Constitutive heterochromatin and micronucleoli in the human oocyte at the diplotene stage. In the diplotene stage of the human oocyte, the processes of elaboration of the nucleolar material are amplified. The principal nucleoli are more voluminous but their relations with the secondary constrictions and the satellites of the D and G chromosomes are not modified. Numerous micronucleoli, frequently to the number of 15-20 this stage. The most remarkable point is their association to various segments of constitutive heterochromatin: centromeric regions, secondary constrictions of the C9 and probably of the A1 and E16. These observations reveal that the human oocyte at the diplotene stage shows an amplification of the ribosomal cistrons. This phenomenon is homologous, to a more reduced scale, of this described from the inferior vetebrates. Besides, the role of heterochromatin in the synthesis of nucleolar material without the intervention of the classic nucleolar organizers is suggested.


Chromosome anomalies in mouse oocytes after irradiation. We investigated the cytogenetic effects of X-rays on unfertilized mouse oocytes. NMRI females received an irradiation with 0,22.2,66.6,200, and 600 R during the preovulatory phase 3 hrs after HCG (human chorionic gonadotrophin). This is a stage during oogenesis in which the oocytes pass from late dictyotene to diakinesis. Chromosome analysis was performed after ovulation at metaphase II. From these experiments we can draw the following conclusions: 1) X-rays induced during the preovulatory phase a high number of chromosome anomalies. Among these, structural anomalies prevail. 7 out of 144 ovulated oocytes in matched controls carried such an abnormality, whereas after irradiation we observed with 22.2, 66.6, 200, and 600 R, 11 out of 72, 34 out of 108, 89 out of 102, and 122 out of 124, respectively. 2) Irradiation seems also to affect the chromosome segregation during the 1. meiotic division, as we observed after 22.2, 66.6, and 200 R a total of 6 oocytes out of 204 with a supernummary chromosome. In controls, however, no hyperploidy was found in 143 ova. This increase, however, was not significant. 3) Chromosome anomalies, e.g. breaks and deletions that go back to a one-break event increased linearly with increasing dose. Exchanges, however, going back to two-break events fittest best to the linear-quadratic dose-response model. 4) The dose of 600 R seems to represents a kind of borderline in this experiment, because nearly all (122 out 124) carried at least one structural chromosome anomaly. It is also this dose after which the highest frequency of reciprocal translocations was observed in a hump-shaped slope in spermatocytes after irradiation of spermatogonia (Preston and Brewen, 1973). With an increasing dosage up to 1200 R the frequency of translocations decrease again. The elimination of cells, crossing this borderline, might be due to genetic or non-genetic effects. 5) The frequency of radiation-induced translocations per oocyte agrees with the frequency of translocations in human lymphocytes (Dolphin and Lloyd, 1974) after in vitro irradiation. 6) Significant, lower frequencies of structural chromosome anomalies were observed irradiating earlier stages of mouse oogenesis. These stages are dictyotene from females at the age of 3, or 6 weeks and prophase I-stages in female embryos on the 17th day of gestation. This result may be due to a lower sensitivity of these stages or to modifying events during the interval between irradiation and preparations.


Evidence for two active X chromosomes in a human XXY triploid. A human XXY chromatin negative triploid culture has been found to have both the A and B isozymes of glucose-6-phosphate dehydrogenase, as well as an intermediately migrating AB (hybrid) band. This finding indicates that the cells in this culture each possess 2 active X chromosomes. Possible mechanisms producing the abnormalities seen in this disorder are presented and discussed.


Translocation +(7p+; Bq-) associated with recurrent abortion. A balanced translocation was found in a normal female with a history of four abortions. On the basis of the Giemsa-banding pattern the abnormality was interpreted as to be a translocation of a part of the long arm of chromosome 13 to the short arm of chromosome some 7:t(7;13)(7qter leads to 7p22::13q14 leads to 13qter;13q14 leads to 13pter::7p22 leads to 7 pter). Problems in genetic counseling are discussed with respect to this case.


Studies on the inhibition of C56-initiated lysis (reactive lysis).  V. The roleof C567-INH in the regulation of complement-dependent haemolysis initiated by cobravenom factor. Activation of the alternative pathway of complement by a factor from cobra venom (CVF) can lead to lysis of unsensitized erythrocytes (E) of some species.  In these studies we observed that alterations in CVF-induced lysis could be produced by manipulation of C567-INH, a naturally occurring inhibitory activity which acts on fluid phase C567 complexes.  Venom lysis of sheep and guinea-pig E was markedly inhibited by serum fractions having C567-INH activity.  Microgram quantities of poly-L-lysine (PLL), molecular weight 180,000, a polycation which is a functional antagonist to C567-INH in serum, potentiated CVF lysis of sheep and guinea-pig E, and permitted the lysis of human E, which are otherwise not suscepticle to CVF lysis.  The potentiation of venom lysis by PLL seemed not to be due to alterations in the target cell membrane; furthermore, it in turn was reversed by substances with C567-INH activity.  This suggests that the generation of fluid phase C567 complexes contributes to the CVF-induced lysis of erythrocytes of these species, and that the haemolytic potential of fluid phase C567 generated during alternative pathway activation by this means is regulated by C567-INH.


Therapeutic effectiveness of paracetamol in rheumatoid arthritis. A single-blind, non-crossover study of the effectiveness of paracetamol, compared with aspirin and indomethacin has been carried out in 143 patients suffering from rheumatoid arthritis. Subjective indices have been employed, and the validity of the present method is discussed. The results of the trial have also been compared with those of a previous study (of prednisone, aspirin and placebo). Paracetamol was not significantly different from placebo, either in terms of pain relief or patient satisfaction rating. Prednisone and indomethacin were significantly better than paracetamol in respect to both parameters, but aspirin was not. On the basis of these results, the frequent prescription of paracetamol as the main therapeutic agent in the treatment of rheumatoid arthritis is not justified.


Pharmacological studies of drug action on CNS, with special reference to effects of maprotiline. The effects of maprotiline, mainly on the CNS, were examined and compared with those of imipramine, methamphetamine and chlorpromazine. In the repeated administration and the stroboscope studies, maprotiline exhibited actions to increase behavioral activities of animals, just like amphetamine and imipramine. This appears to attest to pharmacological effects of maprotiline as an anti-depressant. It is noted, however, that whereas maprotiline shows a slight anti-apomorphine action in dogs and an inhibition of the spinal reflex action potential in cats, such actions are not recognized in other anti-depressants of the tricyclic groups including imipramine, amitriptyline, etc. These differences suggest the tranquilizing action of maprotiline at work, probably forming a specific pharmacological feature of maprotiline. Also, maprotiline increases body weight of rats strikingly, which seems to offer a wide range of clinical applications of this drug.


Ethanol-antibiotic interactions at hepatic level. The authors study the influence of acute and chronic ethanol intoxication on the blood levels of rifampicin and isoniazid as well of aminoglycoside antibiotics in rats. While the acute ethanol intoxication has no influence on blood levels of tested antibiotics, in the chronic inotoxication lower levels of rifampicin and higher levels of isoniazid are observed.  A study was performed also to determine the influence of an acute and chronic treatment with alcohol and the avoe quoted antibiotics on the BSP elimination rate. In the acute as well as in the chronic experiments, a constant significant delay in the elimination of BSP was observed in animals treated with rafampicin either alone or associated with alcohol. Isoniazid modified the dye elimination in comparison to controls only in chronic experiments. Ethanol did not seem to have any influence on this behavior. The implications and the possible significance of these findings are discussed.


Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells. Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.


Chemical oncogenesis in cultured mouse embryo cells in relation to the cell cycle. Using the C3H/10T 1/2 CL8 line of mouse embryo fibroblasts and three different methods of obtaining cell cycle synchrony, namely arginine or isoleucine deficiency and release from postconfluence inhibition of growth, a sensitive phase for oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been found. This sensitive phase is located somewhere between the G1/S boundary and a point 4 hr prior to this marker. Methylation of cellular macromolecules by tritiated MNNG is not cycle-dependent in cells synchronized by arginine deficiency. The capacity of cells to repair DNA single strand breaks produced by MNNG was examined by alkaline sucrose sedimentation analysis in cells synchronized by arginine deficiency and treated with MNNG during phases of the cell cycle sensitive and insensitive to oncogenic transformation. Whereas DNA repair was found to be equally rapid in cells treated just before S phase (I), or just after commencement of DNA synthesis (III), transformation was maximal in I. By contrast, cells treated when blocked by arginine deficiency (II) repaired DNA slowly and were not sensitive to malignant transformation. Cells in I and II, which repaired DNA at very different rates, were equally sensitive to MNNG-induced lethality, while cells in III, which repaired DNA at the same rate as cells in I, suffered greater lethality. Thus, in this system it was concluded that there was no direct correlation between DNA repair, as measured by alkaline sucrose sedimentation analysis of prelabeled DNA, and malignant transformation or lethality produced by MNNG. In preliminary experiments malignant transformation induced by cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) has been found to occur mainly in S phase, indicating that diverse chemical oncogens may have different sites of action, or that activation of chemical oncogens is cell cycle-specific for some agents.


Peroneal nerve paralysis in a heifer.

Peroneal nerve paralysis in a heifer. Peroneal nerve paralysis in a heifer developed during an 11-day period of recumbency following dystocia. The condition did not improve during the next 42 days and the heifer was slaughtered. On the basis of microscopic findings, it was concluded that 3 to 4 months would have been required to reestablish normal limb function.


Intrauterine pressure wave-form characteristics of spontaneous first stage labor. Intrauterine pressure wave-form parameters were measured in 827 contractions obtained from 26 patients in spontaneous labor. The coefficients of correlation between the maximal and minimal rates of pressure change and the maximal pressure amplitude were 0.78 and 0.63, respectively, and greater than or equal to 0.70 in 22/26 patients. Contractions partitioned into decile statistical groups of the pressure amplitude and both maximal and minimal rates. A linear relationship between these parameters has therefore been established. Contractions of greater amplitude tend to be longer, but the relationship between duration and amplitude is nonlinear with a limiting maximum contraction time. The duration of the midportion of the pressure wave appears invariate with respect to wave amplitude and only start-up and termination times increase with increasing amplitude. Mean values and standard deviations of the maximal amplitude (40.4 +/- 16.9mmHg). the maximal (2.4 +/- 0.9 mmHg/s) and minimal (-2.1+/- 0.9 mmHg/s)rates of pressure change, and the total duration of contractions (68.6 +/- 17.8s) were determined.


Reproduction and teratogenesis: current methods and suggested improvements. The risks to human reproduction, particularly in regard to possible induction of birth defects, from present levels of environmental chemicals are thought to be low but in need of closer scrutiny than is now the case. Methods currently used to estimate the hazards to reproduction of new chemicals and drugs are reviewed and some shortcomings and potential sources of error are noted. Specific suggestions are made for improving existing methodology. In addition to the widely used treatment span encompassing all or most of the period of organogenesis, it is proposed that some exposure periods of shorter duration be added to avert possibly adaptative changes in maternal homeostatic systems. Greater diversity of test animals, beyond the rodent-rabbit species largely used today, is urged with the objective of better matching metabolic pathways in a test animal to those of man. The demonstration of an embryotoxicity effect-level must be regarded as essential in all tests, and once found this level should be used as the basis of extrapolating downward to find an acceptable tolerance level of dosage. More attention should be focused on postnatal functional evaluation after prenatal and perinatal exposure of test animals to environmental chemicals, especially those that might interfere with the maturation of the central nervous system, endocrine glands, and immunological mechanisms. The use of nonhuman primates should be reserved for the testing of drugs essential for use during human pregnancy or early pregnancy, because of the scarcity of such animals and the high costs of doing meaningful tests with them.


Characterization of polyadenylate from the fungus Trichoderma viride. Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic acid of the deuteromyceteous fungus Trichoderma viride. Similar findings in two other groups of the true fungi raise the possibility that short polyadenylate sequences may be a feature of these relatively simple organisms.


Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae. Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.


Complement activation by a univalent hapten-antibody complex. The univalent hapten, nonadeca lysyl epsilon-Dnp-lysine, binds tightly to rabbit anti-2,4-dinitrophenyl antibody, and the complex has a sedimentation coefficient of 6.7, characteristic of a single antibody molecule. In this communication, we show that this complex is a good activator of the serum complement system. For activation to occur, the univalent hapten must contain the specific group which binds to the antibody, and also the polycationic chain. In addition, activation requires a functional complement-binding region on the intact antibody molecule. The classical pathway appears to be involved since the first, fourth, and second components of complement are markedly depleted when the complement system is activated by this univalent hapten-antibody complex.


Kinetic properties of pyruvate kinase hybrids formed with native type L and inactivated type M subunits. Bovine type M pyruvate kinase, which normally has hyperbolic kinetics with its substrates, was inactivated by treatment with trinitrobenzenesulfonic acid. The inactivation probably occurs through trinitrophenylation of the epsilon-amino group of a lysine residue in or near the ADP binding site. Although 90 to 95% of the enzymatic activity is lost by this treatment, the molecular weight and sedimentation coefficient of the trinitrophenylated enzyme are quite similar to values obtained with the native enzyme. The inactivated, trinitrophenylated type M pyruvate kinase was hybridized in vitro with the native bovine type L enzyme, which has sigmoidal kinetics with phosphoenolpyruvate but can be activated by fructose 1,6-diphosphate to give hyperbolic kinetics. Four enzymatically active species were produced, designated L4, L3M, L2M2, and LM3, according to their subunit composition. L4 and L3M have sigmoidal kinetics with phosphoenolpyruvate and are activated by fructose diphosphate. Little or no sigmoidicity was seen for L2M2, although this species is activated to a moderate degree by fructose diphosphate. LM3 appears to have hyperbolic kinetics and is activated only slightly by fructose diphosphate. The kinetic results obtained with hybrids containing trinitrophenylated type M subunits are quite similar to the results previously reported by Dyson and Cardenas ((1973) J. Biol. Chem. 248, 8482-8488) using native type M and type L subunits, indicating that the properties of a type L subunit are profoundly affected by the nature of the other subunits present in the tetramer. In fact, type L and type M subunits in a given hybrid seem to have similar kinetic responses toward phosphoenolpyruvate and fructose diphosphate.


Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity. 1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.


Studies on a pyrimidine phosphoribosyltransferase from murine leukemia P1534J. Partial purification, substrate specificity, and evidence for its existence as a bifunctional complex with orotidine 5-phosphate decarboxylase. A pyrimidine phosphoribosyltransferase, previously shown to utilize 5-fluorouracil and possibly also uracil and orotate (Reyes, P. (1969) Biochemistry 8, 2057-2062), has been purified about 100-fold from murine leukemia P1534J. Roughly 20% of the original activity was recovered to yield an enzyme preparation with a specific activity of 7.4 mumol of 5-fluorouracil utilized/hour/mg of protein. Disc gel electrophoresis of this preparation revealed the presence of a major band of protein accompanied by several trace contaminants. Emphasis was placed on a study of the substrate specificity of this enzyme. 5-Fluorouracil, uracil, and orotate phosphoribosyltransferase activities purified in parallel during fractionation with ammonium sulfate and protamine sulfate and eluted together from columns of Sephadex tG-150 and DEAE-cellulose. The three phosphoribosyltransferase activities eluted from the Sephadex columns with an apparent molecular weight of 55,000 to 60,000. In spite of this coordinate fractionation, preferential losses of orotate activity were experienced during DEAE-cellulose chromatography. Orotate activity continued to behave in a unique manner under other conditions, such as during proteolytic digestion. In the latter case, however, all three activities responded in parallel when digestion took place in the presence of 5mM UMP. The following results provided additional evidence to support the view that all three phosphoribosyltransferase activities may be catalyzed by the same enzyme: (a) the apparent Km for 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) did not change significantly when enzyme activity was measured with either 5-fluorouracil, uracil, or orotate; (b) 5-fluorouracil and uracil were found to be mutually competitive inhibitors; the effect of 5-fluorouracil on orotate activity was likewise competitive in nature; (c) in the absence of UMP, orotate was a noncompetitive inhibitor of 5-fluorouracil and uracil activities, but in the presence of 5mM UMP it became a competitive inhibitor of both of these activities; (d) 5-fluorouracil and orotate activities co-sedimented in 5 to 20% sucrose gradients (uracil activity was not examined); and (e) a wide variety of normal mouse tissues displayed virtually the same 5-fluorouracil to uracil to orotate activity ratio as found in P1534J enzyme preparations. The apparent Km and Ki values reported in this study indicate that the preferred pyrimidine substrate is orotate. It seems likely, therefore, that this enzyme functions in vivo as an orotate phosphoribosyltransferase. Orotate phosphoribosyltransferase and orotidine 5'-monophosphate (OMP) decarboxylase activities (a) eluted together during gel filtration on Sephadex G-150, (b) co-sedimented in 5 to 20% sucrose gradients, (c) remained associated during fractionation with ammonium sulfate and protamine sulfate, and (d) separated into a phosphoribosyltransferase and decarboxylase component when enzyme preparations previously subjected to limited proteolysis by elastase were sedimented in sucrose gradients...


Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract. RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.


Effect of chronic ethanol ingestion on fatty acid oxidation by hepatic mitochondria. To study possible factors in the pathogenesis of the ethanol-induced fatty liver, we investigated the effect of chronic ethanol consumption on the metabolism of fatty acids by isolated hepatic mitochondria. Chronic ethanol consumption resulted in decreased fatty acid oxidation, as evidenced by a reduction in oxygen uptake and CO2 production associated with the oxidation of fatty acids. The State 3 rate of oxygen uptake was depressed to a greater extent than the State 4 or the uncoupler-stimulated rate; the respiratory control ratio was also decreased. Therefore, one site of action of chronic ethanol feeding is on oxidative phosphorylation. The reduction in fatty acid oxidation, in general, is not due to an effect on the activation or translocation of fatty acids into the mitochondria. There was no effect by ethanol feeding on the activity of palmitoyl coenzyme A synthetase, whereas carnitine palmitoyltransferase activity was increased. The use of an artificial system (formazan production) to study beta oxidation in the absence of the electron transport chain is described. In the presence of fluorocitrate, which inhibits citric acid cycle activity, ketogenesis and formazan production were increased by chronic ethanol consumption. Thus beta oxidation to the level of acetyl-CoA is not impaired by chronic ethanol consumption. Total oxidation of fatty acids to CO2 is depressed by chronic ethanol intoxication because of effects on oxidative phosphorylation or the citric acid cycle (or both). Neither nutritional deficiency, cofactor depletion, nor the presence of ethanol in vitro explains these effects. Several of the effects of chronic ethanol consumption on fatty acid oxidation are mimicked by acetaldehyde and acetate, products of ethanol oxidation. Chronic ethanol consumption leads to persistent impairment of mitochondrial oxidation of fatty acids to CO2. However, oxidation of fatty acids to acetyl-CoA is not decreased by chronic ethanol consumption.


Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria. D-beta-Hydroxybutyrate dehydrogenase of bovine heart mitochondria has been purified to apparent homogeneity. The membrane-bound enzyme is first released by phospholipase A digestion of the mitochondria. Lithium bromide, 0.4 M, is used to aid release, and dithiothreitol is required to stabilize the enzyme. The membranous material is removed by centrifugation, and the apoenzyme is recovered in the supernatant and precipitated with ammonium sulfate to 50 percent of saturation. The main purification (100-fold) is achieved by selective adsorption and elution on controlled pore glass beads. The purified enzyme has been purified approximately 250-fold from the mitochondria. The purified enzyme is homogeneous as shown by poly-acrylamide gel electrophoresis in sodium dodecyl sulfate or acid-urea systems; a sharp band is obtained which is equivalent to a subunit molecular weight of 31,500. The apoenzyme is devoid of lipid and is completely inactive as isolated. It can be reactivated by adding aqueous microdispersions of lecithin or phospholipids containing lecithin. The apoenzyme is stable, i.e. it has a half-life of about 450 hours at 0-2 degrees in 0.4 M lithium bromide, containing 5 mM dithiothreitol at pH 7, and is soluble at these conditions, existing mainly as a monomer and dimer in dilute solution. It has a tendency to associate into larger aggregates when the salt concentration is lowered. The enzyme does not have a distinctive amino acid composition as compared with other proteins or soluble dehydrogenases. The purified apodehydrogenase is well suited for study of specific protein-lipid interaction, as well as the molecular basis for the role of phospholipid in this lipid-requiring enzyme.


Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids. The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.


Nonequivalence of the metal binding sites of conalbumin. Calorimetric and spectrophotometric studies of aluminum binding. Differential scanning calorimetric experiments show that addition of Al(III) to conalbumin increases its denaturation temperature by 5 degrees, from 60 to 68 degrees. Only one Al(III) bound per conalbumin molecule produces this change in heat stability; additional bound Al(III) does not affect the heat stability. Since Al(III) displaces both Cu(II) bound at the metal binding sites of conalbumin, binding of aluminum takes place at the same metal binding sites. The binding constant for the second Al(III) is at least 100-fold less than that for the binding of the first Al(III), and both are displaced by added iron. The order of increasing heat stability of the metal ion complexes of conalbumin, Cu(II), Al(III), Fe(III), is the order of increasing binding constant for these metal ions.


Iron binding to conalbumin. Calorimetric evidence for two distinct species with one bound iron atom. When thermal denaturation of conalbumin solutions partially saturated with Fe(III) is observed by differential scanning calorimetry, four endotherms are observed between 40 and 100 degrees. The relative size of these four endotherms is determined by the Fe(III) to conalbumin ration. At a heating rate of 10 degrees/min, in Tris buffer at pH 7.5, observed endotherm temperature maxima and enthalpies of denaturation are: conalbumin, 63 degrees, 320 kcal/mol; intermediate I, 68 degrees, intermediate I, 77 degrees; Fe2-conalbumin, 84 degrees, 630 kcal/mol. These four endotherms are observed over a range of protein concentration from 7 to 100 mg/ml and are unchanged when excess bicarbonate is present. Stoichiometric calculations of both total protein and total iron indicate that each intermediate endotherm results from denaturation of conalbumin molecules containing only one ferric ion. These experimental results are thus consistent with the presence of two different monomeric one-iron conalbumin intermediates. They strongly suggest that the two iron binding sites of conalbumin are not equivalent.


Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis. Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.


A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats. Cellular autophagy in convoluted tubules of kidney was studied in 24 rats, killed in pairs at constant time intervals during one diurnal cycle, by (a) morphometric evaluation of tubular cells by the point-counting method in randomly sampled micrographs, and (b) selective search for autophagic vacuoles (AV) directly on the electron microscopy screen. The total area of tubular cells recorded in the electron microscopy sections was 93 X 10(-4) mum2. Since the distal convoluted tubules, covering about 12% of the whole tubulocellular area, contained only 3-4% of all AV, they were omitted from the main calculations. The number of AV per area unit and the total amount of segregated material showed a distinct diurnal rhythm, synchronous for the different types of AV which were distinguished from each other according to their contents. The minimum was found during the night, the maximum during the day. This rhythm appears similar to that described elsewhere in liver cells. The mean segregated fractions were calculated from the relation of segregated to nonsegregated material in proximal convoluted tubular cells. The segregated fraction of the mitochondria was 4.4 X 10(-4). This value could account for the degradation of all mitochondria in a cell within 15 days, i.e., the upper limit of the lifetime of mitochondrial DNA in the cortex of the kidney, if one assumes that a mitochondrion is destroyed within 10 min after being segregated. The degregated fraction of microbodies was 11.7 X 10(-4). This suggests a shorter lifetime of these organelles. It is concluded that cellular autophagy plays a significant role in the turnover of cytoplasmic constituents, including the membranes of the endoplasmic reticulum.


Endogenous peroxidase: specific marker enzyme for tissues displaying growth dependency on estrogen. Data derived from a correlated morphological and biochemical study suggest the following: (a) estradiol-17beta, diethylstilbestrol, the estrogen antagonists nafoxidine (Upjohn 11,000), and Parke Davis C1628 induce synthesis of an endogenous peroxidase in the epithelium of target tissues like the vagina, the cervix, the uterus, and in the acinar cells of the estrogen-dependent rat mammary tumor; (b) peroxidase is a "specific" secretory protein of the estrogen-sensitized uterine endometrium; (c) peroxidase synthesis is not a nonspecific response to steroid hormone action, since progesterone and testosterone do not induce its synthesis; (d) endogenous peroxidase is a possible diagnositc protein for the detection of estrogen-dependent growing tissues, including breast cancer; (e) movement of exogenous horseradish peroxidase from the interstitium to the uterine lumina is restricted by tight junctions located at the apices of epithelial cells. Estrogen and antagonists do not appear to influence the transepithelial movement of exogenous peroxidase into the lumen.


Modulation of uterine morphology and growth by estradiol-17beta and an estrogen antagonist. The estrogen antagonist C1628 maintains sustained hypertrophy of the uterine epithelium and the synthesis of many proteins including peroxidase. C1628 is a progestogen, inducing secretion of the protein by surface epithelial and glandular cells. C1628 is a connective tissue mitogen, inducing DNA synthesis in fibroblasts and the endothelium. C1628 and estrogen share these properties mentioned above. Estrogen, however, induced moderate growth of the mucosa within a 24-h period and massive hyperplasia of the mucosa within a 24-h period thereafter. C1628 alone, or in combination with estradiol, does not have mitogenic effect on the mucosa, and in fact blocks the mitotic response normally induced by estrogen alone.


Estrogen and antagonist-induced changes in endometrial topography of immature and cycling rats. The topographical changes of the luminal surface of the endometrium of immature and ovariectomized rats treated with estrogen, antagonists to estrogen, and progesterone. and during various stages of the estrous cycle and in pregnancy were examined by scanning electron microscopy. Massive increases in numbers and length of endometrial cell microvilli were observed at estrus, after injection of estradiol-17beta, diethylstilbestrol, estrogen plus progesterone. or the inhibitor C1628 to immature and ovariectomized rats. Withdrawal of the estrogen stimulus results in diminution of microvilli, producing a state identical to diestrus, during pregnancy, and after injection of progesterone, The estrogen antagonist appears to have both estrogenic and progestogenic properties, stimulating endometrial cell hypertrophy, secretion of protein, and production of numerous apical microvilli.


Formation of enlarged mitochondria in a liver cell line in response to a synthetic glucocorticoid. For a number of years it has been recognized that glucocorticoids cause alterations in liver cell morphology (6, 9). Several investigators have shown that in liver in vivo mitochondria can be enlarged to many times their normal volume by treatment with cortisone (13, 15). There is a concomitant decrease in mitochondrial number, and the results of Kimberg and Loeb suggest that this is due to mitochondrial fusion (7). However, the exact mechanism whereby mitochondrial volume is altered and whether in fact cortisone is the direct causal agent are not known due to the complexity of studying these questions in a whole animal system. We have found that dexamethasone sodium phosphate (dex), a synthetic glucocorticoid, causes the formation of enlarged mitochondria in a liver cell line RLC-GAI, which grows in defined medium. In this paper we present our observations on the amount of enlargement that occurs after 5 days of treatment. The formation of enlarged mitochondria is reversible upon removal of the hormone from the medium, and we have attempted to determine whether "mitochondrial" or "nonmitochondrial" inhibitors are more effective in blocking the return of mitochondria to their normal size when the hormone is removed.


Cell movement and adhesion in the developing chick wing bud: studies on cultured mesenchyme cells from normal and talpid mutant embryos. Mesenchyme fragments from early wing buds of normal and talpid3 mutant chick embryos were explanted for culture in plastic Petri dishes and the behaviour of individual cells as they moved out on to the plastic surface was studied by time-lapse ciné photography, followed by statistical analysis. Two parameters of cell movement were recorded: (1) the distances moved over measured 100-s intervals and (2) the length of time each cell spent at rest before moving on. The average speed of movement over the whole path tracked for each cell, inclusive of time at rest, was significantly greater in normal than talpid3 cells. There was no significant difference between normal and mutant cells in the average distance mover per 100-s step, equivalent to the speed over the whole path exclusive of time at rest, but the percentage of time spent at rest was significantly less in normal than in talpid3 cells. This difference appears to be related to a difference in cell morphology, since it was observed that the mutant cells were more flattened than normals, with very extensive ruffled membranes and short spiky microvilli all round the cell periphery. The relation of these differences in cell morphology and behaviour in vitro to the production of the characteristically fan-shaped limb bud outgrowth and altered pattern of cartilage elements in the developing mutant limb bud is discussed.


Effect of calcitonin on serum gastrin concentration and component pattern in man. Synthetic salmon calcitonin (sCT, doses of 0.7 Medical Research Council U per kg) was injected into nine normal subjects and three patients with hypergastrinemia (pemicious anemia). sCT depressed basal as well as food-stimulated serum gastrin concentrations without concomitant changes in total and ultrafiltrable concentrations of calcium in serum. Gel filtration of sera revealed that sCT reduced mainly the small components, III (gastrin-17 or "little" gastrin) and IV (gastrin-13 or "mini"-gastrin).


Inhibition by somatostatin of basal levels of serum thyrotropin (TSH) in normal men. To elucidate the effect of somatostatin on basal, unstimulated TSH secretion 6 young healthy nonobese men were studied with blood samples every 15 min from 2300 h to 0500 h. Somatostatin 1 mg was indused over a 2-h period from 0100 h. One of the subjects was also studied with half hourly blood samples throughout a 26-h period without receiving somatostatin. In all cases the high night levels of serum thyrotropin (TSH) were significantly suppressed by somatostatin. The fall of TSH continued throughout the two hour infusion period. Immediately upon termination of the infusion serum TSH increased rapidly to preinfusion values. The 26-h control study showed the typical diurnal pattern of serum TSH with low values in the daytime and high values at night. The serum triiodothyronine (T3) and thyroxine (T4) levels were not changed by somatostatin infusion. From these results we suggest that endogenous somatostatin may be of physiological importance in the regulation of the TSH secretion.


A highly immunoreactive peptide fragment of human luteinizing hormone alpha subunit, discerned with a new, "sequence-specific" radioimmunoassay. hLHa, whose primary amino acid sequence is known, was reduced and s-carboxymethylated (RCM) to remove secondary and teriary structure. RCM-hLHa was utilized for development of a "sequence specific" ria. RCM-hLHa ria revealed that the NH2-terminal tryptic peptide of hLHa (consisting of only 32 amino acid residues) was nearly as immunoreactive as the entire RCM-hLHa molecule (consisting of 89 residues). No other tryptic peptide was immunoactive. Reduced and s-carbamidomethylated hLHa, differing only slight in structure from RCM-hLHa, was weakly active in the RCM-hLHa ria, demonstrating the utility of this ria for precise study of structure-immunologic activity relationships.


Effect of osteoclast activating factor from human leukocytes on bone metabolism. The effects of osteoclast activating factor (OAF) released by normal human peripheral blood leukocytes cultured with phytohemagglutinin have been examined in organ culture. Like parathyroid hormone (PTH), OAF causes a rapid increased in the release of previously incorporated 45Ca from fetal rat bone after brief or continuous exposure; the bones also lose stable calcium and collagen content. The resorption response to OAF also resembles that of PTH in having a steep dose response curve and being only transiently inhibited by calcitonin and partially inhibited by increasing medium phosphate concentration. OAF-stimulated resorption was inhibited more effectively by cortisol than was PTH stimulation. The response to maximally effective doses of OAF was not enhanced by PTH or prostaglandin E2, but submaximal doses gave additive effects. Both OAF and PTH inhibit collagen synthesis in fetal rat calvaria at the concentrations that stimulate bone resorption.


Sensitivity of some human pathogenic yeasts and systemic fungi to myxin. Myxin, a relatively new antibacterial and antifungal antibiotic, produced by a species of Sorangium, was used to investigate its effectiveness against some yeasts and dimorphic fungi associated with human diseases. Results indicated that the minimal fungicidal concentrations (MFC) of myxin for Candida albicans, C. krusei, C. parapsilosis, C. tropicalis, and Torulopsis glabrata were 0.39-6.25 mug/ml, and for C. guilliermondii and C. tropicalis 12.5-25 mug/ml. The MFC for Blastomyces dermatitidis and Sporothrix schenckii was 0.195 and 6.25 mug/ml, respectively. The MFC for these two systemic fungi for amphotericin B (fungizone) was 0.39-0.78 and 6.25 mug/ml. It seems that myxin is more effective against B. dermatitidis than amphotericin B. The isolate of Coccidioides immitis was found to be very sensitive to myxin (MFC, 0.78-1.56 mug/ml).


A presumptive sex pheromone in the hamster: some behavioral effects. Female hamster vaginal discharge (FHVD) contains a presumptive pheromone(s) that affects male hamsters in at least 2 ways. Alone it attracts investigatory interest, and if applied to an appropriate stimulus object, it facilitates copulatory behavior. The latter property was investigated by applying a fixed quantity of the discharge to a series of "surrogate females." The results indicated that the patterning and vigor of the elicited behavior more closely resemble the normal stereotyped mating sequence as the features of the stimulus object more closely approach those of a receptive female hamster. The ability of FHVD to facilitate mating with an otherwise sexually uninteresting stimulus object was unchanged 2 wk. after ovariectomy of the donor female.


Opioid-reinforced operant behavior: selective suppression by alpha-methyl-para-tyrosine. Morphine-addicted rats performed operant responses for opioid or milk rewards. When rats were then denied access to opioids, abstinence symptoms developed. Responding under extinction conditions 2 wk. after opioid withdrawal provided a measure of the "relapse" tendency. Systemic administration of DL-alpha-methyl-para-tyrosine, an inhibitor of catecholamine synthesis, suppressed these responses if they had been reinforced by an opioid but not if they had been established with milk reinforcement. This indicates that the integrity of the catecholamine systems is vital for relapse to occur.


One-trial learning and superior resistance to extinction of autonomic responses conditioned to potentially phobic stimuli. Human subjects were exposed to pictures of potentially phobic (snakes) and supposedly neutral (houses) objects as conditioned stimuli (CSs) in a Pavlovian conditioning experiment with shock as unconditioned stimulus (US), and skin conductance and finger pulse volume as dependent variables. The skin conductance responses conditioned to phobic stimuli were acquired after one CS-US pairing, and showed practically no extinction, whereas the responses to neutral stimuli showed very little resistance to extinction after both 1 and 5 reinforcements. The superior resistance to extinction of the phobic condition was interpreted to be a specific associative effect. In general, the skin conductance acquisition data showed tendencies similar to those during extinction. For finger pulse volume responses, however, there were very weak conditioning effects, and no effect of stimulus.


Differential rates of exhaustion and recovery of several parameters of male rat sexual behavior. Sexual exhaustion of the male rat was studied with respect to several copulatory measures. The ejaculatory latency, intercopulatory interval, and intromission frequency demonstrate a U-shaped curve, exhibiting high values for the initial ejaculatory series, falling to a minimum at an intermediate ejaculatory series, and again increasing at exhaustion. The absolute refractory period of the postejaculatory interval (measured at vocalization termination) increases linerly, whereas the relative refractory period (the remaining portion of the postejaculatory interval) is a postively accelerating function. Partial recovery tests demonstrated that the preejaculatory measures and absolute refractory period have substantially returned to baseline values by Day 6, while the relative refractory period is still extended. The significance of these data to the theoretical modeling of sexual behavior is discussed.


The ultrasonic postejaculatory vocalization and the postejaculatory refractory period of the male rat. After ejaculation, the male rat emits an ultrasonic (22-kHz.) vocalization. This sound is produced repeatedly until about three fourths of the postejaculatory interval has elapsed. In this study, the occurrence of the vocalization was described, and physiological and behavioral evidence was presented that the postejaculatory vocalization reflects an inhibitory state that underlies the postejaculatory refractory period. The vocalization period was characterized by a predominance of slow-wave, spindling, sleep-like electroencephalographic activity. Electrical shock was able to stimulate mating responses only after the cessation of the vocalization period. It was concluded that an absolute refractory period of the postejaculatory interval lasts until the end of the vocalization period and that the time from the termination of the vocalization until the resumption of mating is a relative refractory period.


Aggression and social experience in domesticated rats. Small colonies of rats were established, using adult animals that had either received continuous social experience or had been isolated since weaning. Unfamiliar "intruder" rats--with or without postweaning social experience--were exposed individually to the colonies for a 21-hr. period. Behavioral observations and an assessment of the intruder's physical condition indicated that serious fighting, physical injuries, and large weight losses occurred only when an isolation-reared intruder was placed into a colony of socially experienced rats. These results demonstrate that aggression is a joint function of the rearing history of both the colony and the intruder and that social experience plays an important role in the behavioral development of this species.


Comparison of scotopic sensitivity in diurnal (Anas platyrhynchos) and crepuscular (Dendrocygna autumnalis) ducks. Scotopic visual adaptation curves were obtained from 4 mallard ducks. A curve of best fit was used to compare the mallards' mean adaptation curve to the curve previously reported for the black-bellied tree duck, a crepuscular species. The curves did not differ significantly in either their slopes or base levels (thresholds). The mallards curve had a rod-cone "break" at approximately 25 min. This break is evident in the scotopic curve for pigeons, but is absent from the black-bellied tree ducks' curve. Examination of retinal tissues indicated that the black-bellied tree ducks had significantly more rods and cones, and a larger rod:cone ratio than the mallards. The mallards' scotopic visual threshold is exceeded by the natural illumination present under several nocturnal conditions.


Tonic immobility in the blue crab (Callinectes sapidus, Rathbun) its relation to threat of predation. The tonic immobility. response in the blue crab (Callinectes sapidus, Rathbun) was investigated in a series of seven experiments. Although reported to be a powerful variable in other species, preinduction electric shock produced inconsistent increases in the duration of tonic immobility with the blue crab; Manipulations that were more directly relevant to the fear of predation had considerably greater effects than shock. Physical damage to the chelipeds, mirror image stimulation, and immobilization beneath artificial glass eyes all produced significant prolongation of the immobility episode; Crabs immobilized on a bed of sand rather than on a hard surface showed shorter immobility durations, suggesting that opportunity for escape is an important variable affecting the immobility reactionmthe present results support the contention that threat of predation is the organizing principle behind tonic immobility


Attack, eating, drinking, and gnawing elicited by electrical stimulation of rat mesencephalon and pons. Eating, drinking, and gnawing were electrically elicited from the rat mesencephalon in the vicinity of the lateral branch of the descending medial forebrain bundle, but attack was evoked from the dorsomedial tegmentum adjacent to the central gray. The effective zones continued further caudally to the dorsal posterior pons. Unlike hypothalamically elicited behavior, eating, drinking, and gnawing often persisted 5-40 sec after termination of stimulation. Vocalization and escape activity were obtained principally from the vicinity of central pain pathways originating from the anterolateral cord. Other electrodes produced eating, drinking, gnawing, and grooming, which began only after termination of stimulation.


Male aggression in the presence of an estrous female. The relationship between male rat aggressiveness and the presence of a female was studied. Twenty 100-day-old dominant male albino rats were individually exposed to an inaccessible female rat in a specially constructed apparatus. The males were then given free-forced trials to an empty compartment or a compartment housing a submissive male. The results indicate that following exposure to an estrous female, dominant males increased their percentage of choices to the submissive male. Moreover, the incidences of aggressions by the dominant animal toward the submissive animal increased sharply after prior exposure to the estrous female. These data suggest that one antecedent source of aggression among male rodents may be the presence of a sexually receptive female.


Relationship between activity, rest, and free feeding in rats. General activity and food intake were recorded concurrently in free-feeding rats. Correlations of meal size with associated postmeal interval, activity, and rest were determined. Meal size was significantly positively correlated with both the length of the postmeal interval and the amount of rest in the postmeal interval, but not with the amount of activity in the postmeal interval. This suggests that the amount of energy expended following a meal is not related to the initiation of the subsequent meal and that some alternate mechanism underlies meal-to-meal regulation.


Heredity and experience: their relative importance in the development of taste preference in man. Heritability estimates for sucrose, lactose, and sodium chloride taste preferences were uniformly low in a sample of 311 monozygotic and like-sex dizygotic twin pairs between 9 and 15 years of age. Black children preferred more concentrated solutions of all three tastants than did Caucasian children. This effect was independent of socioeconomic status in the total sample. Males preferred more concentrated solutions of sucrose and lactose than did females- but there were no sex differences in sodium chloride preference. The possibility that early intake experiences may play a role in the determination of enduring taste preferences in Homo sapiens is discussed.


The Wisconsin Allergy Society fee and practice survey. A study for professional standard review organizations. Physicians practicing allegy in the State of Wisconsin were surveyed regarding their fees charged and norms of practice relating to the diagnosis and management of patients with allergic diseases. Over 50% of the physicians surveyed responded and the results were compiled and analyzed. The data obtained provide an over-view of the practice of a medical subspecialty and can thus be used for possible peer evaluation.


Metastatic hemangiopericytoma associated with microangiopathic hemolytic anemia: review and report of a case. The hemangiopericytoma is an invasive tumor of vascular origin. No matter how benign the course and how circumscribed the mass, it must be considered a lesion with high malignant potential. In the case report here, a hemangiopericytoma of the pancreas with metastasis to the liver was associated with microangiopathic hemolytic anemia in a 78-year old woman. The anemia may have been present before the onset of metastasis. If so, it could have been a major factor in the breakdown of host response and could have initiated the malignant dissemination. Under these circumstances it could be an indicator of metastasis in patients with previously diagnosed tumors. However, there are many cases of metastatic malignancy associated with this anemia in which the opposite situation holds. The anemia is usually of extracorpuscular origin. Apparently intravascular coagula1ion caused by injury from tumor-cell aggregates in small vessels induces erythrocyte fragmentation in fibrin strands. A vicious cycle of cell fragmentation, vascular injury and fibrin strand formation completes the course. Analysis of the findings should be based upon more than a strictly pathologic approach; it should also be related to the new discoveries in cancer research.


Aspiculuris tetraptera in wild Mus musculus. The prevalence of infection in male and female mice. A survey was carried out of the levels of infection with Aspiculuris tetraptera and Syphacia obvelata in a wild house mouse population living in the Charles Clore Small Mammals Pavilion at the London Zoo in Regent's Park. The extent of infection with A. tetraptera is analysed according to the sex of the host. It is shown that the prevalence of infection was greater in male than in female mice and frequency distribution studies suggest that this is not only because fewer female mice become infected but also because females resist larvae more effectively than do males.


Immune responses to a soluble schistosomal egg antigen preparation during chronic primary infection with Schistosoma mansoni. Murine schistosomiasis mansoni is characterized by an intense, predominantly cell-mediated, anti-egg, granulomatous response to schistosomal egg antigens (SEA). Anti-SEA responses include lymphocyte blastogenesis, the production of the lymphokine eosinophil stimulation promoter (ESP), hemagglutinating antibody, heat-labile and heat-stable, 72-hr passive cutaneous anaphylaxis (PCA) antibodies, and pronounced peripheral blood eosinophilia. These responses were followed during the course of chronic (1 year) infection and analyzed with specific reference to the observed diminution of granuloma formation, in the presence of continued antigenic exposure, which occurs by 10 to 12 weeks after infection and persists during long-term schistosomiasis. Lymphocyte blastogenesis and peripheral blood eosinophilia were positive from the 8th week of infection until the 50th. Lymphokine production and circulating heat-labile PCA antibody were only positive for a few weeks after 8 weeks of infection. In contrast, hemagglutinating antibody and heat-stable, 72-hr PCA antibody increased during weeks 10 to 14 and remained high throughout chronic infection. The development and regression of these various immune responses to SEA indicate that there are several potential mechanisms that could explain the immunoregulatory interactions that result in specifically diminished lesion formation in this chronic infection.


Detection and quantitation in plasma and synovial fluid of a fragment of human C4 with alpha mobility generated during the activation of the complement system. A newly identified fragment of human C4 was detected, using a particular antiserum, in human serum after activation by heat-aggregated immunoglobulins, but not after activation of the complement alternative pathway. This fragment was shown to have a sedimentation velocity of approximately 2.5S, to be heat stable, and to exhibit alpha mobility in immunoelectrophoresis. This C4alpha mobility fragment was not generated in human C4 deficient serum but was generated in human C2 deficient serum after incubation with heat-aggregated immunoglobulin. After precipitation of native C4 and its higher molecular weight fragments from serum by polyethylene glycol, it was possible to quantitate the lower molecular weight C4 alpha mobility fragment by radial immunodiffusion. In kinetic experiments, it was shown that the C4alpha mobility fragment was generated after some delay when compared to the disappearance of C4 hemolytic activity. Quantitation of the C4alpha mobility fragment may be of further use in human diseases for the evaluation of the catabolism of C4: joint fluids of patients suffering from rheumatoid arthritis contained high levels of the C4alpha mobility fragment, and low concentrations were found in patients with degenerative joint disease.


Cell-mediate cytotoxicity in vitro of human lymphocytes against a tissue culture melanoma cell line (igr3). Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (ADCC) and  microcytotoxicity assays (ma). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosette-formation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes ("K" cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients


Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination. A simple and rapid method utilizing immune adherence hemagglutination has been developed for the detection of antibodies to hepatitis B core antigen (anti-HBc). Hepatitis B core antigen (HBcAG) was prepared from Dane particles that had been isolated from plasma of asymptomatic antigen carriers. The method was specific and about 10 times more sensitive than the conventional complement-fixation method. A total of 215 serum samples obtained from healthy blood donors were surveyed for HBsAG and anti-HEc, as well as for hepatitis B surface antigen (HBsAg) and antibody to HBsAG (anti-HBs). Anti-HBc was found in 36 serum samples, at a prevalence rate higher than that of anti-HBs (31/215)...


Hereditary vestigial pulmonary arterial trunk and related defects in rabbits. In a population of IIIVO/J rabbits at the Jackson Laboratory a vestigial pulmonary arterial trunk is the commonest expression of a malformation involving the heart and great vessels. There appears to be a series of effects from a completely absent pulmonary trunk through a vestigial but patent pulmonary trunk and/or pulmonary valve stenosis on the one hand to a vestigial or absent ascending aorta on the other. In a few cases the pulmonary trunk is bulbous and the ductus arteriosus is vestigial or absent. In almost all cases of this syndrome, there is also a high ventricular septal defect. The animals appear perfectly normal in every other respect. In its extreme forms this mutation is lethal, but those few animals that are somewhat less severely affected may live for a short time. No effect was seen on birth weight or litter size. Inheritance of vestigial pulmonary arterial trunk appears to be due to two autosomal recessive factors both of which must be homozygous for the expression of the condition. We propose the symbols vpt-1 and vpt-2 for the genes responsible for vestigial pulmonary arterial trunk and its related abnormalities.


Effect of freezing and storing on the biological properties of macrophages. Storage of intact macrophages would be a useful procedure for laboratories studying the biological functions of these cells. Oil-induced guinea pig peritoneal cells were harvested and frozen at an ultra-low temperature using a cryoprotective media. Studies of the biological properties of the frozen cells upon thawing indicated that the cells retained viability for a fairly long period of time, were metabolically active and comparable to normal cells in morphology. Indications of activation were obtained in enhanced phagocytic activity and significant decrease in migration of the stored cells.


Effects of antibody to oestrogen or of ovariectomy on the incorporation of [35-S]methionine into brain protein and on gonadotrophin levels during the oestrous cycle in the rat. The incorporation of [35S]methionine into protein of the anterior pituitary and discrete brain areas was measured following the administration of antibodies to oestrogen, ovariectomy, or adrenalectomy on the afternoon of dioestrus. The antibody to oestrogen deleted the circadian rhythms of methionine incorporation normally observed in the various brain areas together with the peaks of incorporation normally observed in the median eminence area and anterior pituitary on the evening of pro-oestrus. The peaks of incorporative activity normally observed in the preoptic area and amygdala (relative to the putamen) at 03.00 h on the day of pro-oestrus were also deleted. Administration of the antiserum on the morning of pro-oestrus failed to alter the pattern of methionine incorporation normally observed on the evening of pro-oestrus. Ovariectomy performed at 16.00 h of dioestrus blocked the preovulatory rise of luteinizing hormone (LH) (as did the antibody to oestrogen) and inhibited the peak of methionine incorporation normally observed in the anterior pituitary on the evening of pro-oestrus. However, for the peak in the median eminence to be inhibited, ovariectomy had to be performed on the morning of the preceeding oestrus. Adrenalectomy alone, or adrenalectomy with ovariectomy, performed on the afternoon of dioestrus did not affect the levels of methionine incorporation in the brain or anterior pituitary at 18.00 h on the day of pro-oestrus. Animals which had been ovariectomized and injected with 2-5 mug oestradiol benzoate on the morning of oestrus showed significantly increased levels of methionine incorporation in all the brain areas and the anterior pituitary at 18.00 h of expected pro-oestrus. The administration of antibody to oestrogen to a similar group of animals on the afternoon of expected dioestrus inhibited the rise at 18.00 h of expected pro-oestrus. The apparent discrepancy between the results obtained with ovariectomy and the antiserum appeared to be due to the ability of the antiserum to neutralize the activity of oestrogens retained by the tissues. The present results suggest that the changes in incorporation of methionine into protein in the brain and anterior pituitary are brought about by the action of endogenous oestrogen: there appears to be a steady summative effect on the median eminence throughout the oestrous cycle together with a short-lived effect occurri4g during pro-oestrus and affecting the anterior pituitary.


Diurnal variation in depression--is it there? In this paper we present the results of an investigation designed to measure diurnal variation of mood in a group of unipolar depressives using a sensitive self-assessment inventory and a behavioral measure of the depth of depression. Both measures correlate well with the Hamilton Rating Scale which was designed to quantify depression during a psychiatric interview. Although all patients included in our sample reported that they experienced diurnal variation in mood, this fluctuation was not manifest on behavioral measures and only one patient showed a significant difference on the depressive inventory. Our data indicate that self-report and behavior may not be affected by diurnal variation in eight of these nine depressed patients. One possible explanation is that self-report in depression is subject to perceptual distortion leading to retrospective falsification.


Clinical and surgical aspects of posterior fossa haemangioblastomata. A retrospective study has been carried out on 67 patients with posterior fossa haemangioblastomata. Clinical details are presented, and the problems of diagnosis discussed. A fresh definition of the von Hippel-Lindau complex is put forward. The results of surgery are good for patients with solitary and sporadic tumours.


Pathological and haematological aspects of posterior fossa haemangioblastomata. A retrospective study has been carried out on 67 patients harbouring 78 posterior fossa haemangioblastomata. The site, macroscopic nature, and histological features are described, and correlations sought with the degree of erythrocytosis in the peripheral blood. The problems of aetiology are discussed and a single hypothesis advanced.


The nuchocephalic reflex.

The nuchocephalic reflex. The nuchocephalic reflex, previously undescribed, was investigated in a controlled study of 146 subjects ranging in age from 11 hours to 94 years. In 110 subjects over 16 years of age, the presence of the reflex was correlated directly with evidence of diffuse cerebral dysfunction as measured by a shortened form of the Wechsler Adult Intelligence Scale. Thirty-six children were studied from the newborn period to the age of 16 years. The reflex was found to be uninhibited in infants and to become inhibited by the age of 4 years. Among the adult subjects, the nuchocephalic reflex was found to be sensitive indicator of the status of higher cortical function. The presence or absence of the reflex appears to have somewhat different predictive value at different points in the adult age spectrum.


Cerebral mucormycosis following open head trauma. Case report. The authors describe a case of human cerebral mucormycosis following open head trauma and craniotomy, and discuss possible roles of steroids and antibiotic therapy in its pathogenesis. They suggest that the common usage of prolonged broad-spectrum chemoprophylaxis in head trauma may require critical review.


Evolution of a primary intrasellar germinomatous teratoma into a choriocarcinoma. Case report. A case of intrasellar teratoma with a germinal structure in a 10-year-old girl is described. A few months after intracranial surgery the tumor differentiated into a choriocarcinoma and finally spread to multiple cerebral, pulmonary, and renal metastases. In the course of choriocarcinomatous evolution, very high urinary levels of luteinizing gonadotropin (HCG) developed, but there was no clinical or anatomical evidence of precocious puberty.


Flavins in chick embryo.

Flavins in chick embryo. During incubation of fertilized hen egg, flavin in egg white migrated into the embryo. The total amount of flavins in the embryo increased with development. On the 7th day of incubation, molar ratio of riboflavin and flavin nucleotides in the embryo was already similar to that of animal tissues. Even though appreciable amounts of FMN and FAD were found in egg white, flavin nucleotides found in the embryo in its early embryonic life cannot be simply ascribed to the migration of these nucleotides originally present in egg white, because egg yolk contains no appreciable amount of flavin nucleotides; the possibility that flavin nucleotides are synthesized from riboflavin in the embryo in its early embryonic life still exists.


The effect of an acute dose of biotin at the pre-implantation stage and its relation with female sex steroids in the rat. An acute dose of biotin (10 mg/100 g body weight) in two subcutaneous injections when given to a rat on day 1 and 2 of pregnancy, caused resorption of fetuses and placentae. Pregnancies under such biotin-treated conditions were maintained by continued estrogen or progesterone therapy. Biotin-treated pregnant rats failed to maintain normal levels of uterine weight, glycogen and protein as well as hepatic protein concomitantly with the loss of pregnancy. Estrogen therapy under such conditions improved all the parameters in these organs including the placenta, but progesterone therapy did not. Glucose-6-phosphate dehydrogenase (G-6-PD) activity in ovary, adrenal, liver and uterus was also reduced following biotin-induced loss of pregnancy which had been improved by estrogen or progesterone therapy. Nevertheless, estrogen was superior to progesterone in stimulating the enzyme activity in these organs excepting the adrenal. As far as the tissue response to biotin, estrogen or progesterone in the nonpregnant rat is concerned, biotin and progesterone exerted a suppressing effect on uterine glycogen and protein and also on liver protein, while estrogen stimulated them. Similarly biotin and progesterone adversely affected G-6-PD activity in all the organs studied except the liver and adrenal. Estrogen stimulated the enzyme activity in all these organs but adrenal. The study suggests that the primary reason for an acute dose of biotin-induced loss of pregnancy is blockage of estrogen production, which probably regulates endogenous progesterone secretion. The associated metabolic derangements are probably secondary to estrogen deficiency and are discussed.


Ulceration and necrosis of vocal cords in hospital and unexpected child deaths. Lesions are described in the vocal cords of an unselected group of 91 infants dying as "cot deaths", 11 stillbirths and 107 infants and children dying from conventional diseases. The lesions have been classified into six types. After allowances for the effects of intubation, the same incidence and type of change was present in all but the stillbirths. This argues against them being a specific pathogenic mechanism confined to the cot death situation. The aetiology and pathogenesis are unexplained but indicate the existence of an unsuspected laryngeal disorder that merits further study in mechanism leading to child death.


An attributional approach to moods and the menstrual cycle. Acceptance of an attribution pattern linking negative moods (depression, irritability) to the approach of menstruation and the likelihood of internal and external attributions were examined in a questionnaire study in which cycle phase (pre- versus postmenstrual), mood (positive versus negative), and environment (pleasant versus unpleasant) were varied. Subjects' rating indicated that (a) biology was judged important for explaining negative moods occurring premenstrually: (b) inconsistency between mood and environment produced more internal (personality) attributions, while consistency enhanced external attributions; and (c) emotionally expressive behavior was thought to reflect underlying personality dispositions despite extenuating situational factors (assumed personal causation). The theoretical relevance of the findings to a new conceptualization of premenstrual emotionality and to an attributional chain relating female self-concept and premenstrual tension is discussed.


Percutaneous absorption of drugs. IV. Percutaneous absorption of drugs from oily vehicles. The percutaneous absorption and retention of salicylic acid and carbinoxamine from four oily vehicles (liquid paraffin, oleic acid, hexadecyl alcohol, and isopropyl myristate) were studied by employing a recirculation apparatus. The absorption followed first-order kinetics, with the exception of the initial period. The vehicle that had a strong affinity to the drug showed a poor drug-releasing effect, and poor absorption and retention of drugs by the skin were observed. Higher absorption rate constants were observed for damaged skin than intact skin. The acceleration of absorption because of skin damage, however, was not so great from liquid paraffin as from the aqueous solution. The amount of drugs retained in the damaged skin declined following the initial increase.


Drug-biomolecule interactions: fluorescence studies on interaction of aminonaphthalenesulfonic acid derivatives with serum albumins. The binding of the three aminonaphthalenesulfonic acid derivatives to human and bovine serum albumins was studied by measuring the fluorescence enhancement of the compounds. The number of binding sites of human and bovine serum albumins for these compounds appears to be one and two, respectively, under the experimental conditions. As the molar ratio of the fluorescent compounds to bovine serum albumin increased, the binding sites appeared to increase for the compounds. The quenching of the native fluorescence of albumin was examined by the successive addition of methanolic solutions of these compounds. 1-Anilinonaphthalene-8-sulfonate quenched the protein fluorescence to a greater extent than the other compounds studied, indicating that 1-anilinonaphthalene-8-sulfonate molecules are bound more closely to the tryptophan residues of albumin. The finding that the three compounds did not quench the fluorescence of tryptophan dissolved in water indicates no direct molecular interaction between tryptophan and the three fluorescent probes. The driving force for binding may be due to the structural characteristics of the amino acid sequence surrounding the tryptophan residues.


Systematic identification of drugs of abuse I: spot tests. More than 40 of the most commonly encountered street drugs were subjected to several spot tests. These tests were carried out in a special sequence leading to the construction of a flowsheet. Thus, with a limited number of simple tests, it is possible to identify tentatively or narrow down the drug. Since each drug investigated was subjected to all selected tests, whether such tests were developed for this type of compound or not, some unexpected and undocumented results were obtained.


Isolation and partial characterization of toxins from the dinoflagellate Gymnodinium breve Davis. Three neurotoxins were isolated from unialgal cultures of the dinoflagellate Gymnodinium breve Davis. Of the three toxins, only one toxin (T1) has hemolytic acitivity. The major toxin (T2), in chromatographically pure form, appears to have a molecular weight of 725. The neurotoxin T2 has no antiacetylcholinesterase activity.


Binding study of sulfonylureas and phenothiazines to bovine serum albumin using difference spectrophotometry. 2-(4'-Hydroxybenzeneazo)benzoic acid is a spectrophotometric probe which shows absorption spectrum changes upon binding to protein. Difference absorption spectra of this probe were used as an indirect measurement of the binding of selected sulfonylurea and phenothiazine drugs to bovine serum albumin. The results obtained using the spectrophotometric probe were similar to data obtained from other methods, especially fluorescent methods. Of the four sulfonylureas studied, tolbutamide showed the highest binding affinity, followed by glyburide, glipizide, and acetohexamide, in that order. The data collected for phenothiazine drugs indicated that chlorpromazine has the highest affinity, followed in order by trifluoperazine, perphenazine, fluphenazine, and promazine. Correlation of these results with chemical composition indicated that the interaction of phenothiazine drugs with bovine serum albumin was of a hydrophobic nature.


Binding of cupric ions to bovine serum albumin. The binding of cupric ions to bovine serum albumin was investigated by using a cupric-ion-specific electrode. When using a modified form of the Scatchard equation, it was determined that there are at least two classes of binding sites on bovine serum albumin for cupric ions. One class has three binding sites of relatively strong affinity, with an average binding constant of 3.0 times 10(6). The other class has about 16 binding sites of relatively weak affinity, with an average binding constant of 2.0 times 10(4).


Synthesis and cytotoxicity of 6-selenopurine arabinoside and related compounds. 6-Chloro-9-(beta-D-arabinofuranosyl)purine served as an intermediate for the chemical synthesis of a series of 6-substituted selenopurine arabinosides. In an in vitro test using murine leukemic cells (L-5178Y) these 6-substituted selenopurine arabinosides showed some cytotoxicity. Lengthening the side chain had no effect on their cytotoxicity. Selenourea was a useful reagent for synthesizing selenopurines, selenopurine nucleosides, and selenopurine arabinoside under mild conditions.


Interactions of acetylcholine mustard with acetylcholinesterase. The hydrolysis of acetylcholine and acetylcholine mustard by acetylcholinesterase was compared over a substrate concentration range of 1-10 mM. Reactions were allowed to proceed for 2 min at 25 degrees. Results of these experiments reveal that the substrates have similar affinities for the enzyme, whereas the maximum velocity for the hydrolysis of acetylcholine mustard was significantly lower than for acetylcholine. These findings suggest that acetylcholine mustard has the ability to inactive acetycholinesterase.


Efficacy and interactions of oxandrolone, halo-fenate and clofibrate in a factorial study on experimental acute nephrotic hyperlipidemia. Nephrotic mixed hyperlipidemia may be associated with accelerated coronary artery disease. To investigate the response of experimental nephrotic hyperlipidemia to therapy, a 2(4) factorial study of sodium clofibrate and beta-benzalbutyrate, halofenate and oxandrolone (250, 150, 100 and 10 mg/kg/day, respectively) was carried out. Nephrotic syndrome was induced by a single i.p. injection of puromycin aminonucleoside (90 mg/kg) in 80 female white rats of average weight 160 g. Oxandrolone proved to be significantly hypotriglyceridemic in combined therapy (average fall, 38%; P less than .05), and also lowered serum total cholesterol and phospholipid concentrations (23% and 21% falls, P less than .01) and less than .05), due largely to synergistic interactions with clofibrate-like drugs. Hypocholesteremic effects (23 and 22% average falls) were also significant for halofenate (P less than .01) and clofibrate (P less than .05) . Serum triglyceride levels actually rose significantly (P less than .05) with drug combinations containing beta-benzalbutyrate. Clofibrate and its analogs (halofenate and beta-benzalbutyrate) produced significant hepatomegaly (mean responses of +18, +18 and +10%, respectively) whereas oxandrolone produced significant hepatic shrinkage (-10%)(P less than .05). Secondary effects (drug interactions) were also found; hypotriglyceridemic synergism (effects more than additive) occurred between oxandrolone and clofibrate or its analogs (P less than .05), whereas antagonism (effects less than additive) was observed within the clofibrate-like group (P less than .01 or less .05).


Brain acetylcholine and choline following acute and chronic morphine treatment and during withdrawal. Levels of brain acetylcholine and choline were measured with a gas chromatograph in Swiss-Webster or ICR mice and Sprague-Dawley rats: a) 30 minutes after various single doses of morphine sulfate s.c.; b) rendered highly tolerant to and dependent on morphine by pellet implantation for 3 days; c) at various times after abrupt withdrawal (pellet removal); or d) during abstinence precipitated by the narcotic antagonist naloxone. The specific activity of brain acetylcholinsterase was determined in treatment d. Brain choline levels generally remained unaffected by the above manipulations. It was found that analgetic doses of morphine did not alter the steady-state levels of brain acetylcholine, but slight increases were observed after high doses and in morphine-tolerant animals. Abrupt withdrawal of morphine in the mouse caused a significant increase in brain acetylcholine levels, which was observed at 6 hours but not at 12 and 24 hours. In rats, abrupt withdrawal had no effect on acetylcholine levels at 6 and 18 hours. Nalonone-precipitated withdrawal significantly lowered the brain acetylcholine in both mice and rats without affecting acetylcholinesterase activity. This lowering was observed in animals that jumped after naloxone, but not in those failed to jump. It is concluded that this decrease in brain acetylcholine may be related to an increased neuronal release.


Head-turning induced by electrical stimulation of the caudate nucleus and its antagonism by anti-parkinson drugs. Intermittent high-frequency electrical stimulation of the caudate nucleus induces contralateralhead-turning in rats. The anti-Parkinson drugs, L-dopa, amantadine and apomorphine, raise the threshold for or completely inhibit head-turning. There was a high correlation between the predicted clinical potency and these drugs based on inhibition of head-turning and their respective clinical anti-Parkinson potency. The centrally acting anticholinergic drugs also antagonized head-turning but there was not a good correlation between the predicted and acutal anti-Parkinson doses used in man. In order to determine if these drugs blocked head-turning by acting on the caudate nucleus, a combination cannula and stimulating electrode was used to administer drugs directly into the same area of the caudate nucleus being stimulated electrically. Dopamine, amantadine and apomorphine each antagonized head-turning when infused into the same site, at doses which did not produce concurrent overt sterotyped behavior. Time- and dose-response data with all three drugs suggest a direct inhibitory action on the caudate nucleus consistent with their proposed mechanism for treatment of Parkinson symptomatology. Head-turning appears to be a useful animal model for the development of new, specific anti-Parkinson drugs and for the study of possible mechanism(s) of action of existing drugs.


The bioelectric parameters of the vagina during the oestrous cycle of the rat. 1. A new in vitro preparation of the rat vagina was devised to allow the measurement of the electrical potential difference (p.d.), short-circuit current (s-c.c.) and tissue resistance. 2. In vivo and in vitro, the magnitude of the transvaginal p.d. (adventitia positive to lumen) was greatest at oestrus and smallest at dioestrus and metoestrus. The p.d. at pro-oestrus was not significantly different from the latter values. 3. The s-c.c. was significantly greater at oestrus than at metoestrus, dioestrus and pro-oestrus. 4. The vaginal resistance was maximal at pro-oestrus and was significantly greater than that at metoestrus, dioestrus and oestrus. The value at oestrus was significantly greater than that at metoestrus and dioestrus. These changes in resistance correlate well with known changes in vaginal morphology during the oestrous cycle. 5. It is proposed that the cellular site for the generation of vaginal bioelectric activity is the basal cell layer of the vagina and that the variations of the transvaginal p.d. measured in vivo are caused by the changes in vaginal current and tissue resistance.


[Electroencephalographic studies on classically conditioned defensive reflex and avoidance lever-pressing in the dog (author's transl)]. Six dogs with implanted cortical and subcortical electrodes were trained to press a lever to avoid electroshock to a hind leg. Intracerebral stimulation at low frequency was delivered as an "indifferent" or a "CD" tracer. Changes of EEG responses to the tracers prior to the voluntary lever-pressing or the conditioned avoidance lever-pressing were prior to the voluntary lever-pressing or the conditioned avoidance lever-pressing were examined by continuous frequency analysis. (1) Evoked potentials to the tracer stimulation were changed just before the conditioned defensive reflex, the voluntary lever-pressing and the conditioned avoidance lever-pressing, regardless of the site of the tracer stimulation. (2) The cortical and subcortical structures (Hippo, RF) seemed to be involved in the neural circuit responsible for the avoidance lever-pressing, whereas the sensorimotor cortex may be essential in the circuit for the alimentary lever-pressing. (3) The avoidance lever-pressing and the EEG response to the "CS" tracer were influenced by the internal inhibition (experimental extinction, inhibitory mechanism was discussed.


Embryo-endometrial relationship in the mouse during activation of the blastocyst by oestradiol. Blastocysts that have been activated for implantation by an injection of oestrogen become separated from the uterine surface by a layer of secretion. By 8 hr after the injection, the trophoblast cytoplasm contains several glycogen granules, groups of ribosomes, and mitochondria of a condensed type. These ultrastructural changes are signs of the increased metabolic activity exhibited by the trophoblast at activation. It is suggested that the uterine epithelium keeps the trophoblast inactive by delivering only a minimal nutritive medium and that the epithelium activates the trophoblast by making more nutrients, probably some carbohydrate, available as a consequence of an oestrogen-induced increase in the secretory activity.


Female mouse maturation: relative importance of social factors and daylength. The present experiment utilized a cross-classified design to test the interactions and relative importance of male presence or absence, female density and three different photoperiods as factors affecting puberty in female house mice. First vaginal oestrus was used as the criterion for sexual maturity. All three factors significantly affected the timing of first oestrus; male presence or absence accounted for 31 percent of the total variation in age at maturity, 9 percent of the variance was attributable to the female density factor and 6 percent to differences in daylength treatments. There were no significant interactions among the treatment variables. These results conform with a general model relating the processes of sexual and morphological development.


The effect of progesterone implants on ovulation and plasma levels of LH, FSH and progesterone in anoestrous ewes. The effects of 100-mg progesterone implants in anoestrous ewes on plasma progesterone and gonadotrophin levels are described. Implant removal resulted in a surge of plasma gonadotrophins and ovulation, but there was no evidence of behavioural oestrus in 90% of the ewes. These results are discussed.


Pregnant mare serum gonadotrophin: rate of clearance from the circulation of sheep. The process involved in the disappearance of PMSG from the blood of sheep, following a single intravenous injection, has been separated into two exponential components. Values (mean plus or minus S.E.) calculated from experiments on five animals were: metabolic clearance rate (37.8 plus or minus 1.6 ml hr-minus 1); rate constant of disposal (0.0315 plus or minus 0.0016 hr-minus 1); half-time of disposal (21.2 plus or minus 1.1 hr). The stage of the oestrous cycle, ovariectomy and the dose of PMSG used had no apparent effect on these values.


Follow-up study of male and female offspring of DES-treated mothers a preliminary report. This is a follow-up study of male and female offspring of mothers who were part of a double-blind placebo controlled investigation during the years 1951-1952, originally aimed at determining the usefulness of DES administration in maintaining pregnancy. So far, 84 DES-exposed females, 43 female controls, 42 DES-exposed males and 37 male controls have been examined. Circumferential ridges of the vagina and cervix were seen in 39% of the DES-exposed females but in none of the controls. Colposcopy revealed vaginal epitheleal changes in 78% of the DES-exposed females 2% of the female controls. Cytology proved to be reliable as a screening test for vaginal epithelial changes in the DES-exposed female. Urine cytology was negative for tumor cells in all patients. The main abnormal finding in the DES-exposed males was that cysts in the epididymis were detected in 10%. No cases of cancer were observed in either the male or female offspring.


Comparison of DNA binding in normal population, general hospital laboratory personnel, and personnel from laboratories studying SLE. Sera from 18 of 56 individuals working in SLE laboratories bound more than 30 per cent of 0.1 mug of 125-I denatured calf thymus DNA, in an ammonium sulfate precipitation assay. Only two of 58 normal non-laboratory personnel and four of 40 sera from routine hospital laboratory personnel bound more than 30 per cent. Statistical analysis of these results with the Kolmogorov-Smirnov nonparametric test indicated this was a very significant difference (p less than .001). Using native DNA as a test antigen a similar pattern was seen, but it was not as clear cut. Part of this increased reactivity is heat labile. Further studies must be carried out before an adequate interpretation of these findings is possible.


New aromatic diamidines with central alpha-oxyalkane or alpha, omega-dioxyalkane chains. Structure-activity relationships for the inhibition of trypsin, pancreatic kallikrein, and thrombin and for the inhibition of the overall coagulation process. A series of omega-amidinophenylalkyl amidinophenyl ethers was synthesized and examined for inhibitory activity against trypsin, pancreatic kallikrein, and thrombin. Modifications of the compounds included lengthening of the alkane chain, variation in the position of the amidino groups, and substitution of halogen on the benzene rings. The compounds act as competitive reversible inhibitors, and many of them possess considerable potency. An outstanding trypsin inhibitor was found in 4-amidinophenylethyl 4 amidino-2-bromophenyl ether (compound 7) with a Ki value of 7.3 x 10(-8) M (pH 8.1, 37 degrees). A number of aromatic diamidines with a central dioxyalkane chain were similarly studied. Here, 1-(4-amidino-2-iodophenoxy)-5-(3-amidinophenoxy)pentane (compound 32) was a highly effective inhibitor of bovine thrombin (Ki = 1.1 x 10(-6) M), of human thrombin, and of the overall clotting process of human plasma.