction. Longitudinal studies of right ventricular performance in these patients continue to be important in assessment of current methods of therapy.


Comparison of radioimmunoassay methods for human luteinising hormone. 1. Four radioimmunoassay methods for the determination of luteinising hormone in serum or urine are described. 2. Evaluation of these methods (one employing laboratory prepared reagents, three utilizing commercial kits) was made to assess their routine laboratory value. Comparable precision and accuracy were found. Great economy is achieved by pruchasing LH and anti-LH preparations and by labelling one's own hormone. 3. Suggestions for future improvements in kit LH assay control and unitage are made.


Electronic timing of a high-speed immunonephelometric continuous-flow system. We describe a high-speed immunoephelometric continuous-flow system. The mechanical timing asembly and cams of the sampler were replaced by an external electronic control unit. Improved sampling precision, speeds of 2 to 900 samples per hour, and a wide variety of sample-to-wash ratios are the most important advantages of the unit. We analyzed albumin and fibrinogen at speeds of 150, 180, and 210 samples per hour. Carry-over studies showed that, in clinical practice, it should be possible to run the system at 150 samples per hour with an interaction of less than 5% and a precision (CV) of around 4%. Comparative studies with a previously described method, run at 60 samples per hour, showed correlation coefficients of 0.968 and 0.935 for albumin and fibrinogen, respectively. The electronic control unit can be used to increase speed and improve sampling precision of any continuous-flow system. An immunonephelometric system equipped with this device provided fast and reliable results, and required less antiserum than other systems described.


Standardization of the alkaline phosphatase determination with human placental phosphatase. The influence of changes in temperature and salt concentrations on the enzymic activity of 3 different alkaline phosphatase preparations has been examined. Beef liver phosphatase is labile; the activity is already lost by lyophilization. Hog intestinal and especially human placental phosphatase are more stable. In contrast to reconstituted sera no preincubation is necessary to restore the enzymic activity. A lyophilized human placental phosphatase, kept at room temperature, showed no change in activity in a period of about one year. A mean value of enzymic activity of 133 U/l was observed with a coefficient of variation of 2.4% for 12 determinations. It is suggested to assign a value to such a preparation and to use it as a primary standard in the determination of the alkaline phosphatase.


[Lipoprotein lipase activity in human milk; inhibition in vitro of the glucuro-conjugation of bilirubin (author's transl)]. The lipolytic activities of esterase, lipase and lipoprotein lipase were measured in 17 samples of human milk. Lipase and esterase activities were the same in all samples. However, lipoprotein lipase activity was increased only in samples provided from mothers whose infants had prolonged neonatal jaundice; after storage these samples inhibited the glucuro-conjugation of bilirubin in vitro and their concentration in non-esterified fatty acids was high. The mechanism of action of this enzyme is discussed in relation to the physico-chemical state of fat globules.


Anticoagulant potency of mucosal and lung heparin. Hog mucosal heparin was compared with beef lung heparin in 24 healthy male and female subjects by double-blind crossover study, by both intravenous and subcutaneous routes, to determine their relative anticoagulant effects as estimated by the activated partial thromboplastin test. Both drugs by both routes induced therapeutically comparable anticoagulation effects, without adverse experience. Thus both products are equivalent, unit for unit.


Radioimmunoassay and pharmacokinetics of saralasin in the rat and hypertensive patients. A radioimmunoassay for the new angiotensin antagonist, saralasin (1-Sar-8-Ala-angiotensin II), was developed and applied to pharmacologic studies in rats and hypertensive man. Specificity of the assay was established using naturally occurring angiotensins, potential saralasin metabolites, and other synthetic angiotensin analogues. Saralasin pharmacologic half-life of 3.9 min after intravenous administration to rats was similar to the biochemical half-life of 4.2 min. The pharmacologic half-life in high-renin hypertensive patients averaged 8.2 with a biochemical half-life of 3.2 min. These observations suggest that metabolites of saralasin do not accumulate in sufficient quantity in man or rat to interfere with the assay. The biochemical half-life of 3.2 min is consistent with infusion time of less than 20 min required to achieve a stable plasma concentration and pharmacologic activity of saralasin during constant saralasin infusion into hypertensive man. These studies provide a rational basis of future experimental design for saralasin and possibly other peptide analogues.


The effect of isometric exercise on the systolic murmur of patients with idiopathic hypertrophic subaortic stenosis. The effect of isometric handgrip exercise on the systolic murmur and bifid pulse in patients with idiopathic hypertrophic subaortic stenosis (IHSS) was evaluated. During handgrip exercise, the systolic murmur became inaudible or decreased in intensity by auscultation and disappeared or decreased in amplitude in phonocardiographic recordings in nine of nine patients with IHSS. During handgrip exercise, the bifid pulse observed in seven patients with IHSS disappeared or markedly decreased. Handgrip exercise may be helpful in the bedside diagnosis of IHSS.


Hypertrophic subaortic stenosis complicated by high degree heart block: successful treatment with an atrial synchronous ventricular pacemaker. The development of high degree atrioventricular block in a patient with hypertrophic subaortic stenosis underscores the importance of a properly timed atrial contraction in this disorder. Atrial synchronous ventricular pacing therapy, by providing, reliably timed atrial systole and increased left ventricular end-diastolic volume, has an important role in this patient and in similar cases.


[Spironolactone and thiabutazide in the treatment of essential hypertension (author's transl)]. The anti-hypertensive effect of spironolactone and thiabutazide was tested on 47 unselected patients with primary hypertension. They were divided into two groups according to the change in plasma-renin activity in response to furosemide administration: those with and those without response to stimulation, and sub-groups with low, normal or high plasma-renin activity (low renin hypertension; normal renin hypertension; high renin hypertension). During both 4-week treatment periods there was a distinct fall in systolic blood pressure, most marked in the patients without plasma renin stimulation (after spironolactone of about 26.2 mm Hg, after thiabutazide 29 mm Hg), the diastolic pressure fall being only slight in all groups (about 5-10 mm Hg). The plasma-renin activity and plasma-aldosterone concentration increased in all groups, after both spironolactone and thiabutazide three-to fourfold compared with the basal value,  even in patients without change in plasma-renin activity after furosemide injections. Serum sodium content decreased after administration of spironolactone, increasing again after subsequent thiabutazide administration. Serum potassium content increased after spironolactone, decreasing after thiabutazide. There was no significant difference between either individual groups or different treatment periods with spironolactone or thiabutazide as far as weight, urine volume or electrolyte excretion in 24-hour urine was concerned.


[Sequential scintigraphy of liver and spleen in patients with polyvinyl chloride disease (author's transl)]. Occurence of severe liver damage including angiosarcoma in polyvinyl chloride production workers necessitates regular control investigations of liver and spleen. Radioisotope techniques with small irradiation doses which give valid results should be used. Investigations of 15 patients with PVC-induced liver disease showed that hepatic perfusion as demonstrated by 99mTc pertechnate does not correlate with the uptake of sulphide colloid in the hepatic reticuloendothelial system. This provides evidence that in VC disease specific damage of the reticuloendothelial system of the liver occurs. Sequential scintigraphy also proved that the liver perfusion quotient can be considered as a measure of portal pressure. In practice this may be used for follow-up controls. Vascularized tumours described in VC disease can also be demonstrated by scintigraphy.


On the role of prostaglandins in parturition in the rat. Treatment of both intact and hypophysectomized pregnant rats on days 19 and 20 of pregnancy with PGF2alpha results in a rapid fall in plasma progesterone concentrations by day 20 followed by premature delivery on day 21. Administration of depoprovera did not prevent the PGF2alpha-induced fall in progesterone levels but premature littering was adverted. Indomethacin, an inhibitor of prostaglandin biosynthesis, postponed the onset of parturition by 20 h when administered in late pregnancy but rats treated with both indomethacin and PGF2alpha delivered at the normal time. Treatment of both intact and hypophysectomized animals with indomethacin delayed the fall in plasma progesterone concentrations which normally occurs between days 21 and 22 of pregnancy. Concomitant PGF2alpha treatment of rats receiving indomethacin caused plasma progesterone levels to fall in a pattern similar to controls. These findings indicate that endogenous PGF2alpha induces a fall in blood progesterone concentrations which is followed by increased myometrial activity.


Serum gonadotropins and follicular development in the Syrian hamster. Hamster serum gonadotropins were measured by RIA at 4 h intervals during the estrous cycle. On the afternoon of proestrus both LH and FSH exhibited a surge, but unlike the situation in the rat and mouse FSH returned to "baseline" with LH by early evening of proestrus. Shortly following this return FSH concentrations increased and reached a second peak by noon on estrus which was equal in magnitude and longer in duration than that occurring on proestrus. FSH fell to its lowest levels on diestrus 2 (D2) and early proestrus. Serum gonadotropins were measured by RIA 6 h following unilateral ovariectomy on D2. A slight elevation of LH resulted while FSH increased to a level equal in magnitude to that found during the proestrous surge. In intact females administration of a total of 45 mug FSH in 5 injections on D2 resulted in ovulation of twice the normal number of eggs. The t1/2 of this rat FSH in the male hamster was found to be 122 minutes. The low levels of FSH during the cycle between D2 and proestrus, the large increase in serum FSH following unilateral ovariectomy, and the "doubled" ovulation in intact hamsters following the administration of FSH on D2, suggest that the serum FSH concentration on D2 is critical in determining the number of follicles which will be available for the subsequent ovulation.


Surgical disconnection of the medial basal hypothalamus and pituitary function in the rhesus monkey. II. GH and cortisol secretion. Eighteen female rhesus monkeys subjected to complete or anterior disconnection of the medial basal hypothalamus (MBH) were studied to assess the effects of these deafferentation procedures on GH and cortisol secretion. Basal serum levels of GH were not disturbed or were slightly elevated following complete or anterior MBH disconnection. GH secretion in response to vasopressin administration or insulin hypoglycemia, however, was abolished by complete isolation of the MBH. In contrast, the elevations in serum cortisol concentrations observed in response to these noxious stimuli were not noticeably affected. The normal diurnal rhythm in cortisol secretion remained fully evident following anterior deafferentation, but was severely attenuated or abolished when all neuronal inputs to the MBH were transected. Such observations suggest that the central components of the neuroendocrine systems which regulate basal GH secretion and which subserve stress-induced elevations in cortisol secretion are resident within the MBH-hypophysial unit. In addition, these data indicate that the mechanisms underlying the diurnal rhythm in cortisol secretion, as well as those mediating the discharges of GH in response to vasopressin administration and insulin hypoglycemia, are dependent on the integrity of neuronal connections between the MBH and other hypothalamic and/or extrahypothalamic areas.


Effects of bovine growth hormone, human placental lactogen and ovine prolactin on intestinal fluid and ion transport in the rat. The influence of bovine growth hormone and human placental lactogen on intestinal absorption was compared with that of ovine prolactin. Administration of each of these hormones in vivo daily for 2 days, resulted in increased fluid and electrolyte transport by the rat intestine, as measured in vitro. Hypophysectomy causes a fall in fluid and ion absorption in the rat jejunum but these changes are prevented by growth hormone treatment. Bovine growth hormone and ovine prolactin produce essentially similar effects in intact rats: significant increases in fluid, sodium and calcium transport in the duodenum; in fluid, sodium and potassium transport in the jejunum; in sodium, chloride, potassium and calcium transport in the ileum. Growth hormone also significantly increased fluid, sodium and chloride transport in the colon. Treatment of hypophysectomized rats with human placental lactogen enhanced fluid and ion transport in the jejunum; however, it failed to restore normal potassium transport in the ileum and  colon at the 1 mg daily dose level. Growth hormone and human placental lactogen appear  to affect jejunal water and electrolyte transport in the same manner as occurs with prolactin, possibly by influencing active ion transport.


The effect of gonadotrophins on the fetal and neonatal rat testis. Testosterone formation was measured in fetal and neonatal (2 h to 20 days) rat testes incubated with and without 100 IU human chorionic gonadotrophin (hCG). At all ages studied, testosterone production was significantly elevated in the presence of hCG. Neonatal testes showed an absolute drop in testosterone production with increasing age, but the percentage increase above baseline produced by hCG stimulation leads to the speculation that this decrease is due to a relative reduction in the number of Leydig cells with increasing age rather than to a lowered sensitivity to hCG stimulation.


The nyctohemeral rhythm of plasma prolactin: effects of ganglionectomy, pinealectomy, constant light, constant darkness or 6-OH-dopamine administration. In male rats maintained on a 12 h light-dark schedule (6 AM-6 PM), there is a nyctohemeral cycle of plasma prolactin which consists of a nadir at 11:30 AM and an apogee at approximately 11:30 PM. In rats exposed to constant darkness, this rhythm persists for 7 days. Seven days of constant light, however, reverses this diurnal variation such that plasma prolactin levels peak at 11:30 AM and reach a nadir at approximately 11:30 PM. In animals maintained on a 12 h light-dark cycle, ganglionectomy and lateral ventricular injections of 6-OH-dopamine (250 mug) also appear to reverse the diurnal variation of plasma prolactin, whereas a single injection of 6-OH-dopamine (250 mug) into the third ventricle decreases plasma prolactin values at all times intervals but does not alter the diurnal rhythm. Both sites of 6-OH-dopamine administration markedly deplete hypothalamic dopamine and norepinephrine, but injection of 6-OH-dopamine into the lateral ventricle destroys the catecholaminergic terminals in the pineal, whereas injection of 6-OH-dopamine into the third ventricle does not. Pinealectomy slightly increases the early morning values of plasma prolactin, but otherwise has no effect on the diurnal variation of prolactin. Five conclusions appear to be justified: 1) there is a nyctohemeral rhythm of plasma prolactin, which is reversed by constant light; 2) the pineal gland probably plays no role in the diurnal regulation of plasma prolactin secretion; 3) the diurnal rhythm of plasma prolactin is controlled by sympathetic input into the brain via the superior cervical ganglion; 4) a rhythm of plasma prolactin develops in constant light which is the exact opposite of the normal diurnal variation; 5) there appears to be a noradrenergic pathway in the hypothalamus or brainstem which stimulates release of prolactin.


Serum prolactin and LH in early phases of delayed versus direct pseudopregnancy in the rat. Delayed or direct pseudopregnancies were induced by electrical stimulation of the cervix at 1400 h on diestrus day 2 (D-2) or on estrus, respectively. Blood samples for measurement of prolactin and LH by radioimmunoassay were collected by rapid decapitation at 5, 15, 30, 45 or 60 min after cervical stimulation or thereafter at 3-h intervals throughout the first 6 days of leukocytic vaginal smears of pseudopregnancy (PSP L-1 to L-6). For comparison, untreated animals were decapitated at the same 3-h intervals throughout the 4-day cycle. Serum LH concentrations in all the experimental animals did not vary from those measured in the cyclic controls. Compared with the normal cycle, prolactin (PRL) levels were not different until 10 h after cervical stimulation during estrus. After stimulation on D-2, PRL secretion did not differ from that in the normal cycle until approximately 37 h later, when there was a short rise following the usual proestrus surge. In both direct and delayed pseudopregnancy, twice-daily PRL surges appeared on L-1 and, except for a missing nocturnal surge on L-2 in delayed PSP, continued regularly through L-6. The absence of any immediate increase of PRL following the D-2 stimulus strongly supports the view that information from the stimulus is retained in the central nervous system to be expressed later after a set of new, competent corpora lutea has been formed.


Effect of an exposure to chloramine-T on the immunoreactivity of glucagon. Various preparations of glucagon treated with chloramine-T under different conditions have been studied with respect to their immunoreactivity toward two different glucagon antisera; one specific for pancreatic glucagon and the other capable of reacting with enteroglucagon as well. The glucagon preparations exposed to chloramine-T for different periods reacted almost identically with the nonspecific antibody whether they were used as tracer or standard. On the contrary, treatment with chloramine-T under severe conditions led to reduced immunoreactivity toward the specific antibody. Inclusion of dimethyl sulfoxide (DMSO) in the chloramine-T reaction resulted in preservation of the immunoreactivity of the treated preparations. The cyanogen bromide cleaved-glucagon, (1-26) homoserine lactone, showed little cross-reactivity with the specific antibody whereas it reacted to a similar extent with the nonspecific antibody as natural glucagon did. Amino acid analysis of the hormone exposed to chloramine-T demonstrated that the methionine residue at position 27 in the glucagon molecule had been oxidized to methionine sulfoxide. In addition, tryptophan had also been affected. DMSO protected methionine and tryptophan from the oxidative action of chloramine-T. We postulate from these results that the change in the immunoreactivity toward the specific antibody of glucagon exposed to chloramine-T is mainly due to oxidation of the methionine residue at position 27 in the molecule. The usefulness of DMSO in the iodination process is also discussed.