Electron microscopy of yeastlike cell development from the microconidium of Histoplasma capsulatum. Fine details of the sequential morphological events occurring during transition of microconidia (spores less than 5 micrometer in diameter) to the yeastlike phase of Histoplasma capsulatum as seen in ultrathin section are described and illustrated by electron micrographs. Masses of microconidia were obtained when the fungas was grown on a garden soil extract medium. Spores were incubated under in vitro environmental conditions conducive for phase transition (an enriched medium at 37 degrees C). Within 48 h of incubation, the microconidia either germinated to give rise to a short mycelium or the germ tube process became a yeast mother cell without further extension. The wall of the yeast mother cell was thin and smooth, and its cytoplasmic content was ultrastructurally complex, consisting of numerous lipid bodies, vacuoles, glycogen-like deposits, and membrane systems. Within 96 h, the mother cell underwent multipolar budding to form simultaneously linear hyphal and/or ovate yeastlike daughter cells. During the transition, new cell wall materials of the germ tube, the mother cell, and yeastlike daughter cells arose by blastic action from the innermost layer(s) of the wall of the precursor form. Lomasome-like vesicles were often seen in association with areas of new cell wall formation. After organellar migration into and septation of the daughter cells, the yeast mother cell's cytoplasmic content underwent marked degenerative changes.


Location of protein synthesis during morphogenesis of Mucor racemosus. The intracellular location of active protein synthesis was examined during the emergence of germ tubes from both sporangiospores and yeast-phase cells of Mucor racemosus. It was determined that protein synthesis occurs in all regions of the cell and not preferentially at the growing tip.


Pentose metabolism in Mycobacterium smegmatis: comparison of L-arabinose isomerases induced by L-arabinose and D-galactose. D-Galactose, which did not serve as a growth substrate, was found to induce an L-arabinose isomerase of similar properties to the L-arabinose-induced L-arabinose isomerase. In both cases the pH profiles, pH stability, optimum temperature, heat stability, substrate specificity, metal ion requirements, mobility on polyacrylamide gel electrophoresis, and kinetic properties of the induced isomerases were identical. It appears possible that D-galactose was incorporated into the cells by an L-arabinose permease system that was alos induced by D-galactose.


Physical properties of plasmid Mor174, which determines bacteriocin production in Proteus morganii 174. Plasmid Mor174 has a molecular weight of 3.6 X 10(6) and a buoyant density of 1.6994 g/cm3. The covalently closed circular form has a sedimentation coefficient of 22S. These are 30 to 40 plasmid copies per genome equivalent, but growth in chloramphenicol results in amplification of the copy number to 600. In Proteus morganii 174, Mor174 coexists with a cryptic plasmid of molecular weight 15.8 X 10(6) and a buoyant density of 1.7170 g/cm3.


Genetic analysis of Acinetobacter calcoaceticus proline auxotrophs. Twenty-six Acinetobacter calcoaceticus proline auxotrophs were isolated after ethyl methane sulfonate mutagenesis. Studies using the efficient transformation system of this organism indicate that the mutations comprise therr genetically distinct groups.


Properties of the halophilic nuclease of a moderate halophile, Micrococcus varians subsp. halophilus. The halophilic nuclease of Micrococcus varians ATCC 21971 hydrolyzed thymidine 5'-monophospho-p-nitrophenyl ester at a rate that increased with the NaCl concentration up to saturation. The nuclease attacked RNA and DNA exonucleolytically and processively, producing 5'-mononucleotides. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 99,000, approximately the same as that previously determined for the native enzyme. Examination of amino acid composition showed that acidic amino acids were in high excess over basic amino acids.


Membrane lipid biosynthesis in Acholeplasma laidlawii b: elongation of medium- and long-chain exogenous fatty acids in growing cells. The chain elongation of a wide variety of exogenous fatty acids and the subsequent incorporation of the chain elongation products into the total membrane lipids of Acholeplasma laidlawii B were systematically studied. Within each chemical class of fatty acids examined, the extent of chain elongation increased with increases in chain length, reached a maximum value, and then declined with further increases in chain length. Depending on chemical structure, exogenous fatty acids containing less than 6 to 9 carbon atoms or more than 15 to 18 carbon atoms were not substrates for the chain elongation system. The substrate specificity of this fatty acid elongation system was strikingly broad, and straight-chain, methyl isobranched, and methyl anteisobranched saturated fatty acids, as well as cis- and trans-monounsaturated, cis-cyclopropane, and cis-polyunsaturated fatty acids, underwent chain elongation in vivo. The extent of chain elongation and the average chain length of the primary elongation products correlated well with the physical properties (melting temperatures) of the exogenous fatty acid substrates. The specificity of fatty acid chain elongation in A. laidlawii B maintained the fluidity and physical state of the membrane lipids within a rather wide but definitely limited range. The fatty acid chain elongation system of this organism could be markedly influenced by the presence of a second exogenous fatty acid that was not itself a substrate for the chain elongation system but was incorporated directly into the membrane lipids. The presence of a relatively low-melting exogenous fatty acid increased both the extent of chain elongation and the average chain length of the elongation products generated, whereas the presence of a relatively high-melting fatty acid had the opposite effect. The extent of chain elongation and nature of the elongation products formed were not, however, dependent on the fluidity and physical state of the membrane lipids per se. The second exogenous fatty acid appeared instead to exert its characteristic effect by competing with the chain elongation substrate and elongation products for the stereospecific acylation of positions 1 and 2 of sn-glycerol-3-phosphate. The similar effects of alterations in environmental temperature, cholesterol content, and exposure to the antibiotic cerulenin on the fatty acid chain elongation and de novo biosynthetic activities suggested that the chain elongation system of this organism may be a component of the de novo biosynthetic system.


Phosphonate utilization by bacteria.

Phosphonate utilization by bacteria. Bacteria able to use at least one of 13 ionic alkylphosphonates of O-alkyl or O,O-dialkyl alkylphosphonates as phosphorus sources were isolated from sewage and soil. Four of these isolates used 2-aminoethylphosphonic acid (AEP) as a sole carbon, nitrogen, and phosphorus source. None of the other phosphonates served as a carbon source for the organisms. One isolate, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen, and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. This is the first demonstration of utilization of a phosphonoalkyl moiety as a sole carbon source. Cell-free extracts of P. putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to release orthophosphate from AEP; acetaldehyde was tentatively identified as a second product. Phosphite inhibited the enzyme system.


Selective restriction endonuclease cleavage of human globin genes. Double-stranded human globin DNA synthesized in vitro from sickle cell mRNA has been used as a substrate for a series of restriction endonucleases. The double-stranded DNA contained full length transcripts of the alpha- and beta- globin genes. Of the 10 enzymes tested, only 3 (Hpa I, Sal I, and Kpn I) failed to cleave either alpha- or beta-DNA; 2 (Eco RI and Bam HI) cleaved only beta-DNA; 3 (HindIII, Hpa II, and Hha I) cleaved only alpha-DNA; and 2 (Hae III and Alu I) cleaved both alpha- and beta-DNAs. The selective cleavage of human globin genes by restriction endonucleases should provide a strategy for the identification and purification of DNA fragments of genomic DNA containing globin genes plus their flanking sequences, simplify the preparation of pure, chain-specific globin probes, and permit the isolation of DNA probes for specific regions of the globin genes.


On the tyrosine residues of ribonuclease A. The six 13Czeta resonances of the 6 tyrosine residues of ribonuclease A have been resolved by high field 13C Fourier transform nuclear magnetic resonance spect=oscopy. Titration studies of these resonances reveal 1 nontitrating or buried tyrosine residue, 1 partially buried residue, and 4 normally titrating residues, which are tentatively identified.


Biosynthesis of P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate in calf pancreas microsomes. Calf pancreas microsomes incubated with UDP-N-acetyl-D-[14C] glucosamine in the presence of Mn2+ incorporated radioactivity into P1-2-acetamido-2-deoxy-D-glucopyranosyl P2-dolichyl pyrophosphate and P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate. The formation of both glycolipids was enhanced to the same extent by exogenous dolichyl phosphate. Labeled P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate was formed from synthetic P1-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P2-dolichyl pyrophosphate and from prelabeled pancreatic P1-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P2-dolichyl pyrophosphate without the addition of divalent cation. Upon thin layer chromatography, it had the same mobility as synthetic P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate recently synthesized by Warren et al. (Warren, C. D., Herscovics, A., and Jeanloz, R. W. (1977) Carbohydr. Res., in press), but was different from the synthetic compound prepared by Wedgwood et al. (Wedgwood, J. F., Warren, C. D., Jeanloz, R. W., and Strominger, J. L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 5022-5026).


On the stereospecificity of microsomal "26"-hydroxylation in bile acid biosynthesis. The stereospecificity of microsomal "26" -hydroxylation in bile acid biosynthesis was studied. Cholesterol was biosynthesized from [2-14C] mevalonate by a rat liver preparation. The cholesterol was converted stepwise into 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid by microsomal and soluble fractions of rat liver homogenate. The 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid was decarboxylated chemically and the carbon dioxide was assayed for 14C. The amount of radioactivity in the liberated carbon dioxide was assayed for 14C. The amount of radioactivity in the liberated carbon dioxide was such as to indicate complete stereospecificity of the microsomal "26" -hydroxylase system. The system hydroxylates the methyl group in position C-26 (the 25-pro-R methyl group) and its stereospecificity is opposite that of the mitochondrial "26" -hydroxylase system which hydroxylates the methyl group in position C-27 (the 25-pro-S methyl group).


Kinetics of carbon monoxide binding to singly reduced human methemoglobin. The kinetics of reaction of singly reduced methemoglobin (HbFe3(3+)Fe2+) with carbon monoxide have been investigated by the pulse radiolysis method. The rate constant for carbon monoxide binding to this form of hemoglobin is 4.1 X 10(6) M-1 S-1 at 24 degrees in our solutions. This value compares with existing values for various forms of hemoglobin ranging from 4 X 10(6) to 6.5 X 10(6) M-1 S-1. Addition of inositol hexaphosphate to the solutions results in a lower rate constant for carbon monoxide binding amounting to 1.1 X 10(5) M-1 S-1.


Activation of adenylate cyclase in cultured fibroblasts by trypsin. Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.


Lack of stereospecificity at carbon 6 of methyltetrahydrofolate transport in Ehrlich ascites tumor cells. Carrier-mediated transport of both stereoisomers. Nonlabeled and tritiated stereoisomers of 5-methyltetrahydrofolate were prepared and were both shown to be substrates for the high affinity H4 folate cofactor membrane transport carrier in Ehrlich ascites tumor cells. Both the enzymically active form and the isomer having the opposite configuration at carbon 6 inhibited the influx of enzymically synthesized (+)-5-methyltetrahydrofolate, methotrexate, and aminopterin. When added to the media of cells preloaded with methotrexate, both isomers stimulated a net efflux of the antifolate from the cell. Influx of the natural and unnatural isomers followed Michaelis-Menten kinetics with comparable Km values. Each isomer competitively inhibited influx of the other.


Sequential methylation of globin mRNA in nucleated erythroid cells and reticulocytes of mice. The order of methylation of the 5'-terminus of globin mRNA of mice was studied by incubation of staged nucleated erythroid cells and peripheral reticulocytes with [methyl-3H] methionine. Methylation of the 5'-termini of alpha and beta- globin mRNAs in enucleated reticulocytes was demonstrated as follows: (a) [methyl-3H] incorporation into poly(A)+ RNA of reticulocytes co-migrated with the alpha- and beta- globin mRNAs on gel electrophoresis, and (b) following digestion of this RNA, radioactivity was localized to the four methyl sites at the 5'-capped structure of mouse globin mRNAs. However, this methylation is only 5 to 8% as efficient as in nucleated erythroid precursor cells, suggesting that most globin mRNA molecules are fully methylated prior to the reticulocyte stage. Incubations of early and late nucleated erythroid precursor cells and pulse-chase experiments with reticulocytes demonstrate that addition of the four 5'-terminal methyl groups follows an orderly sequence. In addition, the pulse-chase experiments suggest the turnover of the N7-methyl group on the 5'-terminal guanosine, but not of the other methyl groups in the 5'-terminus of globin mRNA. Thus, 5'-terminal methylation of globin mRNA is a nonrandom, dynamic process.


Human antithrombin III. Carbohydrate components and associated glycolipid. Human antithrombin III was found to contain covalently linked N-acetylglucosamine, mannose, galactose, and sialic acid in a molar ratio of approximately 1:1:0.6:1. Sialic acid was released upon treatment with neuraminidase. The modified glycoprotein retained the capability to inhibit thrombin and to bind with heparin. Antithrombin III isolated by different procedures was also found to contain glucose in an approximately equimolar ratio with N-acetylglucosamine. Th" glucose-containing component was extractable with lipid solvents and shown to be beta-glucosylceramide. This glycolipid is tightly complexed with antithrombin III and could not be separated by fractional precipitations or ion exchange gels. Although it remains to be established whether the inhibitory actions of antithrombin III are affected by glucosylceramide, the relative amounts which are bound suggest that antithrombin III may be a significant carrier of the glycolipid.


Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen. Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.


Quantitation of messenger RNA levels for rat liver 6-phosphogluconate dehydrogenase. Liver poly(A)-containing RNA isolated from rats in different dietary states was translated in a cell free protein synthesizing system employing reticulocyte lysates. Immunoprecipitation of the cell-free reaction products with goat anti-6-phosphogluconate dehydrogenase followed by sodium dodecyl sulfate-urea-gel electrophoresis showed that the induction of this lipogenic enzyme was accompanied by a corresponding increase in the concentration of its specific translatable mRNA.


Biochemical characterization of a mutant asparaginyl-tRNA synthetase from Chinese hamster ovary cells. The biochemical and physical properties of asparaginyl-tRNA synthetase from wild type Chinese hamster ovary cells and a temperature sensitive mutant strain (lys 65a) are compared. The asparaginyl-tRNA synthetase in the mutant strain exhibits a greater temperature lability in vitro, a higher temperature-independent Km for asparagine, and a lower temperature-dependent catalytic capacity than the enzyme from the wild type strain. The mutant enzyme shows no differences in its molecular weight, its Km for tRNAAsn, or its ability to aminoacylate tRNAAsn isoacceptor species compared to the wild type enzyme. These observations, as well as the growth properties of the mutant cells as a function of temperature and exogenous asparagine concentrations, are consistent with their decreased ability to aminoacylate tRNAAsn in vivo.


Use of the integrated steady state rate equation to investigate product inhibition of human red cell adenosine deaminase and its relevance to immune dysfunction. The analysis of progress curves using the integrated rate equation was applied to the adenosine deaminase-catalyzed conversion of adenosine to inosine. Adenosine deaminase was purified from human red blood cells of phenotypes ADA 1, ADA 2, and ADA 2-1. For all three types, no measurable product inhibition by inosine was observed. These results do not confirm the hypothesis that inosine accumulation in purine nucleoside phosphorylase deficiency causes adenosine deaminase inhibition, resulting in a common mechanism for the immune defects related to these two enzyme deficiencies.


Specific adhesion of rat hepatocytes to beta-galactosides linked to polyacrylamide gels. Rat hepatocytes, isolated by a collagenase perfusion technique, specifically bind to polyacrylamide gel containing covalently immobilized 6-aminohexyl beta-D-galactopyranosyl groups. Less than 5% of these cells bind to polyacrylamide or to gels with the following covalently linked ligands: 6-aminohexanol, or the 6-aminohexyl D-pyranosides of alpha-mannose, beta-glucose, beta-2-acetamido-2-deoxyglucose, beta-cellobiose, beta-maltose, or beta-melibiose. Cell binding to beta-D-galactoside gels occurs after a lag period at 37 degrees and 65 to 100% (depending on the cell preparation) of the cells adhere. The duration of the lag period is inversely related to the beta-D-galactoside content of the gel but preincubation of the cells at 37 degrees reduces the lag period. Cell-gel binding is a threshold phenomenon. Adhesion of cells to gels does not occur when the glycoside concentration is less than about 900 nmol per cm2 x 0.25 mm thick gel piece. Above this critical concentration, cell-gel binding occurs and becomes maximal when the concentration is increased by only 20%. If these in vitro results apply to cellular interactions in vivo, they suggest that slight changes in the levels of cell surface or extracellular matrix carbohydrates may profoundly influence the behavior of neighboring cells.


Isolation and partial characterization of an endogenous inhibitor of ceramide glycosyltransferases from rat brain. A heat-stable factor has been extracted from the microsomal membranes of rat brain that inhibits the activities of rat brain ceramide galactosyl- and glucosyltransferases. It is nondialyzable, susceptible to proteolytic enzymes but resistant to DNase and RNase, and has no effect on lysosomal hydrolytic enzymes. The factor could also be prepared from microsomes of bovine, mouse, and rabbit brains and in much lower concentrations, from systemic organs of rats. This inhibitor might play a role in the regulation of ceramide glycosyltransferases in vivo.


Comparison of atypical and usual human serum cholinesterase. Purification, number of active sites, substrate affinity, and turnover number. Atypical and usual human serum cholinesterases were purified and studied with the fluorescent probe, N-methyl-(7-dimethylcarbamoxy)quinolinium iodide. Four active sites per tetramer were found in each enzyme. The turnover numbers of usual and atypical cholinesterases were the same: 15,000 mumol of benzoylcholine hydrolyzed/min/mumol of active site; 48,000 min-1 for o-nitrophenylbutyrate; and 0.0025 min-1 for N-methyl-(7-dimethylcarbamoxy)quinolinium iodide. They had identical rate constants for carbamylation, (5.0 min-1) and for decarbamylation (0.15 h-1). The major difference between the two genetically determined forms of the enzyme was substrate affinity, KD being 0.16 mM for usual and 5.4 mM for atypical cholinesterase, for the fluorescent probe substrate. Km for the uncharged ester, o-nitrophenylbutyrate, was 0.14 mM for both enzymes, whereas Km for benzoylcholine was 0.005 mM for usual and 0.024 mM for atypical cholinesterase. We interpret these data to mean that the two enzymes differ only in the structure of their anionic site.


Human blood group glycosyltransferases. I. Purification of n-acetylgalactosaminyltransferase. An N-acetylgalactosaminyltransferase, which converts blood group O red blood cells to A cells, was purified to homogeneity from plasma of blood group A1 subjects. The enzyme was adsorbed on Sepharose 4B, and after washing out the impurities, the enzyme was eluted with UDP. This procedure resulted in a 70,000- to 100,000-fold increase in specific activity with recovery of about 80%. Further purification of the enzyme was achieved by Bio-Gel P treatment. The final enzyme preparation showed a single protein band, which coincided with enzyme activity, on acrylamide gel electrophoresis, and revealed a single protein band on sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight (90,000 to 100,000), which was estimated by Sephadex gel filtration, and the subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ and had optimum activity at pH 6.5 to 7.0.


Human blood group glycosyltransferase. II. Purification of galactosyltransferase. A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.


Comparative studies of Hb Lepore Boston, Hb A2, and Hb A. Several functional tests were performed to compare Hb A, Hb A2, and Hb Lepore Boston, which has a delta-beta crossover in the region of residues 87 to 116. Oxygen equilibrium curves determined by an automatic apparatus in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees showed that the p50 was 5.8 mm Hg for Hb Lepore Boston, in contrast to 8.1 and 10.3 mm Hg for Hb A2 and Hb A, respectively. The n values (Hill coefficinets) of Hb Lepore Boston and Hb A2 were slightly smaller than that of Hb A. The effect of 2,3-diphosphoglycerate and inositolhexophosphate on the p50 of Hb Lepore Boston and Hb A2 was less than that on the p50 of Hb A. The molecular stability to mechanical shaking of Hb Lepore Boston and Hb A2 showed that the oxy forms of Hb Lepore Boston and Hb A2 denatured at a rate 3 times faster than that of Hb A. MetHb Lepore Boston was more unstable than MetHb A2 to mechanical shaking. These results indicate that, although the molecular stability of Hb Lepore Boston is more similar to that of Hb A2 than that of Hb A, the oxygen-binding properties of Hb Lepore differ from both Hb A and Hb A2.


Nucleotide sequence of rainbow trout (Salmo gairdneri) ribosomal 5.8 S ribonucleic acid. The nucleotide sequence of rainbow trout (Salmo gairdneri) 5.8 S rRNA was determined to be (formula: see text). The major molecular species is 161 nucleotides long; the sequence differs in only eight nucleotides from that of Man. An estimate of the secondary structure retains essentially all of the features previously described in mammalian 5.8 S RNAs with several interesting variations. The very stable G-C-rich hairpin loop is extended by two additional hydrogen bond pairs; the A-U-rich loop is shorter; and a third 2'-O-methylation occurs at residue 8 close to the under methylated U(m) residue at position 14. Comparisons with other known sequences indicate that in eukaryotes 5.8 S rRNA is more highly conserved than 5 S RNA species.


Human erythrocyte 5'-AMP aminohydrolase. Purification and characterization. Human erythrocyte 5'-AMP aminohydrolase has been obtained using phosphocellulose chromatography and affinity chromatography on a GTP-agarose column to yield a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 285,000, and is comprised of four subunits. Since limited quantities of the homogeneous enzyme were available, the kinetic properties of a nonhomogeneous preparation purified about 20,000-fold over the red blood cell lysate by phosphocellulose chromatography were examined. Like the muscle enzyme, it exhibits a sigmoid AMP saturation curve in the absence of activating monovalent cations; a hyperbolic saturation curve is observed in the presence of 0.15 M KCl. Activation by monovalent cations and ATP, and inhibition by Pi, PPi, GDP, GTP, and 2,3-diphosphoglyceric acid were studied in more detail.


Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis. The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.


5,6,7,8-Tetrahydrofolic acid. Conformation of the tetrahydropyrazine ring. It is suggested from analysis of proton spin-spin coupling constants that the tetrahydropyrazine ring of tetrahydrofolate is a roughly equal mixture of two half-chair conformations, one with the C-6 proton axial and the other with the C-6 proton equatorial. The chemical shifts and spin-spin coupling constants for the carbon-bound protons of (+/-)-L-, (-)-L-, and (-)-L-[6-2H] 5,6,7,8-tetrahydrofolate were measured at 25 degrees and at 300 MHZ. The resonances corresponding to the two C-7 protons in the deuterated compound constituted an AB quartet with JAB of 12 Hz and chemical shift difference of 92 Hz or 0.307 ppm; the C-7 protons are proposed to be a geminally coupled axial-equatorial pair whose rapid equilibration does not result in equivalence due to the adjacent chiral center at C-6. The spin-spin splitting in the C-7 resonances were 3.0 and 6.6 Hz for the low field and high field resonances, respectively, reflecting coupling to the C-6 proton. These coupling constants reflect the conformational equilibrium. The resonances assignable to C-9 protons are nearly equivalent in the 6-2H compound, but exhibit the resonances corresponding to a complex spin system in the 6-H compound.


Gene 4 protein of bacteriophage T7. Characterization of the product synthesized by the T7 DNA polymerase and gene 4 protein in the absence of ribonucleoside 5'-triphosphates. DNA polymerase and gene 4 protein of bacteriophage T7 catalyze extensive DNA synthesis on duplex phage T7 or PM2 DNA templates containing single strand breaks. A variety of physicochemical techniques have been used to characterize the DNA product synthesized in this reaction in the absence of ribonucleoside 5'-triphosphates. Pyknographic and sedimentation analyses reveal that all of the newly synthesized DNA is covalently attached to the template DNA. Analysis by electron microscopy shows the major portion of the product molecules synthesized on duplex T7 DNA templates to consist of a double-stranded branch attached to an intact template molecule. Using PM2 DNA templates, the predominant product consists of a double-stranded branch attached to the circular PM2 DNA template. Analyses of these product molecules indicate that DNA synthesis by the gene 4 protein and T7 DNA polymerase is initiated at single strand breaks in the duplex DNA and that synthesis is accompanied by extensive displacement of one of the parental strands. At later times in the reaction, a portion of the 3'-hydroxyl terminus of the newly synthesized DNA is displaced from the template by branch migration and is used as a primer by the DNA polymerase to copy the displaced 5' single-stranded parental strand to form a duplex branch.


Regulation of macronuclear DNA content in Paramecium tetraurelia. The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.


Functional and morphological characterization of isolated bovine adrenal medullary cells. Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% collagenase in Ca++-free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 X 10(6) cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min. Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenaline-containing and (b) noradrenaline-containing cells. Biochemical analysis showed that the cells contained catecholamines and dopamine-beta-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca++ have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.


Resolution of electron microscope autoradiography. IV. Application to analysis of autoradiographs. The previous publications of this series described the expected grain distributions around model radioactive structures in EM autoradiographs as a function of the specimen resolution. This family of expected distributions was called the "universal curves". In the present study, experiments on 14C-sources were compared, significant differences were found depending on the energy of the isotope. These differences were primarily in the tails of the distributions, and are therefore important in correcting for cross-scatter when analyzing electron microscope autoradiographs. Using the universal curves unique for 125I, 3H, and 14C, we designed three sets of transparent overlays, or "masks", one set for each of these isotopes. The masks can be used by an investigator in a manner similar to that suggested by Blackett and Parry to generate grain distributions in autoradiographs on the basis of any desired hypothesis regarding the levels of radioactivity in different structures. A subsequent comparison between these generated distributions and those obtained from the observed grains in these autoradiographs leads to a determination of the most likely levels of radioactivity in the tissue. A computer (described in an Appendix by Land and Salpeter) can be used to find the "best fit" levels of radioactivity in complex cases. The accuracy of the masks was checked on generated line sources for each of the three isotopes.


Lens differentiation. Crystallin synthesis in isolated epithelia from calf lenses. In the calf eye lens, four morphologically distinct cell types can be detected: three in the epithelial monolayer and one in the cortical part. During differentiation, there is a quantitative change in the synthesis of crystallin subunits. A marked increase in alphaA-chains and several beta-crystallin polypeptides accompanies the transition from epithelial to fiberlike lens cells while synthesis of the non-crystallin proteins diminishes significantly.


Isolation of nuclear envelopes with polyanions. Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei are established and a morphological and biochemical study of such isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4 degrees C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outher leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating the pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37 degrees C, in the presence of phosphate ions.


Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells. The formation and fate of apical endocytic vesicles in resting and isoproterenol-stimulated rat parotid acinar cells were studied using luminally administered horseradish peroxidase (HRP) to mark the vesicles. The tracer was taken up from the lumen by endocytosis in small, smooth-surfaces "c"- or ring-shaped vesicles. About 1 h after HRP administration the vesicles could be found adjacent to the Golgi apparatus. At later times HRP reaction product was localized in multivesicular bodies and lysosomes; in isoproterenol-stimulated cells it was also present in autophagic vacuoles. HRP reaction product was never localized in any structure associated with secretory granule formation. These results suggest that the apical endocytic vesicles play a role in membrane recovery, but that they are degraded and not reutilized directly in secretory granule formation. Additionally, it was found that when isoproterenol was injected before HRP administration, the apical junctional complexes became permeable to the tracer, allowing it to gain access to the lateral and basal intercellular spaces. This permeability may provide an additional route whereby substances in the extracellular fluid could reach the saliva.


Alterations in number of protofilaments in microtubules assembled in vitro. Tubulin from bovine brain was polymerized in vitro using a variety of assembly conditions. Many of the formed microtubules were shown to contain 14 wall protofilaments. The number of microtubules containing 14 protofilaments increased with consecutive repetitions of cold-dissociation followed by reassembly in vitro.


Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles. Experiments were performed to localize the hepatic microsomal enzymes of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol biosynthesis to the cytoplasmic or lumenal surface of microsomal vesicles. Greater than 90 percent of the activities of fatty acid-CoA ligase (EC 6.2.1.3), sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15), lysophosphatidic acid acyltransferase, diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2), and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) was inactivated by proteolysis of intact microsomal vesicles. The phosphatidic acid phosphatase (EC 3.1.3.4) was not inactivated by any of the protease tested. Under conditions employed, <5 percent of the luminal mannose-6-phosphatase (EC 3.1.3.9) activity was lost. After microsomal integrity was disrupted with detergents, protease treatment resulted in a loss of >74 percent of the mannose-6-phosphatase activity. The latency of the mannose-6-phosphatase activity was not affected by protease treatment. Mannose-6-phosphatase latency was not decreased by the presence of the assay components of several of the lipid biosynthetic activities, indicating that those components did not disrupt the microsomal vesicles. None of the lipid biosynthetic activities appeared latent. The presence of a protease-sensitive component of these biosynthetic activities on the cytoplasmic surface of microsomal vesicles, and the absence of latency for any of these biosynthetic activities suggest that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. The location of biosynthetic activities within the transverse plane of the endoplasmic reticulum is of particular interest for enzymes whose products may be either secreted or retained within the cell. Phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol account for the vast majority of hepatic glycerolipid biosynthesis. The phospholipids are utilized for hepatic membrane biogenesis and for the formation of lipoproteins, and the triacylglycerols are incorporated into lipoproteins or accumulate within the hepatocyte in certain disease states (14). The enzymes responsible for the biosynthesis of these glycerolipids (Scheme I) from fatty acids and glycerol-3P have all been localized to the microsomal subcellular fraction (12, 16, 29, 30). Microsomes are derived from the endoplasmic reticulum and are sealed vesicles which maintain proper sidedness. (11, 22). The external surface of these vesicles corresponds to the cytoplasmic surface of the endoplasmic reticulum. Macromolecules destined for secretion must pass into the lumen of the endoplasmic reticulum (5, 23). Uncharged molecules of up to approximately 600 daltons are able to enter the lumen of rat liver microsomes, but macromolecules and charged molecules of low molecular weight do not cross the vesicle membrane (10, 11). Because proteases neither cross the microsomal membrane nor destroy the permeability barrier of the microsomal vesicles, only the enzymes and proteins located on the cytoplasmic surface of microsomal vesicles are susceptible to proteolysis unless membrane integrity is disrupted (10, 11). By use of this approach, several enzymes and proteins have been localized in the transverse plane of microsomal membranes (11). With the possible exception of cytochrome P 450, all of the enzymes and proteins investigated were localized asymmetrically by the proteolysis technique (11). By studies of this type, as well as by product localization, glucose-6-phosphate (EC 3.1.3.9) has been localized to the luminal surface of microsomal vesicles (11) and of the endoplasmic reticulum (18, 19). All microsomal vesicles contain glucose-6-phosphatase (18, 19) which can effectively utilize mannose-6-P as a substrate, provided the permeability barrier of the vesicles has been disrupted to allow the substrate access to the active site located on the lumenal surface (4). An exact correspondence between mannose- 6-phosphate activity and membrane permeability to EDTA has been established (4). The latency of mannose-6-phosphatase activity provides a quantitative index of microsomal integrity (4.) Few of the microsomal enzymes in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol have been solubilized and/or purified, and little is known about the topography of these enzymes in the transverse or lateral planes of the endoplasmic reticulum. An asymmetric location of these biosynthetic enzymes on the cytoplasmic or lumenal surface of microsomal vesicles may provide a mechanism for regulation of the glycerolipids to be retained or secreted by the cell, and for the biogenesis of asymmetric phospholipid bilayers. In this paper, we report investigations on the localization of all seven microsomal enzymes (Scheme I) in the biosynthesis of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, using the protease technique with mannose-6-phosphatase serving as luminal control activity. The latency of these lipid biosynthetic enzymes was also investigated, using the latency of mannose-6-phosphatase as an index of microsomal integrity.


Adhesion and detachment characteristics of Chinese hamster cell membrane mutants. We have investigated the adhesion and detachment properties of wild-type Chinese hamster cells and of variant lines, which possess altered cell surface glycoproteins as detected by galactose oxidase-[3H]borohydride labeling. The wild-type and variant lines tested all adhered to protein-coated glass surfaces at the same rate; however, the variant cells differed from wild type and from each other in terms of the ease with which they were detached by trypsinization. Morphological differences between the various lines were also apparent. Our results suggest that the carbohydrate moieties of the terminal region of surface glycoproteins are not directly involved in the initial phase of cell-to-substratum attachment, but that they may modulate the proteolytic susceptibility of surface components which are involved in cell detachment.


Observations on the disassembly of isolated mammalian neurofilaments. Intact mammalian neurofilaments were separated by centrifugation of osmotically shocked, desheathed segments of rat peripheral nerve. Neurofilament-rich supernates were incubated in different media with varying dilutions or dialysis of samples. Neurofilaments attached to carbon-Formvar-coated grids were exposed to similar incubations. The relative preservation or disruption of neurofilaments during different incubational conditions was monitored through periodic examinations of neurofilaments by negative staining techniques. Maximum structural stability of neurofilaments was manifested during incubation in isotonic NaCl or KCl. Decreasing salinity of incubational media led to increasing disruption of neurofilaments, especially in solutions less than 0.01 M. Hypertonic saline solutions were found to be less disruptive to mammalian neurofilaments. Additional studies examined neurofilamentous alterations effected by pH, protein denaturants, mitotic spindle inhibitors, reducing agents, and freeze-thawing procedures.


A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis. What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.


Particle arrays in earthworm postjunctional membranes. Analysis of freeze-fractured earthworm body wall muscle reveals distinctive trough-shaped concavities in the protoplasmic leaflet of the muscle cell membrane which contain diagonally oriented rows of particles sometimes in highly ordered arrays. The troughs correspond to the concave postjunctional patches of sarcolemma seen previously in thin sections of myoneural junctions identified as cholinergic, and the intramembranous particles within the troughs correspond in concentration and arrangement to granular elements present in the outer dense lamina of the postjunctional membrane which were interpreted as acetylcholine receptors. The freeze-fracture data provide a more accurate picture of the arrangement of these putative receptors within the plane of the membrane, and indicate also that they extend into the membrane at least as far as its hydrophobic layer.


Passage of serum-destined proteins through the Golgi apparatus of rat liver. An examination of heavy and light Golgi fractions. The participation of hepatic Golgi apparatus in the intracellular transport of blood-destined proteins has been analyzed using Golgi fractions enriched in cis and trans components of the Golgi apparatus. SDS-polyacrylamide gel electrophoresis of the liver Golgi fractions showed several proteins corresponding in relative proportions and mobilities with serum proteins. After a pulse injection of labeled leucine, the secretory content of the cis Golgi fraction was labeled earlier than the trans Golgi fraction. Taken together, the results show the participation of the liver Golgi apparatus in the secretion of most of the serum proteins and provide documentation for a sequential progression of secretory protein through the cis and trans components of the Golgi apparatus.


Studies on phosphoglucose isomerase from cultured human fibroblasts: absence of detectable ageing effects on the enzyme. Some properties of the enzyme phosphoglucose isomerase (PGI) from the foetal lung fibroblast strain MRC-5 have been investigated throughout the in vitro lifespan of this cell strain. No significant age-related alterations in specific activity or thermostability of PGI could be detected. Titration of enzymatic activity with antibody directed against purified PGI showed no detectable differences in PGI from extracts of early passage cells compared with enzyme from senescent cells. The effect of p-fluorophenylalanine incorporation on PGI was examined in early passage fibroblasts. Thermostability studies showed increased heat lability of PGI from analogue treated cells when compared with enzyme from control cells at the same passage. However, no inactive PGI protein could be detected by anti-serum titration in extracts from analogue-treated cells. The results indicate that no significant amount of altered or inactive PGI is produced in ageing fibroblasts.


Reversible regulation by magnesium of chick embryo fibroblast proliferation. The rate of 3H-thymidine incorporation and of cell proliferation in chick embryo fibroblast cultures are reduced coordinately when the [Mg2+] of the external medium is reduced below the physiological concentration of about 0.8 mM. These effects of moderately reduced [Mg2+] and the accompanying change in appearance of the cells, resemble the effects produced by lowering the [serum] of the medium. Cells subjected to severe Mg2+ deprivation, especially at low [Ca2+], die and detach from the culture dish. Cells kept at a reduced rate of proliferation for three days by moderate Mg2+ deprivation are quickly restored to rapid proliferation upon restoration of the normal [Mg2+] of the medium. The rate of proliferation of the chick embryo cells is reduced markedly by lowering [Ca2+] about 100-fold, but unlike the case of Mg2+-deprivation this can occur without significant effect on the rate of 3H-thymidine incorporation. More severe Ca2+ deprivation, which does lower the rate of 3H-thymidine incorporation, produces retraction of cells from one another and from the dish, and results in a distinctly abnormal, rounded appearance. The results lend weight to the thesis that free [Mg2+] plays a central role within the cell in the coordinate control of metabolism and growth. They also suggest that the effects produced by varying [Ca2+] in the medium are caused by changes at the external surface of the cell.


Polyamine metabolism in regenerating livers from normal and hypocalcemic rats. There is a marked increase in the concentration of putrescine during the first ten hours following partial hepatectomy in rats. The concentration of spermidine also increases but to a smaller degree. Putrescine levels return to normal between 10 and 24 hours after the operation, whereas the increased spermidine level is maintained. The production of putrescine and spermidine appears to be initiated by the induction of ornithine decarboxylase which shows a single peak of activity at four hours after hepatectomy. The activity of S-adenosylmethionine decarboxylase shows little change following hepatectomy. The changes in polyamine levels and the activities of the enzymes of polyamine metabolism are not affected by thyroparathyroidectomy 72 hours prior to hepatectomy. Thus although these hypocalcemic conditions considerably reduce and delay DNA synthesis and mitosis, the prereplicative changes in polyamine metabolism still occur. These data suggest that the hepatocytes in hypocalcemic animals have become activated and moved to an advanced stage of prereplicative development before being blocked.


Arginase activity and other cellular events associated with epidermal hyperplasia. The unknown biochemical role of arginase in epidermal metabolism was probed by examining the association of elevated arginase activity with epidermal hyperplasia and hyperkeratinization. Epidermal hyperplasia was induced experimentally by topical application of 1-decanol to the right side of male hairless mice while the contralateral side served as control. Arginase activity, incorporation of 3H-thymidine into DNA, DNA and protein content were measured in the separated control and experimental epidermis six hours and on days 1 through 5 and 7 after 1-decanol application. After six hours, the epidermis appears damaged histologically, and DNA synthesis is inhibited. By day 1, incorporation of 3H-thymidine into DNA is elevated and a new hyperplastic epidermis has formed beneath the original epidermis. Epidermal arginase is elevated two through seven days after 1-decanol application and always is associated with continuing epidermal hyperplasia. The stimulation of DNA synthesis, which parallels the induction of epidermal hyperplasia by 1-decanol, precedes the induction of epidermal arginase activity. An attempt to relate these results with polyamine synthesis and other metabolic events is made.


Antibody-dependent cellular cytotoxicity in primary immunodeficiency diseases and with normal leukocyte subpopulations. Importance of the type of target. To gain insight into a possible role for antibody-dependent cell-mediated cytotoxicity in vivo, we examined the ability of leukocytes from 28 patients with primary immunodeficiency and from 20 normal controls to lyse three different types of antibody-coated targets in vitro. Mean cytotoxic indices +/-1 SD elicited by unfractionated mononuclear cells from normal controls were 28.74+/-13.26 for human HLA antibody-coated lymphocyte targets, 42.79+/-8.27 for rabbit IgG antibody-coated chicken erythrocyte targets, and 47.58+/-10.34 for human anti-CD (Ripley)-coated O+ erythrocyte targets. Significantly (P=<0.05) lower than normal mean cytotoxic indices against lymphocyte targets were seen with effector cells from 10 patients with X-linked agammaglobulinemia (3.7+/-4.33), in 10 with common variable agammaglobulinemia (16.05+/-7.74), in 3 with immunodeficiency with hyper IgM (18.41+/-4.88), and in 2 with severe combined immunodeficiency (3.94+/-0.3). Antibody-dependent cytotoxicity against chicken erythrocytes was significantly (P=<0.05) lower than normal only in the common variable agammaglobulinemic group (33.33+/-12.3) and against human erythrocytes only in the common variable (34.36+/-9.59) and hyper IgM (27.54+/-0.66) groups. Rosette and anti-F(ab')(2) depletion studies with normal leukocytes indicated that a nonadherent, nonphagocytic, non-Ig-bearing, non-C receptor-bearing, Fc receptor-bearing lymphocyte was the only effector capable of lysing HLA antiboyd-coated lymphocyte targets. Patients with infantile X-linked agammaglobulinemia and severe combined immunodeficiency appear to have a marked deficiency in this type of effector cell function.


Effects of glyceraldehyde on the structural and functional properties of sickle erythrocytes. The d- and l-isomers of glyceraldehyde are equally effective in the inhibition of SS erythrocyte sickling in vitro. The following compounds at a concentration of 20 mM were ineffective in inhibiting sickling: glyceraldehyde-3-phosphate, d-erythrose, d-ribose, d-fructose, d-glucose, d-sucrose, dihydroxyacetone, and methylglyoxal. Glyceraldehyde does not reverse the sickling of cells in the deoxy state. The properties of purified hemoglobin after treatment with glyceraldehyde and of the hemoglobin isolated from treated cells are very similar; these results suggest that glyceraldehyde itself is the reactive species within the erythrocyte. Erythrocyte glutathione is decreased by treatment in vitro with the aldehyde. Relatively high concentrations of glyceraldehyde (50 mM) lead to a small amount (3%) of cross-linking between hemoglobin monomers as well as to some cross-linking of erythrocyte membrane proteins. Lower concentrations of dl-glyceraldehyde (5-20 mM), which reduce the sickling of erythrocytes in vitro, lead to proportionally less cross-linking of hemoglobin. Cells that have been treated with those concentrations of the aldehyde show little change in their osmotic fragility, exhibit improved filtration properties, and have a lowered viscosity.


Binding and uptake of transcobalamin II by human fibroblasts. We have used purified, (125)I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism-designated cbl C-in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4 degrees C reached a plateau after 3-4 hr; 95% of the bound (125)I was releasable with trypsin. Binding of TC II-Cbl at 4 degrees C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at congruent with5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'- tetraacetic acid. At 37 degrees C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound (125)I. After 2 h at 37 degrees C, increasing amounts of acid-soluble (125)I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.


Hepatic accumulation and intracellular binding of conjugated bilirubin. After the intravenous injection of unconjugated [(3)H]bilirubin into normal Sprague-Dawley and Wistar R rats, radiolabeled bile pigments rapidly accumulated in the liver. By 1.5 min after injection, an average of 36% of the injected isotope was present in liver homogenates. Between 3 and 15 min, 37-64% of the total intrahepatic radiolabeled bilirubin was conjugated, as demonstrated by extraction of label into the polar phase of a solvent partition system. This indicates both rapid conjugation, and accumulation of conjugated bilirubin within the liver cell. Fluorometric determination of the dissociation constants of purified bilirubin and its mono- and diglucuronides for homogeneous preparations of two human and four rat glutathione S-transferases, including ligandin, revealed avid binding of all three bile pigments to this class of proteins. Hence, the observation that the intrahepatic bile pigment pool contains substantial amounts of conjugated bilirubin can be attributed to the high binding affinities observed. Thin-layer chromatographic analysis of the (3)H-pigments produced by p-iodoaniline diazotization of homogenates and cytosol demonstrated that the intrahepatic pool of conjugated bilirubin was almost exclusively monoglucuronide. Examination of radiolabeled bilirubin conjugates excreted in bile during the first 20 min after injection of [(3)H]bilirubin showed no preferential excretion of diglucuronide. These studies indicate that (a) both bilirubin and its monoglucuronide accumulate within the liver cell as ligands with the glutathione S-transferase; and (b) bilirubin diglucuronide does not significantly accumulate within the general intrahepatocellular pool of protein-bound bile pigments. The latter observation is compatible with the formation and excretion of bilirubin diglucuronide directly from the canalicular pool of the liver cell.


Familial hyperglycerolemia.

Familial hyperglycerolemia. A 70-yr-old mildly diabetic white male was discovered to have an elevated level of serum free glycerol in the range of 75 mg/dl and to excrete about 13 g of free glycerol in the urine per 24 h. During a 24-h fast the urine glycerol loss increased to 21.5 g per 24 h. Studies carried out in vitro using leukocytes prepared from the patient's blood which were incubated with [14C]glycerol demonstrated an almost complete absence of glycerol oxidation to 14CO2 and of glycerol phosphorylation, in contrast to control studies with leukocytes collected from normal subjects. Homogenates of the patient's leukocytes contained negligible activity of ATP:glycerol phosphotransferase (glycerokinase EC 2.7.1.30) as measured by a direct spectrophotometric method. Marked hyperglycerolemia has thus far been detected in one brother and in one son of the daughter of this patient. This evidence suggests an x-linked recessive inheritance pattern of the trait. There is a high prevalence of diabetes mellitus in this family.


Evidence that the severity of depletion of inorganic phosphate determines the severity of the disturbance of adenine nucleotide metabolism in the liver and renal cortex of the fructose-loaded rat. To test the hypothesis that in both the liver and renal cortex of the fructose-loaded rat, severity of depletion of inorganic phosphate (P(i)), and not the magnitude of accumulation of fructose-1-phosphate (F-1-P), determines the severity of the dose-dependent reduction of ATP, we intraperitoneally injected fed rats with fructose, 20 and 40 mumol/g, alone, and at the higher load, in combination with (a) sodium phosphate, 20 mumol/g, administered shortly beforehand or subsequently or, (b) adenosine, 2 mumol/g, administered beforehand. The following observations were made: (a) With fructose loading alone, at the higher load, both P(i) and total adenine nucleotides (TAN) were reduced by one third in the renal cortex and (as previously observed) by two thirds in the liver; and at either load, the reduction of ATP (and TAN) and the accumulation of F-1-P were less severe in the renal cortex than in the liver. (b) Prior phosphate loading largely prevented the reductions of ATP and TAN in the renal cortex and significantly attenuated them in the liver, yet doubled the renal cortical accumulation of F-1-P. (c) Adenosine loading substantially attenuated the reductions of ATP, TAN, and P(i) only in the renal cortex. (d) ATP varied directly with P(i) (P < 0.001, r = 0.98) in the domain of control and reduced values of P(i) taken from both liver and renal cortex. (e) As judged from tissue and plasma concentrations of fructose and glucose, and tissue concentrations of fructose-6-phosphate and glucose-6-phosphate, the rate at which the renal cortex and liver converted fructose to glucose was much lower at the higher fructose load. (f) Prior phosphate loading prevented this decrease in rate in the renal cortex and attenuated it in the liver; adenosine loading attenuated it only in the renal cortex. We conclude that in both the renal cortex of the fructose-loaded rat: (a) Depletion of P(i) is critical to the causation of the reductions in both ATP and TAN and, at the higher fructose load, of a decrease in the rate at which ATP is regenerated. (b) The severity of depletion of P(i) determines the severity of these disturbances. (c) By differentially mitigating the depletion of P(i), prior phosphate loading largely prevents these disturbances in the renal cortex, and attenuates them in the liver; and adenosine loading attenuates them only in the renal cortex. The findings provide some basis for the observation that in patients with hereditary fructose intolerance experimentally exposed to fructose, prior loading with sodium phosphate substantially attenuates the renal but not hepatic dysfunction.


Evidence for a parathyroid hormone-dependent influence of calcium on the glomerular ultrafiltration coefficient. Experiments were performed on 36 plasma-expanded Munich-Wistar rats to examine the effects of acute hypercalcemia on the determinants of glomerular ultrafiltration. Elevation of total plasma calcium concentration to an average value of 13.2 +/- 0.5 mg/dl, by acute infusion of calcium chloride into nonthyroparathyroidectomized (non-TPTX) rats, resulted in significant declines in single nephron and whole kidney glomerular filtration rate. These declines were due primarily to a fall in the glomerular capillary ultrafiltration coefficient (Kf), to a mean value approximately 60% below that determined in the pre-infusion period. These changes were not seen in a separate group of sham-treated non-TPTX rats. It is of interest that these effects of acute hypercalcemia were largely abolished in rats that underwent acute TPTX before hypercalcemia. Infusion of a submaximally phosphaturic dose of parathyroid hormone, together with calcium chloride, into a second group of acute TPTX rats, however, had the effect of reproducing the striking declines in filtration rate and Kf noted in non-TPTX rats given calcium chloride alone. These findings suggest that the decline in filtration rate associated with hypercalcemia is due largely to the reduction in Kf, the latter dependent upon the presence of parathyroid hormone.


In vitro action of bombesin on amylase secretion, membrane potential, and membrane resistance in rat and mouse pancreatic acinar cells. A comparison with other secretagogues. Bombesin caused depolarization of rat or mouse pancreatic acinar cell membrane, reduction of membrane resistance, and a steep rise in amylase output from superfused pancreatic fragments. These effects were similar to those previously described for acetylcholine, cholecystokinin, and gastrin. The dose-response curves for these three effects of bombesin were very similar, with effects being detectable at concentrations of about 30 pM and maximal effects at about 10 nM. The equilibrium potential for the membrane action of bombesin, i.e., the membrane potential at which bombesin did not cause any change in membrane potential, was -16 mV. Similar values for equilibrium potential were obtained with acetylcholine, caerulein and pentagastrin. Bombesin in the higher dose range (10 nM) caused electrical uncoupling of acinar cells within an acinus, i.e., a marked increase in junctional membrane resistance. Similar uncoupling effects were observed after acetylcholine, caerulein, and pentagastrin stimulation. In conclusion, bombesin acts on the pancreatic acinar plasma membrane in exactly the same way as acetylcholine and cholecystokinin-pancreozymin. The electrical uncoupling caused by stimulation is evidence for an increase in cytosol free calcium ion concentration.


Permselectivity of the glomerular capillary wall. Facilitated filtration of circulating polycations. To examine the electrostatic effects of fixed negative charges on the glomerular capillary wall, polydisperse [(3)H]DEAE dextran, a polycationic form of dextran, was infused into 10 Munich-Wistar rats. Fractional clearances of DEAE ranging in radius from 18 to 44A were determined in these rats, together with direct measurements of the forces and flows governing the glomerular filtration rate of water. These results were compared with data previously obtained in Munich-Wistar rats receiving tritiated neutral dextran (D) and polyanionic dextran sulfate (DS). Measured values for the determinants of the glomerular filtration rate of water in rats given DEAE were found to be essentially identical to those in rats given either D or DS. In addition, DEAE was shown to be neither secreted nor reabsorbed. Fractional clearances of polycationic DEAE were increased relative to both D and DS, the increase relative to D being significant for effective molecular radii ranging from 24 to 44A. Fractional DEAE clearances were also measured in a separate group of six Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). Fractional DEAE clearances in NSN rats were reduced significantly, relative to values measured in normal rats, for effective DEAE radii ranging from 18 to 42A. Moreover, in NSN rats, fixed negative charges on the glomerular capillary wall were greatly reduced, relative to non-NSN rats, as evidenced by a reduction in intensity of colloidal iron staining. Thus, in NSN rats, DEAE clearances were essentially indistinguishable from values obtained with both neutral D and polyanionic DS.


Humoral and cellular sensitivity to collagen in type II collagen-induced arthritis in rats. We have recently described a new animal model of arthritis induced by intradermal injection of a distinct type of collagen found in cartilage (type II collagen). Since immunologic sensitivity to collagen could play a role in the pathogenesis of this type II collagen-induced arthritis in rats, the ability of purified types of native collagens to induce cellular and humoral responses was quantified by antigeninduced tritiated thymidine incorporation into lymphocytes by collagen and passive hemagglutination, respectively. Rats injected intradermally with native heterologous or homologous type II collagens in adjuvant developed type-specific cellular as well as humoral reactivity. Types I and III collagens were less immunogenic than was type II. The latter collagen induced brisk cellular and humoral responses that were equivalent whether complete Freund's adjuvant or incomplete Freund's adjuvant were employed. Both responses could be induced by native type II collagens modified by limited pepsin digestion, indicating that they are not attributable to determinants in the telopeptide regions of the molecule. Thus, these studies demonstrate the unique immunogenic as well as arthritogenic properties of the type II collagen molecule and indicate that both result from a helical conformation of its structurally distinct alpha-chains. Further, they suggest that type II collagen may, by humoral or cellular mechanisms, provoke or perpetuate inflammation in other arthritic diseases.


Bence Jones proteins and light chains of immunoglobulins. XV. Effect of corticosteroids on synthesis and excretion of Bence Jones protein. The effect of corticosteroids and cytotoxic chemotherapeutic agents on the excretion of Bence Jones protein was determined for periods of 1 - 62 mo in 29 patients with multiple myeloma and Bence Jones proteinuria. The amount of protein present in 24-h urine specimens collected before treatment and at frequent intervals during monthly treatment cycles was determined. Striking variations occurred in the amount of Bence Jones protein excretion; these changes were especially evident when 75 mg of prednisone were given daily for 7 days as part of a monthly chemotherapeutic regimen. Within the 7-day period seven patients showed essentially no decrease (<25%), whereas 13 and 9 patients had a moderate decrease (25-75%) or a marked decrease (>75%), respectively, in Bence Jones proteinuria as compared to pre-treatment values. The decrease in excretion of Bence Jones protein during this period was attributed mainly to corticosteroid therapy because of the transient nature of the response in most patients and the lack of such response in three patients when the hormone was omitted. Biosynthetic studies were performed to determine in vitro the effect of corticosteroids on Bence Jones protein synthesis. Plasma cells obtained from the bone marrow of 13 patients were incubated in a growth medium containing (14)C-labeled lysine and isoleucine and prednisone in concentrations up to 240 mug/ml, and the amount of Bence Jones protein synthesized was determined immunochemically. No differences in viability were apparent between untreated and prednisone-treated cells. The type of response exhibited by an individual patient in the percent decrease of Bence Jones protein excreted after 7 days of prednisone treatment was comparable to the percent decrease in newly-synthesized Bence Jones protein secreted by tumor cells when cultured in the presence of prednisone at a concentration of 120 mug/ml. The marked differences in the capacity of corticosteroids to affect Bence Jones protein synthesis appear to reflect a biochemical heterogeneity among plasma cell neoplasms.


Relationship of serum and myocardial digoxin concentration to electrocardiographic estimation of digoxin intoxication. Serum and myocardial digoxin levels were studied in 18 patients who came to autopsy. An independent analysis of electrocardiograms prior to death was made to ascertain the relationship between serum and tissue levels of digoxin and clinical estimation of drug toxicity. Patients with arrhythmias of digoxin toxicity had higher mean serum and tissue digoxin levels than patients without arrhythmia. There was overlap in the patient groups, however, and the differences were not statistically significant. The tissue to serum ratio was lower in the toxic patients. The latter phenomenon is unexplained but may be related to decreased tissue binding.


Connections of the visual cortex in the hedgehog (Paraechinus hypomelas). II. Corticocortical projections. Cortical subdivisions based on cytoarchitectural and myeloarchitectural observations of normal tissue were correlated with the topography of corticocortical connections in the visual system of the Pakistani hedgehog. Large subpial aspiration lesions were made in both visual and non-visual cortical regions to determine the areal limits of the corticocortical connections of the visual cortex. Subsequently, discrete electrolytic lesions were placed within the visual cortex. After appropriate survival periods, the brains were processed and stained with the Fink-Heimer technique. The results of these studies show that the visual cortex may be subdivided into four distinct regions from lateral to medial; the lateral parastriate cortex, the lateral and medial part of striate cortex, and the medial parastriate cortex. Within these regions, interhemispheric connections between visual cortices arise mainly in the lateral striate and lateral parastriate regions and terminate in a single band within the lateral portion of the cytoarchitecturally defined striate cortex. These corticocortical projections, therefore, substantially overlap with the geniculostriate projections. Lateral striate cortex and lateral parastriate cortex project in a reciprocal fashion that correlates well with the physiologically defined mirror image representation of two retinotopic maps of the binocular visual field on cortex. These connections are reflected about a line that is closely correlated with the medial edge of the band of commissural axon terminals that is located within the lateral striate cortex, instead of corresponding exactly with the striate-parastriate border as they do in other mammals. Medial striate cortex projects to medial parastriate cortex, indicating that the monocular portion of V I is related to a separate secondary area of cortex on the medial wall of the hemisphere.


Interlaminar connections of the visual cortex in the hedgehog (Paraechinus hypomelas). The contribution of each cortical lamina to intracortical circuitry was studied in the visual neocortex of the Pakistani hedgehog. Punctate laminar lesions were made electrolytically within the visual cortex, and after five to seven days the brains were processed and stained with the Fink-Heimer technique. The results of this study suggest that both horizontal and vertical connections are important to the organization of visual cortex in the hedgehog. The horizontal projections originate at three distinct cortical depths. Lesions in layer II reveal projections that traverse the inner one-half of layer I; lesions in layers III and IV reveal projections that traverse layer IV and that enter the cortical white matter; lesions in layers V and VI reveal projections that traverse the outer one-half of layer VI and that enter the cortical white matter. The vertical projections are oriented perpendicular to the pial surface. Layers II and III project to underlying layer V. Layers V and VI in turn contribute to a reciprocal projection back to all superficial cortical laminae. This anatomical data about intracortical circuitry is discussed with reference to the functional organization of visual cortex.


Different patterns of cochlear innervation during the development of the kitten. The postnatal development of neuro-epithelial junctions inside the kitten cochlea has been investigated by electron microscopy, and correlated with previous electrophysiological results. Two main stages of development are described. During the first postnatal days, outer hair cells look very immature with only a few afferent endings adjoining them. The inner hair cells, on the other hand, are surrounded by numerous endings  with mature afferent and efferent synapses. Thus, when the efferent olivo-cochlear system begins to function during the first postnatal day s, it is able to modify only inner hair cell responses. The second postnatal week is characterized by maturation of the large efferent endings below the outer hair cells. At the same time, direct efferent connections become sparce at the level of inner hair cells. The maturation of hearing, at the receptor level, seems to proceed in two steps, one related to inner hair cells and corresponding to a gross and primitive hearing, the other related to outer hair cells and corresponding to more precise and discriminative hearing abilities.


Chemosurgery: microscopically controlled surgery for skin cancer--past, present and future. The development of microscopically controlled surgery, the use of the fixed-tissue technique for advanced, complicated carcinomas and for malignant melanomas, the use of the fresh-tissue technique for less advanced carcinomas, the five-year results, and plans for the future of the method are discussed.


Microscopically controlled excision of malignant neoplasms on and around eyelids followed by immediate surgical reconstruction. Mohs' fresh-tissue technique of ablating malignant neoplasms is a highly reliable, yet conservative method of treating cutaneous cancers. Removal of such lesions on eyelids by this technique, followed by immediate reconstruction is a most efficacious way to manage them. Forty-seven cases of recurrent, invasive, or large carcinomas on eyelids and canthi were excised by this method followed by immediate reconstruction. The excisions were done by dermatologists specializing in Mohs' techniques and the reconstruction by an oculoplastic surgeon. The advantages of the combined procedures are: 1) high cure rate, 2) conservation of tissue, 3) reduced operating time, and 4) maximum utilization of specialized competence.


A case of basal-cell carcinoma in a patient with the Wyburn-Mason syndrome. A young man with the Wyburn-Mason syndrome was found to have a large basal-cell carcinoma. It is not known if the lesion was merely coincidental or in some way related to this rare anomaly.


Sclerosing epithelial hamartoma.

Sclerosing epithelial hamartoma. Two cases of sclerosing epithelial hamartoma are presented. This recently described benign fibroepithelial tumor needs to be distinguished from trichoepithelioma, syringoma and morphea-like epithelioma. The distinctive clinical and histologic features are discussed.


The promotion of wound healing following chemosurgery (Mohs' technique) by dressings with a lotion of benzoyl peroxide. A lotion of benzoyl peroxide was applied postoperatively to chemosurgical wounds in a small group of patients. The technique of utilizing dressings of benzoyl peroxide is described and illustrated. Enhanced wound healing was observed in all cases. The effects appear to be due, at least in part, to antimicrobial activity, debriding activity, hyperbaric oxygenation, and stimulation of granulation.


IgE antibody response to mite antigen in the mouse. Suppression of an established IgE antibody response by chemically modified antigen. The antibody response to mite antigen in several mouse strains was studied. BALB/c, CBA, C3H/He, and C57B1 strains showed good responses against mite antigen. The AKR strain, on the other hand, showed a relatively poor response. When BALB/c mice were immunized with DNP-mite conjugate in aluminum hydroxide gel (alum), anti-DNP IgE antibody and IgG1 antibody were induced. When these mice were boosted with mite antigen alone in alum, both anti-mite IgE antibody and IgG1 antibody were induced, although these antibodies were not observed after the first immunization. Both IgE antibody responses were high and persistent. Mite antigen was denatured by alkylation and reduction in the presence of 8 M urea. Native antigen remaining in the chemically denatured antigen was removed by an immunoadsorbent method. The modified mite antigen could stimulate the carrier-specific helper cells, at least when it was injected with alum. It was also found that repeated injections of the modified mite antigen resulted in the suppression of an established IgE antibody response against mite antigen. These findings suggest possible clinical application for hyposensitization therapy.


Allergy to insects in Japan. I. The reaginic sensitivity to moth and butterfly in patients with bronchial asthma. A high frequency of reaginic sensitivity to moth and butterfly extracts was shown in 66 randomly selected patients with bronchial asthma. Thirty-seven (56.1%) of these 66 patients showed positive intradermal reactions to 1:10,000 dilution of moth allergen and 34 patients (51.5%) had positive reactions to 1:10,000 butterfly allergen. These positive responses were as frequent as those to Candida or house dust which are recognized as the most commonly reactive allergens in Japan. Radioallergosorbent tests (RAST) showed that 20 of 66 sera were positive to moth and 22 sera were positive to butterfly, which indicates that about 1/3 of patients with bronchial asthma had specific IgE to moth and butterfly in the sera. Bronchial testing revealed that skin reaction-positive and RAST-positive patients were commonly provoked by specific allergen but skin reaction-negative and RAST-negative patients were not. These data suggest that moth and butterfly emanations could cause allergy-induced bronchial asthma in certain patients. Since moths are attracted readily to artificial lights and often fly into houses, these insects are especially suspect as important factors in extrinsic asthma.


Adrenergic responses of asthmatic and normal subjects to submaximal and maximal work levels. The response of the adrenergic system of asthmatic subjects to exercise and the role of plasma catecholamines in exercise-induced asthma were investigated. Plasma levels of norepinephrine and epinephrine were measured at rest, during and after exercise in 7 asthmatic and 9 matched normal subjects. Exercise-induced bronchospasm occurred in all asthmatic subjects following exercise, while no significant change was observed in the normal subjects. The results showed that plasma levels of norepinephrine and epinephrine at rest and changes that occcurred during and after exercise were similar in both normal and asthmatic subjects. These data suggest that the adrenergic response of asthmatics to the same relative exercise stress as reflected in plasma catecholamine levels does not differ from that of normal subjects. It appears that changes in the circulating catecholamines do not play a significant role in the pathogenesis of exercised-induced asthma.


Comparison of immune reactivity to polyvalent monomeric and polymeric ragweed antigens. The proteins of an aqueous extract of ragweed pollen (RW) were precipitated at 90% saturation with (NH4)2SO4, solubilized, and sieved through Sephadex G-15. The excluded fraction, termed the monomeric form (MRW), was cross-linked with glutaraldehyde to form polymerized ragweed (PRW). As compared with MRW, PRW is more immunogenic in rabbits in the production of antibody against RW antigen E (AgE). MRW and PRW resulted in equal peak reaginic responses in rabbits but the duration of reagin production was longer after immunization with MRW than with PRW. Both MRW and PRW have exposed antigenic determinants of AgE but PRW had approximately 1/3 the number as shown by neutralization of human antibody against RW AgE. PRW had 100-to 1,000-fold less human cutaneous reactivity than MRW. PRW may be a more easily effective therapeutic agent for human RW immunotherapy than currently used aqueous extracts and may be more readily obtainable than polymerized AgE.


Reappraisal of intracutaneous tests in the diagnosis of reaginic allergy. In an attempt to determine the clinical relevance of positive intracutaneous tests when epicutaneous tests are negative, 34 patients with symptoms of perennial rhinitis who had negative epicutaneous but positive intracutaneous tests were evaluated by radioallergosorbent (RAST) test, nasal provocation (NP) tests and leukocyte histamine release (LHR) assay. Nineteen patients with perennial rhinitis who had positive epicutaneous tests and 13 normal healthy volunteers were also studied as positive and negative controls, respectively. None of the 34 patients with negative epicutaneous but positive intracutaneous tests had positive RASTs or LHR assays, but 1 patient had a positive NP test. In contrast, of the 19 patients with positive epicutaneous tests, 12 patients had positive RASTs, 17 had positive LHR assays, and 17 had positive NP tests. In the negative control group of 13 subjects, none had positive RAST, LHR assays, or NP tests, although 3 subjects showed positive intracutaneous tests. These results indicate that when epicutaneous tests are negative in patients with perennial rhinitis, positive intracutaneous tests are not likely to indicate the presence of reaginic allergy.


The estrogen--cancer controversy.

The estrogen--cancer controversy. Postmenopausal women should not be denied estrogen therapy because of the controversy about the alleged causal relationship of estrogens to endometrial cancer. A prestigious team of biostatisticians reviewed three publications that claimed this relationship and concluded that no such association could be confirmed. Although the incidence of endometrial cancer has doubled in the past 25 years, there is no valid basis for the assumption that the widespread use of estrogens is the cause. The incidence also has increased in Norway and Czechoslovakia where estrogens are rarely used. Moreover, the histologic pattern of endometrial dysplasias is such that many pathologists will make a diagnosis of endometrial cancer as a safeguard for the patient. Thus the statistics for the incidence of endometrial cancer may be inflated. As a test, the administration of 200 mg of an oral progestogen over 5-10 days will convert a histologic pattern of pseudomalignancy, but not of adenocarcinoma, into one of secretory endometrium. Unopposed endogenous or exogenous estrogens may induce hyperplasia, atypical adenomatous hyperplasia or adenocarcinoma in women who are genetically predisposed. To minimize such a possibility, it is advisable to give a 5-7 day course of an oral progestogen so that the endometrium will be shed as monthly intevals. The benefits of hormone replacement therapy for the postmenopausal woman far outweigh the putative risk of endometrial cancer.


Renin and the juxtaglomerular apparatus in diabetic nephropathy. In order to study the role of the renin-angiotensin system in patients with diabetic nephropathy, renin release and the juxtaglomerular apparatus were studied in 17 diabetic patients with proteinuria and in 23 without proteinuria; 8 normal subjects were used for conctrls. Despite hypertension and marked arteriosclerosis, plasma renin activity (supine posture) was normal; however, the renin response to salt restriction and upright posture was less in the diabetic patients with proteinuria than in the controls. Renal renin content, determined at autopsy, was also normal. Examination of the juxtaglomerular apparatus in the diabetic patients with proteinuria revealed hyalinization of the afferent and efferent arterioles in most of the glomeruli and various degrees of destruction of the juxtaglomerular cells. The findings suggest that renin production is not increased in diabetic patients with proteinuria plus marked vascular damage, and that the renin-angiotensin system in patients with diabetic nephropathy apparently does not play an important role in the exacerbation of hypertension or the degree of vascular damage.


Clinical utility of a rapid diagnostic test series for elderly psychiatric outpatients. Psychiatric evaluation of the elderly is especially difficult for several reasons, e.g., the presence of organic impairment, the effects of multiple drug therapy, and the tendency to confuse the normal concomitants of aging with neurotic symptoms. The authors have developed and evaluated a brief series of self-report assessment tests (GRDB) designed to assist the psychiatrist in the process of diagnosis. Coefficients of the internal consistency of the seven scales are presented. A comparison was make between 28 psychiatric patients attending a geriatric clinic and 48 well-functioning elderly persons matched for age and sex, and living in the same community. Results showed that the geriatric outpatients functioned at a lower level of adaptation than did the control group, on all seven scales - depression, daily living skills, social interaction, cognitive skills, number of illnesses, number of life problems, and the use of drugs and alcohol. These findings provide a measure of discriminant validity. The advantages of this brief assessment instrument are discussed.


The social hour for geropsychiatric patients. This study was an attempt to evaluate the effects of a controlled alcohol intake in the socialization of a group of geropsychiatric patients in a clinical setting. Although 47 patients of the Geriatric Unit were involved at various times, 16 remained in the study for the entire eight weeks. The Social Hour consisted of a half-hour session every weekday. The alcohol served was whisky, gin, vodka, and wine. Soft drinks were available for those who preferred them. Each patient was rated on a 5-point scale with regard to ward and off-ward activities. A planned four-week placebo period met with so much negativism that it had to be shortened to three days. According to our observations, the socialization process was increased significantly for these mentally ill geriatric patients during this type of Social Hour.


Death and the elderly patient.

Death and the elderly patient. More attention should be paid to the profound needs of dying patients. The family physician can guide the family members and others in forming a team to help dying elderly patients in this life crisis, so that they may have a "good death." The importance of honesty and cooperation by all those involved is emphasized, as is the need to support the patient emotionally and spiritually.


Optimism in the management of hip fracture in elderly patients. The present generation of those past age 70 grew up with the idea that a fractured hip meant "the beginning of the end" for the elderly. The results reported here on the treatment of 50 consecutive patients over 70 years of age who had sustained a fractured hip indicate that in this situation the orthopedic surgeon can be an important partner in geriatric medicine. He can improve the functioning and health of this special group of elderly trauma victims through reassuring them that the fracture usually can be successfully treated without undue risk to life. The present improved state of the art of anesthesiology, surgery, and pre- and postoperative medical care makes the surgical risk tolerable, and helps to prevent many of the complications that used to occur in elderly patients confined to bed for long periods following hip fracture.


Unusual abdominal complications of a suicidal overdose of analgesic and psychotropic drugs in an elderly patient. A suicidal 67-year-old woman with manic-depressive psychosis took an overdose of asprin, amitriptyline and diazepam. The initial effects were pyrexia, tachycardia, hyperpnea, metabolic acidosis, electrocardiographic changes, hypoprothrombinemia, gastritis, and pancreatitis. Four to six weeks later, she was examined because of persistent abdominal pain with mausea, anorexia anemia, and possibly a malabsorption syndrome. An exploratory laparotomy was performed. The surgeon found several previous adhesions, a small intestinal volvulus, and a nodular pancreas. This suggested previous perforation of the small bowel from enteritis, causing a "blind-loop" syndrone. The invilved section of the small bowel was resected. With appropriate treatment, the patient is well three months after operation.


A multidimensional approach to religiosity and disengagement. This study of 106 elderly Central Missourians examines religious behavior as an indication of "disengagement." It is argued that religiosity must be measured in both organizational forms such as attending religious services and the more subjective nonorganizational forms including prayer and listening to religious services and music on radio and television before an assessment of "disengagement" is made. Following Hochschild (1975) it is argued that from the perspective of researchers and others it may appear "engaged". Data support the thesis that elderly may be disengaged organizationally but engaged non-organizationally.


Thirty years of research on the subjective well-being of older americans. Thirty years of research among older Americans on life satisfaction, morale, and related constructs has yielded a consistent body of findings. Parallel results for measures of these contructs and high intercorrelations justifies considering them in terms of a single summary construct, subjective well-being. As this research has relied almost exclusively on survey measures, interpretations are limited to the social-psychological level of people's day-to-day verbal behavior. This research shows reported well-being to be most strongly related to health, followed by socioeconomic factors and degree of social interaction, for the general population of Americans over 60. Marital status and aspects of people's living situations are also conclusively related to well-being. Age, sex, race, and employment show no consistent independent relation to well-being. In addition to indicating that negative life situation exigencies, such as poor health and low income are related to lower well-being, the results tentatively indicate that these exigencies create a greater vulnerability to the impact of other negative conditions.