The ultrastructure of the accessory sex organs of the male rat. XI. Nuclear alterations of prostatic epithelial cells induced by castration. The fine structure of the nuclei of epithelial cells of the dorsal lobe of the rat prostate were studied 2, 3, 5, 7 and 21 days after castration. The nucleolus appears to undergo a progressive disorganisation with partial fragmentation and dispersion of its normal components. Changes in the nucleoplasm were primarily reflected by a condensation of chromatin, particularly along the nuclear membrane and adjacent to the nucleolus. Later, different types of intranuclear inclusions were observed. After 21 days, the nuclei were characterized by an irregular outline with large indentation. Within the nucleoplasm aggregates of coarse granular chromatin were found. No cell necrosis was observed, indicating that androgen deprivation results in a remodeling of the cell to a less active state with marked cellular alterations and cessation of secretion, but apparently with some of their basic functions still intact. Injections of testosterone completely reverse the castrated-induced alterations. The changes observed are assumed to be due to the withdrawal of the androgenic stimulus, with a direct influence on the secretory function of the cell. The findings support the view that the stimulating secretory effect of androgen is mediated via an intranuclear androgen receptor, probably located in the nucleolus-associated-chromatin. It is also proposed that the secretory function of the epithelial cells of the prostatic complex, initiated by androgens, may be regulated by an intranuclear secretory center.


Exocrine pancreas under experimental conditions. I. Formation of paracrystalline inclusions in vivo. After the application of parachlorophenylalanine (pCPA), an amino acid analogue, paracrystalline inclusions are observed in the exocrine pancreas of the rat. The formation of the paracrystalline structures varies according to the dose and the time of examination. Although the first alterations can be seen in the Golgi apparatus and the condensing vacuoles, the main localization of these structures is within the cisternae of the RER. At the same time as degenerative changes occur in the cells, involving autophagic and heterophagic processes, regeneration also takes place. With the freeze-fracturing method, the paracrystalline inclusions are interpreted as lamellae or plates of probably altered secretory proteins in extremely extended RER-cisternae. The fracture surfaces of the paracrystals show a periodicity of about 80 A running diagnonally to the main axis of the paracrystalline structures, which are mainly oriented from the basal parts of the exocrine pancreatic cells to the cell apices. The mechanism of paracrystalline formation is discussed on the basis of the morphologic results. It could be shown that after pCPA administration the amylase content is decreased concomittantly with degranulation. pCPA seems not to be incorporated into secretory proteins; high intracellular concentrations, however, are required to induce the formation of the paracrystalline structures. This morphological study is the basis for other studies dealing with secretion and intracellular transport in the pancreatic acinar cell under experimental conditions.


The general occurrence of intramitochondrial crystals in Hydra cells. Intramitochondrial crystals are found in normal Hydra as well as in animals undergoing various conditions (budding, regenerating, eserine-treated, and sexual). They appear in all regions of the animal, but seem to be more prevalent at or near the extremities: hypostome, tentacles and basal disk. They are found in all of the seven basic cell types: interstitial, cnidocyte, nerve, epithelio-muscular, gland, mucous and digestive cells. The chemical nature of the intramitochondrial crystals is unknown and their significance remains speculative.


Glucose uptake by normal human breast tissue in organ culture. Influence of insulin and other additives. Normal human breast tissue explants were cultured in a synthetic basic medium with and without additives. The mean daily glucose uptake per explant was measured under six basic conditions. Our results show that glucose uptake is strongly related to the glucose concentration of the medium. On the other hand insulin does not affect significantly glucose uptake in vitro, but does enhance mitotic activity. These findings support a role for insulin in promoting D.N.A. synthesis rather than in controlling glucose metabolism of human mammary tissue in vitro.


The initiation region of the SV40 VP1 gene. The sequence of 15 nucleotides located at the 5' terminus of the plus strand of the SV40 Hind K fragment has been determined as (5') A-G-C-T-T-A-T-G-A-A-G-A-T-G-G (3'). The 3' on OH terminal G of this segment is part of the G-C-C codeword for the N terminal alanine of the VP1 protein. This region therefore presumably corresponds to a ribosome binding site on the 16S late mRNA. Complementarily to the 3' OH of eucaryotic 18S ribosomal RNA and homology with the BMV coat ribosome binding site are discussed.


Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus. In normal human fibroblasts, the synthesis of a cell surface receptor for plasma low density lipoprotein (LDL) is regulated by a sensitive system of feedback suppression. The number of functional LDL receptors declines by more than 20 fold when cellular stores of esterified cholesterol are increased by incubation of cells with an exogenous source of cholesterol. Fibroblasts from patients with the heterozygous form of familial hypercholesterolemia (FH) possess one functional allele and one nonfunctional allele at the LDL receptor locus. In the current studies, we have examined the effect that this deficiency produces upon the pattern of regulation of the single functional allele at the LDL receptor locus. Under growth conditions that induced a maximal rate of LDL receptor synthesis (that is, growth in the absence of an exogenous source of cholesterol), the FH heterozygote cells produced about one half as many functional LDL receptors as did the normal cells. More importantly, when grown in the presence of increasing amounts of exogenous cholesterol, the FH heterozygote and normal cells suppressed their respective LDL receptor activities in parallel. Over a wide range of LDL receptor activities, at each level of cellular esterified cholesterol, the FH heterozygote cells expressed about one half as many receptors as did the normal cells. These data indicate that in the FH heterozygote cells, the receptor regulatory mechanism dictates that the normal allele produce only the amount of gene product that it would normally produce at a given level of cellular esterified cholesterol. The failure of the regulatory mechanism to stimulate the normal allele at the LDL receptor locus to produce twice its normal amount of gene product leaves the FH heterozygote cells with a persistent 50% deficiency in LDL receptors under all conditions of cell growth.


Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease. The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.


In situ hybridization of repetitive monkey genome sequences isolated from defective simian virus 40 DNA. The origin of a repetitive monkey DNA sequence that is incorporated into a defective simian virus 40 genome has been studied. A fragment (about 140 base pairs in length) containing essentially all the repetitive monkey DNA present in the defective and few, if any, SV40 sequences can be cleaved from the purified defective DNA by restriction endonucleases Hind(II and III). Radioactive cRNA prepared with the isolated fragment as template was hybridized in situ to African green monkey chromosomes. The results indicate that all or part of the sequence in question occurs at both centromeric and noncentromeric positions in many, but not all, chromosomes. Of the typical 60 chromosomes, between nine and eleven hybridize with the cRNA in noncentromeric regions.


The arrangement of simian virus 40 sequences in the DNA of transformed cells. High molecular weight DNA, isolated from eleven cloned lines of rat cells independently transformed by SV40, was cleaved with various restriction endonucleases. The DNA was fractionated by electrophoresis through agarose gels, denatured in situ, transferred directly to sheets of nitrocellulose as described by Southern (1975), and hybridized to SV40 DNA labeled in vitro to high specific activity. The location of viral sequences among the fragments of transformed cell DNA was determined by autoradiography. The DNAs of seven of the cell lines contained viral sequences in fragments of many different sizes. The remaining four cell lines each contain a single insertion of viral DNA at a different chromosomal location. The junctions between viral and cellular sequences map at different places on the viral genome.


Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein. The major capsid protein (VP1) of simian virus 40 (SV40) has been analyzed by two-dimensional electrophoresis. This system separates protein according to isoelectric point by isoelectric-focusing, and according to molecular weight by sodium dodecylsulphate electrophoresis (O'Farrell, 1975). VP1 synthesis in infected CV-1 cells can be monitored directly by analysis of unfractionated whole cell extracts; the resolution of VP1 from cellular proteins allows its detection as early as 13 hr after infection. The two-dimensional separation of VP1 reveals that it is heterogeneous, consisting of one major protein (molecular weight 47,000 daltons and isoelectric point of approximately pH 6.8) and five minor protein components. The minor forms of VP1 are 10% of the total VP1 and differ from the major form of VP1 both in molecular weight (by approximately 500 daltons) and isoelectric point (ranging from approximately pH 6.7 to pH 6.9). Evidence is presented to show that two of the minor forms are phosphorylated derivatives of VP1, and it is further suggested that all the different forms of VP1 are the result of modifications of the primary product of translation. A temperature-sensitive mutant of the BC complementation group (BC11) of SV40 results in the synthesis of VP1 with an altered electrophoretic mobility; both the major form of VP1  and the minor forms are shifted in their isoelectric points. In addition to the specific case of SV40, two aspects of these studies should be generally significant to investigators studying eucaryotic gene expression by two-dimensional gel electrophoresis: first, the genetic origin of a protein can be determined by a temperature-sensitive mutation which causes a charge change in the resultant protein; and second, two or more protein spots on a two-dimensional separation may be the products of a single gene.


The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences. The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.


On the nature of 5' termini in nuclear pre-mRNA of Ehrlich carcinoma cells. 5' terminal nucleosides of nuclear pre-mRNA of Ehrlich ascites carcinoma cells were analyzed by a combination of different chromatographic methods and phosphatase treatment. The heavy nuclear pre-mRNA contains mainly unblocked triphosphorylated nucleosides at the 5' end, although some capped 5' ends could also be found. In this respect, it differs from cytoplasmic poly(A)+ mRNA which contains blocked 5' termini and no triphosphorylated ends. The 5' terminal nucleotides in pre-mRNA are pppGp and pppAp (in a ratio of 3:2). The determination of pppNp content in poly (A)+, poly(U)+, and poly (A)-(U)- fragments of RNA has been used as an approach to establish the topography of pre-mRNA. We also established that the technique for isolation of triphosphorylated 5' terminal fragments of RNA based on hydroxyapatite chromatography (Bajszár, Samarina, and Georgiev, 1974) is still valid in the presence of blocked oligonucleotides. The latter do not interfere with fragments containing free triphosphate groups. Using this technique, we showed that a small but significant portion of triphosphorylated 5' end fragments of  100 nucleotides in length contain oligo(U) sequences reacting with poly(A)-Sepharose.


Repair processes and the response of dividing and non-dividing cells to methyl methanesulphonate and dimethyl sulphate. A method for producing a viable non-dividing population of Chinese hamster V79 cells in suspension is described and the characteristics of the population outlined. The stationary population is more sensitive to methylating agents than a similar but exponentially growing population, the increased sensitivity arising from the loss of the shoulder from the survival curve. The extent of reaction of the agent with cellular macromolecules is similar in both cases. The repair capabilities of the two populations was examined. Non-semiconservative DNA repair synthesis occurs whether the cells are in a growth or no-growth condition when insulted. Repair of single-strand breaks, which arise following methylation, also proceeds up to the size of the replicon. The relationship of this stationary population to other no-growth conditions and its utility as a model for carcinogenesis studies is discussed.


Biochemical changes after hepatic injury by allyl alcohol and N-hydroxy-2-acetylaminofluorene. Administration of hepatotoxic doses of allyl alcohol and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) TO adult male rats produced periportal necrosis and functional derangement of the hepatic endoplasmic reticulum within 24 h. The rates of N-demethylation of ethylmorphine and p-hydroxylation of aniline were decreased 6 h following allyl alcohol administration, but cytochromes P-450 and b5 were unchanged. In contrast, administration of NOH-AAF decreased cytochromes P-450 and b5 and the rate of aniline p-hydroxylation, but did not change the rate of N-demethylation of ethylmorphine or the activities of cytochrome c reductase and glucose-6-phosphatase. No decrease was observed in the activity of the cytosol enzyme, DT diaphorase, following allyl alcohol treatment. The changes by these periportal hepatotoxins were compared with those produced both by central and midzonal hepatotoxins and with changes occurring in the liver after surgical partial hepatectomy.


Substrate specificity of tonin from rat submaxillary gland. The substrate specificity of tonin from rat submaxillary gland was examined with a series of synthetic peptides encompassing the C-terminus of the decapeptide substrate angiotensin I. In contrast to angiotensin I-converting enzyme from plasma or lung, only angiotensin I, (des-Asp1)-angiotensin I, and (des-Asp1, des-Arg2)-angiotensin I are substrates of tonin with Km values of 34.5 muM, 39.3 muM, and 54.4 muM, respectively, while the shorter C-terminal peptides are not hydrolyzed. Thus, the N-terminal sequence extending from position 1 to 3 is the enzymatic binding site for tonin. Turnover numbers of 33.4 sec-1, 42.8 sec-1, and 6.5 sec-1 are observed for the hydrolysis of angiotensin I, (des-Asp1)-angiotensin I, and (des-Asp1, des-Arg2)-angiotensin I, respectively. The relative percentage rates of hydrolysis (proportional to V/Km) at low substrate concentrations ([S] less than less than Km) are almost identical for (des-Asp1)-angiotensin I, angiotensin I, and the tetradecapeptide substrate, indicating that these three peptides are equally good substrates at low physiological concentrations. The observed high specificity of the enzyme lends support to the possible important role of tonin for local conversion in tissue. The conversion of (des-Asp1)-angiotensin I to (des-Asp1)-angiotensin II (angiotensin III) is of particular interest in relation to the recently suggested, potential role of the latter peptide in aldosterone release.


Quantification of human atrioventricular nodal concealed conduction utilizing S1S2S3 stimulation. We studied antegrade concealed conduction of atrial extrastimuli (A2) that blocked in the atrioventricular (AV) node in eight subjects, using a third extrastimulus (A3), coupled at decreasing coupling intervals to A2. Three A1-A2 intervals were tested in each subject: late (just shorter than AV nodal effective refractory period), intermediate, and early (just longer than atrial functional refractory period). The curves relating the following variables were constructed for each A2: A1-A3, H1-H3 and A2-A3, A3-H3. The former was compared to the control A1-A2, H1-H2 curve. Concealment of A2 was demonstrated in all eight subjects at the three tested values of A1-A2. The A2-A3, A3-H3 curve allowed analysis of AV nodal conduction time (A3-H3) and AV nodal recovery time (defined as the shortest A2-A3 at which the impulse conducted to the His bundle) at identical values of A2-A3. In all subjects the timing of blocked A2 had minimal effect on both AV nodal conduction time and recovery time. In five of the eight subjects a late A2 sporadically conducted to the His bundle. Conduction of A2 to the His bundle resulted in marked lengthening of both AV nodal conduction and recovery times. Concealed conduction of A2 was always demonstrated, but the degree of concealment was relatively fixed, whether A2 was an early, intermediate, or late blocked premature beat. Slow conduction of A2 had a much greater effect than concealment of A2 on subsequent impulse conduction.


Electrophysiological observations on the digitalis-potassium interaction in canine Purkinje fibers. We studied the effects of elevating potassium concentration on the membrane potential of Purkinje cells exposed to toxic concentrations of acetylstrophanthidin or ouabain. Conventional intracellular microelectrode techniques were employed. Rapid elevation of [K+]o from 2.7 to 5.4 mEq/liter resulted in an initial increase (more negative) in membrane potential of cells demonstrating ouabain-induced phase 4 depolarization. The increase in maximal diastolic potential occurred initially without suppression of phase 4 depolarization. In cells rendered inexcitable by ouabain or acetylstrophanthidin, elevation of [K+]o consistently increased membrane potential and restored excitability. In four experiments automaticity was initiated within 2 minutes after the increase in [K]o. Although automaticity reappeared, as maximal diastolic potential increased, the automatic rate slowed and then pacemaker activity was suppressed. Studies with 3H-ouabain showed that the increase in membrane potential paralleled K+-induced release of 3H-ouabain from Purkinje cells. These studies suggest that elevation of [K+]o reverses digitalis toxic manifestations in canine Purkinje fibers by causing release of cardiac glycosides bound to the membrane. The observed increase in membrane potential of ouabain-treated Purkinje fibers that occurred after [K+]o elevation was considered to be mediated in part by restoration of the Na pump and by electrogenic pumping.


The effects of iron deficiency on the respiratory function and cytochrome content of rat heart mitochondria. We have examined the effects of total body iron deficiency on the function of mitochondria isolated from rat hearts. Male Wistar rats were weaned at 21 days and divided into an experimental iron-deficient group and a control group. Both groups received identical diet but an iron supplement (180 mg of ferrous sulfate per kg of diet) was added for the control group. Rats were studied at 7 and 14 weeks. Iron-deficient rats weighed less than controls but showed significantly increased ventricle to body weight ratio at both 7 and 14 weeks, indicating relative cardiac hypertrophy. Isolated mitochondrial fractions from iron-deficient and control rats contained similar proportions of whole homogenate protein and succinic cytochrome c reductase activity, indicating that the fractions isolated from the experimental and control rats were comparable. In iron-deficient rats NADH cytochrome c reductase, succinic cytochrome c reductase, succinic dehydrogenase, and NADH ferricyanide oxidoreductase activities were all significantly reduced at 7 and 14 weeks. Cytochrome c oxidase activity was significantly reduced only at 14 weeks as were the concentrations of cytochromes a3, c1, and b. The rate of oxygen uptake by mitochondria was significantly lower at both 7 and 14 weeks but the P/O ratio was unaltered. We conclude that iron deficiency is associated with impairment of myocardial mitochondrial electron transport.


Hay fever treated with ACTH gel.

Hay fever treated with ACTH gel. ACTH gel was used as a treatment for hay fever during the 1972-1974 hay fever seasons. Patients suffering from hay fever were given ACTH in different dose schedules and other hay fever patients were used as controls. Subjectively, those treated with ACTH gel were pleased with the results and reported more favourably on the injections than those receiving placebo. Objectively, using presence of any symptoms as the index of measurement, those treated with ACTH gel (80 units once weekly or 80 units twice weekly) had a significantly lower incidence of hay fever. Side effects were few.


Superoxide anion independent reduction of nitrobluetetrazolium by human renal tissue. The rate of nitrobluetetrazolium reduction by extracts from rodent and human kidney tissue has been measured in the presence and absence of exogeneous superoxide dismutase and catalase. These oxygen metabolite scavengers had no effect on that reduction rate. Oxygen was inhibitory to nitrobluetetrazolium reduction whereas anaerobiosis enhanced the rate. These data suggest that the reduction rate in renal tissue is not superoxide anion radical mediated as had been previously suggested.


Detection of inhibitors in reduced nicotinamide adenine dinucleotide by kinetic methods. Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.


Automated radioimmunoassay of choriomammotropin (human placental lactogen). We describe a totally automated procedure for radioimmunoassay of choriomammotropin, in which all phases of the assay are automated in a single system ("Centria"). This system permits the simultaneous incubation and separation of many samples in a nonequilibrium assay, and measurements are obtained in less than 30 min. Results for clinical samples by reference radioimmunossay methodology and with the Centria system compared uniformly well: y=0.91x - 0.87;r=0.94. The coefficient of variation for samples run in duplicate on the same day was 5.2%, 7.4% for samples run on different days. The specificity, sensitivity, simplicity, and speed of this system makes it a useful new tool for kinetic, nonequilibrium immunoassay.


Bile acid and detergent interaction with radioassays based on coated charcoal. We tested the radioassays for cyclic AMP and human gastrin, both involving separation on coated charcoal, for interaction with bile acids and detergents, and found a concentration-dependent interaction of taurocholic and glycocholic acid as well as of the surfactant Triton X-100 and sodium laurylsulfate in both assays. The interaction was detectable from concentrations of 0.5 mmol of bile acid per liter or 625 mg of detergent per liter, giving rise to falsely decreased gastrin values and falsely increased or decreased values for cyclic AMP. The interactions demonstrated may be a general effect on all radioassays that are based on the use of coated charcoal in the separation of free from bound ligands.


Creatine kinase in serum: 2. Interference of adenylate kinase with the assay. Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%.


Nature of the interaction between low-density lipoproteins and polyanions and metal ions, as exemplified by heparin and Ca2+. I examined the effect of lipid concentration on the interaction of the very-low density and low-density  lipoproteins in serum with heparin and calcium. A precipitate forms when partly delipidated serum is subjected to the polyanion-metal ion system. Although this precipitate is less turbid than the complexed lipoprotein-polyanion-metal ion in undelipidated serum, the two precipitates contain identical amounts of apolipoprotein. Totally delipidated serum produces only a slight precipitate with heparin and calcium, and this precipitate contains only a fraction of the appolipoproteins. Cholesterol and triglycerides are the major determinants of turbidity when serum is mixed with heparin and calcium, but have no effect on the precipitation of the protein moiety. Phospholipids contribute a small proportion of the turbidity in the lipoprotein-polyanion-metal ion interaction. Precipitation of the low-density lipoproteins by heparin and calcium depends on the protein moiety and on the chemical composition of the lipid moiety. Phospholipid is required, but cholesterol and triglyceride are not, for total precipitation of the complex.


Plasma high-density lipoprotein cholesterol concentrations determined after removal of other lipoproteins by heparin/manganese precipitation or by ultracentrifugation. The widely used heparin/MnCl2 precipitation procedure for determination of plasma high-density lipoprotein cholesterol has been re-examined in light of recent reports that isolated preparations of the lipoprotein are only partly precipitated under the test conditions. In the present study, the procedure as applied to plasma tolerated rather wide variations in heparin and MnCl2 concentrations without significant effects on the assayed values in several plasma pools tested. The procedure was further tested on 129 individual samples by comparison with an ultracentrifugal method in which high-density lipoprotein-cholesterol is assumed to be represented by the cholesterol content of the plasma fraction of relative (to water) density greater than 1.063. Our results indicate that high-density lipoprotein is not precipitated under the test conditions when applied to unfractionated plasma.


Evaluation of enzyme-multiplied immunoassay technique (EMIT) for determination of serum digoxin. Sixty-seven digoxin-containing sera were analyzed by both radioimmunoassay and EMIT. After some important modifications of the EMIT method, agreement between the two methods was very good. Reproducibility of the EMIT assay was excellent; daily variations in values found for control sera were quite small, and recovery of added digoxin was good. Slight hemolysis hadnegligible effects, but highly hemolyzed specimens gave low recoveries of digoxin.


Gas chromatographic determination of urinary vanilglycolic acid, vanilglycol, vanilacetic acid and vanillactic acid-chemical parameters for the diagnosis of neurogenic tumours and the evaluation of their treatment. A gas chromatographic method is described for the determination of urinary vanilglycolic acid, vanilglycol, vanilacetic acid and vanillactic acid as their trimethylsilyl derivatives. These metabolites are chemical parameters for the dignosis of neurogenic tumours. Their determination is especially recommended in order to evaluate the effect of the therapy. Results in normals and controls are given. Data in a number of selected patients with neuroblastoma, ganglioneuroma and phaeochromocytoma are presented and discussed.


A rapid method for the estimation of beta-galactocerebrosidase, beta-glucocerebrosidase and sphingomyelinase activities in leukocytes. A method for the assay of leukocyte beta-galactocerebrosidase, beta-glucocerebrosidase and sphingomyelinase activities has been developed, based on the separation of the tritiated sphingolipid substrates from their corresponding radioactive hydrophobic product (ceramide) by thin-layer chromatography on Silica gel H coated microscope slides. For the determination of beta-galactocerebrosidase and beta-glucocerebrosidase activities the silica gel is impregnanted with sodium tetraborate. Each chromatogram is easily divided into two distinct zones and the radioactivity content of each is determined by liquid scintillation counting. The technique described, is rapid, less costly than conventional methods and provides an accurate assessment of sphingolipid hydrolase activity. It is suggested that it should be of considerable value in those areas which require the rapid analysis of large numbers of samples, such as in screening for the sphingolipidoses or for enzyme purification studies.


Biokinetics of galactose in the homozygotes and heterozygotes of both forms of galactosemia. 41 heterozygoes and 4 homozygotes with a deficiency of galactose 1-phosphate uridyl transferase and also 3 heterozygotes and 1 homozygous patient with galactokinase deficiency were subjected to intravenous galactose loading tests with a dose of 350 mg/kg body weight in order to answer the question whether it is possible to detect the heterozygotes of both types of galactosemia by this method. For comparison, 38 healthy children and adolescents, 24 children with epidemic hepatitis and 4 children with cirrhosis of the liver, which was verified by histology, were included in the study. The elimination half-life (and also the other pharmacokinetic parameters as inaugurated by Dost) was the same for all the heterozygotes for both types of galactosemia almost without exception, and for the healthy cs, children in the acute stages of hepatitis and patients with cirrhosis of the liver was prolonged 2 to 5 times the normal. In patients with hepatitis, however, the elimination half-life was normal before the transaminases. Accordingly, the galactose clearance was decreased to half and one-fourth of the normal. Hence, heterozygotes with galactosemia cannot be detected with galactose loading tests.


Acute intravascular hemolysis with minimal renal impairment in Clostridium perfringens infection. We report here a case of post-abortal clostridium prefringens infection in which there was severe intravascular hemolysis with black urine, but only minor abnormalities of the clotting mechanism and mild renal failure. The patient recovered following supportive therapy only.


The interrelationships between antidiuretic hormone, adenyl cyclase, tissue cyclic AMP and diffusional water permeability. 1. Physiological concentrations of antidiuretic hormone increase diffusional water permeability but not measurable cyclic AMP content in the isolated papilla of the rat's kidney. 2. Theophylline (6 mM) increases diffusional water permeability and cyclic AMP content in the isolated papilla of the rat's kidney. 3. The increase in water permeability is detected with 5 muunits.ml-1 of ADH and is maximal with 50 muunits.ml-1. The same maximum was achieved with 6 mM theophylline. 4. Cyclic AMP and dibutyryl cyclic AMP both increase water permeability, but to a lesser extent than theophylline or ADH. 5. In the presence of theophylline, ADH causes a dose related generation of tissue cyclic AMP up to a dose of 2,000,000 muunits.ml-1. 6. Adenyl cyclase is increasingly activated by ADH up to doses of 2,000,000 muunits.ml-1. 7. These results suggest that while ADH activates the adenyl cyclase system and changes water permeability there are sufficient disparities to cast doubt on an exclusive role for cyclic AMP as the second messenger.


The effect of pregnancy on mineralo- and gluco-corticoid secretion in the sheep. 1. The peripheral blood concentrations of aldosterone, corticosterone and cortisol were measured during pregnancy in conscious, undisturbed sheep. 2. Aldosterone levels did not change during pregnancy and the mean pregnant value, 1-2 s.d. 1-4 ng/100 ml(n = 12) was not significantly different from the non-pregnant value, 2-1 s.d. 1-7 (n = 16). 3. Cortisol levels likewise were unchanged by pregnancy-non-pregnant values were 0-56 s.d. 0-50 mug/100 ml (n = 12) compared with 0-46 s.d. 0-40 mug/100 ml (n = 16) in pregnant sheep. 4. Sheep of 110-140 days gestation had a 400 mmol greater total exchangeable sodium than non-pregnant sheep. Plasma volume and plasma renin concentration tended to be elevated near to term. 5. Very high aldosterone secretion rates and peripheral blood levels could be produced in pregnant sheep by stress, intravenous ACTH or angiotensin II infusions, and by sodium deficiency. It is suggested that the pregnant sheep may show increased sensitivity in contrast to non-pregnant sheep to these stimuli and the enlarged size of their adrenals may be a contributing factor.


Control of pain by direct electrical stimulation of peripheral nerves. Our results after implanting electrodes around peripheral nerves in 69 patients over a 10 year period are only fair with but 17 individuals maintaining relief until death or until the present time. Thirteen others had weeks or months of temporary relief. A continuing use of transcutaneous and better still of percutaneous electrodes to guide the decision regarding implantation is almost certainly advisable. Intensive efforts in laboratory animal studies with models of chronic pain are needed to improve our understanding of exactly what we should be doing. Empirical sustained work with the individual patient is likely to improve the figures we have presented, even in our present state of ignorance.


Effectiveness of intermediate-term use of secobarbital. Secobarbital, 100 mg, was evaluated in two separate sleep laboratory drug evaluation studies, each with 4 insomniac patients. In both studies, the effect of secobarbital in inducing and maintaining sleep was evaluated, as well as the effects of the drug on sleep stages. Statistical analysis demonstrated that the results of the two studies could be combined. With short-term drug administration of secobarbital (up to 3 nights), there was an improvement in both sleep induction and sleep maintenance. Total wake time was decreased 43% below baseline and was consistently decreased in each third of the night. With intermediate-term drug administration (2 wk), total wake time was decreased only 14% (not statistically significant). Following drug withdrawal, the degree of sleep difficulty returned to baseline levels. The results indicate that secobarbital 100 mg is effective for short-term use but loses much of its effectiveness with intermediate use and suggest that the drug is of limited value for insomniac patients who require nightly medication beyond a period of 1 wk. With short-term administration, secobarbital induced a slight decrease in rapid eye movement (REM) and slow-wave sleep and a significant increase in stage 2 sleep. With intermediate administration, sleep stage values were similar to baseline levels. Following withdrawal, there was only a minimal increase in REM sleep above baseline levels, a significant increase in stage 3 sleep, and a significant decrease in stage 2 sleep. The rebound increase in stage 3 sleep is similar to that reported following withdrawal of pentobarbital.


Feline malignant giant cell tumor of bone associated with C-type virus particles. A malignant giant cell tumor of bone in a cat is described. Numerous C-type particles were found budding from the neoplastic cells. Viruses have not previously been associated with malignant bone noeplasms in cats.


Serial cyclic transmission of bluetongue virus in sheep and Culicoides variipennis. Bluetongue virus (strain 62-45S) was transmitted from sheep to sheep throughout a year by vector bites. A colonized population (SONORA strain, 000 line) of the biological vector Culicoides variipennis (Coquilllett) was used. Fifteen serial cyclic transmissions covered a period of 13 months from October through November of the following year. The mean infection rate of the biting gnats was 37 percent. The clinical response to bluetongue virus was significantly more severe in sheep infected by vector bites than in those inoculated with the virus at the same sheep-serial passage level. A second corroborative serial transmission was conducted for 7 months from June through December. The mean infection rate of the vector was 27%.


Solanum malacoxylon toxicity: inhibition of bone resorption. Young rabbits on high (0.57%) or low (0.24%) calcium were given an aqueous extract of Solanum malacoxylon (S.m.) leaves (20 g dried leaves/200 ml distilled water) intragastrically at 0, 12 and 36 hours. On bothe diets S.m. induced progressive hypophosphatasemia but serum calcium and phosphorus underwent only minor changes. In rabbits necropsied at 0, 12, 36, 60, 84 and 108 hours, S.m. was shown to have a negative effect on the resorbing osteocytes. With retarded osteocytic osteolysis, osteopetrosis resulted. Further regressive changes in the osteocytes resulted in osteonecrosis which was observed within 12 hours after administration of S.m. extract. The osteonecrosis, combined with retarded apposition, later resulted in osteopenia. It was concluded that the recommended dietary calcium for growing rabbits--about 0.6%--is too high. Whereas the histologic appearance of bone in rabbits fed low calcium was normal, bones from rabbits on high calcium showed retarded resorption and the rabbits had a relative hypophosphatasemia.


The etiology of inferior vena caval obstruction and compression. This review of inferior vena caval obstruction and narrowing centers the discussion on the major groups of diseases which produce primary or secondary obstruction and those which cause distortion or narrowing of the inferior vena cava. Although the majority of these diseases which are involved affect both adults and children, a separate discussion of pediatric causes of inferior vena caval involvement is included. The paper has been organized for discussion by the anatomic areas of potential involvement by disease: the lower, middle, and upper thirds of the vena cava. Other subjects which are covered include a description of the various techniques used for performing inferior vena cavography. A section on the normal anatomy, anatomic variations which may be encountered, and collateral venous pathways is presented. There is also a section on possible pitfalls in interpretation of cavography. The relative value of the vena cavogram in the evaluation of abdominal disease is discussed and the cavogram is compared with other methods of evaluating the retroperitoneum such as arteriography, lymphangiography, excretory urography, and B-mode ultrasonography. Numerous illustrations are included to demonstrate various aspects of inferior vena caval involvement by diseases. Where appropriate, short discussions of the clinical aspects are also included.


Some specific radiological findings and consideration of linitis plastica of the gastrointestinal tract. Linitis plastica denotes a diffuse, intramurally infiltrating, anaplastic carcinoma in a hollow structure resulting in a shrunken organ with thickened walls. Microscopically, linitis plastica is characterized by tumor cells in the presence of inflammatory changes with much fibrosis. Linitis plastica is found most frequently in the stomach where it may produce the classical "leather-bottle stomach". Metastases to the colon are frequent via contiguity along mesenteric fascial planes. Therefore, when linitis plastica carcinoma of the stomach or colon is found, the other organ must also be carefully examined. Although rare, primary linitis plastica carcinoma can occur in the colon where it is often characterized by a long stenotic lesion without irritability, sometimes appearing more like an inflammatory lesion than a carcinoma. While the radiological features are not diagnostic, they are, in many cases, suggestive of this entity. The entire spectrum of linitis plastica is reviewed in relationship to the gastrointestinal tract, synthesizing the pertinent literature, with correlation of clinical, pathophysiological, and specific radiological findings.


Analysis of ventilation and performance abnormalities by washout in alveolar air and arterial blood continuous measurement of inert gas. The availability of a rapidly responding arterial catheter and gas analyzer permits the observation of inert gas washout from arterial and alveolar sources. These curves are a logical development of alveolar washout techniques and appear to provide significant new information. While a detailed and formal mathematical treatment of combined alveolar, arterial, and mixed venous washout curves has not yet been derived, inspection and measurement of the y-intercept of the terminal slow space has provided considerable understanding into the distribution of V/Q ratios in the critically ill patient.


Ultrasonically guided percutaneous biopsy of peripheral pulmonary masses. Ultrasonically guided percutaneous biopsy and aspiration of solid and cystic structures in the abdomen, neck, and pericardial space are common practice. The technique may be utilized for biopsy of peripheral pulmonary masses in contact with the chest wall, in order to assure accurate placement of the needle in the center of the mass, reduce the risk of pneumothorax, and eliminate the exposure to radiation that occurs with fluoroscopic guidance. Four patients had successful biopsies in this manner, without complications. Histologic studies revelaed malignant neoplasms in three and lipoid pneumonia in one.


Acute appendicitis and postoperative fecal fistula: symptoms of an unrecognized carcinoma of the colon. This report is a retrospective evaluation of 12 case histories. All patients had been subjected to laparotomy on suspicion of acute appendicitis. The operative situations seemed to corroborate clinical expectations: an inflamed ileocecal process was assumed to be a result of performation of a gangrenous appendicitis. In three patients a primary adenocarcinoma arising from the appendiceal base was the incidental histologic finding postoperatively. In three other patients this inflammatory-looking process was related to a cecal carcinoma. In six patients a carcinoma existed unrecognized as the basic disease, these patients being subjected to intra-abdominal drainage only or in connection with appendectomy. In five instances a fecal fistula was the main persisting symptom of postoperative morbidity, in one patient even as long as two years after laparotomy. The problem in diagnostic verification of the initially unidentified carcinoma is illustrated. Attention is directed towards avoiding a false feeling of security in the presence of inflammatory manifestations in the right lower abdominal quadrant, which may misleadingly suggest a ruptured appendix.


Ischemic colitis secondary to Premarin: report of a case. A case of reversible ischemic colitis in a young woman taking Premarin, with varium-enema confirmation of the initial diagnosis and subsequent clearing after discontinuation of the oral estrogen preparation, is reported. Ischemic colitis may be more common than realized with the increasing use of conjugated estrogen preparations in menopausal women and elderly men treated for prostatic cancer.


[Xantinol-nicotinate in primary type-V hyperlipoproteinaemia (author's transl)]. The effect of xantinol-nicotinate (50 mg/kg body-weight) on serum lipids and lipoproteins was tested in 16 out-patients with primary type V hyperlipoproteinaemia. The lipids and lipoproteins were measured before treatment, during a three-week period of drug administration and ten days after it had been stopped. There were no side effects such as flushing or gastritis, and no notable reduction of weight. Each serum-lipid fraction (triglycerides, nonesterified fatty acids, phospholipids, ester-cholesterol and free cholesterol) decreased significantly, regaining the initial values ten days after the drug had been stopped. While chylomicrons and very low-density lipoproteins (VLDL) decreased, there was a significant increase in low-density lipoproteins (LDL). High-density lipoproteins were not significantly changed. The decrease in chylomicrons and the significant decrease in VLDL with xantinol-nicotinate was opposite to that seen with dietary treatment. In none of the patients did LDL increase to abnormally high levels.


[Electronmicroscopic characterisation of human plasma lipoproteins after isolation by zonal ultracentrifugation (author's transl)]. Human plasma proteins were isolated by zonal ultracentrifugation and the fractions obtained examined under the electron microscope. HDL2 had a high degree of uniformity in normal persons, characterised by numerous two-dimensional, hexagonal arrangements. Optical diffraction analysis of HDL2 gave a centre distance of 11.3 nm, individual HDL2 particles being spherical with a diameter of 9.8 nm. HDL3 represented a somewhat less uniform lipoprotein population with a particle diameter between 6.5 and 9.5 nm. LDL had a diameter of 20-25 nm. In patients with hyperlipoproteinaemia type III a third peak was isolated, in addition to the two normal lipoprotein peaks VLDL and LDL. It lies between them and has been called Lp III. VLDL from type III plasma consisted of spherical particles, diameter 31-81 nm, morphologically indistinguishable from normal VLDL. Lp III consisted of a relatively homogeneous spherical lipoprotein population with a diameter of 25-30 nm. LDL consisted of spherical particles whose size was highly homogeneous (18-23 nm diameter), indistinguishable from normal LDL.


Functional differentiation of the fetal anterior pituitary cells in the rat. The onset of hormone synthesis in the rat fetal pituitary gland was studied with the use of the peroxidase labelled antibody method. On the 16th day of fetal life, cells containing immunoreactive ACTH appeared in the primordial cell outgrowth of the Rathke's pouch. In some fetuses, also prolactin cells could be detected at the same developmental stage and these cells were present consistently on subsequent days. Differentiation of ACTH and prolactin cells was followed by onset of TSH, LH and GH synthesis on the 17th, 18th and 19th day of gestation, respectively.


Prolactin binding sites in the male rat liver following castration. Specific binding sites for prolactin (PRL) have been detected in membrane preparations from the liver of the male rat following castration. The magnitude of the increased binding following castration varied with the age of the animals and with the time after castration. The effect of castration did not appear to be PRL mediated, since increases or decreases of serum PRL levels after pharmacological agents had no effect on PRL binding. The pituitary, however, seems to have a critical role in mediating the increase in PRL binding. Adrenalectomy did not influence the extent of binding of PRL after castration. Testosterone administration, however, completely prevented the increased PRL binding which followed castration. These studies suggest that testosterone has a modulating effect on hepatic PRL binding sites. The maintenance of such binding activity requires not only PRL, but also a functioning pituitary.


Characterization of somatogenic and lactogenic binding sites in isolated rat hepatocytes. Suspensions of rat hepatocytes isolated enzymatically by the method of Berry and Friend were used to study the binding of 125I-labeled human (hGH) and bovine (bGH) growth hormones and ovine prolactin (oPRL). Displacement of these labeled hormones by their unlabeled analogues was analyzed by means of Scatchard plots and affinity constants (K) and the number of binding sites per cell (q) were calculated. Specificity of binding was studied using hGH, bGH oPRL and rat growth hormone (rGH) and rat prolactin (rPRL). Rat hepatocytes contained two types of binding sites which bound hGH. The first, somatogenic, was specific for the growth-promoting hormones bGH and rGH. The second, lactogenic, was specific for lactogenic hormones, oPRL and rPRL. Human GH, which has both lactogenic and growth-promoting properties in rodents, bound to both sites. The somatogenic binding sites were present in both males and females, and the number of sites was similar in females and in males and was not affected by hypophysectomy. The lactogenic binding sites were present only in females, and the number of lactogenic and somatogenic sites was similar (40,000/cell). The affinity of hGH for the lactogenic binding sites was less than for the somatogenic (0.37 X 10(9) vs. 1 X 10(9)M-1). The lactogenic binding sites were lost when female rats were hypophysectomized and could not be restored by estrogen treatment.


Acidosis activation of the pituitary-adrenal-renal glutaminase I axis. Previous studies have demonstrated that the adrenal glands were necessary for acidosis activation of the mitochondrial glutaminase I pathway. The present studies were undertaken to determine if corticosterone levels are elevated in acidotic rats and if so, whether acidosis stimulates the adrenal glands directly or via the pituitary-adrenal axis. Metabolic acidosis induced by NH4Cl, either acute or chronic, increased corticosterone levels 100 to 130% in intact rats. Acute metabolic acidosis did not activate the mitochondrial pathway in adrenalectomized rats; corticosterone levels were not elevated in hypophysectomized rats nor did activation of the mitochondrial pathway occur in response to acidosis. Therefore, acidosis does not stimulate the adrenal gland directly; rather, it requires the intact pituitary. Administering exogenous corticotropin to hypophysectomized rats resulted in elevation of plasma corticosterone levels and activation of the mitochondrial pathway. The pituitary-adrenal cortex-renal glutaminase I axis apparently operates as a functional unit in the homeostatic response to metabolic acidosis.


In vivo hormonal induction of ornithine decarboxylase in rat kidney. Single pharmacological doses of parathyroid hormone, calcitonin, vasopressin, d-aldosterone, or L-triiodothyronine produced a significant increase in the ornithine decarboxylase activity of rat kidney. The activity of kidney ornithine decarboxylase was also enhanced by other hormones, such as pentagastrin and serotonin, which, although they are not known to modify kidney physiology, are secreted by cells having close relationships to the calcitonin-secreting parafollicular cells. The induction of the enzyme was observed in hypophysectomized rats, with or without some other hormone-secreting glands remaining. However, the magnitude of the stimulation elicited by the hormones was somewhat diminished in animals still having the endocrine gland whose hormone was being tested. The maximal stimulation of kidney ornithine decarboxylase activity by parathyroid hormone, calcitonin, vasopressin, L-triiodothyronine, pentagastrin, and serotonin occurred at 4 h after the hormone injection. The enhancement in ornithine decarboxylase activity produced by d-aldosterone was maximal at 3 h after the injection of the hormone. The content of ornithine in the kidney was found to be virtually unchanged whatever the type of hormone treatment. No statistically significant increases in renal ornithine decarboxylase activity of hypophysectomized animals were observed after injection of melatonin or of vitamin D3. Since the stimulating hormones possess clearly different mechanisms of action, the role of cyclic AMP as a general mediator of ornithine decarboxylase induction is questioned.


Oxytocin-stimulated release of prostaglandin F2alpha from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endometrial and myometrial tissues, obtained from Merino ewes on 5 different days of the estrous cycle, were incubated at 37 C in 30 ml of gassed (95% O2:5% CO2) Krebs-bicarbonate buffer containing, 0, 10, 100 or 1,000 muU/ml oxytocin. Aliquots of the medium were removed at 10 min intervals and examined for prostaglandin F2alpha (PGF2alpha) content by radioimmunoassay. Fresh-frozen (-70 C) samples of endometrial and myometrial tissue were homogenized in Tyrode's solution. Particulate fractions from each tissue, sedimenting between 1,000 X g for 10 min and 165,000 X g for 30 min, were prepared and assayed for [3H]oxytocin-binding activity. Endometrium incubated in vitro released PGF2alpha spontaneously and oxytocin enhanced this release in a dose-dependent manner. The degree of enhancement with low doses of oxytocin appeared to increase as estrus approached, reaching a maximum on the day of estrus. High-affinity binding sites (Kd = 5 to 7 X 10(-10) M) were found in both myometrium and endometrium. The number of high-affinity sites rose to a peak at estrus in both tissues but the binding capacity of endometrium was twice that of the myometrium at this time. Although both tissues released PGF2alpha during incubation, oxytocin enhanced release from endometrial tissue only. The results suggest that (i) the endometrium is a target for oxytocin, (ii) synthesis of PGF2alpha by the uterus may involve interaction between oxytocin and its endometrial receptors and (iii) ovarian steroids may influence uterine PG synthesis by regulating the availability of these receptors.


Potentiation of the ACTH response to metyrapone by L-dopa in the monkey. The intravenous injection of L-Dopa (15 mg/kg) to monkeys (Macaca mulatta) failed to alter plasma concentrations of ACTH and of 11-deoxy-cortisol. When cortisol synthesis was blocked with iv metyrapone, potentiation of ACTH secretion by L-Dopa became apparent. Simultaneous injection of L-Dopa and metyrapone resulted in a marked increase in plasma ACTH from 93 +/- 18 pg/ml to 432 +/- 80 pg/ml, whereas plasma 11-deoxycortisol increased from 1.5 +/- 0.2 mug/100 ml to 14.6 +/- 1.0 mug/100 ml 90 min after treatment. Throughout the experiment the rise in ACTH and in 11-deoxycortisol following coadministration of L-Dopa and metyrapone was significantly (P less than 0.01) higher than that produced by metyrapone administration alone. The results suggest that acute administration of L-Dopa in monkeys enhances the response of ACTH to metyrapone. L-Dopa (or one of its metabolites) probably acts upon a noradrenergic or a dopaminergic system located in the hypothalamus to alter the release of hypothalamic corticotropin regulatory factor(s) and thereby enhance the release of ACTH.


Fluorescence polarization measurement of the hormone-binding site interaction. Fluorescence polarization methodology has been applied to the binding of fluorescent-labeled prolactin, growth hormone and estradiol to subcellular fractions prepared from rabbit mammary and uterine tissue. Equilibrium measurements treated by Scatchard plots have shown that there are high affinity sites (K approximately 10(9) 1 mol-1), as well as lower affinity sites (K approximately 10(8) 1 mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions has been shown to be inhibited by the prior addition of native, unlabeled hormone. Kinetic results on the interaction of prolactin with the microsomal fraction are consistent with a bimolecular reaction involving significant structural rearrangements during the reaction (not diffusion controlled). The forward rate constant calculated from data on initial rates was found to be 1.7 X 10(5) 1 mol-1 sec-1. Stopped flow kinetic measurements on the reaction between fluorescent-labeled estradiol and cytosol binding sites show that at low temperatures, the reaction goes in two distinct steps separable in time. The second step may be the reaction found by others (utilizing sedimentation velocity methods) which precedes translocation of the hormone-binding site complex to the nucleus. Fluorescence polarization makes it possible to observe both the formation and dissociation of hormone-binding site complexes over a time scale down to a fraction of a second and at concentrations down to the nanogram per nl range.


Biosynthesis of adrenodoxin in mouse adrenal tumor cells. ACTH and cAMP stimulate steroidogenesis and the mitochondrial electron transport system for steroid hydroxylation in cultured mouse adrenal cortex tumor cells. During this stimulation, the biosynthesis of adrenodoxin, a non-heme iron protein which is one of the electron transport enzymes, was examined. 14C-labeled adrenodoxin was isolated by employing a purified rabbit antibody to bovine adrenodoxin. The antibody-adrenodoxin precipitates were further purified by acrylamide gel electrophoresis. It was observed that the biosynthesis of adrenodoxin was stimulated in response to ACTH induction and that this stimulation was completely inhibited with cycloheximide and partially inhibited with chloramphenicol. As a result, it was concluded that adrenodoxin requires both mitochondrial and cytosol ribosomal activities for its synthesis and integration into adrenal mitochondria.


Diglyceride kinase activity of microtubules. Characterization and comparison with the protein kinase and ATPase activities associated with vinblastine-isolated tubulin of chick embryonic muscles. Vinblastine-isolated microtubule protein from chick embryonic muscles has an enzymatic activity which catalyzes the formation of phosphatidic acid from diglycerides and ATP. The pH optimum (6.4), sedimentation on sucrose gradients (Mr = 85 000), and sensitivity to ions of this diglyceride kinase activity are different to those of a similar enzymatic activity present in 150 000 X g supernatants of chick embryonic muscle homogenates, suggesting that it is a different species which is associated specifically with the microtubules. The reaction requires a divalent ion (e.g. 0.4 mM Mg2+ gives half-maximal stimulation), and GTP can replace ATP rather effectively, especially at nucleotide concentrations lower than 50 muM. The sedimentation of the diglyceride kinase on sucrose gradients coincides with that of the microtubules-associated protein kinase (Mr = 75 000); the heat-stability and sensivitity to proteolysis of both activities are also very similar. Stimulation of one reaction by the addition of the corresponding exogenous substrate does not impair the phosphorylation of the other, and no radioactivity is lost from phosphatidic acid or the protein moiety upon incubation of pre-labelled microtubules with a large excess of unlabelled ATP or GTP. In addition to diglyceride and protein kinase activities (0.2 and 0.3 nmol 32P-transferred X min-1 X mg-1 microtubular protein, respectively), microtubules also contain an associated ATPase (2.8 nmol X min-1 X mg-1), which requires either Mg2+ or Ca2+, can hydrolyze GTP quite effectively, and sediments with a molecular weight of 95000. The results obtained are discussed in connection with the possible relationships existing among these enzymatic activities, as well as their probable role in microtubular functions.


The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase. Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.


An approach to the antigenic structure of arginine kinase and creatine kinase. Physical, chemical and immunological study of some modified derivatives. The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage. Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein-solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross-react fully, while creatine kinase and its oxidized derivative cross-react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of argining kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12%) the homologous antigen-antibody interaction. The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine-modified derivatives.


New methods for the study of complex enzyme kinetics illustrated by analysis of the wavy curves of v versus (S) and non-linear double-reciprocal plots for human-placental 15-hydroxyprostaglandin dehydrogenase. A new method for discovering the minimum degree of rate equations using only the experimental graphs and a straight-edge (transparent ruler) is presented. This method is then illustrated by an analysis of the wavy v vs [S] curves and non-linear double-reciprocal plots reproducibly given by NAD-dependent 15-hydroxyprostaglandin dehydrogenase for which a new improved purification is described. It is shown by this analysis that the enzyme has a complex mechanism involving cooperatively linked dependent sites and requiring a rate equation of at least fourth degree in prostaglandin and NAD+ with no kinetically significant dead-end complexes.


Kinetics of insulin secretion in chronic pancreatitis and mild maturity onset diabetes. (Evidence for "gut hormone" action beyond glucoreceptor and cyclic adenosine monophosphate mediated insulin release). We studied insulin responses to glucose with and without cholecystokinin-pancreozymin and aminophyllin infusions in normal, chronic pancreatitic and genetic (maturity-onset) diabetic subjects. Glucose was given alone as separate 5 and 10 g boluses followed by infusion at 250 mg/min. and 500 mg/min., respectively. Chronic pancreatitis patients and genetic diabetic patients had decreased Imax values, indicating a decreased insulin reserve. Sensitivity to glucose was normal in pancreatitic subjects, but the diabetic subjects had a raised G50 value, compatible with glucoreceptor dysfunction. Infusions of aminophyllin enhanced insulin responses (Imax) to glucose injection in normal subjects and to a lesser degree in pancreatitic subjects, but decreased sensitivity to glucose (increase in G50) in both groups. Although the Imax value in pancreatitic subjects was significantly lower than in the control subjects during the glucose plus aminophyllin infusion, the blood glucose concentration in the pancreatitic subjects was nonetheless decreased. This suggests that pancreatitic subjects have increased endogenous insulin sensitivity. Aminophyllin had no effect in diabetic subjects. Crude cholecystokinin-pancreozymin changed the shape of the glucose/insulin dose response curve in normal, pancreatitic and diabetic subjects. These findings further suggest that the defect in insulin secretion in pancreatitic subjects is partly situated at the cyclic adenosine monophosphate stage of insulin release. Crude cholecystokin-pancreozymin seems to affect insulin release at a point beyond the cyclic adenosine monophosphate stage.


Primary and secondary in vitro generation of cytolytic T lymphocytes in the murine sarcoma virus system. Cell-mediated cytotoxic responses in vitro to surface antigens associated with murine sarcoma virus (MSV)-induced tumors were investigated using mixed leukocyte-tumor cell cultures (MLTC). The source of responding cells was either spleens from normal C57BL/6 mice (primary MLTC) or spleens of C57BL/6 mice carrying or having rejected a MSV-induced tumor (secondary MLTC). Graffi virus-induced GiL-4 leukemia cells, Rauscher virus-induced RB1-5 leukemia cells, and MSV-induced MSV-B16 sarcoma cells were used as stimulating syngeneic tumor cells and/or target cells. Under appropriate culture conditions, cytolytic T lymphocytes (CTL) were generated in both primary and secondary MLTC. As assessed by a quantitative short-term 51Cr release assay system, CTL activity in secondary MLTC populations was at least 10-fold higher than that in primary MLTC populations, and 100-fold higher than that in spleen cells taken at the peak of the in vivo response of MSV-infected mice. The ability of spleen cells to mount a secondary CTL response in vitro could be observed as early as 5 days after virus injection, increased up to the time of maximum tumor size and persisted long after tumor regression. This suggests the development of increased numbers of CTL progenitors and/or the formation of "memory" CTL in spleens of MSV-injected mice.


Studies on the effect of various agents on the maturation of thymus stem cells. Experiments were carried out on 14-day fetal mouse thymic stem cells which were treated with agents known to increase cellular cAMP levels for a period of 90 min at 37 degrees C. When compared to controls, a high proportion of treated stem cells were found to develop Thy-1 (theta) antigen as indicated by dye exclusion tests using anti-Thy-1 serum and complement.


Analysis of human thymocyte subpopulations using discontinuous gradients of albumin: precursor lymphocytes in human thymus. Precursor lymphocytes, obtained from human tissue on bovine serum albumin (BSA) gradients followed by removal of sheep erythrocyte (E)-reactive thymocytes on Ficoll, are the predominant lymphoid subpopulation found in young fetal thymuses. This subpopulation shows a progressive decline in relative numbers during fetal life, accompanied by a rise in both the relative and absolute numbers of E-reactive thymocytes. Precursor thymocytes differentiate in vitro to E-reactive cells resembling T cells in their morphology, sedimentation on BSA gradients, and response to phytohemagglutinin. Agents tending to increase intracellular concentrations of cyclic-AMP accelerate the rate of differentiation of precursor cells to lymphocytes which closely resemble T cells.


Receptors for immune complexes on cells within a polyoma virus-induced murine sarcoma. Cells present in the polyoma virus-induced murine ascites tumor SEYF-a showed the capacity to fix soluble immune complexes of ovalbumin anti-ovalbumin. The fixation could be inhibited by preincubating the cells with antisera directed against H-2 antigens and with syngeneic anti-tumor antibodies. The latter did not react with normal cells. Depletion of phagocytes from the ascites cell population, thus enriching for tumor cells, increased the inhibition of complex fixation by the syngeneic anti-tumor antiserum. The inhibition of complex fixation by the anti-tumor antibodies was not mediated by "third-party" complexes. Pepsin-treated globulin derived from the syngeneic anti-tumor antiserum could still inhibit complex fixation by SEYF-a cells. These results raise the possibility that tumor cells, per se, expressed receptors for immune complexes. Macrophages, apparently of host origin, residing in the SEYF-a tumor, also expressed such receptors.


Naltrexone-induced hypothermia in the rat. Naltrexone, in relatively high doses, has been reported to cause a fall in body temperature in human ex-heroin addicts who had been abstinent for at least 6 weeks. The underlying mechanism of this hypothermic effect has been investigated in rats. The first consideration was that the temperature change was a reflection of delayed withdrawal but rats implanted with a morphine pellet 45 days earlier showed no significant change in temperature after a dose of naltrexone that caused marked withdrawal hypothermia in dependent rats implanted 3 days previously. A fall in core temperature was only induced in rats after doses of 80 and 160 mg/kg i.p. of naltrexone. Behavioral thermoregulatory studies revealed that the animals correct the falling body temperature by increased exposure to a radiant heat source indicating that the central thermostats had not been significantly affected by the drug. These data suggest that the major component in the hypothermic effect of naltrexone is activation of efferent heat loss pathways or peripheral heat loss mechanisms. Due to current suggestions that opiate receptors might represent the receptors for an endogenous transmitter the results are discussed in relation to this consideration. When compared to the sites and mechanism of action of opiates on thermoregulation the results with naltrexone lend little support to the hypothesis that the fall in temperature is due to displacement of an endogenous substance from central opiate receptors.


Pharmacological evidence for a stimulation of dopamine neurons by noradrenaline neurons in the brain. The effects of yohimbine (3 mg/kg i.p.), phenoxybenzamine (20 mg/kg i.p.) and clonidine (0.1 mg/kg i.p.) on the synthesis and the utilization of dopamine and noradrenaline in the central nervous system of rats were investigated. Dopa accumulation following decarboxylase inhibition and the alpha-methyltyrosine-induced disappearance of the amines were used as the measure of these effects. The synthesis and the utilization of dopamine and noradrenaline were accelerated by yohimbine. Clonidine plus phenoxybenzamine inhibited the synthesis and utilization of dopamine and the combination also partly antagonized the effects of yohimbine on the trunover of dopamine. The effects of the three drugs on the synthesis and utilization of dopamine might be secondary to their actions on alpha-adrenoreceptors of noradrenaline synapses since, at the doses used, yohimbine increases the release of noradrenaline, phenoxybenzamine blocks postsynapptic noradrenaline receptors and clonidine reduces the release of noradrenaline. This hypothesis is supported by the findings that yohimbine and phenoxybenzamine did not change the increased synthesis of dopamine in reserpine-treated rats and that clonidine did not inhibit the increased synthesis of dopamine after axotomy or treatment with reserpine.


Effect of decentralization and glucose withdrawal on the potassium-induced cAMP increase in the rabbit superior cervical ganglion. The concentration of cAMP in the isolated rabbit superior cervical ganglion (SCG) increases when the tissue is exposed to high potassium media for short periods. The increase was found not to be affected by agents that are known to potentiate or to antagonize the postsynaptic effects of acetylcholine. Pretreatment of the rabbits with various sympaticolytic substances did not appear to affect the cAMP increase in the isolated SCG. It was found that the K+-induced cAMP synthesis occurred neither in ganglia which had been decentralized 3 days before isolation nor in intact ganglia preincubated in a glucose-free medium. Under both these conditions the effect of potassium declines with a time course which is characteristic for the degeneration of the presynaptic terminals. These terminals therefore, appear to be the most likely site for the cAMP increase.


Block of electrically induced contractions of guinea pig longitudinal muscle by prostaglandin synthetase and receptor inhibitors. Inhibitors of prostaglandin synthetase as well as prostaglandin receptor inhibitors block electrically induced contractions of the longitudinal muscle of the guinea pig ileum. This blockade is selectivety reversed by some prostaglandins, particularly those of the E series. There is a very close parallel between the potency with which the synthetase inhabitors block transmission and inhibit the enzyme. That blockade is actually accompanied by inhibition of synthesis of PG was shown by inhibition of arachidonic acid contractions of the tissue by indomethacin. The inhibition of transmission by indomethacin involves block of acetylcholine release as shown by direct assay and by the fact that physostigmine can reverse the block. Physostigmine also reverses block of transmission by prostaglandin receptor inhibitors and by morphine but not that produced by chlorpromazine or papaverine. Other evidence for a presynaptic site of action for the synthetase and PG receptor inhibitors is indicated by lack of effect of blocking concentrations on response of the tissue to exogenous acetylcholine. That prostaglandin reverses block of transmission by a presynaptic effect was shown by lack of reversal of atropine and papaverine inhibition of electrically induced contractions; both of these drugs produce this effect directly on the smooth muscle. These results are compatible with the previous postulate that a prostaglandin system, comprised of prostaglandin, its synthesizing enzyme and its receptor, is directly involved with the release of acetylcholine in the ileum.


Adenosine 3',5'-cyclic monophosphate as a possible mediator of rotational behaviour induced by dopaminergic receptor stimulation in rats lesioned unilaterally in the substantia nigra. A possible involvement of c-AMP in the rotational behaviour induced by a stimulation of dopamine receptors in corpus striatum of rats was investigated. Rats were lesioned unilaterally in the substantia nigra with 6-hydroxydopamine. Intraventricular injection of dopamine, norepinephrine and apomorphine induced rotational behaviour towards the intact side as did dibutyryl c-AMP (dB-c-AMP). Dopamine, norepinephrine and apomorphine could activate adenylate cyclase in homogenates of caudate nucleus. The activation by dopamine was blocked by haloperidol. I.p. injected apomorphine increased c-AMP content bilaterally in caudate nucleus and caused turning towards the intact side; theophylline potentiated and haloperidol blocked the effect. In non-lesioned rats, dopamine and norepinephrine, when injected unilaterally into the caudate nucleus, elicited truning twoards the non-injected side if the rats were pretreated with reserpine and tranylcypromine. c-AMP and dB-c-AMP given similarly to rats pretreated with theophylline also produced turning towards the non-injected side regardless the pretreatment with reserpine and tranylcypromine. All these results emphasize the possibility that c-AMP acts as a second messenger in the central dopaminergic pathway in rats. The supersensitivity of the dopaminergic system which developed after denervation is also discussed.


Cyclic AMP and alpha-receptor-mediated modulation of noradrenaling release from rat brain slices. Oxymetazoline (an alpha-receptor agonist) reduced and phentolamine (an alpha-receptor antagonist) increased depolarization-induced release (potassium or electrical field stimulation) of 3H-noradrenaline (NA) from superfused neocortical slices in a dose-dependent way. Inhibition of phosphodiesterase also reduced NA release; this effect could be reversed by phentolamine. Phosphodiesterase inhibition potentiated the effect of oxymetazoline. It is suggested that stimulation of presynaptic alpha-receptors may reduce NA release up to about 60% and that increased cyclic AMP formation might be involved in this modulation.


Interaction between ACTH fragments, brain opiate receptors and morphine-induced analgesia. The present study confirms that N-terminal fragments of ACTH have an affinity for rat brain opiate receptors in vitro. Such peptides, devoid of corticotrophic activity, were found to inhibit morphine-induced analgesia if they also possessed affinity for opiate receptors in vitro. The structure-activity relationship for these two parameters is comparable to that observed for the same peptides on the induction of excessive grooming.


Neuronal peptide (enkephalin) receptors in the ear artery of the rabbit. Methionine-enkephalin methylester (MEM) and leucine-enkephalin (LE) inhabit the vasoconstrictor responses of the rabbit ear artery to nerve stimulation by acting on a specific neuronal peptide (enkephalin)-receptor insensitive to opiate agonists. The tetrapeptide: H-Tyr-Gly-Phe-Leu-OCH3 is ineffective. This is the first instance of enkephalins acting in an organ devoid of receptors. In a new test for the analysis of opiate receptors, MEM (ID50=6.9 X 10(-9) M) was a potent inhibitor of transmission. The presence was shown of opiate receptors in the brain which were insensitive to high i.v. or intraventricular doses of enkephalins. It is concluded that enkephalins are not natural ligands to the opiate receptors, but that some of the receptors confuse these structures because of similar characteristics which determine the binding of both opiates and peptides.


Effect of normorphine and enkephalin on spontaneous potentials in the vas deferens. Spontaneous excitatory junction potentials were recorded intracellularly from the smooth muscle of the mouse vas deferens. Exposure of the vas deferens to either normorphine (1 muM) or methionine-enkephalin (0.5 muM) did not alter the frequency or amplitude distribution of the spontaneous potentials. The possible mechanisms by which the narcotic agonists depress the release of noradrenaline on nerve stimulation without inhibiting the spontaneous release of noradrenaline are discussed.


3H-noradrenaline outflow induced from isolated aventitia and intima-media of rabbit aorta by electrical field stimulation. Electrical field stimulation of the isolated adventitial and intima-medial layers of rabbit aorta preloaded with 3H-noradrenaline elicited a 3H outflow. The initial 3H outflow from adventitia was independent of external Ca2+ concentration. Each of the subsequent 3H outflows was mainly Ca2+ sensitive. All stimulation-induced 3H outflows from media were Ca2+ insensitive. It is concluded that the isolated adventitia is a suitable preparation to outflow from aorta is of neuronal and extraneuronal origin.


Dependence liability of enkephalin in the myenteric plexus of the guinea pig. The endogenous ligands of opiate receptors, methionine-eukephalin and leucine-enkephalin, were tested for their ability to maintain or to reinduce dependence in myenteric plexus-longitudinal muscle strips taken from tolerant/dependent guinea pigs. Both peptides fully substituted for the function morphine exerts in this preparation. It is concluded that the enkephalins posses a high degree of dependence liability.


The role of cyclic adenosine monophosphate in the swarming phenomenon of Proteus mirabilis. The levels of cylic AMP and adenyl cyclase in swarming and non-swarming cells of Proteus mirabilis and the effect of glucose on swarming have been investigated. The results indicate the cAMP is required for swarming, but that the flagellar derepression characteristic of swarming does not result from increased cAMP levels.


Adenyl cyclase activity at different environmental temperatures in the isolated rat anterior pituitary membranes. The effect of different environmental temperatures on adenyl cyclase was studied. An increase in temperature appears to increase TRH-induced activity of adenyl cyclase, and possible causes an increases sensitivity to the hormone. Cyclic AMP levels of the pituitaries showed change at different environmental temperatures.


Actinomycin D peritonitis in the rat.

Actinomycin D peritonitis in the rat. Following intraperitoneal injection of actinomycin D rats show a decrease in number of cells present in the peritoneal cavity, reaching the lowest point after 24 hr. At the same time a highly significant increase of free beta-glucoronidase and of the intracellular concentrations of both cyclic AMP and cyclic GMP has been observed. No exudate was present at this time. Measurable quantities of exudate were present 48-72 hr after actinomycin injection concomitantly with an intense cellular immigration, the dominant cell being mononuclears. In this second phase of the reaction the free beta-glucuronidase decreases towards normal values and both the cyclic nucleotides are significantly below the control values. It is suggested that the increase of intracellular cAMP--concomitant with the maximum release of lysosomal enzymes--is a feedback mechanism preventing further release of inflammatory mediators.


Angiotensin antogonists as pharmacological tools. The availability of specific competitive antagonists stimulated investigation of the physiological and pathological role of angiotensin (A-II) and permitted the qualitative and quantitative characterization of numerous angiotensin receptor sites. The specific, competitive antogonists for A-II inhibit both the direct actions of A-II on isolated smooth muscle preparations and the stimulation of specific vascular receptor sites by which A-II evokes prostaglandin biosynthesis and release. Converting enzyme inhibitors a) block the action of exogenous A-I; b) lower blood pressure in conditions associated with high plasma renin levels (e.g., two-kidney renal hypertension, dehydrated diabetes insipidus rats, or in hemorrhagic shock); c) enhance responses to exogenous bradykinin (by inhibiting bradykininase); but d) do not block the effects of A-II at its receptor sites. A-II-receptor antagonists a) block the action of both A-I and A-II, b) lower blood pressure in high renin states, but c) have no effect on bradykinin degradation or action. Angiotensin receptor and synthesis antagonists have been shown to decrease the overall peripheral resistance and to reverse the renal cortical vasoconstriction during hemorrhagic shock and to prolong survival time in hemorrhaged dogs. It is our belief that angiotensin antogonists have therapeutic potential in hemorrhagic shock and would be expected (alone or in combination with alpha-andrenergic blockade) to overcome vascular shutdown and enhance organ perfusion (especially in the kidney).


Structure--activity relations of antagonists of the renin--angiotensin system. In this review, inhibitors of the renin-angiotensin system, both naturally occurring and synthetic, are considered. Inhibition of renin itself, of converting enzyme (dipeptidyl carboxypeptidase), and of interaction with the receptor is characterized from the point of view of mechanism, structure-activity relations, and future developments. The concept of transition state analog and its relevance to the inhibition of renin by pepstatin is discussed. Questions concerning selectivity, increased affinity, and longer duration of action are raised with regard to future design of inhibitors of the renin-angiotensin system.


Renin--angiotensin antagonists and the regulation of blood pressure. The role of the renin--angiotensin system in the regulation of blood pressure in dogs and in human subjects was assessed by the use of the nonapeptide converting enzyme inhibitor (CEI), permitting the following conclusions: 1) In the normal, sodium replete dog, the renin--angiotensin system plays little role in the regulation of blood pressure. 2) As sodium depletion progresses, the renin--angiotensin system becomes increasingly important in the maintenance of blood pressure. In the markedly hypovolemic animal, blocking the conversion of angiotensin I to angiotensin II leads to prolonged hypotension of shock-like levels. 3) The renin--angiotensin system is responsible for the initiation of renovascular hypertension. Blood pressure does not rise during chronic renal artery constriction when the generation of angiotensin II is prevented by the CEI. Although angiotensin II is essential for the initiation of the elevated blood pressure, the renin--angiotensin system plays a decreasing role in the maintenance of the chronic hypertension as sodium and water are retained, and plasma volume increases. 4) In congestive failure induced in the conscious dog by circulatory impairment, the renin--angiotensin--aldosterone system plays an essential role in the compensatory response. During chronic administration of the CEI, the animal cannot compensate even for a relatively mild degree of constriction, and remains hypotensive. In the dog with congestive failure, as in the dog with renovascular hypertension, plasma renin activity (PRA) and plasma aldosterone are elevated early in the syndrome; during this phase, injection of the nonapeptide produces a marked drop in blood pressure. With the retention of sodium and water, and expansion of plasma and extravascular fluid volumes, PRA and plasma aldosterone return to control levels in the new steady state. The inhibitor no longer produces a drop in blood pressure. Thus, the sequential changes in the renin--angiotensin--aldosterone system are remarkably similar in renovascular hypertension and congestive failure. 5) In the normal, salt replete human subject the renin--angiotensin system plays little role in the regulation of blood pressure either in the recumbent or upright posture. However, with relatively mild sodium depletion, the CEI transiently lowers blood pressure even in the recumbent subject. In the absence of angiotensin II such sodium-depleted subjects are unable to compensate when tilted upright, and faint within minutes.


The secretion of lysosomal enzymes.

The secretion of lysosomal enzymes. The studies reviewed in this chapter provide further evidence that the secretion of lysosomal enzymes and other hydrolases is a constitutive function of certain cells whereas in other cells is an inducible process probably contributing to the pathology of a variety of diseases. Little is known of the mechanisms mediating the secretion of lysosomal enzymes. We have summarized evidence suggesting a role of microfilaments and microtubules in controlling enzyme release, but further studies of the biochemical mechanisms which control the activity of these subcellular structures are required. The fusion of lysosomes with the plasma membrane has been observed in several situations and the mechanisms underlying processes of this nature have been studied in lower organisms (Satir et al. 1973; Plattner 1974). Agents, such as concanavalin A,  which interfere with the fusion of endosomes with lysosomes (Goldman 1974; Edelson and Cohn 1974a, b) should also be useful in determining the chemical nature of membrane components involved in the fusion process. New information on the fate of secreted acid hydrolases has been obtained from studies of the uptake of lysosomal enzymes by fibroblasts. Clearly, the mechanisms by which these cells endocytose secreted lysosomal enzymes will be a subject for detailed study in view of the important of directing enzymes and drugs into lysosomes (De Duve et al. 1974). The mechanisms by which extracellular inhibitors inactivate hydrolytic enzymes, particularly proteinases, is also being clarified (for review see Davies 1975) and this should aid in finding new ways for preventing tissue damage caused by the excessive secretion of these enzymes. Further investigation concerning the secretion of lysosomal enzymes should establish the essential physiological role which these enzymes play at both extracellular and intracellular sites. Also, a close examination of the interaction of both endogenous and exogenous stimuli of inflammation with cells resulting in the secretion of hydrolytic enzymes, will clarify the mechanisms underlying the initiation and progression of the inflammatory process in its diverse forms.


[In vitro assay for ACTH-releasing activity using ACTH radioimmunoassay: ACTH releasing activities by various drugs (author's transl)]. Several procedures have been reported for the assay of corticotrophine-releasing factor (CRF), each having its advantages and disadvantages. This report deals with an in vitro assay of ACTH releasing activity utilizing pituitary incubation combined with ACTH radioimmunoassay. Rat half pituitary was preincubated in 2 ml Krebs Ringer bicarbonate buffer containing 0.2% glucose and 0.25 % BSA (KRBG-BSA) for 1.5 hr (45 min X 2). The medium was replaced by 1 ml KRBG-BSA and incubated for 30 min. Then the medium was again replaced by 1 ml KRBG-BSA or KRBG-BSA containing test materials and incubated for another 30 min. The amount of ACTH assayed by radioimmunoassay in the 2nd 30 min incubation was compared with in the 1st 30 min incubation and expressed as percentage. In ACTH radioimmunoassay, anti-ACTH serum was diluted to 1 : 1,500-3,000. The 125I-alpha 1-24ACTH-antibody system was not affected by lysine-vasopressin (LVP), arginine-vasopressin (AVP), rat's pituitary LH, GH and prolactin. Human 1-39ACTH was used as ACTH standard, and the dilution curve of incubation medium was paralleled with the standard curve. Repeatability of immunoassayable ACTH within-assay was 174 +/- 5.0 pg/tube (CV = 2.9%). A log dose-relationship was observed between the amounts of stalk median eminence extracts (SME ; NIAMDD) added to the incubation medium and its ACTH releasing activities. The sensitivity of this assay method was at least 0.1 SME or 10 mU of LVP and AVP. Using this method, it found that LVP, AVP, norepinephrine (100 ng/ml200 ng/ml) and 5-hydroxytryptophane (1 mug/ml) had ACTH releasing activities but LH-RH, TRH, glucagon, dopamine, phentolamine, propranolol, haloperidol, prostaglandin E1 and indomethacin did not affect the release of ACTH.


[The study of physiological chemistry on a subunit of salivary gland hormone (2) (author's transl)]. It had been reported by authors that salivary gland hormone, parotin, was composed with subunit (parotin-subunit) which showed molecular weight of 45,000, and that parotin-subunit had rabbit serum calcium decreasing activity and the cross reactivity with rabbit anti parotin serum. In the present report, in order to study physiological chemistry of parotin-subunit, the influence of parotin-subunit on serum Ca and 45Ca levels relating to calcium metabolism, the distribution of 131I-parotin-subunit, the effect of parotin-subunit, on adenyl cyclase-cyclicAMP system, the anabolic action, C-terminal amino acid sequence and sugar component of parotin-subunit were investigated. The results are summarized as follows: 1) The decrease of rabbit serum Ca after injection of parotin-subunit was related to change of Ca in stable bone, but not to inhibition of bone resorption. 2) A high concentrated localization of radioactivity of 131I-parotin-subunit was found in liver, kidney and spleen, and as much as 60% of administrated radioactivity was localized in liver at 5 min after the injection. The retention of radioactivity was found in testis, seminal vesicle, prostate, parotid gland and submaxillary gland. 3) Cyclic AMP level increased significantly in metaphysial bone, submaxillary gland and plasma after administration of parotin-subunit but in other organs with localized much radioactivities, the level did not changed. Parotin-subunit activated adenyl cyclase of particular fraction of metaphysial bone. 4) The C-terminal amino acid of parotin-subunit was Leu, and its C-terminal amino acid sequence was -Val-Ser-Ala-Thr- Leu-OH by digestion of carboxypeptidase A. 5) Parotin-subunit included 3.3% of sugar which consisted of amino sugar and uronic acid.


In vitro biosynthesis and release of three immuno-reactive insulin-like components by a human insulinoma. Pieces of a human insulinoma were incubated for 120 or 180 min in media containing labelled leucine. In addition to insulin and proinsulin, the tumoral tissue was found to synthesize and release a third immunoreactive insulin-like component of higher molecular weight. No significant amount of the latter component was detected in normal rat islets under identical experimental conditions. The rate of secretion of insulin-like peptides was abnormally high relative to their concentration in the tumoral tissue, and was apparently unaffected by various insulinotropic or inhibitory agents. Only cycloheximide was found to significantly reduce the synthesis and subsequent release of insulin-like peptides by the tumoral tissue. It is suggested that the secretion of large amounts of both the high molecular weight component and proinsulin relative to that of insulin might be due, in part at least, to the inability of the tumoral cells to prevent the release of newly synthetized hormonal peptides, leading to the depletion of the tissue in hormonal products, as judged by both immunological and ultrastructural criteria.