Double-headed protease inhibitors from black-eyed peas. V. Analysis of the energetics of protease-inhibitor interactions. The half-site reactivity of trypsin and chymotrypsin binding to two double-headed black-eyed pea protease inhibitors a trypsin-chymotrypsin inhibitor (BEPCI) had a trypsin inhibitor (BEPTI), is explained in terms of the energetics of these inhibitor-protease interactions. Free energy diagrams are constructed to facilitate interpretation of the energetics. Coupling-free energies are calculated to reflect the magnitude of the interdependence of protease-binding (alloassociation) and inhibitor subunit interactions (isoassociation). The experiment observation of the predominance of liganded monomer complexes for the lima bean inhibitor and the Bowman-Birk soybean inhibitor and the predominance of half-site-liganded complexes for BEPCI and BEPTI is the direct result of the magnitudes and signs of the coupling free energies which result from these protease-inhibitor interactions.


Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes. The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.


Avian mitochondrial glutamine metabolism. Intact avian liver mitochondria were shown to synthesize glutamine from glutamate in the absence of exogenous ATP and ammonia. With L-[U-14C]glutamate as the substrate, there was an approximate 1:1 stoichiometry between glutamate deaminated (as measured by the release of 14CO2 due to alpha-keto-[14C]glutarate oxidation) and glutamate amidated. With L-[15N]glutamate as the substrate, the isolated glutamine was shown by low and high resolution mass spectrometry of its phenylisothiocyanate derivative to contain 15N in both the alpha-amino and amide groups. Thus, for each mole of glutamate taken up, approximately 0.5 mol is deaminated and the other 0.5 mol serves as a substrate for glutamine synthetase previously localized in these mitochondria (Vorhaben, J. E., and Campbell, J. W. (1972) J. Biol. Chem. 247,2763). The permeability of L-glutamine to intact avian liver mitochondria was studied by a rapid centrifugation technique. Efflux as well as influx of L-glutamine were both rapid and appeared to occur by a passive, energy-independent process. These results indicate that the mitochondrial glutamine synthetase present in uricotelic species represents the primary ammonia detoxication reaction in that ammonia released intramitochondrially during amino acid catabolism is converted to glutamine for efflux to the cytosol where it may serve as a substrate for purine (uric acid) biosynthesis.


Exchange of 3-O-methylglucose in isolated fat cells. Concentration dependence and effect of insulin. 3-O-[14C]Methylglucose was used to study the insulin action on the sugar transport in white fat cells. The experiments comprised determinations of the 3-O-methylglucose space at stationary distribution, of the rate constants for 3-O-methylglucose equilibrium exchange under various conditions, and of the 3-O-methylglucose inhibition of the lipogenesis from glucose. The following was found. The intracellular distribution space for 3-O-methylglucose at equilibrium was unaffected by insulin and was identical with the intracellular 3H2O space. The half-time for the equilibrium exchange of 3-O-methylglucose at a concentration of 25 mM was about 240 s in the absence of insulin and about 15 s with insulin (0.7 muM) present. Addition of phloridzin (5 mM) decreased the rate of the exchange process about 25-fold in both cases. The self-exchange of 3-O-methylglucose (1 mM) was at least 50 times faster than the self-exchange of L-glucose (1 mM). The concentration dependence of the 3-O-methylglucose exchange rate was approximately hyperbolic both in the absence and the presence of insulin, although the saturation of the transport mechanism at high concentrations of sugar was not as complete as predicted. In the absence of insulin the estimate of the half-saturation constant (Kt) was about 5 mM; that of the maximal exchange rate (Vmax) varied from 0.07 mmol s-1/liter of intracellular water to 0.2 mmol s-1 liter-1. In the presence of insulin Kt remained about 5 mM, whereas Vmax was increased to about 1.7 mmol s-1 liter-1. The latter estimate was reproducible within about 20%. The incorporation of trace amounts of [U-14C]glucose into intracellular lipids was inhibited by unlabeled 3-O-methylglucose pre-equilibrated over the membrane. The inhibition constant estimated from such experiments was about 5 mM both in the absence and the presence of insulin, and the insulin-induced increase in the rate of glucose incorporation was similar to the increase in the rate of the 3-O-methylglucose exchange process. It is concluded that exchange of 3-O-methylglucose proceeds via a mechanism which shows stereospecificity and saturability and that insulin acts by increasing the maximal transport capacity without changing the half-saturation constant.


Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue. A monoacylglycerol-hydrolyzing enzyme has been purified 2500-fold from rat adipose tissue. The key step was the solubilization of the enzyme, presumably as an enzyme-detergent complex, by sonication with a nonionic polyoxyethylene alcohol detergent. The purification was achieved by ion exchange and gel chromatography, and isoelectric focusing, in the presence of detergent. By sodium dodecyl sulfate gel electrophoresis the enzyme protein was more than 85% pure. This method indicated a minimum molecular weight of 32,900. The preliminary amino acid composition, excluding tryptophan, could best be fitted with a value of 31,800. The purified enzyme had a pI of 7.2, an estimated Stokes radius of 39 A by gel chromatography and a pH optimum of 8.0. Enzyme stability was highly dependent on presence of detergent and free sulfhydryl groups. The enzyme was responsible for the main monoacylglycerol- but only a small part of the p-nitrophenylacetate-hydrolyzing activity of crude adipose tissue extracts and hydrolyzed 1(3)- and 2-monooleoylglycerol at equal rates. Under the assay conditions used it did not catalyze the hydrolysis of emulsified trioleoylglycerol, micellar or emulsified dioleoylglycerol, emulsified cholesterol oleate or micellar lysophosphatidylcholine. It is possible that the enzyme may be a specific monoacylglycerol hydrolase.


Synthesis of human placental lactogen messenger RNA as a function of gestation. It was shown previously that 4 to 5 times more human placental lactogen (hPL) was synthesized in cell-free extracts from term placentae than in comparable extracts prepared from first trimester tissue. In an attempt to define what accounts for this differential rate of synthesis RNA was prepared from first trimester and term placentae. Following purification through an oligo(dT)-cellulose column, these RNA preparations were tested for their ability to direct the synthesis of the hPL precursor in the wheat germ cell-free system. With similar amounts of first trimester and term mRNA, the overall efficiency as defined by the stimulation of total amino acid incorporation was comparable. However, there was approximately 4 times more hPL synthesized in the presence of term RNA. This was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic fingerprinting. The peak of the hPL precursor messenger activity sedimented at 12 to 13 S on sucrose gradient. Analysis of the RNA by formamide-polyacrylamide gel electrophoresis further supported this value. The data indicate that the increased synthesis of hPL at term reflects greater levels of hPL mRNA in term tissue than in first trimester tissue. The data also show that the overall in vivo levels of hPL can be correlated not only with the increase in placental syncytial mass during pregnancy but also in the greater proportion of hPL synthesized per g of tissue. The latter results from the continual differentiation of the placenta occurring throughout gestation.


Alanine and glutamine synthesis and release from skeletal muscle. I. Glycolysis and amino acid release. The synthesis and release of alanine and glutamine were investigated with an intact rat epitrochlaris muscle preparation. This preparation will maintain on incubation for up to 6 hours, tissue levels of phosphocreatine, ATP, ADP, lactate, and pyruvate closely approximating those values observed in gastrocnemius muscles freeze-clamped in vivo. The epitrochlaris preparation releases amino acids in the same relative proportions and amounts as a perfused rat hindquarter preparation and human skeletal muscle. Since amino acids were released during incubation without observable changes in tissue amino acids levels, rates of alanine and glutamine release closely approximate net amino acid synthesis. Large increases in either glucose uptake or glycolysis in muscle were not accompanied by changes in either alanine or glutamine synthesis. Insulin increased muscle glucose uptake 4-fold, but was without effect on alanine and glutamine release. Inhibition of glycolysis by iodacetate did not decrease the rate of alanine synthesis. The rates of alanine and glutamine synthesis and release from muscle decreased significantly during prolonged incubation despite a constant rate of glucose uptake and pyruvate production. Alanine synthesis and release were decreased by aminooxyacetic acid, an inhibitor of alanine aminotransferase. This inhibition was accompanied by a compensatory increase in the release of other amino acids, such as aspartate, an amino acid which was not otherwise released in appreciable quantities by muscle. The release of alanine, pyruvate, glutamate, and glutamine were observed to be interrelated events, reflecting a probable near-equilibrium state of alanine aminotransferase in skeletal muscle. It is concluded that glucose metabolism and amino acid release are functionally independent processes in skeletal muscle. Alanine release reflects the de novo synthesis of the amino acid and does not arise from the selective proteolysis of an alanine-rich storage protein. It appears that the rate of alanine and glutamine synthesis in skeletal muscle is dependent upon the transformation and metabolism of amino acid precursors.


Alanine and glutamine synthesis and release from skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis. The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.


Affinity partitioning of membranes. Cholinergic receptor-containing membranes from Torpedo californica. Affinity partitioning has been employed in the purification of membranes rich in cholinergic receptor from Torpedo californica electric organs. The procedure involves a modification of poly(ethylene oxide)-dextran aqueous phase partitioning systems where a ligand selective for the receptor is conjugated to the poly(ethylene oxide). Specific partitioning of the receptor-containing membranes into the poly(ethylene oxide)-rich phase occurs when bis-alpha,omega-trimethylamino poly(ethylene oxide) or bis-rho-tri-methylammonium phenylamino poly(ethylene oxide) was added to the phase system in low mole ratio. bis-alpha,omega-Methylamino poly(ethylene oxide), which should impart equivalent interfacial electromotive potential to the system but bind poorly to the receptor sites, was much less effective in producing phase distribution changes. The ligand-polymer-dependent phase distribution shifts were blocked by bisquaternary methonium ligands at concentrations consistent with their relative affinities for the cholinergic receptor. Titration or receptor sites with cobra alpha-toxin decreased the phase distribution changes in a linear fashion up to the point of stoichiometry. These observations are consistent with the phase distribution changes being consequent to ligand-polymer association with the pharmacologically important site on the receptor. The affinity partitioning procedure, when employed following an initial purification of the membranes by differential and density gradient centrifugation, yields membrane preparations with a high degree of morphological uniformity and a specific activity between 2.9 and 4.6 nmol of bound cobra alpha-toxin/mg of protein.


Regulation of carbohydrate uptake in gram-positive bacteria. Uptake of glycerol and other carbohydrates by Staphylococcus  aureus cells is sensitive to regulation by sugar substrates of the phosphoenolpyruvate:sugar phosphotransferase system. Inhibition requires an intact phosphotransferase system. In contrast to results obtained with Gram-negative bacteria, it appears that intracellular sugar phosphate is the inhibiting species.


Intermolecular tritium transfer in the transcarboxylase reaction. Transfer of tritium from [3-3H]pyruvate into propionyl-CoA is found during the reaction of transcarboxylase: Methylmalonyl-CoA + pyruvate leads to oxalacetate + propionyl-CoA. About 5% of the tritium counts that are labilized in the reaction are found in a position of the propionate that exchanges rapidly with water in the presence of transcarboxylase. Transfer from [2-3H]propionate of propionyl-CoA to pyruvate is real but only about one-tenth as great. The tritium transfers between reactants on two subunits are difficult to explain by a "carbanion" mechanism of --C--H bond cleavage and support the cyclic mechanism in which carboxybiotin itself is the base and the enol form of biotin is the proton-transferring agent.


Evidence that approximately eighty per cent of the soluble proteins from Ehrlich ascites cells are Nalpha-acetylated. Analysis of soluble Ehrlich ascites proteins by the Sanger procedure revealed methionine, alanine, valine, and glycine as the major NH2-terminal amino acids. The average monomer weights of these proteins calculated from the yields of NH2-terminal amino acids was 144,000. In contrast, the average monomer weight of Ehrlich ascites soluble proteins calculated from the data obtained after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate was 32,500. The explanation for the disparity in the estimates of average monomer weight obtained by the procedures appears to be that extensive blocking of alpha-NH2 groups by acetate occurs in these proteins, i.e. of the acetate present in the acidic peptides isolated from proteolytic digests of ascites proteins, 23.2 nmol/mg of protein appears to originate from N-acetyl amino acids. These results suggest that approximately 80% of the soluble proteins from Ehrlich ascites cells contain acetate at their NH2-terminal residues. The extensive N-acetylation of proteins does not appear to be limited to Ehrlich ascites cells and may be characteristic of eukaryotic proteins.


Synthesis of 1alpha-hydroxy [6-3H]vitamin D3 and its metabolism to 1alpha, 25-dihydroxy [6-3H]vitamin D3 in the rat. 1alpha-Hydroxy [6-3H]vitamin D3 has been synthesized with a specific activity of 4 Ci/mmol, and its metabolism in rats has been studied. It is rapidly converted to 1alpha,25-dihydroxy [6-3H]vitamin D3 in vivo. Following an intravenous or oral dose, a maximal concentration of 1alpha,25-dihydroxy [6-3H]vitamin D3 is found 2 and 4 hours, respectively, before the maximal intestinal calcium transport response is observed. Similarly, 1alpha,25-dihydroxy[6-3H]vitamin D3 accumulation in bone precedes the bone calcium mobilization response. It appears, therefore, that the biological activity of 1alpha-hydroxyvitamin D3 is largely, if not exclusively, due to its conversion to 1alpha,25-dihydroxy[6-3H]vitamin D3 1alpha-Hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appear in intestine equally well after an oral or an intravenous dose of 1alpha-hydroxy[6-3H]vitamin D3. However, much less of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appears in bone and blood after an oral than after an intravenous dose. A much reduced bone calcium mobilization response is also noted following an oral dose as compared to an intravenous dose of 1alpha-hydroxyvitamin D3, suggesting that oral 1alpha-hydroxyvitamin D3 is not utilized as well as intravenously administered material.


Metabolism of 1alpha-hydroxyvitamin D3 in the chick. Chicks convert both orally and intravenously administered 1alpha-hydroxy[6-3H]vitamin D3 rapidly to 1alpha,25-dihydroxy[6-3H]vitamin D3. The maximal accumulation of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine precedes the intestinal absorption response to 1alpha-hydroxyvitamin D3 by at least 2 hours. Oral administration results in the highest concentrations of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine, giving a level about 1.5 times that achieved with an intravenous dose. On the other hand, an oral dose of 1alpha-hydroxy[6-3H]vitaminD3 gives much lower amounts of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 in bone and blood than an intravenous dose, which suggests that the 1alpha-hydroxy[6-3H]vitamin D3 may not be utilized as well by the oral route as by an intravenous route. Liver homogenates from both rat and chick convert 1alpha-hydroxy[6-3H]vitamin D3 to 1alpha,25-dihydroxy[6-3H]vitamin D3. However, intestinal homogenates from chick, but not rat, can also cary out this conversion, which may account for the higher concentration of 1alpha,25-dihydroxy[6-3H]vitamin D3 found in the intestine of chicks given an oral dose of 1alpha-hydroxy[6-3H]vitamin D3.


Effect of dietary protein and tryptophan and the turnover of rat liver ornithine aminotransferase. Under controlled conditions of diet, age, and animal management we confirmed our earlier studies that the fractional turnover rate constant of rat liver ornithine aminotransferase (L-ornithine:2-oxoacid aminotransferase, EC 2.6.1.13) was about 0.4 day-1 when estimated by tracer technique. However, when estimated by the kinetics of enzyme activity perturbation during dietary induction or return to normal levels, a value of about 0.7 day-1 was obtained. This discrepancy may now be attributed in part to a transistory change in the fractional rate constant during enzyme adaption.


Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from the mouse plasmacytoma, MOPC 315. Class III DNA-dependent RNA polymerases were purified from the mouse plasmacytoma, MOPC 315. RNA polymerases IIIA and IIIB were solubilized from a whole cell extract and resolved by chromatography on DEAE-Sephadex. Chromatography on DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose ion exchange resins and sedimentation in sucrose density gradients yielded chromatographically homogeneous Enzymes IIIA and IIIB which were purified approximately 22,000 and 53,000-fold respectively, relative to whole cell extracts. The specific activity of these enzymes was comparable to that reported for other purified eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested a molecular weight of approximately 650,000 for each of the class III enzymes.


Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.


Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin. 1. A detailed study of cytochrome C oxidse activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome C concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD2 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2...


Differential effects of sulfhydryl reagents on activation and deactivation of the fat cell hexose transport system. A rapid filtration method was used to measure initial rates of 3-O-[3H]methylglucose uptake and thus estimate hexose transport system activity in isolated white fat cells. Insulin markedly stimulated the transport system activity and its effect was rapidly and completely reversible. In addition, such oxidants as vitamin K5 (50 muM), hydrogen peroxide (4mM), methylene blue (50 muM), and diamide (20 mM) also maximally activated 3-O-methylglucose transport and their effects were not additive to those of maximal concentrations of insulin. These oxidants had no effect on total cellular ATP levels under these conditions. Hexose transport system activity in either the presence or absence of these stimulatory agents was uniformly sensitive to inhibition by cytochalasin B. Treatment of fat cells with either 0.5 mM N-ethylmaleimide or 3 mM dithio(bis)nitrobenzoic acid abolished the ability of insulin or oxidants to activate hexose transport system activity. Control transport activity was not significantly influenced by these agents. Fat cells treated with dithio(bis)nitrobenzoic acid completely regained the ability to respond to insulin or vitamin K5 after removal of the agent by washing in low concentrations of reductant. Elevated rates of transport due to prior incubation of cells with insulin or vitamin K5 were completely resistant to inhibition by subsequent addition of N-ethylmaleimide or dithio(bis)nitrobenzoic acid. Deactivation of the hormone-stimulated transport system could be achieved by washing cells free of insulin or by destruction of insulin-receptor interaction by trypsin. N-Ethylmaleimide effectively blocked deactivation of insulin-stimulated transport system activity, while dithio(bis)nitrobenzoic acid was without effect. These results suggest that distinct cellular components mediate activation versus deactivation of the fat cell hexose transport system. N-Ethylmaleimide, which effectively penetrates fat cells, inhibits both processes while the layer, more polar dithio(bis)nitrobenzoic acid blocks activation but not deactivation of this transport system.


Modification of the epidermal growth factor affecting the stability of its high molecular weight complex. The epidermal growth factor (EGF) can be isolated from the submaxillary gland of the adult male mouse as part of a high molecular weight complex (HMW-EGF). This complex can be reversibly dissociated into its subunits, EGF AND EGF-binding protein, an arginine esteropeptidase (Taylor, J. M., Cohen, S., and Mitchell, W. M. (1970) Proc. Natl. Acad. Sci. U. S. A. 67, 164-171). The COOH-terminal arginine residue of EGF was quantitatively removed by digestion with carboxypeptidase B...


Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride. ATP-dependent calcium uptake of rat liver microsomes is examined following ingestion of CC14 (2.5 ml/kg). Within 30 min there is an abrupt drop in calcium uptake activity of the liver microsomes. This activity remains down for 48 hours before slowly returning to normal levels. The effect is specific for CC14 as contrasted with CHC13 and CH2Cl2. The CCl4 does not affect similar calcium uptake activity of kidney microsomes. Calcium uptake activity of the liver mitochondria is unaffected. The first 12 hours after CCl4 ingestion there is a relatively slow rise in the calcium content of the liver tissue and mitochondria. After 12 hours a much larger influx of calcium into the tissue and the mitochondria takes place. Forty-eight hours after CCl4 ingestion the process begins to slowly reverse. The following postulated sequence may relate to the CCl4 hepatotocicity. CCl4 is activated to free radicals by the liver endoplasmic reticulum. The free radical inactivate calcium pump activity of the liver endoplasmic reticulum. Calcium levels of the cytoplasm increase and significantly modify ion permeability of the plasma membrane. High levels of external calcium enter the cytoplasm and are sequestered in the mitochondria. The high level of mitochondrial calcium uptake inhibits mitochondrial oxidative phosphorylation. The specific sensitivity of the calcium pump activity of liver microsomes to CCl4 further establishes the identity of a system seperate from the mitochondrial system. The above postulated sequence of events would suggest a critical role in liver metabolism for calcium pump activity of the endoplasmic reticulum.


Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands. The beta subunit of follicle-stimulating hormone (FSH-beta) from human pituitary glands was reduced and S-aminoethylated prior to thermolytic, tryptic, and chymotryptic digestions. Each digest was gel-filtered on Sephadex G-50 to seperate the glycopeptides. The glycopeptides and the peptides were isolated by high voltage paper electrophoresis at pH 6, 3.5, and 2.0. The purity of the isolated peptides was confirmed by amino acid analyses. The amino acid sequences of peptides were determined by Edman degradation followed by subtractive amino acid analysis and, in certain cases, confirmed by dansylation. COOH-terminal sequences of the peptides were determined by digestion with carboxypeptidases A and B and by hydrazinolysis. The tryptophan content of human follicle-stimulating hormone, of the beta subunit of human follicle-stimulating hormone, and of the glycopeptides obtained from the enzymic digests was determined by fluorescence spectra, titration against N-bromosuccinimide, colorimetric estimation with p-dimethyl aminobenzaldehyde, hydrolysis with methane sulfonic acid containing 0.2% tryptamine followed by amino acid analysis, microbiological assay, and sequence analysis. The presence of 1 tryptophan residue in the beta subunit was indicated.


Hydrophilized polyethylene catheters.

Hydrophilized polyethylene catheters. Complications arising in the application of polyethylene catheters in surgery may be reduced by their surface hydrophilization. This study describes a combination of chemical transformation and coating with a hydrophilic gel. Modified catheters were tolerated well by the patients and in contrast with the original catheters, they did not lead to the formation of thrombi or phlebitis.


Effects of solvent casting copolymer materials as related to mechanical properties. Using a well characterized series of styrene butadiene triblock copolymers a systematic study was carried out to determine the influence of casting solvent, used for film preparation, on the final mechanical properties. The results clearly indicate that in the range of styrene contents of 30-40 mol %, the modulus behavior is greatly affected as the solubility parameter of the casting solvent, delta, is varied. Specifically, the modulus increases as delta increases to the value of polystyrene. At lower or higher styrene content, the properties are influenced but to a much lesser degree. Other mechanical properties measured were yield stress and breaking stress. The ramifications of these data are discussed in light of preparing prosthetic material via solvent casting.


The biostability of silicone rubbers, a polyamide, and a polyester. A biostability test program was designed after evaluation of the relevant literature on in vivo aging phenomena in plastics and elastomers. The program comprised macroscopic, microscopic, mechanical, and physicochemical investigations. Five silicone rubbers, one polyester, and one polyamid were tested as to their aging behavior and their suitability for long-term implantation in the human body was assessed. In order to be able to state the applicability of materials used for endotheses, the various aging phenomena had to be divided into three groups, viz. unspecific, relative, and absolute indications of aging or unserviceability. Changes due to aging were found in all types of implanted plastics and elastomers. Thus, the formation of layers on inlay surfaces occurred regularly and the same would apply to changes in mechanical characteristics during the tensile test. Aging processes resulting in total unserviceability were fragmentation and crazing in the polyester and polyamide sheetings. Other aging phenomena which will easily fit into the classification given above are changes in electrical test values, protein, and lipid depositions without stronger absorptive adhesion, crystallizations on the surface of silicone vulcanizates, and chemical changes in the polyester and polyamide sheetings. Following the assessment of the materials used for endotheses, the test methods used have been evaluated with regard to their suitability for the testing of biostability.


Transient diffusion effects in the study of early platelet adhesion. In the study of interactions of blood with artificial surfaces, it is common practice to wet these surfaces with a saline solution before exposing them to blood. The effect of this procedure on subsequent adhesion of blood platelets has been investigated both theoretically and experimentally, with a spinning disc apparatus. A large part of the observed delay in the initiation of platelet adhesion when using this apparatus is attributed to the time involved in displacing the prewetting fluid and bringing platelets near the surface. This observation is relevant to speculations of other researchers about the time course of adhesion-related reactions at the surface-proper. Other theoretical solutions are developed for the transient Leveque problem, which relates to a uniform shear-flow near a surface, and for a stagnation point apparatus. These solutions are compared with delay effects noticed in experimental data published by others.


Wear of polymeric bearing materials: the effects of in vivo implantation. The gross biocompatibility of the materials implanted was generally satisfactory. Weight gain during implantation due to net sorption of fluid was negligible for PPO and UHMWPE. Delrin AF specimens gained less than 0.5%, while SP-1 and SP-21 gained up to 2% in weight. Amoco-AI specimens gained up to 7% in weight. The friction coefficient of implanted samples of UHMWPE was lower by 20 to 40% than for the virgin material. The friction coefficient of the other implanted materials was usually somewhat higher than the friction coefficient of the corresponding virgin material. The largest increase in the friction coefficient was 38% for Delrin AF. Wear characteristics of UHMWPE were not significantly changed by implantation for periods of three years. UHMWPE was the only acceptable bearing material tested. Significantly higher wear rates were observed for implanted blocks of the SP-21, a Delrin AF, and an Amoco-AI material. Because of excellent creep resistance, virgin wear rates, and other favorable properties of the polyimides, further testings are in progress to indicate potential application of these polymers.


Surface hydroxylation of styrene-butadiene-styrene block copolymers for biomaterials. This work pertains to the development of high strength elastomers potentially useful as nonthrombogenic cardiovascular prostheses. Triblock copolymers of the styrene-butadiene-styrene type have been subjected to surface hydroxylation which provide reactive sites at the surface for the subsequent coupling of heparin while retaining the unique mechanical properties of the SBS copolymers. Curves of hydroxyl content versus the copolymer film thickness demonstrate the effect of swelling in the surface region on the product distribution and on the time dependence of the hydroxylation process. In addition, the effect of time, temperature, and the composition of the reaction bath on the diffusion/reaction process is shown. Finally, the general applicability of this surface modification scheme to the development of biomaterials is discussed.


Canine tendon studies. II. Biomechanical evaluation of normal and regrown canine tendons. Some of the mechanical properties of regrown canine tendons are compared to those of normal tendons of young and mature animals. Patellar and Achilles tendons from 12 beagle dogs were removed and studied with their bone origin and insertions. Mechanical tests were performed within 24 hr and test conditions simulated the physiological function of the tendon in vivo at room temperature. Specimens were soaked in Ringers solution and mounted in an Instron testing machine with load deflection curves plotted automatically. The parameters used for analysis were load extension, stress relaxation, elastic limit, and strain rate dependence. The regrown tendons in young animals appeared to quickly adjust in dimension and structure so that their properties were not significantly different from those of normal tendons on a load extension basis. The normal tendons were stiffer than regrown ones but the modulus of elasticity increased with age. The Achilles were stiffer than patellar tendons. Cyclic loading with 25 kg did not affect reconstructed tendon models, although some increase in stiffness was noted. The elastic modulus decreased with an increase in ambient temperature and increasing strain rate.


Influence of application technique on microstructure and strength of acrylic restorations. An investigation was made of the influence of application techniques on the microstructure and properties of an acrylic tooth restorative. Mixtures of acrylic powder and monomer ("Sevriton Simplified") were applied by the brush technique of Nealon (J. Prosth. Dent., 2, 513, 1952) and by two bulk flow techniques. While similar porosities (about 4%) were observed, the brush technique resulted in a greater quantity of grains from the acrylic powder. Despite this, there was little difference in values of compression modulus, compressive yield stress, and diametral compressive strength. The mechanical strength of the materials studied was less than one-half that of high molecular weight poly(methyl methacrylate) (PMMA). Crack propagation studies established that the interface between the grains and matrix was not a source of weakness. However, as the matrix was crosslinked this could not be checked by solution methods of polymer characterization.


Microstructure in gold-containing Ag3Sn-amalgam. The present study was conducted in order to understand the microstructural characteristics in dental amalgam which consists of both spherical and irregular Ag3Sn alloy particles with gold substituted in varying amounts for silver (0, 9, and 15%). Spherical alloy particles were used as obtained from Western Gold and Platinum and irregular alloy particles were prepared in the laboratory and then employed in the present study. The amalgam was prepared from the alloy particles by the trituration and condensation method. The polished and etched samples were studied using the techniques of X-ray diffraction, optical metallography, scanning electron microscopy, and X-ray energy dispersive spectroscopy. The microstructure in both types amalgam studied after two weeks of preparation contains gamma (unreacted Ag3Sn particles), gamma1 (Ag2Hg3), and gamma2 (Sn7-8Hg) phases in pure Ag3Sn-amalgam and gamma, gamma1, and (Au-Sn) phases in gold-containing amalgam. The The (Au-Sn) phase existed in the form of rings surrounding the gamma grains and acted as a barrier for mercury diffusion in Ag3Sn particles.


Hair sampling for chromium content following Charnley hip arthroplasty. The chromium content of hair has been studied by neutron activation analysis of samples from 62 patients following a Charnley unilateral or bilateral hip arthroplasty by an artificial joint of stainless steel against polyethylene, inserted three to five years previously. The patients lived in the North Wales area of the British Isles. Hair chromium content was also studied in 51 control subjects of similar age to that of the patients, from the same geographical area and often from the patients' own households. Chromium content was recorded as parts per million (ppm) of hair weighed and analyzed in the state received, without preliminary ashing or chemical "washing" of the samples in the laboratory. Fifty of the 51 control subjects showed less than 2 ppm chromium in hair; one showed 2-5 ppm; none showed more than 5 ppm. Fifty-nine of the 62 patients showed less than 2 ppm chromium in hair; three showed 2-5 ppm; none showed more than 5 ppm. Attention is drawn to certain practical aspects when collecting hair samples for chromium analysis. The use and validity of hair sampling as one method of screening for systemic chromium accumulation following implant surgery is discussed. The method may be a useful adjunct to monitoring of chromium levels in blood samples. In this preliminary study, hair sampling has given no evidence of any major risk of systemic chromium accumulation in patients using a Charnley arthroplasty for periods of up to five years; no conclusion can be made concerning periods longer than this. The findings do not exclude systemic reactions of the hypersensitivity type, since this type of reaction could develop to only small amounts of chromium.


Chondromalacia Patellae. A prospective study. In a prospective study of 105 arthrotomies for chondromalacia patellae, patella alta or an increased Q angle was found in most of the knees and was considered to be the usual cause of chondromalacia. The chondromalacia on the patella was usually centrally located with extension equally onto the medial and lateral facets. Femoral changes were uncommon. Patellar realignment together with excision of chondromalacic cartilage gave a satisfactory result in 79 per cent of the eighty-seven knees that could be followed.


Non-traumatic dislocation of the radial head in cerebral palsy. To our knowledge, there has been only one previous description of dislocation of the radial head associated with cerebral palsy. This paper describes nine such dislocations, eight of which were bilateral and posterior and one, unilateral and anterior. There was an incidence of this abnormality of 2.4 per cent in 368 of the cases surveyed. Of those patients with cerebral palsy and with flexion contracture of the elbow and pronation contracture of the forearm, 27 per cent had the dislocation.


Fasciotomy in the treatment of the acute compartment syndrome. Sixty-six cases of acute compartment syndrome were treated by fasciotomy in forty-six extremities of forty-four patients. Fasciotomy performed early, that is, less than twelve hours after the onset of the compartment syndrome, resulted in normal function in 68 per cent of the extremities. Only 8 per cent of those having late fasciotomy had normal function. The complication rates for the early and late fasciotomized extremities were 4.5 per cent and 54 per cent, respectively. No significant differences in residual function or complication rate were noted with "open" or "closed" fasciotomy.


Congenital anomaly of the thumb: absent intrinsics and flexor pollicis longus. Congenital absence of the median-innervated intrinsic muscles and flexor pollicis longus was seen in varying degrees of severity in eleven hands of eight patients. Three members were involved in one family in which the anomaly was probably an autosomal dominant. There were no chromosomal abnormalities. Surgical treatment of seven hands, including release of the adduction contracture and transfer of the ring-finger superficialis tendon to provide opposition and to reinforce the ulnar collateral ligament of the metacarpophalangeal joint, resulted in significant improvement in pinch and grasp.


Spinal deformity associated with heritable neurological conditions: spinal muscular atrophy, Friedreich's ataxia, familial dysautonomia, and Charcot-Marie-Tooth disease. Children with progressive neurological conditions such as spinal muscular atrophy, Friedreich's ataxia, familial dysautonomia (Riley-Day syndrome), and Charcot-Marie-Tooth disease have a significant risk of acquiring a serious spinal deformity. As with paralytic scoliosis following poliomyelitis, the curves are difficult to control with bracing, and progression does not cease with maturation. An increasing spinal curvature may lead to loss of ambulation or, for the wheelchair-bound patient, loss of sitting balance. The curvature may further compromise pulmonary function that may already be compromised by the neurological deficit. Twenty patients are reported with an average follow-up of six years (range, one to fourteen years). For the properly selected patient, surgical stabilization of the spine arrested the progress of the curve and improved function. Complications were few; however, pseudarthrosis was more common than in patients with non-neurological problems.


The patterns of spinal deformity in Duchenne muscular dystrophy. In a clinical and roentgenographic study of spinal deformities in sixty-two patients in the later stages of Duchenne muscular dystrophy, many patients had marked scoliosis and kyphosis, while others with hyperextended spines had comparatively little scoliosis. Based on an analysis of the data, it is suggested that the development of spinal deformity in patients with Duchenne muscular dystrophy may progress in two ways: one leading to the early establishment of a position of extension and a maximum intrinsic stability with minor deformity, and the other leading to progressive deformity. It was concluded that management for these patients should be designed to guide the early straight spine toward the late extended pattern by attempting to prevent kyphosis and pelvic obliquity.


Mobility aids for the paraplegic child.

Mobility aids for the paraplegic child. Paraplegic children derive many benefits from upright bracing during their growing years. Physiological advantages include the prevention of joint contractures, decreased osteoporosis, prevention of urinary tract infections, increased cardiopulmonary tolerance, and the development of trunk and head control. The quality of life is enhanced because the child has increased self-esteem and participates in group activities with greater independence. Two new devices, the stand-up wheelchair and the shuttlebug, have been employed with success in the Winnipeg Child Paraplegic Program.


Energy cost of walking of amputees: the influence of level of amputation. A comparison of selected gait parameters and the energy cost of prosthetic walking was made in seventy patients with unilateral traumatic and vascular amputations. Amputations above the knee, below the knee, and at the Syme's level were compared in both groups of amputees, and a control group of forty normal subjects also studied. In both groups of amputees performance was significantly better the lower the level of the amputation. When preservation of function is the chief concern, amputation should be performed at the lowest possible level.


The upper-extremity amputee. Early and immediate post-surgical prosthetic fitting. The results of immediate and early post-surgical prosthetic fitting in eighty-seven upper-extremity amputees as well as the results in nine patients with shoulder dislocation who were fitted with temporary devices were reviewed. No local wound complications occurred and the rate of prosthetic acceptance was high. A practice prosthesis, with a filler insert formed from liquid Silastic foam allowed to set between the walls of the practice prosthesis and the amputation stump, was used extensively in this series. With the Silastic insert and practice prosthesis, prosthetic training could be instituted during healing of the amputation wounds, proximal wounds, or fractures.


Correction of paralytic claw finger in leprosy by capsulorrhaphy and pulley advancement. Paralytic claw-finger deformity in leprosy was corrected by metacarpophalangeal capsulorrhaphy and fibrous flexor pulley advancement in ninety fingers in thirty-seven hands. Sixty-eight of these were analyzed after one to four years' follow-up. The overall results were: good in forty-two, fair in seventeen, and poor in nine. This procedure was done in selected patients in whom for various reasons good results of tendon transfer were not assured.


Topical hemostatic agents to reduce bleeding from cancellous bone. A comparison of microcrystalline collagen, thrombin, and thrombin-soaked gelatin foam. In fifty-three dogs microcrystalline collagen, thrombin-soaked gelatin foam, and thrombin powder were evaluated as hemostatic agents when applied to bleeding cancellous surfaces after osteotomy of the greater trochanter using two quantitative models: one, a single osteotomy; the other, a double osteotomy. All three agents significantly reduced bleeding compared with the controls, the microcrystalline collagen being most effective. At three months there was no evidence that microcrystalline collagen and thrombin-gelatin interfered with bone healing after the greater trochanter was reattached with two wires.


Elastic fibers in human intervertebral discs. Although previous studies failed to demonstrate elastic fibers in intervertebral discs, electron microscopy of twenty human intervertebral discs obtained at autopsy and operation revealed characteristic elastic fibers in both the annulus fibrosus and the nucleus pulposus. Their contribution to the mechanical properties of the intervertebral disc remains to be determined.


Aging of bone tissue: mechanical properties. The mechanical properties of machined cortical bone specimens from human femora and tibiae were determined in tension, torsion, and compression for a population ranging in age from twenty-one to eighty-six years. No significant differences were found between the mechanical properties of male and female specimens. Tibial specimens had greater ultimate strength, stiffness, and ultimate strain than femoral specimens. Consistent decreases with age for all mechanical properties except plastic modulus were found in the femoral but not in the tibial specimens. No consistent significant differences in tension properties were found in specimens from normal, osteoporotic, and corticosteroid-treated individuals.


Recurrent dislocation of the patella. Relation of treatment to osteoarthritis. Eighty-one knees surgically treated and twenty-six conservatively treated for recurrent dislocation of the patella in seventy-eight patients were studied. The average follow-up on the surgically treated knees was eight years and on the conservatively treated knees, sixteen years. In the non-surgical group dislocations tended to become less frequent with advancing age and there was very little evidence of osteoarthritis. After the sixty-nine tibial tubercle transfers there was a 20 per cent recurrence rate; further procedures were often needed, and the incidence of late osteoarthritis was disturbingly high. After the twelve soft-tissue corrections without movement of the tibial tubercle, dislocation recurred in three, but late osteoarthritis was not seen.


Shear strength of the human femoral capital epiphyseal plate. After the determination of the shear strength and modes of failure of the capital femoral epiphyseal plates in twenty-five pairs of hips obtained post mortem from children five days to fifteen years and ten months old, with the plate intact on one side and with the perichondrial fibrocartilaginous complex removed on the other, the gross and microscopic morphology was studied. The shear strength of the human epiphyseal plate varied with age and was greatly dependent on the surrounding perichondrial complex in infancy and early childhood, but less so in adolescence. When this complex was excised, the strength of the epiphyseal plate was diminished, especially in specimens from younger children. The forces necessary to cause slipping were found to be within the physiological range of the force that would be generated in overweight children, suggesting that purely mechanical factors may play a major role in the etiology of slipped capital femoral epiphysis.


Fine structure of chicken thymic epithelial vesicles. This paper describes the fine structure of epithelial vesicles as observed in the thymus of the chicken. Three types of vesicles are found. The largest is made up of a stratified columnar epithelium containing lymphoid cells. The lumen contains a fairly light material and cell debris. The lumen-bordering cells are columnar and their microvilli are sometimes developed to such an extent as to appear as a brush border. The wall of the vesicles contains lymphocytes and granular cells with dense granules 100-500 nm in diameter. Smaller vesicles are found which contain no lymphoid cells. They are made of a simple or stratified epithelium containing typical mucous cells, and columnar cells similar to the bordering cells of the large vesicles. The third type is an intracytoplasmic vesicle in epithelial reticular cells, which differs from usual vacuoles by the presence of microvilli and its often unusually large size. The morphology of the first 2 types of vesicles indicates a secretory capability, the function of which is unknown. Data from the literature suggest that the third type produces the factor(s) conferring cellular immunological competence.


Effects of prostaglandins E1, E2, and F2alpha on the growth of leukaemia cells in culture. Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine, uridine and leucine. At concentrations of 50 and 25 mug per ml, there was significant inhibition of thymidine and uridine incorporation, but not of leucine, PGF2alpha showed significant inhibition of thymidine and uridine incorporation but not leucine incorporation, in all 3 concentrations studied (100, 50, and 25 mug/ml). The ability of the cells to form colonies in soft agar was significantly inhibited by PGE1 and PGE2 at concentrations as low as 1-8 mug/ml. For F2alpha, however, a concentration as high as 56mug/ml was required to show inhibitory effect, but at 1-8 mug/ml it was found to be stimulatory.


DNA repair synthesis in human heterokaryons. III. The rapid and slow complementing varieties of xeroderma pigmentosum. Patients with Xeroderma pigmentosum and defective DNA excision repair can be distinguished as a rapid (r-XP) and slow (s-XP) complementing variety. When fused with normal cells, fibroblasts from the r-XP are complemented rapidly and in the absence of protein synthesis while those from the s-XP are complemented slowly by a process partly, but not entirely, dependent on protein synthesis. Heterokaryons with different ratios of r-XP to s-XP nuclei (i.e. 1:1-5 and 1-5:1) and control heterokaryons containing one normal and 1-5 r- or s-XP nuclei show that if cell fusion and incubation is conducted in medium preventing protein synthesis, the rXP cells do not complement the s-XP partner at all and, conversely, that the latter is not as effective as normal cells at complementing the rXP partner. On the contrary, if protein synthesis is permitted, the 2 types of XP cells complement each other in a gene dose-dependent manner and to an extent similar to that observed in the control heterokaryons. These findings indicate that the r- and s-XP varieties are caused by mutations at different loci and suggest that the products of these loci interact to produce a functional unit which is present in normal control cells but absent in the XP strains. The relationship between the complementation groups described here and those already reported in the literature being investigated.


Template activity of unfixed metaphase chromosomes. Unfixed metaphase and non-metaphase cells were tested for their template activity with RNA polymerase. A device was used which disrupts the cell membrane by centrifugation, and which also ensures that the cells do not continue with their mitotic cycle during the transcription process. The template activity of acid/methanol fixed cells was also tested. None of the unfixed metaphase cells transcribed RNA whereas most of the non-metaphase cells did. In contrast, using fixed cells both classes of cells were transcribed equally well and to a much greater extent. It was concluded that metaphase chromosomes in vivo cannot act as templates for RNA synthesis.


Regulation of interstitial cell differentiation in Hydra attenuata. I. Homeostatic control of interstitial cell population size. Mechanisms regulating the population size of the multipotent interstitial cell (i-cell) in Hydra attenuata were investigated. Treatment of animals with 3 cycles of a regime of 24 h in 10-2 M hydroxyurea (HU) alternated with 12 h in culture medium selectively killed 95-99% of the i-cells, but had little effect on the epithelial cells. The i-cell population recovered to the normal i-cell:epithelial cell ratio of I:I within 35 days. Continuous labelling experiments with [3H]thymidine indicate that the recovery of the i-cell population is not due to a change in the length of the cell cycle of either the epithelial cells or the interstitial cells. In control animals 60% of the i-cell population undergo division daily while 40% undergo differentiation. Quantification of the cell types of HU-treated animals indicates that a greater fraction of the i-cells were dividing and fewer differentiating into nematocytes during the first 2 weeks of the recovery after HU treatment. Therefore, the mechanism for recovery involves a shift of the 60:40 division:differentiation ratio of i-cells towards a higher fraction in division until the normal population size of the i-cells is regained. This homeostatic mechanism represents one of the influences affecting i-cell differentiation.


Mitotic activity in rice seedlings germinating under oxygen deficiency. Rice seedlings germinating in oxygen-deficient environments do not manifest visual evidence of seminal or adventitious root growth or normal leaf growth, the coleoptile being the only seedling structure to emerge under these environmental conditions. Microscopic evidence, however, shows that mitotic activity occurs in the different tissues comprising the shoots of the seedlings germinating under oxygen deficiency. Thus evidence is presented here which shows that mitotic activity in rice seedlings germinating in oxygen-deficient environments is not confined solely to the emerging coleoptiles of the germinating seedlings.


Information activities in support of the EPA pesticide program. The Environmental Protection Agency (EPA) is responsible for regulating the supply and use of pesticides. The EPA Office of Pesticide Programs (OPP) is involved in (1) supply control via product registration, (2) use control, (3) monitoring and hazard evaluation, and (4) research and economic studies. Major information activities in support of these four activities are reviewed.


The toxicology data bank.

The toxicology data bank. The paper describes the Toxicology Data Bank, a new system under development that will provide on-line access to chemical, physical, toxicologic, pharmacologic, use, and manufacturing data on 4000-5000 selected chemicals including drugs.


Improvements in the coupling of SDI system output with document delivery systems. A brief review is given of approaches used to couple SDI system output to the supporting document delivery systems. A coupling system is described which has been implemented for the nine campuses of the University of California to automatically annotate citations retrieved from a central SDI service with location and call number information. Many of the computer-printed citations sent to each UC user are annotated to show the one or more library locations and call numbers for those serial titles on that user's campus. In the event that a cited title is not held on a user's campus, the citations are annotated to identify the nearest UC library that has that title. The location-code segment of the SDI system has been in operation since mid-1974 for over 1500 profiles, and five data bases, and for users on nine UC campuses. This is the first instance of this kind of coupling on a operational basis for a large, multifile, geographically dispersed multilibrary system.


[Vinylogous acyl compounds. XV. Thin-layer chromatographic detection of 2-halovinyl ketones with toxicological relevance (author's transl)]. The basic ring-opening of 1-(2-acylvinyl)-pyridinium salts yielding azaoxonol dyes and the spontaneous dehydrohalogenation of 1-(2-acylvinyl)-4-(4-nitrobenzyl)-pyridinium salts to deeply coloured anhydrous bases underlie two sensitive thin-layer chromatographic detection procedures for 2-halovinyl ketones (R-CO-CH=CH-X). The detection limits are 0.06 mug on successive use of methanolic solutions of pyridine and potassium hydroxide as spray reagents and 0.02 mug on spraying with an acetonic solution of 4-(4-nitrobenzyl)-pyridine. RF values of twenty-five trans- and twelve cis-configurated 1-halovinyl ketones for three solvent systems are given.


Improved recovery of hexachlorobenzene in adipose tissue with a modified micro multiresidue procedure. Using the described methodology the recovery of hexachlorobenzene from adipose tissue was significantly increased over that normally obtained with other multiresidue procedures. The recovery of other commonly encountered chlorinated hydrocarbon pesticides was not affected nor was the "background" from adipose tissue intolerable. Although extensive work has not been done, it is likely that improved recovery of hexachlorobenzene could be expected from other tissues.


Gas chromatographic determination of thiabendazole in fruits as its methyl derivative. Quantitative determination of thiabendazole, as its methyl derivative, has been achieved by gas-liquid chromatography with flame ionization detection and a column of 10% of DC-200 on Gas-Chrom Q at 240 degrees. Determination was possible with 10 to 200 ng per mul of the methylated reaction mixture. For determining thiabendazole in fruits, clean-up of the crude extracts by liquid-liquid partition allows satisfactory elimination of interference and permits determination in concentrations down to 0.1 ppm. The recovery of thiabendazole added to fruits at the 0.5-ppm level ranges from 90.3 to 94.9%. The methyl derivative was identified as N-methylthiabendazole by its elementary composition, by its melting-point, and by ultraviolet, infrared, mass and nuclear magnetic resonance spectroscopy.


Gas-chromatographic determination of camylofine dihydrochloride in tablets and suppositories. A gas-chromatographic method for the quantitative determination of camylofine dihydrochloride, a spasmolytic agent, is described. The analysis is made on a porous polymer packing material, by determining the 3-methyl-1-butanol formed on alkaline hydrolysis of the drug. The method has been applied to the quantitative determination of the drug in two galenical forms, namely tablets and suppositories, in the presence of papaverine hydrochloride, codeine phosphate, novalgin and aminopyrine.


A rapid gas chromatographic method for the determination of chlorophenoxyisobutyric acid in plasma and urine. A rapid gas chromatographic method is described for the determination of chlorophenoxyisobutyric acid (the active metabolite of clofibrate) in plasma and urine. The assay involves an extraction into toluene and back-extraction of the chlorophenoxyisobutyric acid and the internal standard (2-naphthoic acid) into the methylating reagent (trimethylanilinium hydroxide). Concentrations of 1 mug/ml in plasma and urine can easily be measured; the precision of the method is 3.3 +/- 0.7% for plasma and 2.7 +/- 0.4% for urine. There is no interference from endogenous compounds or from drugs commonly prescribed together with clofibrate.


Separation of 1,2-dimethylhydrazine metabolites by high-pressure liquid chromatography. The separation of 1,2-dimethylhydrazine, azomethane, azoxymethane, methylazoxymethanol, methylazoxymethanol acetate, formaldehyde and methanol by high-pressure liquid chromatography on columns of C18/Corasil, muBondapak C18 and Aminex A-27 is described. The separations are highly reproducible and rapid and may be used for kinetic studies. An example of an application of these methods to the analysis of metabolites in rat urine derived from (14C)-1,2-dimethylhydrazine is reported.


Method for the detection of diethylamine, a metabolite of disulfiram, in urine. Disulfiram is a drug used in the treatment of chronic alcoholism in man. Accurate assessment of patient compliance is important in this treatment. This paper describes a method for the detection and quantitative analysis of diethylamine, a metabolite of disulfiram, in urine. The method involves conversion of the water-soluble diethylamine in the urine to a derivative, N,N-diethyl-3,5-dinitrobenzamide, that is soluble in an organic solvent. This derivative is extracted from urine with diethyl ether and then subjected to thin-layer chromatography. A spectrophotometric procedure is used for quantification. This method provides a means of determining whether or not a patient is taking his prescribed disulfiram.


Concentration technique for determination of air pollutants at sub-micro level. A new technique for concentration of carbon monoxide. The advantages, scope and limitations of a step-wise technique for concentrating permanent gases, particularly carbon monoxide, up to 2000 times before gas chromatographic, spectrophotometric or mass spectrometric determination are described. Determination of less than 0.01 ppm of carbon monoxide has been carried out with an error of +/-10% by using this pre-concentration technique, followed by gas chromatography, with catalytic conversion of the carbon monoxide into methane before flame ionisation detection. The system used without pre-concentration could not detect less than 1 ppm of carbon monoxide.


Application of tributyltin chloride to the separation and column chromatography of thiols. Tributyltin chloride (TBT) has the properties of a liquid anion exchanger with a special affinity for sulphydryl groups. Thiols such as cysteine can be separated by extraction from aqueous solution with organic solvents in the form of TBT mercaptides. The graphs of extraction coefficient verus pH have maxima whose positions depend on the nature of the thiols. Thiols can be separated from each other by using TBT on polyamide as the column packing and elution with buffers of gradually increasing or decreasing pH.


Simple method for the isolation and purification of hemoglobin components. A simple method for the isolation and purification of hemoglobin components from starch gel by electrophoresis is described. An inverted bottle with a cut off bottom is used. Pieces of gel containing the hemoglobin are embedded in potato starch, wetted with buffer, and placed in the neck of the inverted bottle, the mouth of which has been closed with filter paper and tape. Connection with the negative tank buffer is achieved by inserting a layer of starch gel between the potato starch and the negative tank buffer. By electrophoresis, the hemoglobin migrates and accumulates in a dialysing tube tied around the mouth of the bottle and hanging into the positive tank buffer.


Causes and effects of sample loss using a dry capsule injection method for the automated gas chromatographic analysis of pesticide samples. The automatic capsule sampling system is said to have many advantages for accurate gas chromatographic analysis. One is that the solvent may be removed by evaporation before injection, so that no solvent peak is obtained to interfere with the separation. However, working with aldrin and dieldrin, we have shown that up to 50% of the material can be lost from the capsules by air-drying at normal ambient temperature before injection. These losses occurred only with aldrin and it is suggested that they are connected with its relatively high vapour pressure. Significant amounts of material enter the top third of the capsule which is removed during the sealing process. The losses appear to be due to a combination of adsorption to the capsules, creep-up the capsule walls, co-distillation with the solvent, and direct volatilisation. Silylation of the capsules was quite effective in reducing these losses.


Plasma chromatography of heroin and cocaine with mass-identified mobility spectra. Plasma chromatography detects and identifies compounds in trace quantities at atmospheric pressure through characteristic positive and negative mobility spectra. To facilitate use of the technique to detect gas chromatographic effluents, a number of reference mobility spectra for different classes of compounds have been reported. Reference spectra for two more compounds, heroin and cocaine, are presented in this study. The primary ions found in these mobility spectra were determined to be M+, (M - H2)+, and (M - CH3CO2)+ for heroin and M+, (M - C6H5CO2)+ and (M - C6H5CO2 - CO2CH3)+ for cocaine using a directly interfaced plasma chromatograph-mass spectrometer. The identified ions agree closely with those predicted in the ion mobility spectra using mass-mobility correlation data coupled with chemical ionization mass spectrometry data. Also, an independent check demonstrating the reliability of reduced mobility values reported in earlier reference spectra was made.


Fluorescence densitometric method for the determination of gluconic and lactobionic acids ("sugar acids") in pharmaceutical preparations. An in situ fluorimetric method has been developed for the quantitation of gluconic and lactobionic acids and their salts in tablet formulations. The method is based on glycol cleavage with lead tetraacetate followed by treatment with dichlorofluorescein. Calcium gluconate and lactobionate were determined in Calcium-Sandoz and Ca-C 1000 Sandoz effervescent tablets. The reproducibility corresponded to relative standard deviations between 0.7 and 3.5% (usually below 2%). Detection limits of 0.2 mug per spot can be obtained. Interfering compounds such as citric acid, sugars and ascorbic acid can be separated from the "sugar acids". The linearity of the calibration graphs between 0.5 and 5 mug per spot is satisfactory (r = 0.994-0.999). The method is simple and could be applied to the routine analysis of suitable pharmaceutical formulations. Other compounds with glycol structures should also be adaptable to this technique.


Fatty acids. VII. The gas-liquid chromatographic properties of all dimethylene interrupted methyl cis,cis-octadecadienoates. The gas-liquid chromatographic behaviour of all the dimethylene interrupted methyl cis,cis-octadecadienoates was studied on polar (Carbowax 20M, FFAP, DEGA, DEGS and Silar 10C), semi-polar (XE-60) and non-polar (SE-30, OV-101 and Apiezon L) stationary phases. The equivalent chain length of each isomer is recorded; the possibility of separation and the retention pattern of these isomers are discussed.


Studies on the formation of cyclohexylamine and N-methylcyclohexylamine from bromhexine in animals and man, and simultaneous determination of cyclohexylamine and N-methylcyclohexylamine by gas chromatography. In order to detect cyclohexylamine and N-methylcyclohexylamine simultaneously in urine, a gas chromatographic method was developed in which Chromosorb 103 (porous polymer) was found to be suitable as the column packing. The detection limits were 0.1 mug/ml of cyclohexylamine and 0.4 mug/ml of N-methylcyclohexylamine in urine. In tests on animals administered bromhexine, which contains N-methylcyclohexylamine as part of its side-chain, neither cyclohexylamine nor N-methylcyclohexylamine was detected in the urine.


The determination of gamma-amino-beta-(p-chlorophenyl)butyric acid (baclofen) in biological material by gas-liquid chromatography. Baclofen and the internal standard are isolated from biological samples by adsorption on charcoal. After elution, the compounds are converted into butyl esters and purified by base-specific solvent extraction. The butyl esters are then N-acylated with heptafluorobutyrylimidazole, and the neutral derivatives are isolated by solvent extraction and subjected to gas-liquid chromarography, with use of an electron-capture detector. Baclofen can be determined in concentrations down to about 30 ng per sample.


High-pressure liquid chromatographic determination of 2-aminoethyl hydrogen sulfate in plasma and urine. A sensitive and specific method for the analysis of 2-aminoethyl hydrogen sulfate in biological fluids by high-pressure liquid chromatography is described. Aliquots from deproteinized plasma or from acidified urine were subjected to ion-exchange clean-up by eluting with water through two columns packed with Dowex 1-X4 (Cl- form) and Dowex 50W-X8 (H+ form) resins. Recoveries in the eluates were consistent and quantitative (95-100%). Effluents from such treatment were chromatographed on a Durrum cation-exchange column with 0.05 M citrate buffer containing 0.15 N Na+ (pH 2.6). Detection was accomplished with an automated fluorescamine detection system. Using 2 ml of plasma or urine, 2-aminoethyl hydrogen sulfate could be assayed at concentrations of 0.3 mug/ml. Linear responses were observed up to at least 133 mug/ml. The method is both accurate and reproducible with a relative standard deviation of about +/- 4.0%.


Serum protein fractionation by isotachophoresis using amino acid spacers. Analytical and preparative isotachophoresis has been carried out using amino acids and peptides as discrete spacers in contrast to the usually employed continuous mobility spectrum Ampholine. Analytical isotachoresis in free solution, using the LKB Tachophor, demonstrated the separation of human serum into distinct mobility subgroups, n spacers giving rise to n + 1 protein subgroups. Preparative fractionation on polyacrylamide gel was carried out on the LKB Uniphor using threonine and glycine as spacers. Immunoelectrophoretic analysis showed that eight out of ten proteins assayed were clearly resolved in the three subgroups obtained, thus demonstrating the sharpness of isotachophoretic resolution.


Determination of plasma amitriptyline by electron-capture gas chromatography after oxidation to anthraquinone. A selective procedure is described for the determination of amitriptyline in plasma. The method involves extraction, separation of amitriptyline from its metabolites and subsequent oxidation by ceric sulphate in 5.4 M sulphuric acid. The oxidation product, anthraquinone, is determined by means of electron-capture gas chromatography. The metabolites were separated by a column chromatographic extraction technique. The choice of oxidation reagent, optimum conditions for the oxidation, and the electron-capture properties of anthraquinone are discussed. The method can be used to determine down to 2 ng of amitriptyline in a plasma sample; the relative standard deviation at the 50-ng level was 4.0% (n = 8). The levels of amitriptyline found in a series of plasma samples are compared with those obtained by gas chromatography with use of nitrogen-specific detection; the two techniques gave coincident results.


Iodination and purification of oxytocin. Oxytocin was iodinated using the thallium chloride method. Purification of iodination reaction mixture on Sephadex G-10 and G-15 was compared. Sephadex G-10 effected the separation of iodo-oxytocin from excess free radioiodide. Sephadex G-15 succeeded in separating iodinated from non-iodinated oxytocin as well as from readioiodide. It was found that the storage of unfractionated iodination mixture resulted in an apparent increase in the molecular size of iodinated hormone and that this process was accelerated at temperatures of -20 degrees in comparison with 4 degrees.


The metabolic clearance and blood production rates of estriol in normal, non-pregnant women. The metabolic clearance rate (MCR) and blood production rate (PB) of estriol have been measured in normal, non-pregnant women 21 to 65 years old. 6,7-3H-Estriol was administered as a pulse injection to 4 women between days 5-7 of their menstrual cycle. The disappearance of radioactivity as unconjugated estriol can be described as a function which is the sum of two exponentials. The initial component represents spread into and transfer from a space with a volume of 20.6 +/- 5.4 (SE) l. The mean value for the rate constant of total removal (reversible and irreversible) was 290.2 +/- 78.5 units/day of which 0.34 +/- 0.06 was irreversible. The mean MCRR was 990 +/- 70 l/day/m2. 4-14C-Estriol was infused at a constant rate for 3 1/2 hours to 13 women between days 5-7 of their cycle. The mean MCR was 2,100 +/- 100 l/day or 1,240 +/- 40 l/day/m2. Thirteen women received a constant infusion of 4-14C-estriol between days 20-22 of their cycle. The mean MCR was 2,100 +/- 115 l/day or 1,280 +/- 65 l/day/m2. The mean values for the two phases of the cycle were not significantly different (P greater than 0.1). The mean value for the MCR in 4 post-menopausal women studied in similar fashion was 1,890 +/- 95 l/day or 1,060 +/- 35 l/day/m2. The mean concentrations of estriol were 7.0 +/- 0.7 and 10.9 +/- 0.8 in the follicular and luteal phases of young women, respectively. The mean PB for women in the follicular phase was 14.0 +/- 1.6 mug/day and in the luteal phase was 22.7 +/- 1.9 mug/day. These values were significantly different (P less than 0.01). When the PB's for the 11 women studied in both phases of the cycle were compared the luteal phase values were significantly higher 0.02 greater than P greater than 0.01) using the paired t test. The PB in the 4 post-menopausal women ranged from 5 to 22 mug/day. While there was no difference between the MCR of estriol measured in the two phases of the cycle, the PB of estriol was significantly greater in the luteal phase. Estriol probably contributes little to the overall estrogenic activity in normal, non-pregnant, premenopausal women but could make a more significant contribution in some post-menopausal women.


Use of lithium as an adjunct to radioiodine therapy of thyroid carcinoma. The effect of lithium on iodine kinetics after oral 131I-iodide was studied in an athyreotic patient with follicular thyroid carcinoma. Lithium decreased the disappearance rate of 131I from the whole body and from a tumor mass in the patient's thigh from control values of 0.126/day, respectively, while having only a minimal effect on the rate of 131I disappearance from blood. The increased tumor 131I retention would be expected to increase the therapeutic:toxic ratio of 131I. However, a subsequent therapeutic dose of 131I-iodide given with lithium was accompanied by an unanticipated increase in blood 131I and, therefore, in whole body radiation, resulting in significant bone marrow depression. Although lithium may be a useful adjunct in 131I therapy of functional thyroid carcinoma, it must be used cautiously in future studies.


Prolactin-related testosterone secretion in normal adult men. The sleep-related increase of plasma testosterone (T) in adult men appears to be related not only to plasma luteinizing hormone (LH) levels but to prolactin (PRL) levels as well, suggesting that PRL may have a stimulatory influence on Leydig cell function. To further investigate the influence of PRL on T secretion, five young adult men were studied on three separate days one week apart. Blood samples were taken every 20 min between 0900 and 1800. At 1000 on each of the three days they received an intramuscular injection of saline, haloperidol 0.25 mg or haloperidol 0.50 mg, in a double-blind design. The blood samples were analyzed for LH, follicle stimulating hormone (FSH), PRL and T. It was hypothesized that there would be a dose-related increase in both PRL and T following drug administration. Mean PRL levels rose promptly and significantly in a dose-related manner in response to the haloperidol, which has strong dopamine blocking effects. By 1600, PRL had returned to control values. In contrast to the PRL response, neither LH nor FSH levels were affected by haloperidol. On the saline control day mean T levels showed the normal decline during daytime hours. After 0.25 mg haloperidol, mean T levels were maintained for several hours, and after 0.50 mg haloperidol, T levels were increased for several hours. These alterations in the normal diurnal pattern of T were statistically significant. They began about 60 min after the corresponding drug-induced increases in PRL levels. This delay between increased PRL and increased T is consistent with the similar delay between the increases of these two hormones that occur at night during sleep. The results of this study lend further support to the hypothesis that PRL is another pituitary hormone that stimulates T secretion in adult men.


Catecholamines metabolism in thyroid diseases. I. Epinephrine secretion rate in hyperthyroidism and hypothyroidism. We have measured the secretion rate of epinephrine in 6 euthyroid, 6 hyperthyroid, and 6 hypothyroid subjects infused at a constant rate for a one hour period with tritiated epinephrine (.01 muc/kg/min) (New England Nuclear Inc.). Plasma and urinary levels of epinephrine were measured by modifying the fluorometric method of Anton and Sayre. Plasma levels of epinephrine were 3.0 +/- 3.0 ng/100 ml (mean +/- SD) in normal subjects, compared to 4.4 +/- 3.5 ng/100 ml (mean +/- SD) in hyperthyroid subjects. In urine, epinephrine values ranged from 1.3 mug/day to 6.1 mug/day in normal subjects. Mean value observed in hyperthyroidism was 4.9 +/- 2.6 mug/day and 3.8 +/- 1.0 mu/day in hypothyroidism. Plasma secretion rates averaged 48 +/- 27 mug/kg/day in normal subjects, compared to 54 +/- 18 mu/kg/day in hyperthyroidism and 43 +/- 20 mug/kg/day in hypothyroidism. Likewise, the mean urinary secretion rate was 55 +/- 27 mug/kg/day in normal subjects compared to 60 +/- 22 mug/kg/day in hyperthyroidism and 50 +/- 28 mug/kg/day in hypothyroidism. There is no statistical difference between the values found in the three groups of subjects (plasma and urine). Therefore, these results would indicate that the signs and symptoms encountered in hyperthyroidism are not secondary to a high secretion rate of epinephrine.


Influence of surgical stress under general anesthesia on serum gonadotropin levels in male and female patients. Effects of surgical stress under the same general anesthesia (nitrous oxide, oxygen and halothane following induction with thiopental and succinylcholine chloride) on serum LH levels were studied in 18 postmenopausal females, 15 menstruating females and 17 males. In addition, serum FSH levels in female patients and testosterone levels in male patients were estimated. As controls without surgery and anesthesia, serum levels of LH, FSH and testosterone were estimated at identical time intervals to the experimental group in 15 normal postmenopausal females, 15 normal menstruating females and 10 normal males. In male patients, serum LH levels at 30 min and 1 hr after onset of anesthesia increased significantly over those of pre-anesthesia, while no significant intra-operative increase in LH levels was found in female patients. In male and female patients, the LH levels decreased slightly 5-6h and 2 days after onset of anesthesia, though the decreases were not statistically significant except in postmenopausal females. On the 7th postoperative day, the LH levels returned toward the baseline. In female patients, no significant intra-operative changes in FSH levels were found. In male patients, there were significant intra- and post-operative decreases in testosterone levels. In control subjects, no significant changes in serum levels of LH, FSH and testosterone were demonstrated. These observations suggest that significant intraoperative increases in serum LH levels occur in males but not in females.


Prolactin response to arginine in normal subjects and in patients with hyperthyroidism. The effect of arginine on serum prolactin concentrations was studied in 18 normal subjects and in 7 patients with hyperthyroidism in normal subjects, arginine infusion produced an increase of serum prolactin at least 6 ng/ml from the baseline, and the mean peak level (25.2 +/- 3.3 ng/ml, mean +/- SE) was significantly higher than the basal level (8.6 +/- 5.2 ng/ml, P less than 0.001). There was no significant difference in the peak levels between sexes. Unlike prolactin, concomitant serum thyrotropin levels did not change after the arginine infusion. In hyperthyroid patients, the increment of serum prolactin after arginine infusion at 30 min (3.9 +/- 1.5 ng/ml) was significantly lower than that of the normal controls which were matched by age and sex (17.1 +/- 4.4 ng/ml, P less than 0.05). After treatment when these patients were euthyroid, the increment of prolactin after arginine infusion at 30 min was significantly increased (16.3 +/- 4.3 ng/ml, P less than 0.05) and had reached the level of control subjects. These data indicate that the prolactin response to arginine in hyperthyroidism is diminished.


Urinary androstanediol and testosterone in adults. Data are presented on the daily urinary excretion of androstanediol and testosterone in healthy adults using a sensitive radioligand assay. In nine men, the average urinary androstanediol (79 mug/day) was not significantly different from the urinary testosterone (84 mug/day). However, in women the average values of urinary androstanediol excretion (12 mug/day) were significantly higher than the urinary testosterone (4.2 mug/day). In each of the females, the urinary androstanediol was greater than the urinary testosterone. The data do not support the hypothesis that the daily urinary androstanediol excretion is a measure of the 5alpha-reduction of testosterone in androgen target tissues.


Changes in drug metabolism in children with thyroid disorders. The rate of disappearance of antipyrine from the plasma is a useful indicator for the in vivo capacity of mixed function oxidation. The half-life of antipyrine was measured before and after treatment in three hypothyroid and three hyperthyroid children, aged three months to 14 years, in order to examine the effect on drug metabolism of thyroid disorders in children. The half-life of antipyrine decreased in all three hypothyroid subjects and increased in all three hyperthyroid subjects after treatment. The mean half-life decreased from 34.5 h to 8.6 h after treatment of the hypothyroid subjects and increased from 6.1 to 10.1 h after treatment of the hyperthyroid subjects. The mean metabolic clearance rate of antipyrine increased from 11.7 to 25 ml/h in the hyothyroid patients while in the hyperthyroid children there was a decrease from 43 to 25 ml/h. The apparent volume of distribution did not change significantly in the treatment, thus changes in the half-life of antipyrine were most likely attributable to alterations in the metabolic clearance rate of antipyrine.


Heterogeneity of plasma glucagon: patterns in patients with chronic renal failure and diabetes. Immunoreactive plasma glucagon (IRG) in normal subjects and patients with chronic renal failure, diabetic ketoacidosis and diagetic hyperosmolar syndrome circulates in several forms. In the diabetic patients most IRG eluted coincidentally with the extracted, purified pancreatic hormone (MW3500), while in normal subjects a high molecular weight component predominated. In striking contrast, the major component of plasma IRG in patients with chronic renal failure was of intermediate size (MW +/- 9000), consistent with proglucagon. The accumulation of this form of IRG suggests that the kidney plays an important role in its metabolism. If there are differences in the biological activity of the various circulating components of IRG, the significance of immunoreactive glucagon levels in some disease states will require reassessment.


Catechol estrogen formation by the human fetal brain and pituitary. Homogenates of central neuroendocrine tissues from 2 male and 1 female midtrimester fetuses were incubated with (23H) estradiol-17beta. Metabolism at the C-2 position was monitored by measuring the tritium incorporated into water in the incubate. Liberation of tritium from the substrate by hypothalamus, limbic tissues, parietal cortex and pituitary occurred to the extent of 5.1-12.7%, 1.6-8.5%, 4.7-10.8%, and 0.9-4.1% of starting radioactivity, respectively. The nature of the product was confirmed by the isolation of 14C labelled 2-hydroxyestrone derivative from separate incubations with 14C-estrone as the substrate. With or without correction for weight of  tissue incubated, catechol estrogen formation under these conditions occurs at levels similar to those seen in rat brain homogenates except that in contrast to the rat, the human cortex is also highly active.


Effect of sulpiride on serum prolactin levels in humans. The effect of acute and chronic administration of sulpiride sulphate on serum prolactin levels in humans was studied. Six normal women and four normal men received an i.m. injection of 100 mg sulpiride at 0800 h. A control group received saline solution 0.9%. Blood samples were taken before and 30, 60 and 120 minutes after the injection. Serum prolactin was determined by a double antibody radioimmunoassay technique. Sulpiride induced in all subjects a quick and marked increment of serum prolactin levels with peak values at 30 minutes. At 120 minutes the levels remained high. In another experiment six post menopausal women received 150 mg of sulpiride orally during 10 days. Five of them had marked increments of circulating levels of prolactin at the end of treatment. It is suggested that sulpiride could be used in the dynamic functional exploration of hypothalamic-pitutiary system.


Human serum melatonin changes during the menstrual cycle. Serum melatonin concentration in early morning during the menstrual cycle, studied in five healthy women, showed that melatonin was elevated at the time of menstrual bleeding and had its nadir at the time of the menstrual cycling in humans.


Effects of alcohol on growth hormone secretion in acromegaly. In 3 normal subjects and in 4 acromegalic patients pretreatment with an alcohol infusion for 4 hours at a constant rate reduced the GH response to Arginine, when comparison was made with pretreatment with saline. The reduction in acromegalics was more marked and sustained than in normals. Though it is likely that the effect of alcohol is exerted on hypothalamic centers, a direct influence on the pituitary cannot be excluded.


Effect of caloric restriction and dietary composition of serum T3 and reverse T3 in man. To evaluate the effect of caloric restriction and dietary composition on circulating T3 and rT3 obese subjects were studied after 7-18 days of total fasting and while on randomized hypocaloric diets (800 kcal) in which carbohydrate content was varied to provide from 0 to 100% calories. As anticipated, total fasting resulted in a 53% reduction in serum T3 in association with reciprocal 58% increase in rT3. Subjects receiving the no-carbohydrate hypocaloric diets for two weeks demonstrated a similar 47% decline in serum T3 but there was no significant change in rT3 with time. In contrast, the same subjects receiving isocaloric diets containing at least 50 g of carbohydrate showed no significant changes in either T3 or rT3 concentration. The decline in serum T3 during the no-carbohydrate diet correlated significantly with blood glucose and ketones but there was no correlation with insulin or glucagon. We conclude that dietary carbohydrate is an important regulatory factor in T3 production in man. In contrast, rT3 concentration is not significantly affected by changes in dietary carbohydrate. Our data suggest that the rise in serum rT3 during starvation may be related to more severe caloric restriction than that caused by the 800 kcal diet.


A kinetic study of plasma renin and aldosterone during changes of posture in man. Cardiovascular parameters, hematocrit (Ht), plasma electrolytes, renin activity (PRA) and aldosterone concentration (PAC) were measured in 12 normal human subjects (6 males and 6 females) eating an ad lib diet. At 8 AM, volunteers assumed the following postural changes: 1 hour supine, then 2 hours upright and finally 1 hour supine. Orthostatism induced the following changes: heart rate, systolic and diastolic blood pressure increased immediately; Ht rose significantly at the 5th min in males but not in females; Plasma sodium showed no variations but potassium increased after 30 min; PRA rose significantly at the 5th min and, after 120 min of orthostatism, was found to be 3 times greater than its value after recumbency; and PAC increased significantly at the 15th min and exhibited a plateau 4.5 times its basal values after 90 min of upright posture. When subjects returned to the supine position all the parameters, except PAC decreased. During active orthostatism, a significant correlation was found between PAC and plasma potassium, but correlation was closer between PAC and PRA and between PAC and PRA + potassium. It can be concluded that the renin-angiotensin system is a more potent stimulus for aldosterone secretion than plasma potassium in normal man assuming postural changes. The results presented here can be applied to the development of a short posture test in non-hospitalized patients.


Depressed plasma FFA turnover rate in Cushing's syndrome. The turnover rate of plasma FFA measured by constant infusion of albumin-complexed labeled palmitate in a group of 16 patients with Cushing's syndrome was significantly lower than in a group of 6 controls. Seven patients reexamined after correction of the hypercortisolism showed a normalization of the plasma FFA turnover rate. By contrast, 7 patients with simple obesity had a normal or increased plasma FFA turnover rate depending on the way this parameter is expressed.


Adrenal imaging with 131I-19-iodocholesterol in the diagnostic evaluation of patients with aldosteronism. The results of adrenal scintiscans, venograms and venous aldosterone levels are compared with the histologic findings in 33 patients submitted to operations for primary aldosteronism. Standard and suppression scintiscans were performed 2-14 days following intravenous administration of 2mCi of 131I-19-iodocholesterol. The adrenal lesions were histologically classified into four categories: 25 patients had adenomas, 6 had macronodular hyperplasia, 1 had microscopic hyperplasia and 1 had an adenocarcinoma. Asymmetrical uptake between the two adrenals seen on standard scintiscans did not differentiate between a tumor or asymmetrical hyperplasia, unless the tumor was greater than 2 cm in diameter. During suppression scintiscans, unilateral uptake visible within five days of tracer injection was consistent with adenoma. Patients with nodular hyperplasia  demonstrated early uptake in both adrenal glands during suppression scintiscans, while the patient with microscopic hyperplasia did not. The type of adrenal lesion was correctly identified in 20/26 (77%) of patients by suppression scintiscans; 21/28 (75% of patients by venograms and 12/16 (75%) of patients who had adrenal venous aldosterone measurements attempted. The majority of surgically correctible lesions could be identified on suppression adrenal scintiscans. Adrenal vein catheterization can be reserved for those patients in whom the results of suppression scintiscans are inconsistent with the clinical degree of aldosteronism.


Hyperthyroidism with normal concentrations of total serum thyroxine and triiodothyronine. Normal concentrations of total serum thyroxine (T4) and triiodothyronine (T3) were found in a patient who was hyperthyroid because of a hyperfunctioning thyroid adenoma. After surgical removal of the adenoma, the patient became clinically euthyroid; the abnormally high free thyroxine (FT4), triiodothyronine resin uptake (T3R) and rapid achilles reflex time (ART) returned to normal. A low-normal concentration of thyroxine binding globulin (TBG) determined by polyacrylamide gel electrophoresis and a low level of TBG determined by radioimmunoassay were found in the patient. The TBG remained low-normal after the restitution of euthroidism. Low TBG levels were found in the patients 5 brothers and maternal uncle, and a low-normal concentration was found in her mother. It is postulated that the patient was heterozygous carrier for a genetically determined partial (non-zero) TBG deficiency and that a low-normal TBG concentration decreased total T3 as well as T4 when the patient was hyperthyroid. To the author's knowledge, this is the first case of hyperthyroidism associated with both normal total T4 and T3 concentrations. The diagnosis of hyperthyroidism in the presence of low TBG is difficult, and determination of FT4 and free triiodothyronine (FT3) may be necessary to establish the diagnosis.