Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation. The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.


Synthesis of phospholipids in mitochondria and other membrane fractions of rabbit reticulocytes. 1. Reticulocytosis of 40-50% was obtained in rabbits by daily bleeding. Reticulocytes (plus erythrocytes) were subfractionated into plasma membrane fraction, mitochondria and the post-mitochondrial fraction. 2. In all fractions, fatty acids were incorporated into phospholipids. This process was ATP dependent and represented acylation of lysophospholipids. 3. Incorporation of fatty acids into lysophosphatidic and phosphatidic acids occurred only in the presence of sn-glycerol 3-phosphate and was observed in mitochondria and the post-mitochondrial fraction. It represents a two-step acylation of sn-glycerol 3-phosphate. 4. Incorporation of phosphorylcholine from CDPcholine into phosphatidylcholine was observed in the mitochondrial and the post-mitochondrial fractions. This activity was correlated with NADPH-cytochrome c reductase and was probably connected with the remnants of the endoplasmic reticulum.


Hydrolysis of phosphatidylcholine by phospholipase A2 does not cause dissociation of apolipoprotein C from rat plasma very low density lipoprotein. To test the hypothesis that hydrolysis of glycerophosphatides causes displacement of apolipoprotein C from very low density lipoprotein, we have studied the effects of a snake venom phospholipase A2 on very low density lipoprotein labeled with [125I]apoC, [3H]cholesterol, [14C]palmitate and [32P]phospholipids. In spite of hydrolysis of 97% of the phosphatidylcholine, only small amounts of labeled apoC and labeled cholesterol were displaced from the very low density lipoprotein. With purified lipoprotein lipase in contrast, 80-90% of the labeled apoC and cholesterol were removed from the lipoprotein. It is concluded that hydrolysis of phosphatidylcholine does not cause an appreciable dissociation of apolipoprotein C from very low density lipoprotein.


Studies on adenosine 3',5'-phosphate-binding and adenosine 3',5-phosphate-dependent protein kinase activities associated with subcellular fractions of Chinese hamster ovary cells. Both cyclic AMP-binding and cyclic AMP-dependent protein kinase activities exist in Chinese hamster ovary cell extract. Competition experiments demonstrate that the binding is specific for cyclic AMP. All cellular elements including nucleus, mitochondria, plasma membrane, microsome, ribosome and cytosol contain both activities. Binding activity is highest in the cytosol and lowest in the nucleus. Each fraction contains endogenous protein kinase activity which is insensitive to cyclic AMP activation. When histone was used as a substrate, protein kinase activity in all fractions was stimulated by cyclic AMP (with the highest in cytosol and lowest in the nucleus) and inhibited by Walsh's protein kinase inhibitor.


Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium. Embryonic chick (7-9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionic strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7--9 day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio of newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanied by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.


A simple and defined method to study calcification by isolated matrix vesicles. Effect of ATP and vesicle phosphatase. A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 microgram of protein, were incubated for 2 h in a 45Ca-labelled solution with 2.2 mM Ca2+, 1.6 mM PO 3/4-and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO4 hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca2+ or Mg2+ was found to stimulate matrix vesicle ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, beta-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, vesicles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.


Studies on mannose-containing glycopeptides from a normal and an SV40 transformed human cell. The oligosaccharide moiety of cell-surface mannose-labelled glycopeptides from a normal (WI38) and an SV40 transformed cell (W118Va) have been investigated using specific glycosidases. Partially purified mannose-containing glycopeptides were separated into acidic and neutral species by high voltage paper electrophoresis. Endo-beta-N-acetylgucosaminidase D, in the presence of three exoglycosidases, released from the acidic glycopeptides of non-growing cells a product completely absent in growing cells. However, the acidic species from growing WI18 Va and WI38 were found to be similar in the products released by enzyme digestion. The neutral species from growing normal cells contained a proportion of the glycopeptides resistant to endoglycosidase D while those from the non-growing cells were almost free of these resistant species. The SV40 transformed cells were further enriched, when compared to normal cells (WI38), in these neutral resistant species. We suggest that the oligomannosyl core of the majority of the susceptible species contains three mannose residues while that of the resistant species contains between six and eight.


[Study of mechanisms of photoreceptor membrane destabilization under modifying action of oxygen]. Induction of lipid peroxidation in the rod outer segments (ROS) of frog retina results in fragmentation of photoreceptor disc membranes and solubilization of lipoprotein rhodopsin complexes (sedimentation coefficient 2.7S). The substrates of lipid autooxidation are mainly docosahexa- and docosapentaenoyl residues of phosphatidyl ethanolamine. Large fragments (precipitation at 5000 gX30 min) and small vesicles (precipitation at 40 000 gX60 min; average diameter 1000 A) formed from lipoperoxidized ROS differ both in their chemical composition and structural organization. In small vesicles the content of O2-modified polyenoic acyls in the phospholipids is 3,7 times higher as compared to large fragments. Correspondingly, the capacity of hydrophobic areas in the small vesicle membranes evaluated by EPR spin-probing technique is lower than in the large fragments. A mechanism of the photoreceptor membrane destabilization under modification by molecular oxygen is proposed. It is based on a decrease in the value of surface tension upon accumulation of lipid peroxidation products in ROS, the former possessing the properties of non-ionic detergents.


[Some characteristics of mitochondrial multienzyme systems from rat liver mitochondria after heating of rats]. A character of rat liver mitochondria degradation after the heat treatment of animals is studied. It is found that mitochondria under the effect of elevated temperature do not considerably change their functional characteristics and thus they are capable to provide the normal rate of ATP synthesis, the rate of succinate oxidation being slightly increased. At the same time the heating caused the degradation of mitochondria which results in the decrease of their thermostability, in the increased susceptibility to lytic effect of trypsin and phospholipase D, and in the activation of succinate dehydrogenase and cytochrome c oxidase. The mitochondria degradation is due to the formation of "latent impairments" in the structure of mitochondria.


[Investigation of Endomyces magnusii respiratory system. Characteristics of mitochondria from cells grown in the presence of antimycin A]. Mitochondria from AA-cells are a unique model with separately finctioning 1st and 3rd points of energy coupling and with completely blocked 2nd coupling point. In vivo the terminal segment of the respiratory chain does not operate, cytochromes c and a+a3 remain oxidized, and dominating terminal oxidase appears to be a component inhibiting by salicylhydroxamate and bound with the respiratory chain via cytochromes b or ubiquinone pool. The blocking of the 2nd coupling point is due to firm and, probably, quantitative binding of AA with cytochromes b, especially with b559, and also to a partial denaturation of the complex III.


[NAD-dependent formate dehydrogenase from methylotrophic bacteria. Isolation and properties]. NAD-Dependent formate dehydrogenase (FDH) has been isolated from methylotrophyc strain Bacterium sp 1 by (NH4)2SO4 fractionation of cell extract, ion-exchange chromatography and preparative isotachophoresis. Preparation of FDH is homogeneous in analytical polyacrylamide gel electrophoresis and under ultracentrifugation. Sedimentation coefficient of FDH is 4.9S. Mikhaelis constants are 1.1-10(-4) M for NAD and 1.5-10(-2) M for formate. In the absence of sulfhydril compounds FDH is unstable, but it is stable in the presence of mercaptoethanol or ditiotreitol.


The biochemical basis of memory.

The biochemical basis of memory. Several lines of research have indicated that the metabolism of both RNA and protein in the brain are important for permanent learning. One problem is to distinguish learning from stimulation per se, and whether it is meaningful to do so. Another problem is the biochemical interpretation of experiments using tracer precursor techniques. Evidence for the involvement of hormones such as ACTH as mediators of the biochemical responses and perhaps as stimulators of learning is discussed.


Histochemical studies on the EBV-determined nuclear antigen (EBNA). The EBV-determined nuclear antigen (EBNA) was studied with regard to several histochemical properties. Proteolytic enzymes destroyed EBNA staining. RNAse DNAse and hyaluronidase had no effect on the number of EBNA positive cells. Intensive treatment with DNAse weakened the chromosomal fluorescence of EBNA, whereas the staining of interphase nuclei was relatively enzyme resistant. When EBV-associated soluble complement-fixing antigen of Raji cells (CFA-R) was added to methanolacetic acid-fixed chicken red blood cells, brilliant and specific EBNA staining was obtained by anti-complement fluorescence (ACIF) with anti-EBNA positive sera. DNAse treatment abolished the ability of the nuclei to bind the antigen (CFA-R), whereas DNAse treatment following CFA-R antigen binding had no effect on the ACIF staining. EBNA was relatively heat stable. Somewhat weakened, but still fully positive EBNA reaction was obtained after 56 degrees C heating for 30 minutes in BSS. Periodate destroyed the EBNA reaction. Formaldehyde also abolished the staining, whereas methanol, acetone, methanol-acetone and methanol-hexyleneglycol-water preserved the staining relatively well.


Poland syndrome: a concept of pathogenesis based on limb bud embryology. A study of 15 patients as well as a review of the literature was made to determine the etiology of Poland syndrome. Neither the associated pathologic features nor any pattern of prenatal factors were helpful in revealing an etiology. A theory of primary nerve root dysgenesis was disproven. However, a concept of a localized insult at a particular embryonic stage based on known limb bud development is presented as a possible explanation of pathogenesis.


Clinical aspects of gene expression.

Clinical aspects of gene expression. 1. Expression and nonexpressin of genetic information may be viewed in relationship to the biologic structures that express or do not express the genetic information. We suggest defining expressivity as the quality of expression of genetic information in cells, tissues, organs, etc, of individuals, defining penetrance as the quality of expression of genetic information in an individual organism as a whole, and using a new term, phenotrance, to describe the quality of expression of genetic information in generations. 2. Decreased phenotrance may be indicated by "incompletely dominant" inheritance, by conditions for which "dominant" as well as "recessive" inheritance has been reported, or by disorders with sporadic occurrence in most and familial occurrence in some instances. The human conditions with decreased phenotrance that we have studied indicate that there are different types of decreased phenotrance. 3. The mechanisms for decreased phenotrance in man may correspond to certain genetic mechanisms that have been studied in lower organisms, such as delayed mutation, replicating instabilities, controlling elements, extrachromosomal inheritance, and others.


[Isoenzyme pattern of acid phosphatase in epstein-barr-virus-DNA positive permanent growing lymphoid cell lines (author's transl)]. The expression of acid phosphatases is cytochemically one of the most important features in permanent growing B-cell-lines. In few cell lines acid phosphatase is resistant against tartrate. Tartrate resistant isoenzyme 5 with components a and b can be demonstrated in monocytes, lymphocytes, chronic lymphatic leucemic cells and especially in hairy cells as well as in cell lines derived from a healthy donor. Fractionation of acid phosphatase by gelelectrophoresis in separated lymphocytes demonstrates especially isoenzyme 3, in separated macrophages isoenzyme 4. Isoenzyme 4 could not be detected in several cell lines. It is therefore concluded that these cell lines are probably derived from lymphocytic precursors. Cell lines with isoenzyme 4 may be the result of a facultative hybridisation between lymphocytes and monocytes. Profiles of acid phosphatases in virus-negative cell lines (Ramos, BJAB) were not significantly altered by conversion with EBV.


[Fluorinated pyrimidines administered by percutaneous arterial perfusion in primary or secondary cancers of the liver]. The results of a study based on intraaterial therapy applied to 65 cases of hepatic cancers (among which 42 cases of metastases of recto-colitic origin and 8 cases of primitive hepatomas) are reported. The catheters are insected percutaneously through the left brachial artery. After achort term treatment with Fluoro-Uracil FUDR is given continuously for over 20 weeks on the average, in ambulatory practice, using a Watkins chronofusor. A subjective response is reported in 88 p. 100 of the cases, an objective one in 74 p. 100. The mean survival time reckoning from the date of diagnosis is 16.6 months (it is somewhat longer in the cases of primitive hepatoma). The signs of systemic toxicity are frequent but mild; they seldom compel to discontinue the treatment. No death due to therapy has been register-d. The results are at least as good and even better with transcutaneous catheters than with catheters surgically inserted in the abdomen. They should encourage a wide spread of the method.


The effects of alloxanate, nicotinic acid and imidazole on secretory processes and the activities of adenylate cyclase and 3',5'-AMP phosphodiesterase in cat pancreas. 1 Nicotinic acid and alloxanate inhibited water and electrolyte secretion in a dose-dependent fashion when added to the perfusate of the isolated saline-perfused pancreas of the cat stimulated by a supramaximal dose of secretin.2 There were no changes in the concentration of sodium or potassium secreted into the juice, but the anions exhibited changes which were related to flow rate. As the flow rate declined the chloride concentration increased with a reciprocal decrease in bicarbonate concentration.3 Nicotinic acid and alloxanate inhibited enzyme secretion stimulated by carbachol.4 Imidazole inhibited pancreatic electrolyte secretion, but stimulated amylase secretion. Atropine (0.14 muM) reduced the secretion of amylase but did not abolish the effect.5 Adenylate cyclase prepared from cat pancreas, was stimulated by the octapeptide of cholecystokinin-pancreozymin, secretin and sodium fluoride.6 Alloxanate strongly inhibited both basal and hormone-stimulated adenylate cyclase activity. Nicotinic acid and imidazole stimulated basal adenylate cyclase activity but had little effect on secretin-stimulated activity.7 Alloxanate, nicotinic acid and imidazole were all without effect on phosphodiesterase when tested in the presence of micromolar concentrations of adenosine 3',5'-monophosphate (cyclic AMP). At higher cyclic AMP concentrations (2 mM) alloxanate and nicotinic acid were without effect, whereas imidazole had a slight stimulatory effect at 10 mM which was more marked at 50 mM.8 Alloxanate (10 mM) strongly inhibited both basal and secretin-stimulated adenylate cyclase activity.9 It is concluded that the effects of nicotinic acid, alloxanate and imidazole on pancreatic secretion are not mediated entirely through their effects on the adenylate cyclase or phosphodiesterase enzyme systems.


Opiate binding and effect in ileum preparations from normal and morphine pretreated guinea-pigs. 1 Dose-response curves for normorphine in the absence and presence of naloxone have been obtained from myenteric plexus-longitudinal muscle strip preparations from normal and morphine pretreated guinea-pigs. In addition, the high affinity stereospecific binding of [3H]-etorphine has been measured in homogenates of the same tissue. 2 Higher concentrations of normorphine were required to produce 50% inhibition of the electrically stimulated contractions of strip preparations from morphine pretreated animals. There was also an increase in the slope of linearized dose-response curves in opiate-tolerant preparations. Maximum opiate effect was unchanged, and responses to exogenous acetylcholine were not affected by the pretreatment. 3 There was a slight increase in the apparent equilibrium constant for naloxone after morphine pretreatment. 4 Tolerance to opiate effect was not accompanied by a change in the affinity or number of stereospecific binding sites for [3H]-etorphine. Hill plots of [3H]-etorphine binding in both control and morphine pretreated preparations gave slopes close to unity. 5 Most of these results can be explained by the assumption that in tolerant preparations, a certain fractional opiate receptor occupation threshold must be exceeded before opiate effects become apparent. It is suggested that the tissue adapts toward a threshold equivalent to the mean receptor occupancy attained during the period of opiate drug pretreatment.


Identification of respiratory motor neurons in the carp and determination of their firing characteristics and interconnections. Motor neurons of the respiratory muscles of the carp, located in the medulla oblongata, are identified with a signal averaging technique. Analysis of the firing characteristics of these neurons provides data on their activity patterns under different circumstances. The results show that, on the one hand, the number of spikes a motor neuron fires per respiratory cycle depends on the respiratory intensity, and can even be as low as one action potential per respiration. On the other hand, with constant respiratory intensity, the activity of individual motor neurons can stop and be resumed again in periods of several minutes. During mechanical loading of respiration, action potential synchronization between specific respiratory muscles takes place. Possible neuronal circuits causing this synchronization, which is thought to increase the coordination of the muscular effort, are discussed.


Modulation of electrical activity and cyclic nucleotide metabolism in molluscan nervous system by a peptide-containing nervous system extract. A peptide-containing extract (PE) from Helix nervous system modifies the endogenous bursting pattern of electrical activity in Helix neurone F-1. This effect is similar to that induced in neuron F-1 by certain phosphodiesterase inhibitors and cAMP derivatives. The PE, and the vertebrate peptide hormones vasopressin and oxytocin, also cause an accumulation of cAMP in Helix ganglia in vitro. The factor in the PE which causes the cAMP accumulation is destroyed by Pronase, is lost on dialysis, and is stable to boiling. In all these respects it is identical to the factor which causes the change in neuronal electrical activity. The PE also stimulates adenylate cyclase activity in a crude membrane fraction prepared from Helix ganglion homogenates. This stimulation is abolished by prior dialysis of the PE, or pretreatment of the PE with pepsin, but is not affected by boiling of the PE. Pepsin-treated PE has no effect on electrical activity in neuron F-1. The adenylate cyclase-stimulating activity of the PE, like the factor which modifies neurone F-1 electrical activity, elutes in the void volume of a Sephadex G-10 column. The included volume of this column contains a factor which inhibits PE modification of neuronal electrical activity, and also inhibits both basal and PE-stimulated adenylate cyclase activity. The data are consistent with the possibility that cAMP mediates the effects of the PE on electrical activity in molluscan neurones.


A morphine-like factor in mammalian brain: analgesic activity in rats. A partially purified morphine-like peptide 'enkephalin' (PPE) extract from bovine brain elicited pronounced apparent analgesia after injection into the periaqueductal gray matter of rat brain. This analgesia was reversed by the opiate antagonist naloxone in a dose-dependent fashion. Analgesia was more rapid in onset and much shorter in duration after PPE than after morphine administration. Analgesia was elicited only by those ion exchange column fractions of PPE that competed potently for opiate receptor binding. No analgesia could be detected when PPE or morphine injections were administered at a site 2 mm lateral to the periaqueductal gray matter. The potencies of synthetic methionine- and leucine-enkephalin in eliciting analgesia were less than 1% of those of partially purified enkephalin extracts when doses of equivalent ability to compete for opiate receptor binding were compared.


Analyses of relative protein content and distribution of histones in euchromatic and heterochromatic fractions. Euchromatic and heterochromatic fractions obtained by autodigestion of mouse TLT (taper liver tumour) hepatoma chromatin (Paul, I. J & Duerksen, J. D. (1976) Arch. Biochem. Biophys. 174, 491-505) were analyzed for relative protein content and histone content. With one exception, all fractions had the same DNA to protein ratio. Similarly, the total histone to DNA ratio was also constant in all fractions. In addition, the relative contents of the major histones, H1, (H2A + H2B + H3), and H4, were also constant in all fractions.


Protein content and enzyme levels of cultured chick embryo cells treated with camptothecin and actinomycin D. The alkaloid camptothecin uncouples the growth and adivision of chick embryo cells. At a moderate dose (0.5 microgram/ml) it inhibits the incorporation of thymidine but not of uridine and leucine and the cell protein content increases and reaches twice that of control after 4 days of treatment. Twelve hours after addition of the drug, the activities per cell of the mitochondrial enzymes poly A hydrolase (EC 3.1. 4.21), cytochrome c oxidase (EC 1.9.3.1), and succinate dehydrogenase (EC 1.3.99.1) are greater than that of the control and keep increasing for at least 96 H. The increase in the activities of the mitochondrial enzymes precede that of NADPH-cytochrome c reductase (EC 1.6.2.4) and cytidine triphosphatase (EC 3.6.1.15), which are microsomal and plasma membranes enzymes respectively. Actinomycin D (0.01 microgram/ml) also inhibits the multiplication of the chick cells and the synthesis of DNA. The protein content of the actinomycin D treated cells decreases to 70% of the control by day 2. Nevertheless, the activities of the mitochondrial enzymes increase over that of the control but to a smaller extent that with camptothecin. The activities of the enzymes of the other organelles are not stimulated. Camptothecin at a higher dose (5.0 microgram/ml) induces effects similar to those of actinomycin D.


Transmissible gastroenteritis: demonstration of the virus from field specimens by means of cell culture and pig inoculation. Isolation of transmissible gastroenteritis virus was attempted from segments of jejunum collected from piglets submitted for diagnosis of transmissible gastroenteritis. The virus was isolated more frequently in susceptible piglets than in pig kidney or pig thyroid cells. Practically, both cell systems were equally capable of demonstrating the virus when the tissue suspensions were sonicated. The pig thyroid cells prepared with glands collected from minimal disease pigs were preferred to the pig kidney cells for initial virus isolation because of their ability to respond to transmissible gastroenteritis virus with a progressive cytopathic effect. However, the pig thyroid cells, prepared from pool of glands collected in abattoirs, were often contaminated with parvoviruses and could not be used for diagnostic work. Controlled ultrasound treatments of the inoculum increased the frequency of virus isolation in both cell systems.


A monosynaptic fiber track studied in vitro: evidence of a hippocampal CA1 associational system? An excitatory afferent system previously undescribed in the in vitro slice was found to be present in the hippocampal CA1 stratum oriens. Evidence was provided that the system makes monosynaptic, en passage contact with CA1 pyramidal cells in the region of their basal dendrites. Slices from partially deafferented hippocampai were used in evaluating the possibility that the s. oriens pathway originated in the contralateral hippocampus. This possibility was not confirmed and the results were discussed in terms of an ipsilateral associational system in CA1 s. oriens.


Cell mediated immunity in equine herpesvirus type 1 infection I. In vitro lymphocyte blastogenesis and serum neutralization antibody in normal parturient and aborting mares. Blastic transformation of peripheral blood mononuclear cells and serum neutralization antibody levels for equine herpesivurs type 1 were measured in 19 mares from three farms at the time of termination of their pregnancy by normal foaling or viral abortion. The stimulation indexes of lymphocytes obtained from the mares from two farms (Farm 1 and 2) which had virus abortions, ranged from 2.1 to 10.8. But there was no significant difference in stimulation index levels between the aborting and normal foaling mares on these two farms. Equine herpesvirus type 1 was isolated from the mononuclear cells of one mare (No. 5) about two months after she aborted. The stimulation index of lymphocytes from that mare was not significantly different from that of other mares on these farms. Stimulation index of lymphocytes from the mares on one farm (Farm 3) where there was no virus abortion or previous history of virus abortion but were exposed to virus antigen from vaccination, ranged from 1.6 to 2.9. The serum neutralization antibody levels were low in most mares ranging from 1/4 to 1/20 and in three mares these were higher. There was no direct correlation between the levels of serum neutralization antibody and stimulation index of lymphocytes from the mares on these farms.


Medullary unit responses to changes in local and hypothalamic temperatures in the cat. Single unit activity was recorded with glass microelectrodes in the reticular formation of the medulla oblongata of cats lightly anesthetized with urethan, while medullary temperature was changed by surface irrigation with warm or cold artificial cerebrospinal fluid. In addition, water-perfusion thermodes were implanted over the preoptic anterior hypothalamus (POAH) region, and the effects of heating and cooling of the POAH on firing rate of medullary units were studied. One hundred and twenty-five temperature-sensitive neurons were studied in the medullary reticular formation. Out of these 125 neurons, 80 were warm-sensitive and 45 were cold-sensitive. Sixteen warm-sensitive medullary neurons were examined in responses to changes of POAH temperature. Ten units (62.5%) responded, and the remaining 6 units were not responsive to changes of the POAH temperature. Of 13 cold-sensitive neurons examined, 10 units (77.0%) responded. On the other hand, only 1 out of 7 (14.3%) temperature-insensitive neurons tested did respond to changes in the POAH temperature. These results suggest that the temperature signals sent out from thermosensitive structures in the hypothalamus might be transmitted to a major portion of temperature-sensitive neurons in the medullary reticular formation.


Detection and localization of aleutian disease virus and its antigens in vivo by immunoferritin technique. Tissues from mink infected with aleutian disease virus were examined by the electron microscope for the presence of virus particles. Virus-like particles, measuring 22 nm in diameter, were observed in macrophages of spleen, mesenteric lymph node and in Kupffer cells in liver of mink ten to 13 days after infection. The virus-like particles were usually present in vacuoles inside the cytoplasm of macrophages and Kupffer cells and, occasionally, similar particles were observed inside the nucleus. Cells from uninfected mink did not contain such patricles. To correlate the existence of these virus-like particles with the presence of aleutian disease virus antigen in infected cells, tissues were processed for immunoferritin technique. It was found that aleutian disease virus antigen was present in vacuoles inside the cytoplasm of cells from the infected spleen, lymph node and liver, and that the location was similar to that of the 22 nm virus-like particles. In addition, some viral antigen was also detected as cytoplasmic granular material. The nuclei of some cells also contained aleutian disease virus antigen. The pattern of aleutian disease virus antigen was similar to the distribution of virus-like particles in cells of infected tissue. It is suggested that virus replication occurs inside the nucleus with subsequent accumulation of virus in the vacuoles of the cytoplasm.


Necrotizing encephalitis in skunks caused by Herpes simplex virus. Herpes simplex virus was isolated from the brain of a wild skunk (Mephitis mephitis) which had clinical neurological disease. In the brain of this skunk and in brain of skunks inoculated intracerebrally, there were areas of necrosis in the grey matter of the cerebral cortex and to a lesser extent in the brain stem. Intranuclear inclusions occurred in neurons and glial cells with vascular cuffing by mononuclear cells and a few neutrophils. Skunks inoculated intravenously had extensive focal necrosis in the liver and adrenal glands.


Effect of age and pregnancy on the antibody and cell-mediated immune responses of horses to equine herpesvirus 1. The cell-mediated immune response and antibody response of horses of varying ages and of pregnant horses to equine herpesvirus 1 antigen were examined. Six to eight month old horses showed either no increase or slight increases in anti-equine herpesvirus 1 serum neutralizing antibody following vaccination and revaccination with a modified live equine herpesvirus 1 vaccine. However, these same horses showed a marked increase in the cell-mediated immune response to equine herpesvirus 1 as measured by the lymphocyte transformation test. Eighteen to 21 month old horses showed four to 64-fold increases in anti-equine herpesvirus 1 serum neutralizing antibody titer following vaccination, but the cell-mediated immune response to equine herpesvirus 1 was low or absent. Only after revaccination did they show an increased cell-mediated immune response to equine herpesvirus 1. The cell-mediated immune response of mares in the latter stages of pregnancy to equine herpesivurs 1 was suppressed although antibody titers increased as much as 16-fold following exposure to virulent equine herpesvirus 1.


Metabolic clearance rate of adrenocorticotropin in the rat. The metabolic clearance rate (MCR) of adrenocorticotropin (ACTH) was estimated after the intravenous infusion of graded rates of the hormone (40-2560 muU/min per 100 g body weight) in rats pretreated with chlorpromazine, morphine, and Nembutal, a preparation which proved effective in blocking endogenous ACTH release. The hormone was infused over a period of 45 min, at which time the plasma ACTH concentration had reached a steady state. A specific and sensitive bioassay, based on the corticosterone production of dispersed adrenal cells, was used to measure the plasma ACTH concentration. With increasing infusion rates of ACTH, a threefold decrease in the MCR of ACTH was observed. Previous studies of our group have shown that the MCR of corticosterone increases as a function of the infusion rate of the steroid. It appears, therefore, that the metabolism of these two hormonal links of the hypothalamo-pituitary-adrenocortical axis vary in opposite fashions as a function of the secretion rate of the hormone.


Quantitative study of alpha-adrenergic receptors in circular and longitudinal muscle of isthmus of rabbit fallopian tube and in rabbit aorta. The affinity of phenylephrine (pD2 value) and KB value for phentolamine for the alpha-adrenergic receptors of circular and longitudinal muscle of isthmus of rabbit fallopian tube and rabbit aorta have been estimated. The affinity of both phenylephrine and phentolamine was significantly more for the alpha adrenoceptors of the longitudinal muscle of isthmus of rabbit fallopian tube and rabbit aorta than circular muscle of isthmus of fallopian tube. The results of this investigation suggest that alpha adrenoceptors of circular muscle of isthmus are atypical. If this holds good for the human fallopian tube, the development of selective alpha-adrenergic-receptor agonists and antagonists may be of potential practical application for the control of fertility.


Effect in vivo of beta-adrenergic stimulation, angiotensin II, dibutyryl cyclic AMP, and theophylline on tonin concentration in rat saliva and submaxillary gland. Tonin (an enzyme present in rat submaxillary gland and saliva) has previously been shown to be able, unlike renin and reninlike substances, to release angiotensin II either directly by acting on an appropriate substrate or from angiotensin I. The administration of a beta-adrenergic drug, isoproterenol, produces a rise of tonin concentration in saliva without affecting its concentration in the submaxillary gland. Prior administration of a beta blocker, propranolol, partially prevents this effect. The administration of theophylline increases the tonin concentration in both saliva and the submaxillary gland, whereas dibutyryl cyclic AMP increases tonin concentration in the former. These results suggest that beta-adrenergic stimulation enhances both tonin release into the saliva and tonin synthesis in the submaxillary gland, and that these effects might be mediated by cyclic AMP. Infusion of angiotensin II blocked the stimulatory effect of isoproterenol on salivary tonin. 1Sar-8Ile-angiotensin II is both a weak antagonist of angiotensin II in this respect and a strong agonist in terms of blocking the effect of isoproterenol another role mirrored in other physiological mechanisms of derivatives of angiotensin II.


Effect of exogenous nucleotides on the candicidin fermentation. Addition of cyclic-AMP (c-AMP) to Streptomyces griseus fermentations inhibited candicidin formation. In a phosphate-free resting cell system, c-AMP inhibited net candicidin formation and incorporation of labeled propionate and p-aminobenzoic acid into the antibiotic but did not inhibit protein synthesis. All nucleotides tested, regardless of the position of the phosphate ester, were effective inhibitors; nucleosides and free bases were not. Inhibition occurred whether the nucleotide was added early or late. The results indicate that inhibition of antibiotic formation by exogenous nucleotides, including cyclic nucleotides, is similar to the effect produced by inorganic phosphate.


The effect of ionic surface-active agents on macroconidial plasma membrane of Fusarium sulphureum. A method was developed for direct assessment of changes in cytoplasmic volume and permeability of plasma membranes of intact cells to divalent cations. This technique, which ultilized an amphipathic spin label partitioning between intracellular aqueous and hydrophobic environments, allowed estimates of the proportion of cells in a homogenous population sustaining membrane damage associated with Ni+2 and water permeability and the rate at which such damage was induced. Several specific effects of cationic and anionic surfactants on the macroconidial plasma membranes of Fusarium sulphureum Schlect (isolate 1) were distinguished on the basis of detergent concentration and charge. The induction of water uptake by the cells was found to be an effect of dodecylguanidine acetate (Dodine), a cationic fungicide, at low concentration. At higher concentrations (greater than 5 X 10(-5) M) both the impermeability of the plasma membrane to divalent cations and the ability to accumulate actively L-phenylalanine were lost irreversibly and cell lysis occurred above 5 X 10(-4) M. Sodium dodecyl sulfate eliminated divalent cation impermeability more rapidly than Dodine but was less effective in inhibiting active transport function. An antagonistic effect between cationic and anionic detergents was observed.


Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli. A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolic repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce beta-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3',5-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.


Epitheliocystis disease in the striped bass Morone saxatilis from the Chesapeake Bay. Epitheliocystis disease in the gills of the striped bass Morone saxatilis from Chesapeake Bay was studied using light and electron microscopy. The epitheliocystis infection appeared synchronous in that all capsules on a single host were at the same stage of development. The disease appeared to begin in a single cell on the gill lamella, which gradually enlarged to form a large cyst, encapsulated by a thick cellular capsule of epithelial origin. The epitheliocystis inclusion was filled with cells of the general morphology and size of rickettsiae; however, the infection was atypical for rickettsiae in that the cells had a dense central nucleoid region and they developed within an inclusion separated from the host cytoplasm.


Inhibition of antigen and mitogen-induced lymphocyte transformation by fetal calf serum. Use of fetal calf serum as a serum supplement in whole blood microcultures of human lymphocytes resulted in a significant suppression of in vitro stimulation with herpes simplex virus (type 1) antigen, purified protein derivative, and phytohemagglutinin. If the response to viral antigen is weak in autologous serum it may be completely missed if cultures are carried out in fetal calf serum.


Protective effect of polymyxin B sulfate in experimental meningococcal infection in mice. The mouse model of intraperitoneal meningococcal sepsis was used to evaluate the antiendotoxic activity of polymyxin B sulfate independent of its antibiotic effects. Administered either before or after the infective challenge therapeutic doses of polymyxin B sulfate produced small but significant increases in survival over unprotected animals. These results also suggest that endotoxin contributes to the outcome in this variety of Gram-negative infection.


The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects. Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.


Chemotherapy of malignant fibrous histiocytoma: a Southwest Oncology Group report. Malignant fibrous histiocytoma is a rare tumor, which constitutes 3-4% of the soft tissue sarcomas. It occurs with maximum frequency in the sixth and seventh decades of life and has a distinct male preponderance. In two-thirds of the patients an extremity is the primary site and approximately one-half develop local recurrences and one-half, distant metastases. Response to combination chemotherapy occurred in 33%, a rate similar to that seen in other sarcomas.


Human leukemic "null" cell line (NALL-1). A human lymphoblast cell line, NALL-1, was established from the peripheral blood of a patient with acute lymphoblastic leukemia (ALL). NALL-1 cells had neither properties of T and B cells nor Epstein-Barr virus (EBV). Many characteristics of the NALL-1 line were distinct from those of numerous EBV-positive lymphoblastoid cell lines previously reported. NALL-1 cells are considered to have originated from the donor's leukemic cells on the basis of their cytogenetic, morphologic and functional features. The NALL-1 line is the first human leukemic "null" cell line derived from ALL. The significance of this cell line is disscussed.


Bronchiolo-alveolar carcinoma: a Clara cell tumor? The cell of origin, and indeed the very existence, of bronchiolo-alveolar carcinoma have long been the subject of debate. Published cases have demonstrated the presence of type II pneumocytes in some tumors, while others have been composed of mucus-secreting bronchiolar cells. A case of bronchiolo-alveolar carcinoma is presented in which the ultrastructure indicates an origin from Clara cells, but in which some cells contain polymorphous and lamellar bodies, It is concluded that bronchiolo-alveolar carcinoma is a primary lung tumor derived from the terminal airways and in the majority of cases from the bronchiole. The value of electron microscopy in assessing such tumors is emphasized.


Studies of the tumorigenic effect of two goitrogens. The nature of tumors appearing in the thyroid gland and in the lungs of mice fed two standard goitrogenic drugs (MTU and MII) has been studied. These tumors have been considered malignant on the basis of their morphological appearance and their occurrence in abnormal locations. Some investigatiors, however, have questioned that they are actually malignant. The present results indicate that these tumors are most likely not malignant even if it is shown that the pulmonary nodules are of thyroid origin. The thyroid adenomas disappear once the goitrogen is withdrawn, but thryoid enlargement pesists, and event 6 months after discontinuation of the goitrogenic treatment , pulmonary nodules are still produced. Evidence is presented that these nodules are emoli from hyperplastic thyroid tissue and not tumors.


Glycogen-rich Clara cell-type bronchiolo-alveolar carcinoma: light and electron microscopic study. A clear cell type of bronchiolo-alveolar carcinoma removed from the lung of a 63-year-old man was studied by light and electron microscopy. The tumor was composed exclusively of Clara cells identified by the presence of secretory granules, an abundance of granular endoplasmic reticulum, a moderate number of mitochondria and prominent Golgi complexes. The tumor cells were tall columnar and had clear cytoplasm due to the presence of large pools of intracytoplasmic glycogen. This feature has not heretofore been described. Ultrastructural features distinguishing this tumor from benign clear cell tumors of the lung and clear cell carcinomas of the kidney are described.


Müllerian adenosarcoma of the uterus: an ultrastructural study of four cases. Four cases of uterine müllerian adenosarcoma, a distinctive form of mixed müllerian tumor, were studied by light and electron microscopy. All tumors showed the characteristic histologic pattern of benign neoplastic glands within sarcomatous stroma. Ultrastructurally, both mesenchymal and epithelial cells were seen. The mesenchymal cells showed some features of endometrial stromal cells, including the presence of intracytoplasmic collagen fibers. The epithelial cells formed glands, which resembled benign endometrial glands and were separated from the stroma by a well-defined basal lamina. No transitional cells between the epithelial and mesenchymal cells were seen. The ultrastructural features of these tumors suggest that the sarcomatous portion is of endometrial stromal origin. The glandular portion may arise, along with the stroma, from multipotential stem cells, or the glands may be non-neoplastic entrapped endometrial glands stimulated by the stroma and thus appearing to form an integral part of the tumor.


Nasopharyngeal cancer in patients under the age of thirty years. There is evidence that patients under 30 years of age with nasopharyngeal cancer present a somewhat different form of disease than patients of the older age group. Serum immunologic reactivity in the former was quantitatively different. Histologically, tumors in the younger age group are always of the undifferentiated type, and clinically, the disease is aggressive, characterized by frequent bone and lung metastases. The lymphatic spread into the mediastinum is accompanied by a paraneoplastic syndrome consisting of finger clubbing and hypertrophic osteoarthropathy, which is reversible after successful therapy. High rates of recovery have been obtained, even in the presence of advanced disease or metastases. This warrants a radical and persistent radiotherapeutic and or chemotherapeutic approach.


Progression of intraepithelial Paget's disease of the vulva to invasive carcinoma. Sequential progression from intraepithelial Paget's disease of the vulva to invasive carcinoma after an interval of 10.8 years is documented in this report. Infiltration of the dermis with permeation of lymphatics and metastasis to regional lymph nodes are believed to have resulted directly from foci of intraepidermal Paget's cells that persisted after a simple vulvectomy. No underlying primary sweat gland carcinoma was present. Hence, this case establishes the invasive potential of intraepithelial vulvar Paget's disease and substantiates the notion that it is indeed a type of carcinoma in situ.


Inhibits of of colon carcinogenesis.

Inhibits of of colon carcinogenesis. Disulfiram (tetraethylthiuram disulfide, Antabuse) and sodium diethyldithiocarbamate, when added to the diet, inhibit 1,2-dimethylhydrazine (DMH)-induced neoplasia of the large bowel in female CF1 mice. Ethylene bis(dithiocarbamato)manganese (Maneb) and bis(ethylxanthogen), two pesticides with structural similarities to disulfiram, produce comparable inhibition of DHM. In other work, disulfiram was found to inhibit the carcinogenic effect of azoxymethane (AOM) on the large bowel. Under comparable conditions the inhibition of AOM was considerably less than that obtained with DMH as the carcinogen. The data suggest that disulfiram inhibits DMH metabolism at more than one oxidative step.


Investigations into the metabolism and mode of action of the colon carcinogens 1,2-dimethylhydrazine and azoxymethane. Colon cancer can be induced reliably in rodents with 1,2-dimethylhydrazine and azoxymethane (AOM). Our studies deal with the mode of action of these compounds and their organotropism. A partial summary of our previous work on the metabolism of 1,2-dimethylhydrazine and its inhibition by disulfiram, carbon disulfide and other thiono-sulfur compounds is presented. On-going studies with AOM-14C indicate that in male F-344 rats, this carcinogen is rapidly metabolized to 14CO2 (37%, 48 hours), and to methylazoxymethanol-14C (MAM) (0.6--1%), which, along with other metabolites, appears in the urine. Pretreatment of rats with phenobarbital or chyrsene increased exhaled 14CO2 to 53% and 65%, respectively. Pretreatment with disulfiram or CS2 causes a complete, although transient, inhibition of exhaled 14CO2, decreases urinary MAM, and increases significantly the levels of unmetabolized AOM in the exhaled air and in urine. Thus, phenobarbital and chrysene appear to stimulate, while disulfiram and CS2 appear to inhibit, the metabolism of AOM. In vitro hydroxylation of AOM to MAM was demonstrated with rat liver homogenates and microsomal fractions. A hypothetical scheme for the endogenous formation of AOM is presented.


Colorectal carcinoma in adolescents implications regarding etiology. Between October, 1974 and December, 1976, 13 adolescent patients with far-advanced, poorly differentiated colorectal carcinoma had been referred to a pediatric cancer center. All patients received chemotherapy with vincristine, methyl-CCNU and 5-fluorouracil. Five of 13 patients are living, one of whom remains disease-free after 12 months of chemotherapy. Four of the patients were from urban areas and nine from rural areas. One of four from urban areas had intimate exposure to chemicals used in the production of cotton and soy beans. Eight of nine patients from rural areas also had exposure to farm or agricultural chemicals, and three of these patients were intimately involved with the spraying operations. Suggestions regarding etiology and causative factors for the development of carcinoma of the colon in adults have previously been advanced. Results of these studies suggest that alternate etiologies must be suggested for adolescent colorectal carcinoma.


Overview: molecular changes associated with large bowel cancer and their potential as markers and chemotherapeutic agents. The search for molecular changes that may be diagnostic of malignancy in the colonic epithelium is complicated by the diversity of cell types and complex cell kinetics of a tissue in which most of the cells are destined to leave within hours or days. Methods for cell separation and nuclear fractionation now permit biochemical studies of those cells that retain or regain the capacity for DNA synthesis and that are likely to include the transformed cell population. Among the changes associated with malignant transformation to be described are alterations in nuclear protein composition and metabolism, qualitative and quantitative differences in adenosine deaminase activities, activation of the guanylate/cyclic GMP system, and modification of both DNA and chromosomal proteins by alkylating carcinogens. DNA modification to produce O6-methylguanine correlates well with the incidence of tumor induction by methylazoxymethanol. Modifications of chromosomal proteins to produce methylated derivatives of lysine and arginine have been observed after the administration of 1,2-dimethylhydrazine. Such changes are likely to lead to aberrant interactions between DNA and regulatory elements in chromatin, and may not be subject to repair.


Selective toxicity induced by picolinic acid in simian virus 40-transformed cells in tissue culture. When cultured normal and SV40-transformed normal rat kidney and BALB/3T3 cells were exposed to picolinic acid, cell proliferation ceases. Most of the normal cells remained in a quiescent G1 (G0) state and viable for prolonged periods of time. In contrast, SV40-transformed cells progressed to the S and G2 phases of the cell cycle and remained viable only up to 90 to 120 hr. Then, most of the cells began to die. However, a very small fraction of the cell population (approximately 0.01 percent) developed into variants resistant to picolinic acid. Prevention of development of variants, and therefore destruction of all transformed cells, was obtained by addition of glycerol to picolinic acid-treated cells. Untransformed cells were unaffected by the same treatment. These results suggest that differential tumor toxicity should be feasible.


Radioimmunoassay for glycoprotein gp47 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera. Murine mammary tumor virus main glycoprotein (gp47), prepared by diethylaminoethyl cellulose and hydroxyapatite chromatography of detergent-mercaptoethanol-KCI-disrupted virion, was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Seven other (glyco) proteins of the virus were antigenically distinct from gp47. Serum and organs of unifected C57BL mice did not contain gp47, but sera of infected Swiss and RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of gp47 in other organs was identical in male and female mice. The titer of gp47 in serum was high in tumor-bearing females, but it varied with the mouse strain. Anti-gp47 immunoglobulins could not be detected. The investigation included 314 human sera (107 normal, 65 benign mastopathy, 89 breast vancer, and 53 digestive cancer). None contained an antigen related to gp47. One of 20 human mammary cyst fluids was positive.


Membranous effects on adenosine triphosphatase activities of mitochondria from rat liver and Morris hepatoma 3924A. Adenosine triphosphatase (ATPase) activities of sonically prepared submitochondrial particles of rat liver and Morris Hepatoma 3924A were compared as a function of changes in temperature. On Arrhenius plots, a discontinuity at 18 degrees was observed for the rat liver mitochondrial ATPase, while the hepatoma mitochondrial ATPase revealed a discontinuity at 20.4 degrees. Values for energy of activation of the rat liver and hepatoma mitochondrial ATPases were comparable below the break (34.5 and 35.5 kcal/mole, respectively) and above the break (11.6 and 9.2 kcal/mole, respectively). Solubilization of the mitochondrial membrances with Triton X-100 resulted in constant and similar values of energy of activation for the ATPases Km values of hepatoma and rat liver mitochondrial ATPases for adenosine triphosphate were similar in both the membrane-bound and solubilized states. The lack of uncoupler-stimulated ATPase activity in hepatoma mitochondria is apparently not due to membranous effects on the affinity of the ATPase for adenosine triphosphate.


Scanning electron microscopy of the surfaces of hamster embryo cells transformed by herpes simplex virus. The surfaces of normal hamster embryo fibroblast (HEF) cells were examined by scanning electron microscopy. Surface characteristics of HEF cells were compared to those of cells derived from a primary tumor induced in hamsters following s.c. inoculation of herpes simplex virus type 1-transformed HEF cells (14-012-8-1) and to the surfaces of cells derived from a metastatic tumor to the lung induced by the same cells. The most obvious difference in the surface characteristics of the examined cells was the morphology of the microvilli. In the few HEF cells that possessed microvilli, the distribution was uneven, and the lengths of the microvilli and the filopodia were variable. However, the surfaces of both tumor cell lines showed large numbers of microvilli which were evenly distributed over the surface of the cells, giving an almost "hairy" appearance. Long filopodia were occasionally observed on the surface of the primary tumor cell line and on the cell line derived from the metastatic tumor. Ruffles and blebs were occasionally observed on HEF cells and on the primary tumor cells but were not seen on the cells of the metastatic tumor.


Tumor-associated immunoglobulins in pulmonary carcinoma. In view of the uncertainty of location and significance of immunoglobulin in tumors found by elution or rosette formation (as reported in the literature), the presence of IgG, IgM, and IgA in human carcinoma of the lung was studied by means of the peroxidase-antiperoxidase method. Surgically obtained specimens from patients with known survival times were used in this study. Membranous as well as cytoplasmic location of IgG was demonstrated more frequently than was that of IgA or IgM. The number of tumor cells carrying immunoglobulin varied greatly, even within a given case. Albumin could be demonstrated in tumor cells in 10 of 20 specimens, but there was poor correlation with immunoglobuin. In some instances, only the necrotic part of the tumor or the stroma was immunoreactive. The results are discussed and suggest that Fc receptors are not involved in the binding of immunoglobin by pulmonary carcinoma cells.


Long-term effects of prenatal and neonatal administration of 5beta-dihydrotestosterone on normal and neoplastic mammary development in mice. The long-term effects on mammary glands of 5beta-dihydrotestosterone, considered to be biologically inactive, were studied in female SHN mice with mammary tumor, virus. 5beta-Dihydrotestosterone was adminstered to mothers for 4 days from Day 12 to Day 15 of pregnancy (prenatal treatment) and to pups for 5 days of postnatal life (neonatal treatment) at daily doses of 1 mg and 200 mug, respectively. Neonatal treatment resulted in marked stimulation of spontaneous mammary tumorigenesis; all neonatally treated mice had palpable mammary tumors by 6.2 months of age, when mammary tumor incidences in the control mice and in mice treated prenatally with 5beta-dihydrotestosterone were 21.1 and 6.3 percent, respectively. Furthermore, neonatal treatment promoted normal and preneoplastic mammary growth and pituitary prolactin secretion and induced ovarian anovulatory syndrome in all mice. Prenatal treatment also increased the number of mammary hyperplastic alveolar nodules and induced an delayed anovulatory syndrome. These results have demonstrated that perinatal treatment with 5beta-dihydrotestosterone can induce such irreversible changes in the mammary glands, pituitary gland, ovaries, and genital tracts as those seen with other biologically active steroid hormones.


alpha-Lactalbumin levels in human mammary tumors, sera, and mammary cell culture lines. Antiserum to purified human alpha-lactalbumin was produced in rabbits and used to develop a radioimmunoassay capable of detecting 0.1 ng of alpha-lactalbumin per ml of sample. Human breast diseases were analyzed for alpha-lactalbumin levels. A high percentage of breast carcinomas contained varying levels of alpha-lactalbumin. Lymph node metastases from primary carcinomas that synthesized alpha-lactalbumin also contained it. Analysis of serum from breast cancer patients indicated that approximately 25 percent had measurable levels of alpha-lactalbumin before surgery, but no alpha-lactalbumin was found in postsurgery sera. alpha-Lactalbumin was not detected in the urine of early lactational women, although it was present in the sera. Human cell culture lines derived from pleural effusions of mammary carcinomas contained little, if any, alpha-lactalbumin. Other human cell lines derived from mammary carcinomas and grown as solid tumors in athymic mice did not contain measurable levels of alpha-lactalbumin.


Inhibition of concanavalin A stimulation of feline lymphocytes by inactivated feline leukemia virus. In a lymphocyte blast transformation assay, the response of feline lymphocytes to concanavalin A was suppressed 20 to 65 percent in the presence of inactivated feline leukemia virus. The decrease was not due to viral cytotoxicity, as determined by trypan blue viability counts, nor was the virus binding the concanavalin A and interfering with its mitogenic stimulation. The virus may be biochemically repressive in itself, interfering with cell-mediated immunity within the feline system.


Biological characterization of an Epstein-Barr nuclear antigen-positive American Burkitt's tumor-derived cell line. Two distinct cultures derived from a lymphoid cell line designated NAB were characterized immunologically, morphologically, and cytogenetically. Both cultures were positive for Epstein-Barr nuclear antigen. NAB I cultures were negative for virus capsid antigen and early antigen and were not affected by treatment with 5-iododeoxyurdine. NAB II cultures were positive for virus capsid antigen and early antigen, which increased with 5-iododeoxyuridine treatment. Both cultures were superinfected with virus prepared from P3HR-1 cells. Cell-free virus concentrates prepared from both cultures were inactive for transformation and infectivity. NAB I and NAB II cells were lymphoid as determined by light and electron microscopy. NAB II cells showed morphological alterations characteristic of herpes infection. 5-iododeoxyuridine-treated cells from both cultures revealed ultrastructural characteristics of cells infected with herpes-viruses but without particles. In addition, the induction of tubuloreticular structures within the endoplasmic reticulum was observed. Cytogenetic analysis of both cultures revealed a rearranged chromosome 14 and several other chromosome aberrations, three of which may be used as a reliable means of identifying NAB cultures.


Role of radiation therapy in combination with chemotherapy in extensive oat cell cancer of the lung: a randomized study. After 3 months of chemotherapy with procarbazine, vincristine, cyclophosphamide, and CCNU, patients with extensive oat cell carcinoma of the lung were randomly allocated to receive 3000 rads of involved-field irradiation and further chemotherapy or to continue with chemotherapy only. In addition, all patients received 3000 rads of prophylactic whole-brain irradiation. The addition of involved-field radiation, as given in this trial, not only failed to improve survival but was unable to alter the pattern of sites of initial relapse. There were no cases of extension to the brain in 31 consecutive patients who received prophylactic whole-brain irradiation. This was in strong contrast to a preceding trial in which six cases of brain extension occurred among 19 patients who did not receive prophylactic whole-brain irradiation.


Autoradiographic studies of 3H-dexamethasone uptake by immunocytochemically characterized cells of the rat pituitary. 3H-Dexamethasone (10 microgram/kg) was injected intravenously in adrenalectomized rats and after survival times of 5, 30, 60, and 180 min its uptake within the pituitary was studied by autoradiography. Radioactivity was concentrated in cell nuclei in the pars nervosa and pars distalis. Within the pars intermedia, only cells of the marginal zone were labeled. In the pars distalis, some cells showed a weak nuclear accumulation of radioactivity as early as 5 min after injection. The tissue radioactivity was nearly maximal at 5 min, and the proportion of radioactivity in nuclei reached a maximum of 60-70% by 30 min. In competition experiments, non-radioactive steroids (1 mg/kg) were injected 5 min before 3H-dexamethasone and sacrifice was 30 min later. Dexamethasone markedly diminished the nuclear accumulation in the pars distalis, but corticosterone and progesterone did not. In the pars nervosa, corticosterone and progesterone competed for nuclear uptake of 3H-dexamethasone, although less effectively than dexamethasone itself. Different cell types in the pars distalis were characterized by treating autoradiograms with an immuno-peroxidase bridge procedure. Cells treated with anti-ACTH 17-39 had the greatest nuclear concentration of radioactivity, and those stained with anti-TSH were least heavily labeled. Cells treated with antisera to GH, PRL, and hCG were moderately labeled.


Filaments and rod-shaped tubulated bodies in the endothelia of anterior cerebral arteries in young rats. Endothelia of the anterior cerebral arteries in rats aged 1 to 3 days were studied. Thin (about 50-90 A) and thick (about 100-110 A) filaments are present in the endothelia. Numerous spherical- or rod-shaped bodies, measuring approximately 0.07 to 0.3 micrometer in diameter and up to 0.6 micrometer in length occur in the endothelial cells. These bodies contain a tubular structure. The diameter of the individual tubules is about 200 A. The present observations suggest that spherical- or rod-shaped inclusions are first synthesized in the rough endoplasmic reticulum and thereafter these materials are transported into the Golgi complex for maturation. A small number of the inclusions, however, may originate directly from the rough endoplasmic reticulum and not pass through the Golgi apparatus.


Effect of cyproterone acetate on cells of the pars distalis of the adenohypophysis in the beagle bitch. The effects of oral administration of 100 mg per kg per day cyproterone acetate (CPA) for four weeks on cells of the pars distalis, as revealed by the immunoperoxidase technique and chemical staining, were studied in the ovariectomized beagle bitch. For immunochemical staining antisera to the following hormones were used: canine GH, canine PRL, procine ACHT, bovine TSH beta, bovine  LH beta and human FSH beta1. The most striking effects of the treatment were an overall increase in the relative proportion of GH cells and a marked morphological indication of high secretory activity in these cells. In contrast, PRL cells were not affected significantly. In all ovariectomized control bitches a marked atrophy of the cells stained for FSH beta (FSH cells) and hypertrophy of the cells shown to contain LH beta (LH cells) were observed. FSH cells became enlarged, while LH cells appeared reduced in size by administration of CPA. In some treated bitches ACTH/MSH cells showed atrophy and regressive changes, whereas TSH cells seemed to become enlarged and were more densely arranged. These structural responses indicate that, in addition to its partial antigonadotropic properties, CPA as a synthetic progesterone derivative may stimulate GH secretion and possibly suppress CRH-ACTH activity in the ovariectomized beagle bitch.


Autoradiographic study of the distribution of LH(HCG) receptors in the ovary of untreated and gonadotrophin-primed immature rats. The LH(HCG) receptors in the ovaries of immature rats which were either untreated, or primed with PMSG and HCG, have been studied with a histochemical method which has proved to be as effective as when earlier used in the rat testis. This method, which consists of the topical application of 125I-HCG to picric acid-formaldehyde (PAF) fixed frozen sections followed by autoradiography, is also suitable for quantitative studies on the distribution of receptors. In the ovary of the immature 26 days old rat, the LH(HCG) receptors are localized exclusively in the interstitial and thecal tissues. After PMSG treatment many receptors appear in the granulosa of the large antral follicles. These receptors are most numerous in the outer layers of cells and least numerous in the inner. At the same time there are fewer receptors in the thecal and interstitial cells which have undergone the process of luteinization. After PMSG and HCG treatment the newly formed corpora lutea have few receptors, but these become progressively more numerous on subsequent days. It is suggested that, in the rat, the luteinization of the ovarian LH-target cells is associated with an initial decrease in the number of their LH(HCG) receptors.


Formaldehyde-ozone-induced fluorescence in pituitary ACTH cells: effect of adrenalectomy. ACTH and MSH cells of the pituitary are rich in peptides with NH2-terminaltryptophan, as revealed by fluorescence histochemistry. Adrenalectomy stimulates the ACTH cells but not the MSH cells. As a result, ACTH as well as tryptophyl-peptides disappear from the ACTH cells but not from the MSH cells. It is concluded that the tryptophyl-peptides are stored together with the respective hormone in the ACTH and MSH cells and that tryptophyl-peptides in the ACTH cells are released together with the hormone.


Comparative cellular localization of corticotropin and melanotropin in lerot adenohypophysis (Eliomys quercinus). An immunohistochemical study. Corticotropin and melanotropin producing cells were localized in the adenohypophysis of normal Lerots by using antibodies against synthetic corticotropins (anti beta1-24 ACTH, anti alpha17-39 ACTH, anti alpha25-39 ACTH), and melanotropins (anti alpha MSH, anti beta MSH). All the anticorticotropin sera stained the same cells both in the anterior lobe and in the intermediate lobe. The anti alpha MSH serum only stained a few cells, exclusively located in the intermediate lobe. These alpha MSH cells were not stained with anticorticotropin antibodies. The anti beta MSH serum revealed all the cells stained with anticorticotropin and anti alpha MSH sera. Absorption tests showed that the 4-10 heptapeptide common to ACTH and MSH, is not responsible for the immunohistochemical staining. The staining of only some corticotrophs with the anti 4-10 ACTH serum might indicate the presence in these cells of a peptide with an accessible 4-10 site. These results are discussed.


The display of microtubules in transformed cells. Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.


Abelson virus-transformed lymphocytes: null cells that modulate H-2. A-MuLV-transformed lymphoid cells from Balb/c mice had the properties of null lymphocytes. They did not secrete Ig and all but one did not have detectable cell-associated Ig; one line synthesized, but did not secrete, the mu chain of IgM. The cells expressed H-2D and H-2K, but not H-21 histocompatibility antigens or theta-antigen; they had Fc receptors. Most cell lines grew to form donor cell tumors after inoculation into (Balb/c X C57B1/6)F1 mice. The tumor cells have more H-2Dd than cells passaged in vitro. Cell lines carried in vitro progressively lost H-2Dd. A line in which 5-30% of the cells were lysable by anti-H-2Dd was cloned; all eleven clones had H-2Dd (13-69% lysable) demonstrating that H-2 modulates in vitro. A clone with little H-2Dd (10-15% lysable) was tumorigenic even after treatment with anti-H-2Dd sera; at least 50% of the tumor cells were lysed by anti-H-2Dd. Thus A-MuLV-transformed lymphocytes modulate H-2 in vivo to higher levels and in vitro to lower levels.


Inhibition by aldehydes as a possible further mechanism for glucose-6-phosphatase inactivation during CCl4-poisoning. Diffusable aldehydes are known to be produced during lipoperoxidative deterioration of unsaturated fatty acids. Malealdehyde (MLA) and 4-hydroxy-2,3-trans-penten-1-al (4-HPE) inhibit rat liver glucose-6-phosphatase activity in vitro. With MLA inhibition is significant at 0.25 mM concentration. With 4-HPE inhibition takes place at 0.5 mM. 1 mM MLA inhibited by about 89%, 6 mM -HPE by about 67%. Maximal inhibition is present as early as 5 min after addition of both aldehydes. Preincubation of aldehydes with 2 mM cystein or glycine in the absence of microsomes almost completely prevents the inhibitory influence. Previous incubation of microsomes with 2 mM glutathione or 2 mM dithiothreitol or 2 mM cysteine affords a good protection towards the inhibitory action of the aldehydes; on the contrary, no protection is seen when microsomes are preincubated in the presence of either 2 mM glycine or asparagine. The total content of microsomes -SH groups is strongly decreased after incubation with 2mM malealdehyde. These results support the idea that the two aldehydes inhibit glucose-6-phosphatase mostly through interaction with protein -SH groups. The possibility that aldehydes derivated from the peroxidative decomposition of lipids may play a cooperative role in the inhibition of glucose-6-phosphatase occurring early after CCl4-poisoning is discussed.


[Inhibition of corticotropic function by administration of GABA in carp (Cyprinus carpio L.)]. The intracardiac injection (50 microgram/g) of GABA to Carps clearly inhibits the rise of the plasma cortisol level which occurs usually in response to the blood sampling manipulations. The inhibition starts thirty minutes after injection and lasts till the end of the experiment two hours later. The hypothesis that this neurotransmitter has a regulatory function on the corticotrop cells, especially on the excretion of ACTH has to be considered.


Influence of salts on Michaelis-constant values for NADH. We previously observed [Clin. Chem. 22, 1648 (1976)] that values of the Michaelis constant for NADH for the conversion of pyruvate to lactate with lactate dehydrogenase (EC 1.1.1.27) in the presence of 0.1 mol/liter buffers at 25 degrees C showed first-order dependence on enzyme concentration. This is now recognized to be the result of an inhibitory influence exerted by buffers [NH4HCO2, tris(hydroxymethyl)aminomethane, and phosphate] and salts [(NH4)2SO4 and NaCl] present in the reaction mixtures. Inhibition constants for the enzyme/inhibitor complexes formed with these substances are about 0.3 mol/liter for competition of NH4HCO3 with NaOH and 0.4 mol/liter for competition of NH4HCO3 with pyruvate; they are 0.6 mol/liter for NaCl, 1.0 mol/liter for sodium phosphate, 0.3 mol/liter for (NH4)2SO4, and 0.8 mol/liter for tris(hydroxymethyl)aminomethane when these substances compete with NADH. Because of the large molar ratio of buffer to substrate (about 10(9):1) in enzymatic assays, the buffer concentration significantly influences the Michaelis constant, despite the large value for the inhibition constant. Attention to the concentrations of these substances may be required for decreasing variability in clinical assays in which lactate dehydrogenase and possible other enzymes are used.


4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography. We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.


A method for measurement of low levels of guanosine 3',5'-cylic monophosphate in blood plasma. A very sensitive method is described for the assay of guanosine 3',5'-cyclic monophosphate (cGMP) based on a competitive protein-binding technique. The procedure is simple to carry out, and does not require the purchase of expensive reagents. The binding-protein used is extracted from rat lungs and the bound and unbound cGMP are separated by ammonium sulphate precipitation. Although prior extraction of the cGMP is required, the time for this extra step is easily compensated for by the shortness of the assay time. The mean cGMP found in the blood plasma of normal young males and females is 9.9 +/- 2.4 (S.D.) nmol/l (range 5.9--14.3 nmol/l) and 9.5 +/- 2.1 (S.D.) nmol/l (range 6.9--12.7 nmol/l), respectively.


Cerebrotendinous cholestanolosis in relation to other cerebral xanthomatoses. A synopsis is given of 37 reported patients with cerebrotendinous xanthomatosis; divided in 11 possible, but not proven cases of C.T.Ch (cerebrotendinous cholestanolosis), and 26 proven cases. An increased content of cholestanol in serum or tissue, was used as the main criterion. The ophthalmological and neurological signs are tabulated. Attention is drawn to the fact that cholestanol is not mentioned in the extensive literature on related diseases with possible cerebrospinal xanthomas, such as eosinophilic granulomatosis and hyperlipoproteinemia. The importance of a possible role of cholestanol in these diseases is stressed. A hypothetical form of cerebrotendinous cholesterolosis is discussed. A family with a sibship of parents and twelve children, three of which showed C.T.Ch is described. The development of the clinical picture confirmed the variable expression of the autosomal recessive disease. Two patients showed disturbances of steroid metabolism. A first contribution to the search for linkage with the H.L.A. system is presented. Cholestanol over cholesterol ratio, appeared to be independent of the type of lipoprotein. In search for therapy we found in patients with hyperlipoproteinemia type II A, that cholestyramine alone gave dangerously high blood levels of cholestanol; in combined treatment of cholestyramine with clofibrate cholestanol, levels were comparable to those of the controls. The possibilities of therapy are discussed.


Concepts in undergraduate neurological teaching. Common neurological problems in family practice were determined to assess what knowledge of neurology is essential to the graduating medical student. The family practitioner's difficulties in managing these problems and needs for referral, were studied. Types of problems seen in a teaching hospital neurology service were also examined. Neurological problems are important if they are common, require therapy, or demand emergency management. However, dealing with these problems family practitioners report considerable difficulties. An approach to medical training is outlined in an effort to assist future physicians in handling the neurological problems of daily practice with competence and confidence.


Computed tomography of intracerebral hematoma. The C.T. findings of 33 intracerebral hematomas are described. During the first week intracerebral hematomas were visible as a sharply demarcated lesion with markedly increased density without contrast enhancement. Thereafter the density decreased gradually and after 30 days the density was always lower than that of cerebral tissue. Depending on the etiology of the hemorrhage, differences were found in the localization, shape and density of the lesion on the C.T. scan. Secondary mass effects could be evaluated precisely. On first interpretation, 94% of the hematomas were recognized as focal lesions (2 false negative) and 85% as intracerebral hematomas (2 false negative and 3 incorrect). False positives were absent. The review C.T. diagnosis was correct in 94%. In the diagnosis of intracerebral hematomas, C.T. was found to be superior to all other investigations both quantitatively and qualitatively. C.T. also has therapeutic consequences since the operability can be much better judged than before.


Computed tomography of extracerebral hematoma. The appearance on C.T. of 30 extracerebral hematomas is described. Acute subdural hematomas were seen as bandlike lesions with markedly increased, homogenous density. Epidural hematomas demonstrated a high density and a biconvex, lenticular shape. During the process of liquefaction the density decreases and three hematomas appeared as isodense lesions. The majority of chronic subdural hematomas presented a mildly to moderately decreased, nonhomogenous density, the clearly identifiable inner wall of the lesion being either convex or concave. Subdural hygromas showed a homogeneous, markedly decreased density. Almost all the hematomas caused compression of the homolateral lateral ventricle and displacement of midline structures, particularly of the septum pellucidum. In 60% of the hematomas a dilatation of the contralateral lateral ventricle, predominantly of the posterior part was seen. Acute hematomas presented oedema in varying degrees, in chronic hematomas oedema was usually absent. In the original interpretation extracerebral hematomas were recognized as a lesion on C.T. in 97% (1 false negative result), in 90%, the lesion was correctly diagnosed as an extracerebral hematoma (1 false negative and 2 incorrect). The series contained one false positive C.T. scan and 2 intracerebral hematomas were interpreted incorrectly as subdural hematomas. The review C.T. diagnosis proved to be correct in 100%. C.T. and angiography appeared to have about the same diagnostic accuracy. However, C.T. is a non invasive investigation and it provides valuable additional information with regard to all the intracranial contents. Therefore C.T. should be the method of first choice in the diagnosis of extracerebral hematomas.


Neuroaxonal dystrophy, a juvenile-adult form. This paper is an anatomoclinical study of a neuroaxonal dystrophy case, starting in childhood and evolving up to maturity, which may be considered as a juvenile-adult form of this syndrome. Several of the morphologic emphasized aspects suggest that this juvenile-adult form is a primary affection of the axon and therefore, may be considered as a separate entity, differing from the Hallervorden-Spatz disease.


Paraganglioma of cauda equina.

Paraganglioma of cauda equina. After a brief introduction to the problem of the nature and localisation of sympathetic and parasympathetic paragangliomas a new case of paraganglioma of cauda equina is reported.


Noninvasive monitoring of beta-adrenergic tone during isoproterenol infusions. Sphygmo-Recording is a simple, noninvasive technique for analysis of pulse wave contour and timing which has been used to evaluate the change in cardiac dynamics during isoproterenol infusion. The QKd interval, i.e., the time interval between the onset of the QRS complex and the onset of the Korotkoff sound at the brachial artery when the sphygomomanometer cuff is at diastolic pressure, is normally 205 +/- 15 msec. Continuous intravenous infusion of isoproterenol at 0.01, 0.02, and 0.03 microgram/kg/min into 12 euthyroid normotensive adult volunteers for 10-min intervals resulted in decreases of 55, 79, and 89 msec in QKd and increases of heart rate of 14, 27, and 43 beats/min, respectively. The corresponding changes in dP/dt, i.e., slope of the pulse wave upstroke at the brachial artery determined noninvasively from the same records, were 0.65, 1.47, and 2.26 mm Hg/msec. These results confirm previous studies which indicate that the chronotropic response of normal subjects to isoproterenol infusion is comparable to that previously reported in patients with the putative "hyperdynamic beta-adrenergic state."


Collagen and non-collagen protein synthesis in the lungs of rats exposed to a trypsin aerosol. In rat lungs, 24 h after a 10 min inhalation of a nebulized 1% (w/v) trypsin solution, there was a 25% increase in lung weight. The incorporation of 3H-tryptophane and 2,3-[3H]-proline into trichloroacetic acid insoluble material was decreased although there was no alteration in prolyl hydroxylase activity. Although hydroxyproline formation was decreased, this decrease was probably due to the general decrease in protein synthesis. Ninety-six hours after inhalation of the trypsin solution there was an increase in non-collagen protein biosynthesis. Proline incorporation and hydroxyproline formation were both increased more than the tryptophane incorporation increase at this same time point. These increases were accompanied by an increase in prolyl hydroxylase activity. These experiments indicate that major changes in protein biosynthesis occur in lung tissues after inhalation of proteolytic enzymes and demonstrate the temporal biochemical changes which occur in lung injury.


Extractable collagenase and carcinogenesis of the mouse skin. Collagenolytic activity has been demonstrated in the early phase of chemical carcinogenesis of mouse skin following 3-methylcholanthrene application dropwise in acetone or painted on the skin in benzene. In addition very high levels of collagenase could be detected in mouse skin papillomas and carcinomas. In all the tissues investigated, collagenase activity was extracted from the 6000 X g sediment of tissue homogenates with 5 M urea in 50 mM Tris-HCl buffer, pH 7.5. After dialyzing the extract, the enzyme was precipitated with ammonium sulfate and the activity determined against 14C-collagen substrate in solution. This procedure was found suitable for the detection and estimation of collagenase activity in skin tissues with high turnover of collagen and thus offers an attractive alternative to tissue culture methods.


Dream report following commissurotomy.

Dream report following commissurotomy. This paper addresses the question of right hemisphere involvement in the visual components of dreaming. The rationale derives from an observed relation between reports of visual agnosia accompanied by dream cessation and the literature on right hemisphere specialization for visuo-spatial processes. All night sleep EEGs were recorded from subjects with partial or complete section of the corpus callosum and anterior commissure. Upon entering a EEG-, EOG- and EMG-defined REM episode, the subjects were awakened and questioned about dream content. All subjects examined in this fashion were able to recount some visual dream content. This result fails to support any notion of selective right hemisphere visual dream mediation.


Practical problems in clinical enzymology. It is possible to carry out precise determinations of the activity of the clinically important serum enzymes with continuous monitoring (kinetic) methods. Care must be taken that the induction period has passed before the first measurement is taken and that the reading is taken from the zero-order, linear part of the curve. Special precautions must be taken with highly active sera that are also turbid. There are no suitable enzyme standards available at present; therefore, standardization of the instrument is of utmost importance. The instrument must be able to produce the theoretical molar absorptivity of the substrate or cofactor. The instrument must be calibrated for wavelength and absorbance accuracy with nationally accepted standards. Some reference methods are beginning to appear; if widely adopted, they should improve the interlaboratory comparability of enzyme assays. Instrument and method standardization plus well-characterized enzyme standards, when available, will reduce the variability of intra- and interlaboratory enzyme assays.


Cytochrome C as antidote in mice poisoned with the mushroom toxin alpha-amanitin. The curative potencies of cytochrome c applied 8 hours after lethal doses of alpha-amanitin in mice are confirmed. In a comprehensive series of experiments, various dosage schedules were explored. Optimal effects with cure rates approaching 100% were obtained either with repeated doses of cytochrome c or by combining it with penicillin. Male mice are more sensitive to alpha-amanitin than female mice but it was established that the antidote is effective in both sexes. Substantial cure rates are seen even when the treatment after supralethal doses of alpha-amanitin is withheld for 12 hours after the poisoning.


[The diuresis of cyclic adenosine-3'5'-monophosphate (cAMP) in primary and secondary disorders of the parathyroid glands]. The diuresis of cAMP in primary hyperparathyroidism was significantly higher at 7.3 +/- 2.5 mumol/g creatinine X 24 h (P less than 0.005) than that in healthy subjects (3.5 +/- 0.7 mumol/g creatinine X 24 h). After successful operation on the parathyroid gland, cAMP diuresis usually decreased within 24 hours to normal or subnormal values. In primary or secondary hypoparathyroidism subnormal amounts of cAMP (P less than 0.005) were excreted. The method gives false-negative results in functional disorders of the parathyroid glands accompanied or caused by renal failure.


Biochemical pathogenesis and therapeutical prospectives in degenerative chorea. In the last few years both neuropathological and neurochemical data have contributed towards a classification of the pathogenesis of degenerative chorea of which Huntington's chorea shows clear evidence of heredity. The role of GABA, DA and Ach in determining chorea seems clear. In 7 patients with degenerative hypotonic-hyperkinetic chorea (the acid metabolites into the cerebrospinal fluid are not available) a neuropharmacological study was conducted. The symptomatology was evaluated both through clinical rating and through phasic and tonic performance tests. The neuropharmacological tests were both acute and chronic. In the acute tests the effect of modification of various neurotransmitters (Ach, DA, NA, GABA) was evaluated. In chronic treatment the effectiveness of haloperidol, PCPA and bromocryptine was evaluated. The results of the acute study were positive both as regards increment of DA (receptors stimulation) and GABA. The chronic study shows any effect of PCPA, positive results both with bromocryptine and with haloperidol. Our results confirm a DA/GABA interaction in the pathogenesis of degenerative chorea.


Failure of neurotransmitter blockers to alter PGE2-induced LH release. The purpose of this study is to ascertain whether or not prostaglandin (PG) E2 induces LH release by modifying or modulating the release or action of neural transmitters. PGE2 injected iv into spayed rats primed two days earlier with 10 microgram estradiol benzoate increased the plasma levels of LH 10 min later as measured by radioimmunoassay. The peak of plasma LH was not changed by prior treatment with beta- or alpha-adrenergic receptor blockers, propranolol or phenoxybenzamine. The peak level of plasma LH did not alter in rats treated with DL-alpha-methyl-p-tyrosine methyl ester HCl (alpha-MPT) or sodium diethyldithiocarbamate (DDC). Similarly, the peak of plasma LH was not changed by prior treatment with imipramine. Administration of PGE2 produced an increase in anterior pituitary and plasma, but not hypothalamic cyclic AMP concomitantly with the elevation in plasma LH. Although it is possible that the effect of PGE2 could be mediated by another transmitter system, as yet unknown, or that the effect of PGE2 on LH release could be mediated via the adenylate cyclase-cyclic AMP system, the results indicate that PGE2 does not act trans-synaptically, but probably acts directly on LH-RH neurons.


Effects of biogenic amines on the formation of adenosine 3',5'-monophosphate in human thyroid slices. The effects of various concentrations of biogenic amines on the formation of adenosine-3', 5'-monophosphate (cyclic AMP) and their interactions with other thyroid stimulators were investigated in human thyroid slices from normal and Graves' disease. Most of biogenic amines were found to have the stimulatory effects to some extent. Among the biogenic amines tested, histamine was the most potent thyroid stimulator, norepinephrine and serotonin, the intermediate in terms of cyclic AMP formation. The effect of histamine was almost as potent as TSH in thyroid slices from Graves' disease. This stimulatory effect of histamine was blocked by metiamide, a histamine H2-receptor antagonist, but not by chlorpheniramine, a histamine H1-receptor antagonist. The effect of norepinephrine was completely inhibited by propranolol, but not by phentolamine. Polyphloretin phosphate did not inhibit norepinephrine- or histamine-induced cyclic AMP formation, while it significantly depressed cyclic AMP formation induced by prostaglandin E2. The maximal effect of histamine was additive to that of TSH. It is suggested that biogenic amines, histamine and norepinephrine, in particular, have the thyroid receptors different from that of TSH or prostaglandin E2 and could play an important role in thyroid physiology.


Correlation of ovarian binding of 125I-HCG with formation of cAMP, estradiol and progesterone during pregnancy. The content of ovarian gonadotropin receptors in rat was found to change during pregnancy. The specific binding of 125I-HCG to ovarian homogenates rose to its maximal values on days 13 and 16. Thereafter, the binding declined as parturition approached. No consistent changes in responsiveness of rat ovary to LH in synthesis of cAMP and estradiol were observed during pregnancy. Plasma estradiol concentration increased on day 16 and remained high throught days 21 and 22. Progesterone levels increase steadily during the first half of pregnancy and then fall, especially sharply on day 21 and 22. The results demonstrate that the secretion of progesterone correlates with gonadotropin receptors during pregnancy.


Fructose transport in Bacillus subtilis. The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems. The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.


The modification of nuclear proteins by ADP-ribosylation. When incubated in vitro purified mouse nuclei incorporate NAD into poly(ADP-Rib). Analysis of the product on CsDl/urea gradients showed that a large proportion of the poly(ADP-Rib) was not attached to protein. The free poly(ADP-Rib) did not appear to arise through degradation and its chain length was significantly longer than the poly(ADP-Rib) attached to proteins. Fractionation of the proteins on hydroxyapatite revealed that tissue-specific modification of both the histones and non-histone proteins, had occurred. In the case of one protein species there was evidence for the existence of several forms with different numbers of ADP-Rib residues.