Structural investigation of the antibiotic ristomycin A. 13C-NMR spectral analysis of the interglycosidic linkages of the heterotetrasaccharide side-chain. By 13C-NMR studies on Ia, IIa, IIb and IVa obtained by the chemical degradation of ristomycin A and on several synthetic model compounds it has been proved that an O-beta-D-arabinopyranosyl-(1 leads to 2)-O-alpha-D-mannopyranosyl-(1 leads to 2)-O-[alpha-L-rhamnopyranosyl-(1 leads to 6)]-D-glucopyranosyl heterotetrasaccharide moiety is connected to the aglycone of the antibiotic.


Effects of polymyxin antibiotics on iodohippurate accumulation in rabbit renal cortical slices. The in vitro effects of polymyxin antibiotics on 0-125I-hippurate (OIH) accumulation in rabbit renal cortical slices were studied using incubation media with pH ranging from 6.9 to 7.9 and containing polymyxin B sulfate, colistin sulfate, sodium colistimethate and antibacterially inactive N-succinyl colistin in concentrations ranging from 1 to 2,000 microgram base/ml. Polymyxin B, colistin and colistimethate depressed OIH accumulation significantly in concentrations greater than or equal to 300 microgram/ml. The effects on accumulation were clearly pH-dependent and most pronounced at alkaline pH. N-Succinyl colistin had only a marginal influence on accumulation, even in high concentrations. Colistimethate produced a significantly smaller decrease in accumulation at all pH values than both polymyxin B and colistin. The results suggest that the presence of free amino groups is necessary to obtain a decrease in accumulation and correlate with the known in vivo nephrotoxicity of these antibiotics.


Role of small molecules in regulation of D-serine deaminase synthesis. Cyclic AMP is required for optimal synthesis of D-serine deaminase synthesis from dsdO+ templates and for optimal hyperinducible synthesis from low constitutive dsdO templates both in vitro and in vivo. Neither D-serine, cyclic AMP, nor dsdC activator has an effect on expression of a high constitutive dsdO template. The synthesis of the dsdC activator itself in vitro is independent of cyclic AMP. Guanosine tetraphosphate does not have a significant effect on in vitro D-serine deaminase synthesis from dsdO+ or dsdO templates. A previously described class of dsdO mutants showing partial catabolite sensitivity of constitutive D-serine deaminase synthesis proved to be low dsdO types. They all contain a low constitutive dsdC mutation; the two effects are additive with regard to level of constitutivity, but only that portion of synthesis attributable to the dsdC mutation is cyclic AMP dependent.


Levels of cyclic GMP in dormant, germinated, and outgrowing spores and growing and sporulating cells of Bacillus megaterium. The level of cyclic GMP was less than one molecule per organism in dormant, germinated, and outgrowing spores of Bacillus megaterium. A significant level (approximately 8 pmol/g, dry weight) of cyclic GMP was found in early to mid-log phase cells, but the level fell to below 0.2 pmol/g, dry weight, in late-log phase and only rose slightly to approximately 0.9 pmol/g, dry weight, in stationary phare. No significant amount of cyclic GMP was detected in the growth medium at any time.


Role of the dsdC activator in regulation of D-serine deaminase synthesis. The activator of the D-serine deaminase operon, the product of the dsdC gene, has been partially purified. It is reasonably stable to routine purification procedures in the presence of its ligand D-serine, but not in its absence. It loses activity upon dialysis in amino acid-free buffer, but activity is completely restored upon readdition of D-serine. It apparently functions purely as an activator, no repressor function could be demonstrated at suboptimal D-serine concentration. It is a transcriptional control element. The time required for in vitro transcription of D-serine deaminase mRNA, nearly 4 min, is similar to that for beta-galactosidase. Since the beta-galactosidase monomer is a much protein, this is surprisingly long.


Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources. The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.


Changes in composition of envelope proteins in adenylate cyclase- or cyclic AMP receptor protein-deficient mutants of Escherichia coli. Synthesis of several envelope proteins in Escherichia coli K-12 is regulated by cyclic AMP and cyclic AMP receptor protein.


Binding sites for angiotensin II in human mononuclear leucocytes. Angiotensin II binding sites were demonstrated in human mononuclear leucocytes by use of [125I]angiotensin II. The binding of [125I]angiotensin II to mononuclear leucocytes was rapid and reversible. The abilities of unlabeled compounds to displace [125I]angiotensin II were proportional to their abilities to displace labeled hormone in adrenal and smooth muscle membrane preparations. The Scatchard plot revealed two apparent orders of binding sites. The affinity constants were comparable with those for binding sites in other main target tissues of angiotensin II.


Specific cleavages inflicted by venom phosphodiesterase on superhelical phiX174 DNA. A single strand specific endonuclease, venom phosphodiesterase, acting on superhelical phiX174 DNA, inflicts at least seven specific cleavages. Six of these were located to within approximately +/-40 base-pairs by mapping with restriction endonucleases. They are at 12%, 27.4%, 42.7%, 47.5%, 76.1%, and 82.5%. All of these sites lie within regions containing runs of 16 or more base-pairs, of which 12 or more are A and T. Other similarly (A + T)-rich regions in the genome are not cleaved by the enzyme. Increasing the superhelical density of the substrate did not alter the locations of the cleavage sites. There is no correlation between the locations of the cleavage sites and the three known major promoter sites. Only two of five postulated transcription termination sites are cleaved by phosphodiesterase even though all of these terminators contain (A + T)-rich regions. The area containing the origin of viral DNA replication, which includes an (A + T)-rich sequence, is not cleaved.


Evidence for the noninvolvement of sulfogalactosylceramide (cerebroside sulfate) in the enkephalin (opiate) receptor. Mouse neuroblastoma clonal cell line N4TG1 has 20,000 enkephaline (opiate) receptor binding sites/cell, but contains undetectable levels of sulfogalactosylceramide as judged by quantitative and isotope-labeling studies. Mouse glioma cell lines G26-20 and G26-24 contain substantial quantities of sulfogalactosylceramide, but do not bind enkephalins. Preincubation of cells with sulfogalactosylceramide or other anionic glycosphingolipids did not enhance or induce opiate receptors. Thus, sulfogalactosylceramide is not an integral part of the opiate receptor of N4TG1 cells.


Use of a double-headed peptide substrate to study the specificity of cAMP-dependent protein kinase and posphorylase kinase. A peptide containing 2 seryl residues, (1)Leu(2)Ser(3)Tyr(4)Arg(5)Aly(6)Tyr(7)Ser(8)Leu, was chemically synthesized and used as a substrate for phosphorylase kinase and cyclic AMP-dependent protein kinase. The sequence, TryArgGlyTyr, makes up a beta turn in the native protein. Phosphorylase kinase was found to phosphorylate specifically seryl residue2 and protein kinase seryl residue7. Km and Vmax values were obtained and compared with natural substrates. The differences in the specificity of the two enzymes might be explained by a different requirement for organized structure. As a working hypothesis, it is suggested the results could be explained if the two enzymes interacted with seryl residues at different sides of a beta turn.


Interchange of apoprotein components between the human plasma high density lipoprotein subclasses HDL2 and HDL3 in vitro. The major apoproteins of human high density lipoproteins (HDL) labeled with 125I have been shown to exchange between the two major HDL subclasses HDL2 and HDL3 in vitro. This bidirectional exchange process is inhibited by cross-linking with bifunctional reagents and is apparently dependent upon the formation of collision complexes. This exchange has been demonstrated both when the subclasses of HDL are free in solution and also when one of them is covalently bound to Sepharose. Using system involving Sepharose-bound HDL, it could be shown that not only free apoprotein molecules but subunits consisting of lipid-apoprotein combinations were exchanged between HDL2 and HDL3. The rate of exchange in these processes is significant in the lifetime of the protein particles in vivo equalling approximately 2.5% per h for apoprotein exchange. These experiments suggest that there is a dynamic relationship between HDL2 and HDL3 even though each of them exists alone in vitro as stable separate entities; when they are placed together in solution significant interaction occurs between the particles. Apoprotein exchange occurs between HDL2:HDL2 and HDL3:HDL3 as well as between HDL2 and HDL3 molecules. These data also suggest that the interconversion of HDL2 and HDL3 may be affected by the availability of lipids.


Electronic state of heme in cytochrome oxidase III. The magnetic susceptibility of beef heart cytochrome oxidase and some of its derivatives from 7-200 K. Direct evidence for an antiferromagnetically coupled Fe (III)/Cu (II) pair. The temperature dependence of the paramagnetic susceptibility of cytochrome oxidase and some of its derivatives has been measured from 7 to 200 K. The results obtained for the fully oxidized (resting) enzyme correspond exactly to the requirements of the model recently proposed by Palmer et al. (Palmer, G., Babcock, G. T., and Vickery, L. E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210) in which the enzyme possesses two magnetically isolated spin S = 1/2 centers and a spin-coupled S = 2 center. The S = 2 center paramagnetism has been interpreted as arising from a [cytochrome a33+(S = 5/2)--Cuu2+(S = 1/2)] antiferromagnetically coupled iron.copper binuclear complex of total spin S = 2 with -J greater than or equal to 200 cm-1. In addition, the wide temperature range used in the present studies has permitted an analysis of present and other available data (T less than 4K measurements) which readily accommodates results from this and other laboratories (Moss, T.H., Shapiro, E., King, T.E., Beinert, H., and Hartzell, C. R. (1978) J. Biol. Chem 253, 8072-8073) so that a fully consistent picture of the magnetic centers in cytochrome oxidase now appears to be available. Furthermore, anomalous magnetic behavior for the oxidized enzyme.cyanide complex has been interpreted in terms of an antiferromagnetic exchange interaction operating in the binuclear complex [cytochrome a33+.CN-(S = 1/2)--Cuu2+(S = 1/2)] with -J congruent to 40 cm-1. A structural model for the [cytochrome a3(3+)-bridge-CUu2+] center is advanced in which an imidazolate ion serves as the bridging ligand in a manner similar to that found in superoxide dismutase.


The magnetic susceptibility of cytochrome oxidase in the 4.2-1.5 K range. Sixteen low temperature measurements on eight independent cytochrome oxidase samples from two separate laboratories have yielded magnetic susceptibility data compatible with a model of spin-coupled iron and copper ions, as presented in the preceding paper (Tweedle, M.F., Wilson, L.J., García-Iñiguez, L., Babcock, G. T., and Palmer, G. (1978) J. Biol. Chem. 253, 8065-8071). The data in the 1.5-77 K range match those attained at higher temperatures and the predictions of the spin-coupled model. Measurements on reduced samples confirm the high spin nature of one iron atom. No obvious uncoupling of the antiferromagnetic Fe-Cu interaction is detected in partly reduced samples.


Transport of phosphate in membrane vesicles from mouse fibroblasts transformed by simian virus 40. Membrane vesicles were prepared from mouse fibroblasts transformed by SV40 virus (SV3T3). Following disruption of the cells by nitrogen cavitation, the membrane vesicles were obtained by differential centrifugation. As measured by enzyme markers, they consist mainly of membrane from the plasma membrane and smooth and rough endoplasmic reticulum. The vesicles transport Pi by two separate, mediated systems: one is independent of Na+, and the other is secondary active transport driven by a Na+ gradient. Electrical and chemical energy can be provided by a Na+ gradient to drive the concentrative uptake of Pi by the vesicles, one or both forces being used to energize transport. Evidence is provided that both the electrical and chemical potentials produced by the asymmetric distribution of Na+ across the membrane of SV3T3 membrane vesicles are utilized to concentrate phosphate in the vesicles. Phosphate transport by the vesicles cannot be accounted for by a small contamination of this fraction with mitochondria (1 to 4%). The Pi transport properties of the membrane vesicles differ from those of the fraction enriched in mitochondria in the following respects: their kinetic properties, and their responses to a Na+ gradient, N-ethylmaleimide, mersalyl, and succinate/acetate. However, the membrane vesicles share some properties of Pi transport with mitochondria. Cyanide, azide, oligomycin, 2,4-dinitrophenol, and carbonyl cyanide m-cholophenylhydrazone, inhibitors of Pi transport by mitochondria, also inhibit membrane vesicle, Pi transport. The vesicles retain all the features of Pi transport by SV3T3 cells that have been examined. They provide a simplified system for a determination of the details of the mechanism of Pi transport under conditions where transport is dissociated from intracellular reactions and in the presence of a defined electrochemical driving force.


Preparation of crystalline carbamyl phosphate synthetase-I from frog liver. Ammonia- and N-acetylglutamate-dependent carbamyl phosphate synthetase-I (EC 2.7.2.5), the mitchondrial enzyme involved in the initial step of urea biosynthesis, was purified to homogeneity from frog liver and crystallized. The purification involved extraction of a particulate fraction with cetyltrimethylammonium  bromide in the presence of the protease inhibitors antipain, leupeptin, chymostatin, and pepstatin; acetone precipitation; and affinity chromatography with Cibacron blue F3GA-coupled agarose. The enzyme was adsorbed to the gel at pH 8.3 in the presence of 5 mM MgCl2 and eluted with magnesoum-free buffer. The enzyme crystallized as either elongated, thin, rectangular plates or as clusters of small crystals from 37 to 40% saturated ammonium sulfate. The enzyme moved as a single polypeptide band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with a molecular weight of 160,000. In the absence of protease inhibitors, proteolysis of the enzyme occurred with the formation of an enzymatically active fragment with a subunit molecular weight of 139,000.


Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase. Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator weight of 125,000 +/- 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a 2-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.


Metabolite control of L-fucose utilization. Thyroid fucokinase is responsive to a number of metabolites which might serve in a regulatory capacity. In addition to inhibition by ADP and stimulation by GMP, fucokinase responds selectively to a series of nucleotide sugars. Of those studied, only guanine nucleotide sugars moderate the activity of the enzyme. GDP-alpha-D-mannose, GDP-alpha-D-glucose, GDP-alpha-D-rhamnose, and GDP-alpha-L-fucose on the other hand is strongly inhibitory. In the case of GDP-alpha-D-mannose stimulation, a physiological role seems possible, but the rationale is not entirely clear. The effects of GDP-beta-L-fucose, on the other hand may represent physiological control effected through feedback inhibition by and end product.


Amino acid sequence of a peptide containing an essential cysteine residue of Escherichia coli GMP synthetase. The amino acid sequence of a 51-residue tryptic peptide of citraconylated [1-14C]carboxamidomethyl-labeled Escherichia coli GMP synthetase was determined by sequenator analyses of the intact peptide and fragments obtained by cleavage of the peptide with cyanogen bromide, trypsin, and Staphylcoccus aureus strain V8 protease. The cysteine residue of this peptide fragment is essential for glutamine-dependent GMP synthesis activity and is implicated in formation of a hypothetical covalent glutamyl-enzyme intermediate. There is essentially cysteine-containing regions of two other glutamine amidotransferases, Pseudomonas putida anthranilate synthetase Component II and chicken liver formylglycinamide ribonucleotide amidotransferase. There is, however, a cluster of amino acids with "antipathy" for helix formation and a "nonessential" cysteine of anthranilate synthetase Component II.


Effect of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate on RNA release from isolated nuclei. The addition of physiological concentrations of either cAMP or cGMP stimulated the release of RNA from isolated prelabeled rat liver nuclei to a fortified cytosol in a cell-free system. The released RNA was shown to be primarily mRNA by its binding to oligo(dT)-cellulose and its sedimentation profile. Treatment of rats with cAMP or cGMP 30 min prior to the preparation of cyclic nucleotides on the cell-free system. Cyclic nucleotides stimulation of RNA release occurred in systems prepared from resting rat liver, Novikoff hepatoma, and Morris hepatoma 5123D, but not the 18-h regenerating liver. The response of the cell-free system to added cyclic nucleotides reflected the in vivo concentration of these substances in the tissues from which the system was prepared. Those with high in vivo levels were not stimulated while those with lower levels did respond to added cyclic nucleotides. Neither cAMP nor cGMP had an appreciable effect on rRNA release.


Stimulation of the protein synthetic process by adenosine 3':5'-monophosphate and hexose phosphates in gel-filtered rabbit reticulocyte lysates. The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.


Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle. The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.


Two Epstein-Barr virus-associated DNA polymerase activities. We have partially purified and characterized two separate DNA polymerase activities associated with Epstein-Barr virus (EB virus). One activity is present in EB virus producer cell lines but not in nonproducer or negative cell lines. It adheres more strongly to DEAE-cellulose than any host cell enzymes, eluting at 210 to 270 mM potassium phosphate buffer. Further elution from phosphocellulose and sedimentation in glycerol gradients yields an enzyme purified 900-fold with an S value of 8.3. The second DNA polymerase activity co-purifies with EB viral particles, elutes at low salt from DEAE-cellulose (40 to 60 mM potassium phosphate buffer) and phosphocellulose (100 mM), and has an S value of 9.5 on glycerol gradient sedimentation. These two enzymes are referred to for convenience as the EB virus-induced DNA polymerase and the EB virion-associated DNA polymerase. The EB virus-induced polymerase can be distinguished from host alpha, beta, and the virion-associated polymerase in 1) being resistant to salt inhibition, 2) having a more basic pH optima in Tris buffer (pH 9.5), and 3) having a 10-fold lower saturating concentration for the activated DNA template. The EB virion-associated polymerase is distinguished from host alpha, beta, and the EB virus-induced polymerase, because it cannot utilize synthetic deoxy- and ribohomopolymer primer-templates in place of the activated calf thymus DNA template in DNA polymerase assays. Neither of the EB virus-associated polymerases can copy the ribohomopolymers dT10poly(rA) or dG12-18(poly(rC) efficiently and therefore can be distinguished from host gamma polymerase and reverse transcriptase. The activity of the EB virus-induced and virion-associated polymerases are unaffected both by antibody to alpha polymerase, and by antiserum with high antibody titers to EB early antigen and viral capsid antigen.


Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells. Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.


Relationship between the accumulation of pituitary growth hormone and nuclear occupancy by triiodothyronine in the rat. Studies were undertaken in hypothyroid rats in an effort to define the kinetics of growth hormone (GH) accumulation in response to i.v. pulse injections of triiodothyronine (T(3)) and to calculate the relationship between nuclear occupancy by T(3) and the instantaneous rate of accumulation of pituitary GH. Results were contrasted to the findings in previous studies of the induction of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and malic enzyme (ME) by T(3). The dose of T(3) required to achieve half-maximal accumulation of GH in 24 h was 0.6 mug/100 g body wt, a value 15-fold less than the half-maximal dose for alpha-GPD and ME induction at a comparable time after injection. Although significant increase in pituitary GH were evident as early as 3 h after injection of maximally effective doses of T(3), the rate of increase became linear only 12 h after injection. After achievement of peak values, the pituitary content of GH decayed with a similar terminal t((1/2)) of 3.9 days and 4.1 days in two groups of animals injected with a single dose of 1.0 and 50 mug T(3)/100 g body wt, respectively. In vivo isotopic displacement studies carried out at the equilibrium time point indicated that the pituitary nuclear binding capacity was 5.5 ng T(3)/g tissue and that the plasma concentration at which one-half of the nuclear sites are occupied is 1.0 ng/ml. Nuclear occupancy as a function of time was calculated from the estimated plasma T(3) concentration after injection of the dose and the half-occupancy plasma concentration. These data were then analyzed by application of the mathematical model previously developed to ascertain the relationship between nuclear occupancy and the rate of hepatic enzyme induction. Results indicated that the pituitary nuclear occupancy-response relationship was generally linear, in marked contrast to the highly amplified relationship between nuclear occupancy and the response of ME and alpha-GPD to T(3) in the liver. In supplementary experiments, euthyroid rats received daily injections of 200 mug of T(3) for 7 days to keep nuclear sites nearly saturated for the duration of the experiment. No significant increase in the pituitary GH content above euthyroid base-line levels was noted. This also contrasts with the marked increase above euthyroid levels in alpha-GPD and ME observed in previous studies. Our findings suggest the existence of major differences between the specific mechanisms which lead to the induction of pituitary GH and the hepatic enzymes by T(3).


Cytochemical localization of Na+, K+-ATPase in the rat hepatocyte. The enzyme Na+,5+-ATPase was cytochemically localized in the rat hepatocyte by a modification of the Ernst potassium-dependent nitrophenyl phosphatase technique. Measurement of nitrophenol release from 50-micrometer liver slices confirmed the presence of ouabain-inhibitable nitrophenyl phosphatase activity that increased over the 30-min incubation period. Electron micrographs demonstrated that sinusoidal and lateral membrane reaction product deposition was K+-dependent, Mg++-dependent, inhibited by ouabain but not by alkaline phosphatase inhibitors, and was localized to the cytoplasmic side of the membrane. In contrast, canalicular reaction product was K+-independent, Mg++-dependent, inhibited by alkaline phosphatase inhibitors but not by ouabain, and was localized to the luminal side of the membrane. These findings indicate that Na+,K+-ATPase is localized to the sinusoidal and lateral portions of the rat hepatocyte plasma membrane and is not detectable on the bile canaliculus where alkaline phosphatase is confined. This basolateral localization of Na+,K+-ATPase is similar to that found in epithelia where secretion is also directed across the apical membrane.


Bihormonal regulation of the thyrotropin-releasing hormone receptor in mouse pituitary thyrotropic tumor cells in culture. Receptors for thyrotropin-releasing hormone (TRH) are present on mouse pituitary thyrotropic tumor cells. Incubation of thyrotropes with 100 nM TRH or 4 nM L-triiodothyronine (T3) for 48 h decreased the number of TRH receptors to approximately equal to 50 and 20% of control, respectively. There was no effect on the equilibrium dissociation constant which was 3-5 nM. The depletion in the number of available TRH receptors was time- and dose-dependent. TRH, 100 nM, decreased the receptor number to 70% after 24 h, 50% after 48 h, and 45% of control after 72 h. T3, 4 nM, decreased the receptor number to 52% after 24 h, 20% after 48 h, and 17% of control after 72 h. After 48 h, half-maximal depletion occurred with 1-2 nM TRH and approximately equal to 0.15 nM T3. Incubation with 100 nM TRH and 4 nM T3 caused a significantly greater reduction in the receptor level than either hormone alone. The decrease in the receptor level was reversible within 72 h after removal of TRH, 100 nM, but was only partially reversed, from 20 to 40% of control, after removal of T3, 4 nM, after 120 h. By regulating the number of available TRH receptors on the thyrotrope. TRH and T3 interact to control thyrotropin release.


Activation in vitro of rheumatoid synovial collagenase from cell cultures. Rheumatoid synovial cells dissociated from matrix and adherent to culture dishes released a latent form of collagenase into culture medium. Previous studies have shown that the latent enzyme does not complex with alpha2-macroglobulin and binds to fibrillar substrate. We now show that serum-free culture medium of the synovial cells contains an inhibitor of collagenase as well as latent enzyme; the two were separated on a column of acrylamide/agarose. Latent collagenase (estimated mol wt 45,000-49,000) was transformed by trypsin to active collagenase of approximately equal to mol wt 33,000. When mixed with inhibitor the active enzyme formed an inactive complex again with approximately equal to mol wt 45,000-49,000. The inhibitor(s) itself was found in one major peak of mol wt 33,000-35,000 and several minor peaks eluting with lower apparent molecular weight. Mersalyl, an organic mercurial compound, effectively activated latent collagenase producing an active enzyme with approximately equal to mol wt 33,000. Bacterial collagenase did not activate latent enzyme. We suggest that latent rheumatoid synovial collagenase, as it is harvested from synovial cells in culture, is an enzyme-inhibitor complex.


Incomplete anaerobiosis as a cause of metronidazole 'resistance'. Clostridium welchii, used as a control, was found to grow well in a microaerophilic jar used for campylobacters but appeared resistant to a metronidazole disc although the campylobacter was sensitive. Minimum inhibitory concentrations for six strains of Cl. welchii were up to 64-fold higher in these conditions than when grown anaerobically. Zones of inhibition with both Cl. welchii and Bacteroides fragilis varied with the amount of air admitted to anaerobic jars.


A comparison of complement fixation, indirect immunofluorescence for viral late antigens, and anti-complement immunofluorescence tests for the detection of cytomegalovirus specific serum antibodies. It was shown that, on average, the titres of the sera obtained by both the anti-complement immunofluorescence (ACIF) and indirect immunofluorescence for viral late antigens (IFA-LA) procedures were approximately eight times higher than those obtained by complement fixation. There was no significant difference between the mean ACIF and IFA-LA titres, but the former method was preferred because it was not affected by non-specific staining. When 406 sera were screened for the presence or absence of antibodies, only five sera (1.2%) gave discordant results with the three tests. None of the tests could detect all of the sera that contained specific antibodies while each test detected over 98% of these sera. Thus, the increased sensitivity of the immunofluorescence procedures did not result in an appreciably increased rate of detection of cytomegalovirus seropositive sera.


Identification of thorium dioxide in human liver cells by electron microscopic x-ray microanalysis. Thirty-two years after injection of thorium dioxide (Thorotrast) for diagnostic x-ray studies in a female patient deposits were found by light microscopy in the liver macrophages (Kupffer cells). They were shown by electron microscopy to be located inside secondary lysosomes, and by autoradiography and x-ray microanalysis they were identified as thorium.


Multifocal histogenesis of a cystosarcoma phyllodes. An unusual multifocal cystosarcoma phyllodes is reported. It presented as a small, circumscribed tumour mass, which was associated with two diffuse neoplastic lesions which arose sequentially in two geographically separate parts of the mammary disc. Microscopically the tumour appeared to have been enlarging by the formation of isolated satellite tumour nodules within the adjacent normal breast tissue, which represents a third, though rarer way in which a cystosarcoma phyllodes may enlarge.


Case report. Hypoglossal palsy: computed tomography demonstration of denervation hemiatrophy of the tongue associated with glomus jugulare tumor. In a patient with left glomus jugulare tumor and left hypoglossal nerve palsy, computed tomography demonstrated focal lucency of the left half of the tongue. This was interpreted to represent postdenervation fibrosis and fatty replacement of the intrinsic and extrinsic lingual muscles supplied by the hypoglossal (XIIth cranial) nerve.


Consumer acceptability of day care after operations for hernia or varicose veins. The opinions of patients and of caring persons (usually relatives) were sought in this trial of different methods of providing care for 360 patients after operations for hernia or varicose veins. Analysis of patients' opinions suggested that day care was the most acceptable of the three types of care examined. The reactions of caring persons did not reveal any major criticisms or disadvantages.


Economic aspects of day care after operations for hernia or varicose veins. In a trial of 360 patients with hernia or varicose veins, day care surgery provided an economic alternative to the provision of surgical aftercare either in the surgical wards of a district hospital or in a convalescent hospital. There was only slightly more work for general practitioners. Most of the additional work for the community services was carried out by district nurses, with an average contact time in the postoperative period of 325 minutes for day care patients, compared with 186 minutes and 204 minutes respectively for patients admitted for 48 hours to the surgical or convalescent wards. Day care produced estimated savings of 30 pounds compared with the costs of a 48-hour stay in the surgical wards, and savings of 22 pounds compared with a 48-hour stay in the convalescent wards.


On the quantitation of SV40 virus by plaque assay and immunoperoxidase technique. Procedures are described for the quantitation of SV40 virus infectivity by plaque formation within 7 days and T antigen assay by the sensitive and economical indirect immunoperoxidase technique.


Quantitative histological studies on the lateral geniculate nucleus in the cat. IV. Numerical aspects of the transfer from restinal fibers to cortical relay. The numerical data, gained both from the quantitative histology and Golgi stained material was completed with those of optic fibers. The number of X and Y fibers was estimated on the basis of our results, on the data of literature and from the ratio of the large and medium size relay neurons. The crossed and uncrossed fibers were counted and compared with the number of relay neurons in layer A and A1. Knowing the different numerical and metrical counts of neuronal elements in LGN of cat some calculations were carried out on the overlaping of optic fiber arborizations, on the intersections of dendritic ramification points of relay neurons and on the spatial arrangement of both the optic terminals and the relay neurons. The comparison of the neumerical data on relay neurons and optic fibers and their relations were the basic factors of considerations on divergence and convergence of retinal input.


Differences in activation of human and guinea pig complement by retroviruses. C type murine leukemia viruses (retroviruses) have been shown previously to possess a receptor for human C1 that activated human but not guinea pig complement. In the present study we provide evidence that the viral receptor also binds guinea pig C1 but that such binding does not lead to activation. However, incorporation of human C1s into guinea pig C1 to form a C1 hybrid results in activation of that hybrid and in viral lysis. In contrast, incorporation of guinea pig C1s into human C1 abolishes activation by the virus. These results demonstrate that C1s governs the activation of C1 of the viral receptor.


In vitro immune response of human peripheral lymphocytes. III. Effect of anti-mu or anti-delta antibody on PWM-induced increase of cyclic nucleotides in human B lymphocytes. Stimulation of human peripheral blood lymphocytes (PBL) with pokeweed mitogen (PWM) induced consistent increases of intracellular levels of cyclic AMP and cyclic GMP within 15 min. Increases of cyclic AMP were observed in both B and T lymphocyte populations, but increase of cyclic GMP was observed only in the B lymphocyte population. The addition of anti-mu antibody to B cells abolished PWM-induced increase of cyclic GMP without any effect on cyclic AMP response. Anti-delta antibody did not show any inhibitory or stimulatory effect on PWM-induced increase of cyclic GMP or cyclic AMP. Pretreatment of B cells with anti-mu antibody at 37 degrees C for 1 hr inhibited PWM-induced increase of cyclic GMP, whereas pretreatment with anti-mu antibody at 4 degrees C did not show any inhibitory effect on PWM-induced increase of cyclic GMP. The effect of anti-mu-pretreatment was reversible and pretreated cells were recovered from the inhibitory effect of anti-mu antibody after 36 hr culture.


Functional differentiation of B lymphocytes in agammaglobulinemia. III. Characterization of spontaneous suppressor cell activity. The addition of fresh peripheral blood mononuclear cells (PBL) from four of seven patients with agammaglobulinemia to generated hemolytic plaque-forming cells (PFC) resulted in a dose-dependent suppression of PFC. This spontaneous suppressor cell activity (SSA) was restricted to the four patients who could generate a PFC response in vitro. SSA was mediated by a small subset of E-rosetting T lymphocytes characterized by theophylline-sensitive E-receptors and surface receptors for Fc-IgG. The effects of SSA were temperature dependent and reversible, and pokeweed mitogen could prevent the rapid decline of SSA observed during culture. Augmentation of SSA was achieved by agents known to increase intracellular levels of cyclic AMP, whereas lithium chloride abrogated SSA, including the drug-induced effects. Cells mediating SSA may play a role in preventing the normal transition of pre-B cells to B cells in patients with agammaglobulinemia without B lymphocytes.


Enhancement of neutrophil chemotaxis and alteration of levels of cellular cyclic nucleotides by levamisole. Levamisole, an antihelminthic agent reported to enhance nonspecifically various parameters of the immune response, was examined for its effect on chemotaxis of human neutrophils and on levels of cellular cyclic nucleotides. This agent was found, in most instances, to enhance chemotactic responses of neutrophils to a bacterial chemotactic factor derived from Escherichia coli. At similar concentrations, levamisole produced increases in levels of guanosine 3':5'-cyclic phosphate in neutrophils. In contrast, a decrease in concentrations of adenosine 3':5'-cyclic phosphate was observed when neutrophils were incubated with levamisole. Neutrophil chemotaxis, with and without the addition of levamisole, was assessed in 10 patients with recurrent infections. The illnesses of these patients included Job's syndrome, Wiskott-Aldrich syndrome, eczema with an increased level of IgE and recurrent abscesses, chronic mucocutaneous candidiasis, and diabetes mellitus. Levamisole significantly enhanced chemotaxis of polymorphonuclear leukocytes from these patients. Levamisole appears to have a profound effect on chemotactic responses of neutrophils which probably results from alterations in cellular cyclic nucleotide levels. Levamisole may prove to be useful therapeutically in certain patients with defective neutrophil chemotaxis.


Diagnosis and etiology of nongonococcal urethritis. The observation of more than four polymorphonuclear cells (PMN) per high-power field (hpf) in gram-stained smears of urethral secretions was found to differentiate patients with urethritis from patients without urethritis. A urethral discharge was present in 78% of patients with nongonococcal urethritis (NGU). Dysuria without demonstrable urethral discharge and with fewer than four PMN/hpf did not appear to fit into the NGU spectrum. NGU is now defined to include men who have negative urethral cultures for Neisseria gonorrhoeae with a urethral discharge and/or more than four PMN/hpf in their urethral smears. The findings of more than four PMN/hpf in the urethral smears of 22%of asymptomatic sexually active men with more than one sexual partner (polygamous controls) suggests that asymptomatic NGU is not uncommon. Chlamydia trachomatis was isolated significantly more frequently from the NGU study group than from the control group (P less than 0.001). This study adds Corynebacterium vaginale (Haemophilus vaginalis), group B streptococci, and yeasts to the list of sexually transmitted microorganisms that are not etiologic determinants of NGU.


Restriction endonuclease fingerprinting of herpes simplex virus DNA: a novel epidemiological tool applied to a nosocomial outbreak. In a blind study, 14 isolates of herpes simplex virus type 1 (HSV-1) that included nine isolates from a temporal cluster of HSV infections in a hospital Pediatric Intensive Care Unit and five unrelated isolates were analyzed by digestion of their DNA with four restriction endonucleases. These enzymes (HsuI, BglII, EcoRI, and HpaI) cleave the DNA in about 52 sites. To date, at least 16 sites have been found to be variable in the sense that they may be present or absent independently of any other cleavage site. This characteristic is stable, and no change was observed on serial propagation of the strains in culture or following repeated isolation, as long as 12 years apart, from humans. Analyses of the isolates readily discriminated between those belonging to the temporal cluster of hospital infections and the unrelated strains. They also showed that there were two independent introductions of HSV-1 into the Pediatric Intensive Care Unit resulting in two clusters of epidemiologically related infections. This type of analysis has the potential of becoming a powerful tool for tracing the spread of HSV-1 and very likely of other herpesviruses in the human population.


Inhibition of mitogen- and antigen-induced lymphocyte blastogenesis by herpes simplex virus. Lymphocytes were separated from peripheral blood of adult human donors by Ficoll-Hypaque gradient centrifugation and cultured in the presence of nonspecific mitogens (phytohemagglutinin[PHA] and concanavalin A) or specific microbial anatigens (herpes simplex virus [HSV], mumps virus, streptococcal enzymes, and Candida albicans). Exposure of lymphocyte cultures to infectious HSV resulted in almost complete inhibition of blastogenesis ([3/H]thymidine uptake) induced by each of the mitogens and antigens, a finding which suggests that a common mechanism may underlie the inhibitory effect. Several characteristics of the effect of virus on blastogenesis were noted: (1) virus inactivated by heat or ultraviolet irradiation was ineffective; (2) inhibition (is greater than 90%) was greatest in cultures exposed to HSV on or before the addition of PHA; (3) lymphocyte preparations washed free of HSV continued to be refractory to stimulation, an observation indicating that the presence of unabsorbed virions or viral products was not essential; and (4) inhibition was independent of the cell donor's state of humoral immunity to HSV.


Effect of CP-20,961, an interferon inducer, on upper respiratory tract infections due to rhinovirus type 21 in volunteers. Topically administered CP-20,961 is known to stimulate nasal interferon. Studies in volunteers given the drug prior to challenge with rhinovirus have yielded both fairly good results and only fair or poor results. This study was undertaken in an attempt to settle the differences between the results of these trials and to evaluate the possible effectiveness of CP-20, 961 when given after virus challenge. Sixty volunteers were randomly divided into four groups. One group received placebo, the second received the drug on the day before and the day of challenge, the third was given the drug for two days beginning 24 hr after challenge, and the fourth received the drug for two days beginning 48 hr after challenge. The average number and severity of symptoms in the group that received CP-20,961 prior to challenge were about half of those in the control group. There was no decrease, however, in the number and severity of symptoms in the groups that received the drug after challenge.


Sensitivity of the virus isolation and immunofluorescent staining methods in diagnosis of infections with herpes simplex virus. Tissues from marmoset monkeys infected with herpes simplex virus (HSV, Herpes-virus hominis) were utilized to evaluate the relative sensitivity and limitation of the virus isolation technique and the immunofluorescent (fluorescent antibody or FA) staining method for diagnosis of HSV infection. HSV encephalitis and/or disseminated infection in marmosets were established by intracerebral, intramuscular, or intravenous inoculation of the virus. Brain tissues, liver, spleen, kidney, adrenal gland, lymph node, and lung were harvested and prepared for the virus isolation procedure in tissue culture and for direct FA staining. Data from six marmosets infected with HSV type 1 and two infected with type 2 indicated that the virus isolation method was more sensitive and reliable than the FA staining technique. False-negative results by FA staining were found in two situations: (1) presence of focal lesions that were missed by the frozen sections, and (2) presence of low concentrations of virus in tissue (is less than or equal to 3.5 log10 50% tissue culture infective doses/g). FA staining provides a rapid method for detection of viral antigens, but isolation of virus in tissue culture is required for a conclusive diagnosis of active infection.


Cell-mediated immunity to murine cytomegalovirus. Cell-mediated immunity to murine cytomegalovirus (CMV) was tested by an in vitro lymphocyte proliferation assay. CMV antigen was prepared from infected mouse embryo fibroblasts by ultracentrifugation. Control antigen was prepared in a similar fashion from uninfected fibroblasts. Splenic lymphocytes from CMV-infected mice underwent significant proliferation when incubated with CMV antigen. Lymphocytes from uninfected mice did not proliferate when incubated in vitro with CMV antigen. The proliferation was shown to be specific, since lymphocytes from CMV-infected mice did not proliferate when incubated with control antigen or an indifferent (encephalomyocarditis) viral antigen. Lymphocyte proliferation was directly related to the number of lymphocytes in each culture and to the amount of viral antigen. However, at higher numbers of cells and with large amounts of viral antigen, the backgrounds increased, and the standard errors were larger. The optimal incubation time was eight days. Lymphocyte proliferation reached a maximum 15 days after CMV infection but was still significantly elevated above background 60 days later. This type of lymphocyte proliferation test with human lymphocytes and human CMV antigen can be used to study clinical questions regarding cell-mediated immunity to CMV in immunosuppressed patients, in children with congenital CMV, and in transplant patients after vaccination with attenuated CMV.


A long-term survey of rotavirus infection in Japanese children with acute gastroenteritis. Human rotavirus was detected by electron microscopic examination of the stools of 320 (63%) of 506 infants and young children hospitalized with acute gastroenteritis between December 1974 and March 1977. Serologic responses to infection with the rotavirus were revealed by the complement-fixation test in 130 (70%) of 185 patients examined. During the study period three epidemics of human rotavirus infection occurred during the winter months. The peak incidences occurred in January 1975 (88% of patients positive by serologic analysis or electron microscopy of stools), January 1976 (92%), and February 1977 (96%). Rotavirus was detected in the stools of 288 (79%) of 365 patients tested during the cooler months (December to March) and 35 (25%) of 141 during the rest of the year. In the summer (June to August), rotavirus infection occurred rarely. The frequency of human rotavirus infection was highest among patients aged six to 11 months. These results indicate that human rotavirus can be regarded as a major etiologic agent of acute gastroenteritis in infants and young children, of which wintertime epidemics are common in Japan.


Resistance of visna virus to interferon. Visna is a slow infection of sheep caused by a retrovirus. The persistence of virus despite the immune response of the host is best explained by restricted genetic expression of the virus and consequently prolonged periods of residence inside cells. The purpose of this investigation was to determine whether the restriction in genetic expression of visna virus is mediated by interferon. Sheep interferon induced by polyriboinosinic-polyribocytidylic acid in fetal lambs inhibited the growth of herpes simplex virus, vesicular stomatitis virus, and vaccinia virus, but even highly concentrated interferon did not affect the replication of visna virus in sheep choroid plexus cells. The same results were obtained whether the effects of interferon were assessed in single of multiple cycles of growth and when interferon was added at later times in the growth cycle of the virus. This unusual resistance of visna virus to interferon suggests that restriction of viral expression by the host is probably not mediated in this way.


Experimental congenital infection with cytomegalovirus: a guinea pig model. An animal model permitting study of congenital infections with cytomegalovirus (CMV) has been developed in guinea pigs. Fifteen Hartley strain guinea pigs in the latter half of pregnancy were inoculated intraperitoneally with 10(5.5) 50% tissue culture infective doses of guinea pig CMV. Forty percent of infected mothers delivered litters containing at least one infected newborn, as defined by a positive culture of lung, spleen, or brain. All tissues were cultured by an explant technique. The three mothers who had no detectable complement-fixing antibody to CMV prior to experimental infection delivered infected litters, whereas three of 12 immune mothers delivered infected litters (P less than 0.01). A low-passage, tissue culture-adapted virus produced neonatal infection as frequently as did salivary gland-passaged virus. No congenital abnormalities were found in any of the seven infected newborns. CMV was isolated from lung, spleen, or brain in the four newborns of nonimmune mothers; CMV was isolated from lung only in the three newborns of immune mothers. These preliminary experiments demonstrate that the guinea pig is a suitable animal for further study of maternal-fetal CMV infections.


Isolation of viruses from cultures of bovine endometrial cells. Endometrial cells from the uteri of pregnant and nonpregnant cattle were cultured. The presence of one or both of two viruses, noncytopathic mucosal disease virus and bovine syncytial virus, was demonstrated in seven of 19 endometria investigated. It was necessary to subculture the cells an average of four times to detect the viral infections. Difficulties were encountered in producing endometrial cell cultures from cows at term or near the end of term and also from older animals. The infections detected may be significant because both of the viruses isolated are capable of infecting the bovine fetus in utero and mucosal disease virus has been associated with bovine fetal diseases.


Seroconversion against Epstein-Barr virus in two nonhuman primate species infected by the oropharyngeal route. Transforming Epstein-Barr virus (EBV) was inoculated into eight squirrel monkeys (Saimiri sciureus) by spraying of viral concentrates into the nose and throat. Virus was inoculated into three marmosets (two Saguinus oedipus and one Calithrix jacchus jacchus) by direct injection into nasopharyngeal tissue. Two of these squirrel monkeys seroconverted after repeated inoculation as determined by the presence of EBV-specific antibodies to early antigen and viral capsid antigen. One squirrel monkey had antibody to EBV nuclear antigen only. One S. oedipus marmoset also seroconverted. None of the animals developed disease. The results show that seroconversion can be accomplished in squirrel monkeys after spraying of EBV into the nasopharynx, and in marmosets by direct inoculation of the virus into nasopharyngeal tissue.


Comparison of myeloperoxidase activity in leukocytes from normal subjects and patients with chronic granulomatous disease. A study was undertaken to compare myeloperoxidase (MPO) activity in leukocytes from normal subjects and those with chronic granulomatous disease (CGD) and to eliminate further consideration of MPO as the oxidase responsible for the post-phagocytic respiratory burst and subsequent oxidase microbicidal sequence lacking in leukocytes from patients with CGD. With use of granule fractions isolated from a number of samples, MPO activity in several MPO-mediated biochemical systems (peroxidase assays, protein iodination, and amino acid decarboxylation) was measured. No difference was demonstrated between granule fraction preparations from normal subjects and those with CGD. These data show that MPO activity is normal in CGD leukocytes and are inconsistent with a role for MPO in initiating the post-phagocytic respiratory burst.


Weakness, neuropathy, and coma following total parenteral nutrition in underfed or starved rats: relationship to blood hyperosmolarity and brain water loss. The continuous infusion of a concentrated, high-caloric glucose solution intravenously into underfed or 3-day-starved rats at a rate of 390 kcal/kg/day results in hypophosphatemia, muscular weakness, neuropathy, lethargy, occasional convulsions, and eventual coma and death. This sequence of events is not observed in similarly infused normal rats. It is a model of a fatal parenteral nutrition syndrome which occurs in undernourished patients. Rats in coma had an eightfold increase in the blood glucose level, a 1.6-fold increase in serum osmolarity, a 16% to 20( decrease in brain water content, and normal blood ketones. A lag phase of at least 8 hr and often 12 to 24 hr occurred following the start of the hyperosmotic glucose infusion before the blood glucose began to accumulate progressively and the syndrome developed. The onset of the syndrome could be prevented by the administration of large amounts of insulin required to keep the blood sugar from exceeding 250 mg/dl. Thus the rat model of the fatal hyperalimentation syndrome is a form of hyperglycemic, hyperosmolar, nonketotic coma caused by brain dehydration.


Characterization of high-affinity beta2-adrenergic receptor binding of (-)-[3H]-dihydroalprenolol to human polymorphonuclear cell particulates. Human PMNs have well-described responses to beta-adrenergic catecholamines; these include elevation of cellular levels of cyclic AMP and inhibition of the release of lysosomal contents. Using the radioactive beta-adrenergic antagonist (-)-[3H]DHA in direct ligand-binding studies, we have identified and characterized beta-adrenergic receptors on particulate preparations of PMNs. These particulates bind DHA rapidly (t1/2 less than 1 min) and reversibly (t1/2 = 8 to 9 min). DHA binding is saturable and of high affinity (dissociation constant = 1 to 5 nM) and low capacity (870 +/- 128 receptors/cell, mean +/- S.D.) to a single class of binding sites. Competition for DHA binding sites by both beta-adrenergic agonists and antagonists is stereoselective [(-)-isomers more potent that (+)-isomers]. The rank order of potency of adrenergic agents in such competition studies indicates that these receptors are of the beta2 type. Since PMNs can be obtained in high purity with relative ease, the combined use of pharmacologic and ligand-binding studies in PMNs provide a useful system for studying beta-adrenergic receptors and their function in human subjects.


A calibrated delivery system for inhalation challenge of toluene di-isocyanate vapor. The vapor of TDI, a chemical commonly used in the plastics industry, may be associated with pulmonary symptoms at various concentrations. Because of the high volatility and adsorptivity of TDI, it has been difficult to deliver known, dilute concentrations of TDI to test subjects in inhalation challenge studies. A delivery system has been developed which can be calibrated to deliver an air-TDI mixture having a given flow rate and TDI concentration. The delivered gas stream is produced by diluting a gas stream having a low flow rate and a fixed concentration of TDI with a high-flow-rate stream of TDI-free air. A TDI detector continuously monitors the concentration of the resultant mixture, which is delivered to the subject by means of a face mask. For dilute mixtures, the output concentration is proportional to the ratio of the input flow rates; the proportionality factor depends on temperature and the geometry of the system. The flows for the described system could be regulated to produce concentrations in the range 0.0 to 0.08 ppm which remain stable to within +/- 10%.


Effects of secretagogues and theophylline on canine gastric mucosal cyclic nucleotides. cAMP and cGMP concentrations were measured in gastric mucosa in 18 anesthetized mongrel dogs receiving infusions of histamine (16 microgram/kg/hr), pentagastrin (6 microgram/kg/hr), bethanechol (10 microgram/kg/min), and theophylline (150 mg bolus, then 10 mg/min). The possible synergistic effect of continuous theophylline infusion with the secretagogues was studied in 15 other acute experiments. Antral and fundic mucosa were biopsied and measured by radioimmunoassay for cyclic nucleotides. All the secretagogues did not significantly change antral or fundic mucosal cyclic nucleotide concentrations compared to basal levels. In contrast, phosphodiesterase inhibition by theophylline significantly increased cAMP 11-fold and cGMP threefold in both antrum and fundus. Concomitant infusion of each secretagogue with theophylline did not potentiate the mucosal nucleotide response compared to theophylline alone. In a separate series of 19 experiments in three anesthetized dogs with vagally denervated fundic (Heidenhain) pouches, all the secretagogues which were infused in the same doses as in the acute biopsy studies produced maximal acid secretory responses. Theophylline, on the other hand, failed to stimulate acid secretion in five Heidenhain pouch experiments. These studies fail to implicate cyclic nucleotides as mediators of stimulated canine gastric acid secretion.


Osteoclastoma of the petrous temporal bone. This paper has described an unusual case of osteoclastoma of the petrous temporal bone which presented with multiple cranial neuropathies. A radical course of radiotherapy reversed the neuropathies but it is too early to comment on possible cure.


Idiopathic sensory trigeminal neuropathy. A series of seven cases of isolated facial numbness is presented in whom the final diagnosis is considered to be idiopathic trigeminal neuropathy. Although this is a benign condition, careful work-up is needed, preferably by a team of specialists, in order to exclude more serious pathology. The team should include a neurologist, an otolaryngologist and a dental surgeon. By this means all other possible underlying diagnoses can be excluded. A list of differential diagnoses is discussed and the need for careful long-term follow-up is stressed.


Infratemporal fossa approach to tumours of the temporal bone and base of the skull. In spite of the development of a superior (middle cranial fossa) and posterior (translabyrinthine) approach to the temporal bone, tumours situated in the infralabyrinthine and apical compartments of the pyramid and surrounding base of the skull were still a challenge for neurosurgeons and otologists as well. The infratemporal fossa approach closes the existing gap in the surgical management of the most hidden lesions of the temporal bone. The approach features the permanent anterior transposition of the facial nerve, resection of the mandibular condyle and mobilization of the zygoma and lateral orbital rim. Obliteration of the pneumatic spaces of the temporal bone, with permanent occlusion of the Eustachian tube and blind sac closure of the external auditory canal, avoids the danger of post-operative infection and leads to primary wound healing in the shortest time. Three types of infratemporal fossa approach are presented and dicussed on the basis of 51 operated patients.


Intracisternal tubular inclusions in nasopharyngeal carcinoma. Biopsy specimens obtained from 20 cases of nasopharyngeal carcinoma were examined under electronmicroscope for the presence of Intracisternal Tubular Inclusions (ITI). In eight (40 per cent) of them these inclusions could be identified. Although the morphogenesis and significance of these ITI are not clearly understood, they have been found in several autoimmune and viral diseases, including infectious mononucleosis, a disease caused by EBV, and Burkitt's lymphoma, which is closely associated with EBV. Hence, the presence of ITI in NPC, another disease associated with EBV, seems to be of some significance. It is proposed that these inclusions could be the result of an immunological reaction initiated by injured cells infected with EBV, which acted as an antigen. The presence of another type of cytoplasmic inclusion, hitherto unknown, has also been reported.


The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin. Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.


Evaluation of a radioimmunoassay for plasma adrenocorticotrophin. A radioimmunoassay for plasma ACTH has been described and evaluated. Rabbit antiserum produced by immunization with [Asp25, Ala26, Gly27,]-alphah-corticotrophin-(1--28)-octacosa-peptide (a sequence analogue of alphah1--28-ACTH) bovine gamma globulin conjugate was used. The antiserum is specific for the NH2-terminal portion of the ACTH molecule and cross-reactivity of human, porcine and rat ACTH in the system has been demonstrated. Reasonable agreement was found between estimates obtained by bioassay and radio-immunoassay of the ACTH content of rat pituitary gland incubation media, indicating a close relationship between the sequence of ACTH recognized by the antibodies and the sequence possessing the steroidogenic activity. Measurement of the amount of ACTH in the plasma required the preliminary extraction and concentration of the hormone. Over a range of concentrations between 3.5 and 3600 pg/ml, extraction recovery was independent of the initial concentration of ACTH in the plasma. Extraction gave rise to no changes in the immunological properties of standard ACTH. The concentration of immunoreactive ACTH in rat plasma was 48 +/- 3.6 (S.E.M.) pg/ml in the morning and 106 +/- 9.9 pg/ml in the afternoon. Exposure to either for 5 min and subsequent laparotomy gave rise to a significant increase in the concentration of immunoreactive ACTH in the plasma. The resting level of ACTH and the ACTH response to stress were both significantly higher 1 and 7 days after adrenalectomy. Intravenous injection of a hypothalamic extract elicited a considerable rise in the concentration of immunoreactive ACTH in the plasma, but no response was seen after oral administration of this partially purified extract. The sensitivity, precision and specificity of this ACTH radioimmunoassay make it a useful tool for studying pituitary--adrenal physiology.


Activity of the pituitary-adrenocortical system and thyroid gland during the oestrous cycle of the rat. The concentrations of LH, progesterone, ACTH, corticosterone and thyroxine in the plasma were estimated at various times during the oestrous cycle of the rat. The well-established patterns of LH and progesterone secretion were confirmed. On each day of the cycle the plasma concentrations of ACTH and corticosterone were lowest in the morning and rose in the afternoon. Conversely, during oestrus and dioestrus, the plasma concentrations of thyroxine were higher in the morning than in the evening. However, during the afternoon of pro-oestrus the concentrations of ACTH, corticosterone and thyroxine in the plasma rose and, like the concentrations of LH and progesterone, all reached levels far higher than those attained at any other time of the cycle.


Effects of human thyroid-stimulating hormone and immunoglobulins on adenylate cyclase activity and the accumulation of cyclic AMP in human thyroid membranes and slices. The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices. Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS--immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly. Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond. In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.


Distribution of androgen metabolizing enzymes in isolated tissues of human forehead and axillary skin. The distribution of androgen metabolism in human skin was studied using tissues isolated either by direct dissection of axillary skin or by dissection of collagenase-digested forehead and axillary skin. All tissues (epidermis, sweat glands, sebaceous glands, hair follicles and dermis) were found to contain 17beta-, 3beta- and 3alpha-hydroxysteroid dehydrogenase (HSD) activities, 3beta-hydroxysteroid dehydrogenase-delta4--5 isomerase (delta5-3beta-HSD) activity and 5alpha-reductase activity. All tissues converted testosterone into 5alpha-dihydrotestosterone. In confirmation of previous histochemical studies, over 90% of the delta5-3beta-HSD of forehead skin was found in the sebaceous glands. In forehead skin, 40--66% of the 5alpha-reductase activity was in the sebaceous glands, while in axillary skin 50--70% was in the sweat glands, especially the apocrine glands. There was a more even distribution of 17beta-HSD activity in skin tissues than histochemical studies have indicated previously. Knowledge of the distribution of these enzymes has helped in the understanding of the function of androgen metabolism in skin.


Na++K+-ATPase in the osmoregulating clam Rangia cuneata. Of six tissues sampled from the osmoregulating clam Rangia cuneata, mantle contained the highest concentration of Na++K+-ATPase activity and ouabain binding sites. Acclimation to low salinities was accompanied by adaptive increases in Na++K+-ATPase activity in mantle but not in gill. Since the number of ouabain binding sites did not show parallel increases, the mechanism of acclimation to reduced salinity in Rangia appears to involve activation of pre-existing pump sites in the mantle epithelium.


Some structural antigens of herpes simplex virus type 1. Several of the major structural polypeptides of herpes simplex virus were obtained in purified form by polyacrylamide gel electrophoresis of purified virus particle polypeptides. Antisera made by footpad inoculation of these polypeptides into rabbits were used to study the antigenic properties of two envelope glycoproteins and of the major capsid protein.


The fate of protein subunits of parainfluenza (Sendai) virus after adsorption to NIL8 hamster embryo cells. Adsorption of u.v.-inactivated Sendai virus on to NIL8 hamster cells causes fusion of the cells into polykaryocytes within 2 h. "Infected" cells were incubated at 37 degrees C for periods of 10 min to 8 h and their surface proteins iodinated with [125I] catalysed by peroxidase. Structural components of the viral envelope, such as haemagglutin-neuraminidase (HN) and probably also the fusion protein (F) were detected in the cell membrane for periods up to 4 h post infection.


Poliovirus polypeptides examined in more detail. The pattern of viral polypeptide synthesis and cleavage in poliovirus-infected cells was shown by autoradiography to be considerably more complex than previously thought. In normal growth, at least 26 distinct polypeptides were found, and various modifications of the cleavage process revealed a total of at least 34. Most of the new polypeptides were minor components that were unstable during a chase. Different cultural modifications led to different polypeptide ratios, and it appeared likely that several cleavage activities were involved . Minor differences were found in the polypeptide contents of cytoplasmic extracts and whole infected cells. The complexity of the cleavage pattern necessitated a new nomenclature based on mol. wt. (e.g. Pp110, "poliovirus protein" of 110000). Particular attention was paid to mol. wt. determinations, notably in the use of internal protein standards and more fully denaturing gel conditions. The size of the "primary translation product" of poliovirus RNA was found to be 210000 daltons, so that either 20% of the viral genome is not translated in vivo, or some is read as a smaller independent translation unit.


Relations between poliovirus polypeptides as shown by tryptic peptide analysis. Poliovirus proteins were labelled in vivo with [35S]-methionine, and the major products of translation and cleavage were separated by electrophoresis and compared in terms of two-dimensional tryptic peptide maps visualized by autoradiography. The main intermediates p110 and p90 had few or no methionine-labelled sequences in common, but were both contained in, and therefore almost fully account for, the presumed primary translation product p210. The sequences of p79, a major stable product of cleavage and a non-structural protein, were almost completely contained in p90, which in turn is the major component of the larger intermediates p168 and p155. P110 is confirmed as the precursor of virus particle protein, and VP0 as the precursor of VP2. However, the sequences of p31, the other major product of translation that is not a stuctural protein, were not contained in any of the viral polypeptides mentioned above.


A high density component in several vertebrate enteroviruses. In addition to the major infective component, which bands at a density of 1:34 g/ml in caesium chloride ("light component"), a component with a density of 1:44 g/ml ("heavy component") has been found in harvests of poliovirus (type I), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1 . 34 g/ml but approx. 2% was present as a peak at 1 . 44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.


Complementation of adeno-associated virus growth with temperature-sensitive mutants of human adenovirus types 12 and 5. Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesis, were able to support growth of adeno-associated virus type I at the non-permissive temperature.


Effect of isoleucine deprivation of rabbitpox virus DNA synthesis in mouse L cells. Synthesis of rabbitpox DNA was inhibited in mouse L cells deprived of isoleucine. Time-course patterns of incorporation of radiolabelled precursors into viral DNA revealed that synthesis of viral DNA began about 6 h after reversal of the isoleucine-deficient state.


Hyperpolarizing potentials induced by Ca-mediated K-conductance increase in hamster submandibular ganglion cells. The mechanisms of three types of hyperpolarizing electrogenesis in hamster submandibular ganglion cells were analyzed with intracellular microelectrodes. These included (1) spike-induced hyperpolarizing afterpotential (S-HAP), (2) spontaneous transient hyperpolarizing potential (HP), and (3) the hyperpolarizing (H) phase of postsynaptic potential (PSP). Most of these hyperpolarizing potentials were due to conductance increases and reversed polarity at membrane potential (Em) between -70 and -85 mV, which was close to the K-equilibrium potential. The average resting potential of ganglion cells was -53 mV. Action potential overshoot increased slightly in high [Ca2+]0 and decreased in low [Ca2+]0. In most neurons action potentials were completely suppressed by 10(-7)-M tetrodotoxin (TTX). The S-HAP has an initial component due to delayed rectification and a late component. The late component is enhanced by increasing [Ca2+]0, or by applying Ca-ionophore (A23187), TEA, caffeine, or dibutyryl cyclic (DBc-) AMP; it is suppressed by decreasing [Ca2+]0, or by applying Mn2+. Perfusion with Cl--free saline reduced membrane potential slightly but did not modify the S-HAP. Depolarizing pulses also induced hyperpolarizing afterpotential (D-HAP), similar to the S-HAP. Spontaneous transient HPs occurred in some neurons at irregular intervals. HPs were insensitive to TTX but were suppressed by Mn2+. Caffeine induced low frequency rhythmic HPs in many neurons, often alternating with periods of repetitive spiking. The PSP was a monophasic depolarizing (D-) potential in some neurons, but in others the D-phase was followed by a small H-phase. Perfusion with A23187, caffeine or DBc-AMP increased the H-phase of the PSP. Perfusion with K+-free saline or treatment with 10(-5)M ouabain did not abolish the H-phase of PSPs. These membrane potential-dependent phenomena appear to be induced mainly by Ca-mediated K-conductance increases. This mechanism contributes to the regulation of low-frequency repetitive firing in submandibular ganglion cells.


A second brain tumour and irradiation.

A second brain tumour and irradiation. Three patients are described in whom irradiation of 2750 rad or more was used in the management of primary brain tumours, and 21 years or more later a second brain tumour of a different type occurred. One of the new tumours was a meningioma and the other two were cerebral astrocytomas. There is evidence to show that moderate doses of ionising radiations given in childhood for tinea capitis are associated with a late risk of developing a meningioma. Higher doses of radiation used for tumours in childhood are followed also by a late hazard of meningioma. There is insufficient evidence to implicate ionising radiations in the aetiology of gliomas. The oncogenic hazards of radiotherapy to the brain do not outweigh its therapeutic value in brain tumour.


Myasthenia in patients with dermatomyositis: clinical, electrophysiological and ultrastructural studies. In 4 patients with clinical signs of dermatomyositis, confirmed by electromyography and muscle biopsy, a form of muscle fatigue was detected which was expressed clinically by predominantly proximal motor deficit, with phonation and deglutition disturbances, slightly influenced by prostigmine. In all patients, stimulation of the ulnar nerve at 3--10 Hz induced a decrement of muscle-evoked potentials in abductor digiti minimi and at 15--50 Hz an increment at the end of the trains (1.2 sec in duration) of repetitive stimulation (preceded in two cases by a decrement in the response to the fifth stimulus in the train). Stimulation at 30 Hz for 10 sec resulted in a transient facilitation, followed (at 3 Hz stimulation) by postactivation exhaustion which disappeared after 5--15 min. The post-tetanic facilitation, the incremental response and the myasthenic symptoms reverted to normal under treatment with corticosteroids, an immunosuppressor agent and guanidine hydrochloride. A mixed, pre- and postsynaptic mechanism is presumed to underlie the muscle fatigue in our patients. Electron microscopy of muscle biopsies disclosed zones of necrosis and, in incipient stages, large agglomerations of glycogen that had disorganized the structure of myofibrils. The end-plates in the biopsies were larger than normal and the cholinesterase reaction was hyperactive. Serum immunoelectrophoretic and electrophoretic data--increase of IgG and IgM, decrease of IgA and hypergammaglobulinaemia -- point to a possible autoimmune mechanism of the neuromuscular disorders in our patients.


Lipid storage myopathy in von Gierke's disease: a case report. An 18-year-old girl with von Gierke's disease associated with a lipid storage myopathy is reported. The diagnosis of von Gierke's disease was made from decreased activity in glucose-6-phosphatase in the jejunal biopsy specimen. Neurologically she showed generalized hypotonia of the muscles, atrophy of bilateral proximal muscles of the lower extremities, weakness in neck flexors, deltoid and lumbar girdle muscles, and a positive Gower's sign. Muscle biopsy from flexor femoris muscle revealed fatty deposition in type 1 fibers and atrophy of type 2 fibers and the diagnosis of an accompanying lipid storage myopathy was made. This case also had a ventricular septal defect confirmed by right cardiac catheterization.


Periodic nocturnal myoclonus in a patient with hyperexplexia (startle disease). The periodic nocturnal myoclonus of a patient with hyperexplexia has been studied. Evidence has been given that the jerks are spontaneous arousal reactions. The temporal characteristics of the jerks have been analysed. The jerks appear to be correlated with the respiratory rhythm and the data suggest a correlation of the jerks with circulatory and respiratory higher order waves. The results are discussed with relation to the literature concerning spontaneous sleep jerks.


Hypothalamic hypothyroidism and hypogonadism in prolonged traumatic coma. Prolonged coma afterhead trauma is associated with depletion of 3', 5' cyclic adenosine monophosphate (cAMP) in the cerebrospinal fluid (CSF). Because cAMP has previously been implicated in neurorendocrine secretion, this study examines the pituitay-hypothalamic function in 15 adult male patients (to exclude the effects of puberty and menses) with traumatic coma lasting longer than 2 weeks. Ventricular CSF cAMP was measured at 2- to 4-day intervals for 10 to 25 days. Simultaneously, plasma hormone concentrations were also determined. In all 15 cases, CSF cAMP and plasma levels of thyroid-stimulating hormone (TSH), thyroxine (T4), free T4, triiodothyronine (T3), luteinzing hormone (LH), follicle-stimulating hormone (FSH), and testerone became subnormal. In 11 patients whose level of consciousness fluctuated, the reduction in plasma T4 and testerone were proportional to both severity of coma ( r greater than 0.81, p less than 0.05) and depletion of CSF cAMP (r greater than 0.81, p less than 0.05). In four patients who remained deeply comatose for 17 to 25 days, the hypothyroidism and hypogonadism persisted. In six patients who regained consciousness, both endocrine defects improved partially or completely. Injection of 1) thyrotrophic-releasing hormone and 2) gonadotrophic-releasing hormone elicited normal or supernormal increases in plasma concentrations of 1) TSH, and 2) LH and FSH, reduced, respectively, suggesting a suprahypophyseal deficiency. These observations demonstrate that suprahypophyseal hypothryoidism and hypogonadism may occur regularly in patients with traumatic coma lasting longer than 2 weeks.


The effect of vitamin D3 and dietary calcium level on the cadmium-induced morphological and biochemical changes in rat intestinal mucosa. The effect of vitamin D3 and dietary calcium level on the cadmium-induced changes was observed in the duodena of rats raised on various diets differing in vitamin D and calcium levels. Observation with scanning electron microscopy revealed that vitamin D and dietary calcium were required for normal intestinal villi and microvilli formation. The damaged cells were observed in the intestinal villi of cadmium-exposed rats. Furthermore, dietary cadmium reduced the enzyme activities in microvilli. Especially, alkaline phosphatase activity was reduced in the cadmium-exposed groups, even though it was still responsive to vitamin D3. These effects with cadmium were modulated by vitamin D3 and dietary calcium level. That is, in the presence of vitamin D3 and calcium, the effect of cadmium on intestinal villi and microvilli was reduced.


Wear of combinations of acrylic resin and porcelain, on an abrasion testing machine. Wear tests of various combinations of acrylic resin and porcelain were made using a machine which was designed to test materials under conditions similar to those of masticatory function by simulating the loads, sliding distances, and contact times encountered in the human masticatory cycle. The results showed that the amount of wear of the two materials worn in combination depended on the nature of the surrounding medium and on the surface roughness of the opposing material. Acrylic resin showed good wear resistance provided no third party abrasive or opposing hard, rough surface was present. When a mild abrasive was incorporated in the system, the acrylic resin vs acrylic resin combination wore almost seven times more than porcelain vs porcelain. Clinical experience would suggest that this is a reasonably sound order of wear.


Urinary phosphate and cyclic AMP excretion following citrate-induced hypocalcemic stimulation of the neonatal parathyroid glands. Sixteen neonates, ranging in gestational age from 27 to 41 weeks and in postnatal age from birth to 8 days, were evaluated for their renal response to an endogenous PTH stimulus in 22 separate experiments. The PTH stimulus was generated by the decreased serum ionized Ca that accompanies exchange transfusion with citrated blood. The neonates increased their serum PTH from 95.8 +/- 13.1 to 133.9 +/- 15.4 microliterEq/ml (mean +/- SEM) during the transfusion, while increasing their urinary cAMP from 0.77 +/- 0.11 to 1.45 +/- 0.22 nmol/ml, and their urinary P from 12.9 +/- 2.6 to 30.6 +/- 6.1 mg/dl in the four hours following the exchange transfusion. This response was not related to postnatal or gestational age. We speculate that lack of renal responsiveness to PTH does not play a major role in the pathogenesis of early neonatal hypocalcemia.


Peripheral median nerve damage secondary to brachial arterial blood gas sampling. Examination at 18 months post-term of 139 infants of birth weight less than or equal to 1,500 gm revealed 18 instances (13%) of persistent median nerve damage. All affected infants had received frequent percutaneous brachial artery punctures as neonates. Block sections of the cubital fossa done at autopsy on 12 randomly selected very low-birth-weight infants showed perineural hemorrhage, and Wallerian degeneration or traumatic neuroma of the median nerve in eight patients. It is recommended that brachial artery punctures be avoided whenever possible in the neonatal period.


Species differences in sodium-potassium adenosine triphosphatase activity in the smooth muscle of the guinea-pig and rat vas deferens. The acitvities of sodium-potassium-activated adenosine triphosphatase (Na+,K+-activated ATPase) and ouabain-inhibited, sodium-potassium-activated adensoine triphosphatase (Na+,K+-ATPase) in subcellular fractions of guinea-pig and rat vasa deferentia were compared to determine whether the ineffectiveness of ouabain and reduced extracellular potassium in the rat vas deferens observed in the preceding paper occurs because of a relatively low level of Na+,K+-ATPase and/or an insensitivity to ouabain. The results indicate that the specific and total activities of Na+,K+-activated ATPase and Na+,K+-ATPase (i.e., the transport enzyme) in the individual subcellular fractions and in the tissue were higher in the vas deferens of the rat than in the guinea pig. The percentage of inhibition of Na+,K+-activated activity by ouabain (8 x 10(-5) M) varied in the subcellular fractions; it was higher in the guinea-pig (range 31--87%) than in the rat (nonsignificant effect to 40%). A greater percentage of total Na+K+- activated ATPase activity was inhibited in the vas deferens of the guinea pig (56%) than the rat (30%). Differences in the effects of lowered extracellular potassium concentration or ouabain on resting membrane potential (preceding paper) are apparently unrelated to the amount of transport enzyme in the vasa deferentia or the two species, or to its relative sensitivity to ouabain.


Clonidine and related imidazolines are postsynaptic alpha adrenergic antagonists in dispersed rat parotid cells. Alpha adrenergic stimulation elicits a rapid release of K+ from dispersed rat parotid cells. Surprisingly neither clonidine, oxymetazoline, naphazoline, ST 600 nor ST 91 elicited K+ release from parotid cells at concentrations up to 100 micrometer. By contrast, these agents were able to block epinephrine-stimulated K+ release with the relative potencies: oxymetazoline more than naphazoline more than clonidine more than ST 600 more than ST 91. [3H]-Dihydroergocryptine (DHE) binds to sites on parotid membranes and intact dispersed cells with the characteristics of alpha adrenergic membrane receptors. The ability of these imidazolines to displace [3H]DHE binding from parotid membranes correlated with their potencies as alpha adrenergic antagonists in the dispersed cells. Although clonidine, oxymetazolin and naphazoline are agonists in other peripheral alpha adrenergic receptors, they are antagonists in the parotid. This observation explains the most common side-effect of clonidine administration, dry mouth, and is consistent with the proposition that different groups of alpha adrenergic receptors exist in mammalian tissues. These data also suggest that the central actions of clonidine must be interpreted with caution since alpha adrenergic receptors similar to those in the parotid may be present in brain.


Contribution of adrenergic and "purinergic" neurotransmission to contraction in rabbit detrusor. In the presence of propranolol, norepinephrine produced an alpha adrenoceptor mediated contraction in isolated rabbit detrusor. Phenoxybenzamine (3.3 x 10(-8) M) antagonized this response but failed to affect the contraction produced by field stimulation either in normal or in hemicholinium-3-treated tissue. Higher concentrations of phenoxybenzamine were antagonistic to carbachol. Electrically induced contractions were also unaffected by guanethidine (1 x 10(-4) M) in vitro. Reserpine pretreatment produced no change in the contractile response although the tissue was depleted of catecholamine fluorescence on histology. It is concluded that adrenergic neurotransmission does not account for noncholinergic excitatory neurotransmission in rabbit detrusor. In rabbit detrusor adenosine 5'-triphosphate (ATP) produced a transient contraction which was not antagonized by tetrodotoxin (1 x 10(-7) M), atropine (4 x 10(-7) M) or phenoxybenzamine (3.3 x 10(-7) M). Adenosine, adenine phosphate and adenosine 5'-monophosphate had little or no effect, while sodium tripolyphosphate and adenosine 5'-diphosphate produced a smaller response than ATP. Dipyridamole (1 x 10(-8)-1 x 10(-5) M) did not unmask a response to adenosine and did not potentiate the response to ATP or field stimulation. Theophylline (5 x 10(-5) M) and 2, 2'-pyridylisatogen (PIT) (1 X 10(-5) M) depressed responses to ATP without antagonizing those to carbachol. At these doses, theophylline and 2, 2'-pyridylisatogen also antagonized the electrically induced contraction. Desensitization with ATP (1.5 X 10(-3) M for 30 min) selectively depressed responses to ATP but not to carbachol, and also depressed the response to field stimulation, particularly at frequencies of 10 Hz and lower. It is at these frequences that the noncholinergic component of the contractile response is most significant. Combination of the desensitization procedure with atropine produced an additive effect, suggesting that the two mechanisms affected are independent. Combination of the desensitization procedure with hemicholinium-3 produced less than an additive effect, suggesting an interference between the two treatments. It is concluded that ATP plays a role in the noncholinergic component of excitatory neurotransmission in rabbit detrusor.


Presynaptic modulation of beta adrenergic receptors in rat cerebral cortex after treatment with antidepressants. Treatment with desmethylimipramine (DMI), a tricyclic antidepressant, for 7 to 21 days resulted in a 35 to 45% decrease in the accumulation of adenosine cyclic 3':5'-monophosphate (cAMP) in response to a maximally effective concentration of (-)-isoproterenol (ISO) in rat cerebral cortical slices. The EC50 for ISO-stimulated cAMP accumulation was not affected by DMI administration. The diminution in responsiveness to catecholamines was accompanied by a 35 to 40% decrease in the density of beta adrenergic receptors as measured by the binding of [125I]iodohydroxybenzylpindolol. Decreases in ISO-sensitive cAMP accumulation and in beta adrenergic receptor density were temporally correlated, maximal decreases being observed within 5 to 7 days. Within 7 days after cessation of chronic DMI treatment ISO-stimulated cAMP accumulation and beta adrenergic receptor density returned to normal. The role of presynaptic nerve terminals in mediating these phenomena was also investigated. Treatment of newborn rats with 6--hydroxydopamine inhibited the development of noradrenergic nerve terminals in the cerebral cortex and blocked the effects of DMI on cortical cAMP accumulation and on beta adrenergic receptor density. The administration of the beta adrenergic receptor antagonist propranolol led to increases in maximal ISO-stimulated cAMP accumulations and beta adrenergic receptor density in the rat cerebral cortex. This increase was not affected by the simultaneous administration of propranolol and DMI. Thus, the effect of DMI appears to be mediated through an action of norepinephrine at beta adrenergic receptors. Chronic treatment with two other clinically effective antidepressants, pargyline and iprindole, led to effects similar to those observed with DMI administration. Pretreatment of neonates with 6-hydroxydopamine blocked the effect of iprindole on beta adrenergic receptors. Preincubation of cortical membranes with guanosinetriphosphate before determination of the density of beta adrenergic receptors had no effect on the decreased number of receptors had no effect on the decreased number of receptors seen in DMI-treated animals. These experiments suggest that antidepressants, acting presynaptically, increase the concentration of transmitter at noradrenergic synapses and induce a compensatory decrease in the density of beta adrenergic receptors.


Evidence for the involvement of cerebroside sulfate in opiate receptor binding: Studies with Azure A and jimpy mutant mice. The role of cerebroside sulfate in opiate action and binding was studied by examining the effect of decreasing the availability of the glycolipid in vivo on morphine analgetic activity and receptor affinity. Available cerebroside sites were decreased either by injecting Azure A at a dose with selective high affinity for sulfo-lipids, or by using "jimpy" mice, a genetic leukodystropic mutant mouse with a deficiency in brain sulfatides. Injections of Azure A (4.4 MG/KG) intracerebroventricularly produced a 2-fold increase in the AD50 of morphine (tail-flick test) within 2 hr. The genetic mutation also resulted in a decreased sensitivity to morphine; the morphine AD50 was 6- to 11-fold higher in jimpy mice than in their normal littermates. Azure A produced a dose-dependent inhibition of opiate binding to synaptosomal plasma membranes. There was 85% inhibition of [3H]morphine binding with 0.2 micrometer Azure A, and 40% inhibition of [3H]naloxone binding with 5 micrometer Azure A. The inhibition of morphine binding was competitive and was demonstrated to be different from that elicited by Na+. Synaptic membranes from jimpy mice exhibited a decrease in number of binding sites for morphine. Binding of Azure A to cerebroside sulfate, associated with or in the proximity of the receptor sites, was suggested by the fact that in the presence of 0.2 micrometer Azure A, no inhibition of [3H]morphine binding to synaptosomal plasma membranes of jimpy mice could be demonstrated whereas 31% inh-bition was observed in membranes from control littermates. Based on the findings, it is concluded that cerebroside sulfate strategically located may have a role in binding morphine and mediating its effects.