Some properties of Streptomyces viridochromogenes spores. Dry spores of Streptomyces viridochromogenes exhibited no endogenous metabolism when tested by the sensitive radiorespirometric technique. Wetting the spores resulted in a sharp increase in endogenous respiration followed by a gradual decrease to a constant level, whereby 0.02% of the spore carbon was respired to CO(2) per h. The rate of endogenous metabolism increased slowly as unactivated and heat-activated spores germinated in a defined germination medium, reaching rapid rates only after germination was completed. Components of the defined germination medium, adenosine, l-alanine, and l-glutamic acid, were oxidized to CO(2) at appreciable rates only after germination was complete. The QO(2) (microliters of O(2) uptake per hour per milligram [dry weight] of spores) values for endogenously respiring spores were 3.9 for unactivated and 7.8 for activated spores. Various sugars, amino acids, and organic acids were oxidized only slowly or not at all by unactivated and activated spores. The dry spores contained 5.2 x 10(-2) mumol of ATP per g (dry weight). The ATP content of spores increased approximately 4-fold after suspension in buffer and approximately 11-fold after heat activation. During germination, the ATP level increased to a level of 1 mumol of ATP per g (dry weight) and remained constant. Germination was accompanied by excretion from the spores of approximately 8 and 12% of the total spore carbon from unactivated and activated spores, respectively. A potent germination inhibitor was released from the germinated spores. The germination inhibitor had no effect on heat-activated spores or spores which had begun germination for as short a time as 5 min.


Freeze-fracture study of the filamentous, segmented microorganism attached to the murine small bowel. A freeze-fracture study has provided new information about the filamentous, segmented microorganism known to live in the murine small bowel. The intracellular bodies produced by this microbe appear to arise by a modified sporogenesis so that they are enclosed in an envelopment membrane at least prior to release by the filament mother cell. At least some of the intracellular bodies divide while still within the mother cell, suggesting a reproductive role for these structures. The host epithelial membrane remains intact at the site of attachment, but does appear to have a reduced concentration of intramembrane particles. Changes in the host cytoplasm adjacent to the attachment site are documented and interpreted to be a sol-gel transformation which may stabilize the attachment socket.


Ribulose bisphosphate carboxylase from methanol-grown Paracoccus denitrificans. Paracoccus denitrificans grows on methanol as the sole source of energy and carbon, which it assimilates aerobically via the reductive pentose phosphate cycle. This gram-negative bacterium grew rapidly on 50 mM methanol (generation time, 7 h, 30 degrees C) in excellent yield (3 g of wet-packed cells per liter of culture). Electron microscopic studies indicated that the late-log-phase cells were coccoid, having a thick envelope surrounding a layer of more diffuse electron-dense material and a relatively electron-transparent core. Ribulose bisphosphate carboxylase in the 15,000 X g supernatant of fresh cells had specific activities (micromoles of CO2 fixed per minute per milligram of protein) of 0.026, 0.049, 0.085, 0.128, and 0.034 during the lag, early, mild-, and late log, and late stationary phases, respectively. The enzyme was purified 40-fold by pelleting at 159,000 X g, salting out, sedimentation into a 0.2 to 0.8 M linear sucrose gradient, and elution from a diethylaminoethyl-Sephadex column. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels polymerized from several acrylamide concentrations and sedimentation behavior. The molecular weight of the native enzyme, as measured by gel electrophoresis and gel filtration, averaged 525,000. Sodium dodecyl sulfate dissociated the enzyme into two types of subunits with molecular weights of 55,000 and 13,600. The S20,w of the enzyme was 14.0 Km values for ribulose bisphosphate and CO2 were 0.166 and 0.051 mM, respectively, and the enzyme was inhibited to the extent of 94% by 1 mM 6-phosphogluconate.


Association of long surface appendages with adherence-related functions of the gram-positive species Actinomyces naeslundii. Electron microscopy of new isolates of gram-positive Actinomyces naeslundii demonstrated long, fragile appendages. Removal of the appendages impaired attachment to epithelial cells and reaggregation, thus implicating them in attachment-related functions.


Transfer from Rhizobium japonicum to Azotobacter vinelandii of genes required for nodulation. A mutant strain of Azotobacter vinelandii that is unable to fix N2 (Nif-) was transformed to Nif+ with DNA from Rhizobium japonicum. Of 50 Nif+ transformants tested, 3 contained the O antigen-related polysaccharide that is present on the cell surface of a nodulating R. japonicum strain, but is absent from a non-nodulating mutant strain.


Relationship between the heat resistance of spores and the optimum and maximum growth temperatures of Bacillus species. Heat resistance of spores of Bacillus strains was compared with the temperature adaptation of each strain as measured by the optimum and maximum growth temperatures and the heat resistance of vegetative cells. Maximum growth temperatures ranged from 31 to 76 degrees C and were little affected by the nature of the growth medium. The temperature giving maximum growth rate was closely correlated to the maximum temperature for growth, and about 6 degrees C lower. Vetetative-cell heat resistance, determined on exponential-phase cells, was also correlated with maximum growth temperature. The temperature at which spores were inactivated with a decimal reduction time of 10 min was in the range of 75 to 121 degrees C. This temperature was 46 +/- 7 degrees C higher than the maximum growth temperature and correlated with it and the other cell parameters. Spore heat resistance can be considered to have two components, the temperature adaptation characteristic of the species and the stabilization conferred by the spore state.


Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2. Pseudomonas putida mt-2 carries a plasmid (TOL, pWWO) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. Characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m-toluate, whereas the inducers of the enzymes responsible for oxidation of m-xylene to m-toluate include m-xylene and m-methylbenzyl alcohol but not m-toluate. A regulatory mutant is described in which m-xylene and m-methylbenzyl alcohol no longer induce any of the pathway enzymes, but m-toluate is still able to induce the enzymes responsible for its own degradation. Among revertants of this mutant are some strains in which all the enzymes are expressed constitutively and are not further induced by m-xylene. A model is proposed for the regulation of the pathway in which the enzymes are in two regulatory blocks, which are under the control of two regulator gene products. The model is essentially the same as proposed earlier for the regulation of the isofunctional pathway on the TOL20 plasmid from P. putida MT20.


Complementary functioning of the component proteins of nitrogenase from several bacteria. The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity.


Clinical assessment of affect in schizophrenia. The disorder of affect has been a traditional, but subjective, diagnostic criterion for schizophrenia. This paper offers a more precise definition. A brief review of Bleuler's concepts is included as background. The face as an important indicator of schizophrenic affect is discussed. The concept of affect disorder is then reconsidered in terms of four parameters: range, mobility, appropriateness, communicability.


Schizophrenia, rheumatoid arthritis and trytophan metabolism. Rheumatoid arthritis and schizophrenia have been described in early surveys as mutually exclusive disorders. Such claims are seen as especially interesting in view of: (1) indications that both illnesses often follow prodromes of severe psychological stress, (2) theories regarding hypermethylation of indoleamines producing endogenous psychotogens in schizophrenia, and (3) studies of rheumatoid arthritis reporting excessive binding of L-tryptophan to plasma protein, abnormalities of urinary tryptophan metabolites, decreased serotonin binding capacity of thrombocytes, and decreased MAO activity in joint fluid. Further comparative studies of tryptophan metabolism in schizophrenia and rheumatoid arthritis might enhance knowledge of pathogenesis in either or both diseases.


Private psychiatric practice: image and reality. This study reports data collected from an individual private psychiatric practice. Despite the paucity of information concerning private practice, a popular image has evolved. An analysis of a sample of 400 patients' records confirmed certain aspects of this image, but suggested that other aspects of the image are misleading. A short-term model of private practice may be more effective than other competing models.


The psychiatrist and health insurance claims review. Expanded health insurance benefits for the treatment of mental illness have obliged psychiatrists to devote more time to justifying psychiatric treatment to claims reviewers. The author, drawing from experience in utilization review and peer review, summarizes factors contributing to the denial of payment for psychiatric services by health insurers, and gives practical advice on protecting against claims denials and on the process of appealing denials. Psychiatrists should make greater use of peer review committees to justify legitimate treatment services in the face of claims denials. Greater coverage for psychiatric treatment under national health insurance will increase the psychiatrist's responsibility for public accountability. This accountability will require better communication between psychiatrists and claims reviewers.


Sleep deprivation and electroencephalographic abnormalities. Thirty-three patients suspected of having a seizure disorder had routine electroencephalograms (EEG's) followed by post-sleep deprivation (P-SD) EEG's. The P-SD EEG's revealed abnormalities in 33% of those patients with a normal routine EEG. All of those patients with P-SD activation had histories highly suggestive of a seizure disorder. Most patients with normal P-SD EEG's were not highly suspect for seizures, however, several patients with probable seizures were also normal P-SD. Most patients with abnormal routine EEG's continue to show comparable abnormalities P-SD, with enhancement of abnormality in some cases and diminished abnormality in few cases.


Munchausen's disorder: a case of a factitious neurological emergency. Few signs are more ominous than the dilated pupil which is unreactive to light. The deliberate use of this fact and the simulation of other neurologic signs to enhance the aura of a serious illness will often expose a patient to hazardous procedures. A patient with Munchausen's Disorder who made unsuccessful attempts to simulate a neurological emergency is presented. A thorough knowledge of the pathophysiology of neurologic disease will enable the physician to detect the possibility of malingering when confronted with this problem. A strong cholinergic solution will produce pupillary constriction in normals and neurologic disorders but often will not alter the pupil in cases of pharmacologic pupillary paralysis.


Cerebral vasculitis in a drug abuser.

Cerebral vasculitis in a drug abuser. A drug abuse patient with acute intracerebral hemorrhage is presented. Brain tissue obtained at surgery corroborated angiographic evidence of cerebral vasculitis. The possible relationship between drug usage and the occurrence of intracerebral bleeding and/or cerebral vasculitis in such patients is discussed.


Pseudoperiodic bilateral EEG paroxysms in a case of phencyclidine intoxication. A 23-year old white man presented an acute PCP intoxication. His EEG showed a monomorphic nonreactive generalized theta rhythm which is the typical activity of PCP overdose. This background was interrupted by periodic bilaterally synchronous high voltage slow paroxysms similar to those described in subacute sclerosing panencephalitis. This unusual finding supports the hypothesis that PCP may act by reversible deafferentation of cortical neurons.


Inhibition of globin synthesis by rabbit reticulocyte rRNAs. The effect of concentration of rabbit reticulocyte rRNAs on the translation of rabbit globin mRNAs was studied by using a rabbit reticulocyte lysate system. Globin synthesis was studied using L-[U-14C]leucine. Both the 18S and 28S rRNA inhibited globin synthesis. The 18S rRNA inhibited the synthesis of alpha-globin chain more than that of beta chain, but the 28S rRNA inhibited the synthesis of alpha- and beta-chains almost equally. Nascent chains prelabelled with [14C]Leu or f[35S]Met were released at various concentrations of rRNAs. Release of the nascent chains was not inhibited at various concentrations of rRNAs. The ratios [14C]alpha/[14C]beta and [35S]alpha/[35S]beta in the released chains were almost constant at various concentrations of rRNAs. It therefore appears that the inhibition of globin synthesis by these rRNAs was at the step of initiation.


Characterization and in vitro polymerization of Tetrahymena tubulin. Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.


Studies on luciferase from Photobacterium phosphoreum. X. Heat of formation of the intermediate in the bioluminescent reaction studied by stopped-flow calorimetry. The heat production in the reaction of luciferase-FMNH2 complex with O2 in the absence of aldehyde was measured by stopped-flow calorimetry. deltaH of the reaction, luciferase-FMNH2+ O2 leads to intermediate X1, is -1.3 X 10(2) kJ.mol-1 and the calculated deltaS for the reaction is -180 J.mol-1.K-1 at 20 degrees C. The heat production in the bioluminescent reaction was also measured in the presence of a saturating concentration of aldehyde, and it was estimated that 43 and 79% of the C10 and C13 aldehydes, respectively, bound with the intermediate X1 are converted to carboxylic acid yielding energy for photon emission.


Amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase. The amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase was determined in order to compare it with those in phosphorylases from other sources having different regulatory properties. The potato enzyme was reduced by NaBH4 in the presence of urea, carboxymethylated, and digested with chymotrypsin and trypsin. Pyridoxyl peptides were isolated by the differential procedure using paper electrophoresis or DEAE-cellulose column chromatography. In Edman degradation of these peptides, pyridoxyllysine was identified as the phenylthiohydantoin derivative of pyridoxyllysine using a combination of thin-layer chromatography and the Pauli reaction. The sequence around pyridoxyllysine, comprising 57 amino acid residues, was determined except for a region with 6 amino acid residues. The pyridoxal 5'-phosphate binding site in potato phosphorylase showed a high homology with those of the rabbit muscle and yeast enzymes. This finding suggests that the cofactor should be directly related to the essential process of phosphorylase action.


Difference in form of sialic acid in red blood cell glycolipids of different breeds of dogs. Hematoside from dog erythrocyte membrane was previously considered to contain a mixture of N-acetyl- and N-glycolyl- neuraminic acids. However, the hematoside preparation used in the previous study was obtained from pooled blood of several dogs, and individual variation in hematoside was not examined. In this work, hematosides of erythrocytes from 31 mongrel dogs and 108 dogs of 23 breeds were examined individually by thin-layer chromatography, and the component sialic acids were analysed by gas-liquid chromatography. Individual dogs had either NAN-hematoside or NGN-hematoside: dogs with N-glycolyl-neuraminic acid also had a trace of N-acetyl-neuraminic acid, but dogs with N-acetyl-neuraminic acid had no detectable N-glycolyl-neuraminic acid. A few mongrel dogs, some Kai dogs, Kishu dogs, Japanese spaniels and most Shiba dogs had NGN-hematoside, whereas all European dogs had NAN-hematoside and no NGN-hematoside. From pedigrees of some families, inheritance of NGN-hematoside was found to be autosomal dominant. NGN-hematoside is possibly one of dog blood group substances. The sialic acid of delipidized ghost protein of dogs with NGN-hematoside was N-glycolyl-neuraminic acid, and that of dogs with NAN-hematoside was N-acetyl-neuraminic acid. The sialic acid of plasma protein was mainly N-acetyl-neuraminic acid in all dogs.


Deoxyribonucleoproteins of herring sperm nuclei. I. Chemical composition. The chemical composition of deoxyribonucleoproteins from herring sperm nuclei was analyzed and the results are summarized as follows: 1. Chemical analysis of nuclear proteins and nucleic acids revealed that arginine/P molar ratio in herring sperm nuclei is unity but the ratio of arginine residues in protamine to phosphorus in the total DNA is 0.86. 2. The deoxyribonucleoproteins were isolated and their composition showed that about 14% of the total DNA in herring sperm nuclei is free from protamine and is bound with nonprotamine proteins in the weight ratio of nonprotamine proteins to DNA of 0.25-0.30. The remaining 86% of the total DNA is combined mainly with protamine and a small amount of nonprotamine proteins; the weight ratios of protamine and nonprotamine proteins to DNA are 0.75 and 0.08, respectively. In the latter complex, the molar ratio of arginine residues in protamine to phosphorus in DNA is unity.


Glycopeptides isolated from ovine submaxillary mucin. Desialized ovine submaxillary major mucin was digested extensively with pronase. The digestion product was gel-filtered through Sephadex G-25 then fractionated by ion-exchange column chromatography on Hitachi custom resin No. 2614. Of the resulting fractions, the fractions containing relatively simple glycopeptides were purified by rechromatography on the same ion-exchange column, followed by preparative high-voltage paper electrophoresis. The isolated glycopeptides, A12d, A14a, A17c, and B12, were shown to be homogeneous by high-voltage paper electrophoresis at pH 1.9, 3.7, and 6.5 and by paper chromatography. The results of chemical analysis, alkali treatment, determination of the amino acid sequence and digestion with alpha-N-acetylgalactosaminidase of these glycopeptides indicated their structures to be as follows: glycopeptide A12d, O-alpha-N-acetylgalactosaminyl-Ser-Glx-Pro-Gly; glycopeptide A14a, O-alpha-N-acetylgalactosaminyl-Ser; glycopeptide A17c, O-alpha-N-acetylgalactosaminyl-Ser-Gly-Gly-(O-alpha-N-acetylgalactosaminyl-)Thr-Glx; glycopeptide B12, Gly-(O-alpha-N-acetylgalactosaminyl-)-Ser-Ala.


Purification and properties of superoxide dismutase from Thermus thermophilus HB8. Manganese-containing superoxide dismutase was isolated from an extreme thermophile, Thermus thermophilus HB8. About 150 mg of the enzyme was obtained from 500 g of wet cells. The enzyme was easily crystallized in octahedra from ammonium sulfate solution. The molecular weight of the enzyme was determined to be 8.2 X 10(4) and 8.4 X 10(4) by sedimentation equilibrium and gel-filtration, respectively. The enzyme contains 2 atoms of manganese per mole and consists of four subunits of identical molecular weight, about 2.1 X 10(4). The amino acid composition of the enzyme is similar to that of the superoxide dismutase of Thermus aquaticus. Proline was detected as the N-terminal amino acid. The isoelectric point was determined to be pH 6.0 by the electrofocusing method. The enzyme has maxima at 283 nm and 480 nm in the absorption spectrum. The CD spectrum suggests that the enzyme has a high alpha-helical content.


Structural studies on glycolipid of shellfish. II. Occurrence of 3-O-methylgalactosamine in oyster glycolipid. 3-O-Methyl hexosamine was found for the first time and characterized as one of the sugar components of oyster glycolipid. The alditol acetate of this sugar was identified as 3-O-methyl-N-acetylgalactosaminitol acetate by comparing its retention time on gas chromatography and mass spectrum with those of the authentic sample synthesized in this laboratory. The new sugar, 3-O-methylgalactosamine, occupied the non-reducing terminal position of the carbohydrate moiety of the lipid and was linked to the penultimate hexose by a (1 leads to 3) bond.


Preparation and properties of antisera to glycolipid of guinea pig erythrocyte membrane. Antibodies against a glycolipid of guinea pig erythrocyte membranes were prepared in rabbits by immunization with guinea pig erythrocyte stroma or the purified glycolipid, gangliotriaosylceramide. The antibodies agglutinated guinea pig erythrocytes. The specificity of antibodies could be revealed by several immunochemical methods, including inhibition of hemagglutination, immunodiffusion, agglutination of liposomes, and complement fixation. The antibodies were specific for gangliotriaosylceramide.


Changes in chromatin template activity for in vitro transcription during the development of the rat mammary gland. The template activities of chromatin isolated from virgin, pregnant, lactating, and regressed rat mammary glands were compared by using exogenously added bacterial and mammalian RNA polymerases. Template activity increased during the stage of pregnancy, then decreased during the stage from early lactation to regression. It showed the highest level at early lactation when intracellular levels of rRNA and mRNA are known to be at their maximums. These results suggest that transcriptional activity in the mammary gland is regulated, at least in part, by a modulation of chromatin template activity.


Isolation of a novel sphingoglycolipid containing glucuronic acid and 2-hydroxy fatty acid from Flavobacterium devorans ATCC 10829. A new acidic sphingoglycolipid has been isolated from a Gram-negative, glucose-non-fermentative (obligatory aerobic) bacterium, Flavobacterium devorans ATCC 10829, by thin-layer chromatography on silica gel after mild alkaline hydrolysis of the cellular lipids. Chemical degradation studies, thin-layer chromatographic behavior, IR and mass-spectrometric analysis of the original and reduced glycolipid with LiA1H4 revealed that the lipid contained glucuronic acid, long-chain bases, and fatty acids in a molar ratio of approximately 1:1:1. The major long-chain bases were identified by gas chromatography-mass spectrometry as dihydrosphingosine (d-18 :0) and longer homologues, while the N-acyl group was exclusively 2-hydroxy myristic acid. The most probable structure of this glycolipid appeared to be a ceramide glucuronic acid (N-acyl dihydrosphingosine 1-glucuronic acid).


Structure of linkage region between chondroitin polysulfates and peptides. Three different types of chondroitin polysulfate-peptide, chondroitin sulfate D-peptide, chondroitin sulfate E-peptide, and chondroitin sulfate K-peptide, all contained xylose, galactose, and serine in a molar ratio of about 1 : 2 : 1. After treatment with alkali in the presence of NaBH4 and PdCl2, they produced alanine and xylitol in amounts equivalent to the decrease in the amount of serine. Consequently, it was proved that these chondroitin polysulfates are all linked to peptides by O-glycosidic bonds between xylose and serine, as in chondroitin sulfates A and C. It is suggested that the carbohydrate-peptide linkage regions have the same structure in all the chondroitin sulfates, regardless of differences in the structure of the polysaccharide chains, such as the position of sulfate groups and the degree of sulfation.


Kinetics of conformational change of troponin-C induced by binding or removal of calcium ion. The kinetics of conformational change of troponin-C (TN-C) induced by binding or removal of calcium ion were studied in the presence or absence of magnesium ion by measuring the fluorescence of tyrosyl residues by stopped-flow spectrofluorometry. The result was analyzed in terms of first-order kinetics. Two phases were observed both in pCa-up and in pCa-down experiments. The dependence of the rate constants on pCa was explained by a simple mechanism as follows; (see article). The dissociation constants of calcium bound to TN-C, K and K', calculated from the experimentally determined rate constants were K = 3.16 X 10(-7) M, K' = 1.58 X 10(-6) M in the absence of magnesium ion, and K = K' = 1 X 10(-6) M in the presence of 2 mM MgCl2.


Analysis of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells. The molecular size and poly-A content of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells were measured. KCl was found to be essential for synthesis of high molecular weight RNA: when 0.4 M KCl was added to the reaction mixture, the average molecular size of the RNA formed was 14S; without KCl the average molecular size was 5S. A significant amount of poly-A sequences was found in RNA synthesized in the presence of alpha-amanitin, suggesting that RNA polymerase I and/or III may synthesized some RNA containing poly-A in isolated nuclei.


Biochemical and ultrastructural evaluation of isolated rat liver systems perfused with a hemoglobin-free medium. The viability of hemoglobin-free perfused rat liver was examined with respect to several liver functions and to the intactness of subcellular structures under electron microscopic observation. Provided that rat livers were perfused with the oxygenated buffer solution at a flow rate between 3 and 3.5 ml/min per g of liver, all the biochemical parameters measured in the perfused liver system, i.e. the rates of glucose, pyruvate, and lactate production, the rate of oxygen consumption and the tissue contents of adenine nucleotides, were similar to those observed with perfusion systems containing erythrocytes or albumin. The perfused liver showed a sensitive response to norepinephrine, involving a reduction of pyridine nucleotides and enhancements of glucose production and oxygen consumption. On electron microscopic examination, changes in hepatic-structure indicative of hypoxic injury particularly vacuolar degeneration and mitochondrial swelling, were not detected in the liver after 70 min of perfusion; the fact that the fine structure of the hepatocyte was preserved in all parts of the organ confirmed that the supply of oxygen to the perfused liver was sufficient under the conditions employed. From viewpoint of the generally accepted criteria for the viability of perfused liver, therefore, the results confirmed that the perfusion of liver with a hemoglobin- and albumin-free medium is a convenient and reliable tool for biochemical investigation of the reactions occurring in whole liver.


Amino acid sequences of two ferredoxins from pokeweed, Phytolacca americana. The amino acid sequences of two ferredoxins isolated from pokeweed, Phytolacca americana, were determined. Tryptic peptides of maleyl-carboxymethyl-ferredoxin I and carboxymethyl-ferredoxin II were prepared and analyzed. The large peptides were further digested with staphylococcal protease and chymotrypsin. Ferredoxins I and II were composed of 96 and 98 amino acid residues, respectively. Though ferredoxin I lacks tryptophan and methionine, ferredoxin II contains both of them. In a comparison of the amino acid sequences with those of other higher plant ferredoxins, ferredoxin I is one residue shorter than others at the carboxyl-terminus and ferredoxin II one longer than others at the amino-terminus. Ferredoxins I and II differ in 23 sites from each other and in 27 to 37 sites from other higher plant ferredoxins. This suggests that duplication of the ferredoxin gene occurred after the divergence of pokeweed from other higher plants. A phylogenetic tree including all other ferredoxins was constructed.


Amino acid sequence of a ferredoxin from Chlorobium thiosulfatophilum strain Tassajara, a photosynthetic green sulfur bacterium. We have determined the amino acid sequence of a ferredoxin from a photosynthetic green sulfur bacterium, Chlorobium thiosulfatophilum strain Tassajara. It contains 61 amino acid residues with 9 cysteines, and 8 of the 9 were located at positions corresponding to those in clostridial-type ferredoxins. Other structural features were closer to those of ferredoxins from another photosynthetic bacterium, C. limicola, than to those of non-photosynthetic bacteria. Compared with ferredoxin from Chromatium, a photosynthetic purple sulfur bacterium, all photosynthetic bacterial ferredoxins have a common region in the carboxyl-terminal half with several extra residues and a unique cysteine residue. We compared all the photosynthetic bacterial ferredoxins that have been sequenced and concluded that C. thiosulfatophilum ferredoxin is most closely related to C. limicola ferredoxin I.


Interaction of porcine alpha2-macroglobulin with chemically modified proteinases. The interactions of porcine alpha2-macroglobulin (alpha2M) with native proteinases, their zymogens and the chemically-modified enzymes were compared. The alpha2M did not bind to chymotrypsinogen, or to most of the chemically modified derivatives of alpha-chymotrypsin, trypsinogen, DIP- and PMS-trypsins, but it could interact with anhydrotrypsin, PMS-subtilisin, and O-acetylated neutral subtilopeptidase. Anhydrotrypsin appeared to bind very tightly to alpha2M, as does native trypsin, whereas the binding of PMS-subtilisin to alpha2M was weaker than that of the native enzyme, judging from exchange experiments with labeled enzyme and from competitive enzyme assay. There are, however, some differences in the mode of interaction with alpha2M between native and anhydrotrypsins. (1) The shape and the magnitude of ultraviolet difference spectra caused by the interaction with alpha2M were significantly different. (2) The interaction of alpha2M with active proteinase led to the formation of new amino-terminal amino acids, while that with anhydrotrypsin did not. (3) In vivo experiments showed that radioactivity of 3H-labeled trypsin-alpha2M complex was rapidly cleared from the plasma of rats, whereas the anhydrotrypsin-alpah2M complex was cleared very slowly. These results suggest that the proteolytic activity of the enzyme is not obligatory for the first phase of alpha2M-proteinase interaction (formation of Michaelis-type complex), but only the proteolytically modified complex is cleared rapidly from the blood circulation system.


Susceptibilities of various myofibrillar proteins to muscle serine protease. The ability of serine protease of skeletal muscle to degrade native myofibrillar proteins, such as myosin, actin, troponin, tropomyosin, alpha-actinin, and M-protein from rabbit skeletal muscle was studied. The amino acids or peptides liberated from these proteins by the protease were determined fluorometrically using o-phthalaldehyde. The order of their susceptibilities at a molar ratio of the serine protease to substrate of 1:100 was: myosin greater than tropnin greater than tropomyosin greater than actin. Alpha-Actinin and M-protein were not degraded. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the myosin heavy chain was degraded into two fragments, having molecular weights of 100,000 and 88,000, whereas the light chains were scarcely degraded. The serine protease degraded troponin-T rapidly and troponin-I slowly, but did not degrade troponin-C. Tropomyosin was degraded rapidly into two components with molecular weights of 21,500 and 19,000. Actin was degraded slowly, but no liberated fragment could be detected.


Enhancement of fatty acyl-CoA oxidizing activity in rat liver peroxisomes by di-(i-ethylhexyl)phthalate. The effect of di-(2-ethylhexyl)phthalate (DEHP) administration on cyanide-insensitive palmitoyl-CoA oxidizing activity in liver was studied. Two weeks of DEHP treatment increased the activity by one order of magnitude in male Wistar rats. A similar effect was also observed in male and female Sprague-Dawley rats and mice, but not in guinea pigs. When the liver was fractionated by differential centrifugation, the activity was concentrated in the light mitochondrial fraction. On the subfractionation of this fraction by sucrose density gradient centrifugation, the activity was distributed in a pattern similar to that of urate oxidase, but not resembling that of glutamate dehydrogenase. These data suggest that a fatty acyl-CoA oxidizing enzyme system which is located in peroxisomes is induced by the administration of DEHP.


Stimulation of Chromobacterium lipase activity and prevention of its adsorption to palmitoyl cellulose by hydrophobic binding of fatty acids. Fatty acids prevented adsorption of purified Chromobacterium lipase [triacylglycerol acylhydrolase, EC 3.1.1.3] onto palmitoyl cellulose (Pal-C) and also increased the activity of the purified lipase. These effects increased with increase in the concentration and chainlength (up to 16 carbon atoms) of the fatty acids, and long-chain unsaturated fatty acids, such as oleic acid, linoleic acid and erucic acid, were most effective. When the lipase was adsorbed (immobilized) on Pal-C, its activity was elevated to 20 times that of the free lipase in detergent-free reaction mixture (olive oil-buffer system). Thus lipase was adsorbed to Pal-C through a hydrophobic site distinct from its catalytic site and the binding of fatty acids to the hydrophobic site seems to result in stimulation of the lipase activity.


Tropomyosin fragments obtained by tryptic digestion. Rabbit skeletal alpha-tropomyosin, separated by hydroxyapatite chromatography, was treated with trypsin (1/100 wt/wt) at 0 degrees C for 24 h. Trypsin-resistant fragments of tropomyosin were separated into the precipitate and supernatant fractions at pH 4.3 in 1 M KCl, and these were subjected to QAE-Sephadex A50 column chromatography for further purification. SDS-gel electrophoresis showed 16,000 and 14,000 dalton bands for the supernatant (s-fragment) and an 11,500 dalton band for the precipitate (p-fragment). We obtained a 13,500 dalton chain (13,500 dalton fragment) in addition to the s- and p-fragments upon treatment with more dilute trypsin (1/500 wt/wt) for 48 h at 0 degrees C. Both the p- and 13,500 dalton fragment had the same C-terminal portion as intact alpha-tropomyosin, and could form an intra-chain disulfide bond on oxidation. Therefore, these two fragments were deduced to be polypeptides from some points on the N-terminal side of Cys 190 to the intact C-terminal. The s-fragment, on the other hand, did not contain any cysteine, Phe, or His residues according to amino acid analysis, suggesting that the fragment is derived from the N-terminal side from Cys 190. Tentative assignment of the fragments was carried out by amino acid analysis, and C- and N-terminal determination. The p-, s-, and 13,500 dalton fragments appear to be in coiled-coil form in solution, having alpha-helical contents of 77,71, and 64%, respectively, and are able to interact with intact tropomyosin to reduce the viscosity of tropomyosin solution. The s-, p-, and 13,500 dalton fragments have little binding capacity individually to troponin, but the mixture, i.e., the s- and p-fragments, the 13,500 dalton fragment and the N-chain, which was obtained by cleavage at Cys 190, showed clear binding with troponin independent of Ca2+ in solution as detected by gel electrophoresis. The p-fragment showed some binding to troponin, since cross-linkage to troponin was possible by treatment with dimethyl suberimidate. From the result, it can be inferred that the troponin binding regions in tropomyosin are located on both sides of Cys 190, where trypsin attacks more easily than at other parts of the molecule, leaving two trypsin-resistant fragments.


Sinefungin, a potent inhibitor of virion mRNA(guanine-7-)-methyltransferase, mRNA(nucleoside-2'-)-methyltransferase, and viral multiplication. Sinefungin (A9145) and a related metabolite, A9145C, were found to be potent inhibitors of Newcastle disease virion and vaccinia virion mRNA(guanine-7-)-methyltransferase and vaccinia virion mRNA(nucleoside-2'-)-methyltransferase. Both Sinefungin and A9145C were competitive inhibitors of these S-adenosyl-L-methionine-dependent enzymes having inhibition constants substantially less than S-adenosyl-L-homocysteine. These compounds also inhibited plaque formation by vaccinia virus in mouse L-cells.


Ligandin. Bilirubin binding and glutathione-S-transferase activity are independent processes. Physical methods and chemical modifications were used to discriminate between the bilirubin-binding capacity and glutathione-S-transferase activity of ligandin which was purified from rat liver. Binding of bilirubin occurs at a primary high affinity site (KA = 5 X 10(7) M-1) and at a secondary, lesser affinity site (KA = 3 X 10(5) M-1). Circular dichroism and fluorescence-quenching methods were used to distinguish between these sites. Cross-linked as well as reduced and alkylated ligandin lost high affinity bilirubin-binding capacity, but retained glutathione-S-transferase activity, bilirubin binding at a secondary site, and immunological reactivity. Succinylation of ligandin abolished catalytic activity and bilirubin binding at high and low affinity sites, but not immunological reactivity. Catalytic activity was unaffected by concentrations of bilirubin which saturated the primary binding site. These results suggest that the high affinity site at which bilirubin is bound to ligandin is independent from the site at which catalytically reactive substrates bind. The latter substrates probably interact at the secondary bilirubin binding site where bilirubin competitively inhibits glutathione-S-transferase activity.


Structural relationships of low molecular weight viral RNAs synthesized by RNA polymerase III in nuclei from adenovirus 2-infected cells. Previous studies have shown that endogenous class III RNA polymerase(s) in nuclei from adenovirus 2-infected cells synthesize virus-coded RNA species which are approximately 200 (V200), 156 (V156), and 140 (V140) nucleotides in length (Weinmann, R., Brendler, T. G., Raskas, H.J., and Roeder, R. G. (1976) Cell 7, 557-566). The V156 nuclear RNA is identical in sequence to the major virus-associated RNA (VA RNA1 or 5.5 S RNA) synthesized in intact cells (Ohe, K., and Weissman, S. M. (1971) J. Biol. Chem. 246, 6991-7009). The V140 RNA contains several components, one of which appears identical to a minor virus-associated RNA (VA RNAII) which is synthesized in infected cells (Mathews), M. B. (1975) Cell 6, 223-229). Thus transcription of the VA RNAI and VA RNAII genes in vitro accurately reflects the in vivo transcription of these genes. The V200 RNA contains all the nucleotide sequences found in V156 RNA plus an additional 38 to 40 nucleotides on the 3' terminus. Transcription of the gene encoding this RNA species terminates within a stretch of 6 deoxythymidylic acid residues which are located 38 nucleotides beyond the predicted termination site for VA RNAI and which are preceded by a GC-rich sequence of nucleotides. These data suggest either that the V200 RNA is a precursor to the VA RNAI or that the RNA polymerase III occasionally reads-through the presumptive VA RNAI gene termination signal and stops at a potentially stronger downstream termination site.


Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis. After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.


Control of fatty acid metabolism in ischemic and hypoxic hearts. The effects of whole heart ischemia on fatty acid metabolism were studied in the isolated, perfused rat heart. A reduction in coronary flow and oxygen consumption resulted in lower rates of palmitate uptake and oxidation to CO2. This decrease in metabolic rate was associated with increased tissue levels of long chain acyl coenzyme A and long chain acylcarnitine. Cellular levels of acetyl-CoA, acetylcarnitine, free CoA, and free carnitine decreased. These changes in CoA and its acyl derivatives indicate that beta oxidation became the limiting step in fatty acid metabolism. The rate of beta oxidation was probably limited by high levels of NADH and FADH2 secondary to a reduced supply of oxygen. Tissue levels of neutral lipids showed a slight increase durning ischemia, but incorporation of [U-14C]palmitate into lipid was not altered significantly. Although both substrates for lipid synthesis were present in higher concentrations during ischemia, compartmentalization of long chain acyl-CoA in the mitochondrial matrix and alpha-glycerol phosphate in the cytosol may have accounted for the relatively low rate of lipid synthesis.


Kinetics of CO binding to and dissociation from microsomal cytochrome P-450 induced by phenobarbital in rat liver. The kinetics of CO binding to hepatic microsomes from phenobarbital-treated Sprague-Dawley rats, measured by stopped flow spectrophotometry, can be resolved into three components with second order velocity constants of 2.23 +/- 0.35 X 10(5) M-1 S-1, 1.59 +/- 0.18 X 10(6) M-1 S-1, and 8.7 +/- 1.7 X 10(6) M-1 S-1. The three CO-binding species were present in ratios of 1:1.25:1.39 as judged by the relative amplitude of the change in absorbance at 450 nm associated with each of the kinetic components. Similar results were obtained in a range of [CO] from 10 to 700 micron when CO recombination was followed subsequent to flash photolysis of the CO-associated microsomes. In contrast, the dissociation rate of CO from its cytochrome P-450 complex measured by the NO replacement method was biphasic. Approximately 40% of the bound CO dissociated at a rate of 0.40 +/- 0.071 s-1, whereas the remaining 60% dissociated at a rate of 0.049 +/- 0.008 s-1 at 20 degrees C.


Kinetics and mechanism of bilirubin binding to human serum albumin. The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.


Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method. A method was developed which enables the characterization and fractionation of chromosomal proteins according to their chromatin binding properties. The method is based on the ability of hydroxyapatite to bind native chromatin in solutions which do not dissocciate chromosomal proteins from the DNA. The proteins are then selectively dissociated from the immobilized chromatin by treatment with NaCl, urea, or guanidine HCl. The hydroxyapatite dissociation method represents a rapid one-step fractionation procedure which results in the quantitative recovery of chromosomal proteins devoid of nucleic acids and is suitable for large preparations. The hydroxyapatite dissociation method provides a versatile procedure for the study and preparation of chromosomal proteins. The patterns of dissociation of both histones and nonhistone chromosomal proteins by NaCl and urea from chicken oviduct chromatin were characterized by this method. In addition, this technique enabled the purification of the major histone species in a single operation. Partial purification of specific nonhistone proteins, including the estrogen receptor, was also achieved. We suggest that this method will be a useful tool in elucidation of the chemical and biological properties of the proteins from chromatin.


On ferritin heterogeneity. Further evidence for heteropolymers. Tissue ferritins from the horse, rat, and human consist of multiple isoferritins some of which are common to more than one tissue in the same individual. Subunit analyses indicate that the ferritins from all three species are similarly composed of only two types of subunit with an approximate Mr of 21,000 and 19,000, designated H and L. The relative amounts of these subunits vary progressively throughout the isoferritin spectrum. Amino acid analyses and tryptic peptide maps indicate that the H and L subunits have extensive sequence homologies and that both are species-specific. Both subunits have been identified as the primary products of apoferritin synthesis in a wheat germ lysate programmed by rat liver mRNA. These results substantiate our proposal (Adelman, T. G., Arosio, P., and Drysdale, J. W. (1975) Biochem. Biophys. Res. Commun. 63, 1056-1062) that tissue ferritins are not unique homopolymers but families of hybrid molecules consisting of different proportions of two subunit types.


Human hypoxanthine-guanine phosphoribosyltransferase. Evidence for tetrameric structure. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been purified 23,000-fold from normal human erythrocytes. The purification includes affinity chromatography on a GMP column. The subunit molecular weight of the enzyme obtained from this purification is 24,000. The finding of four protein species after cross-linkage of the highly purified enzyme with dimethylsuberimidate, dimethyladipimidate, and glutaraldehyde suggests that the enzyme may exist in the native state as a tetramer.


Adenosylhomocysteine hydrolase. Crystallization of the purified enzyme and its properties. Adenosylhomocysteine hydrolase (EC 3.3.1.1) from calf liver was purified to homogeneity by crystallization. The purified enzyme exhibited one single component in polyacrylamide gel electrophoresis. But by Ampholine gel electrophoresis, two isoelectric focusing variants were observed, with pI values at 5.8 and 6.0. when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, one major subunit with a molecular weight of 60,000 was found; five other minor subunit variants were also observed, with molecular weights ranging between 50,000 and 57,000. These minor subunit variants comprised approximately 15% of the total protein applied. The molecular weight of the native enzyme was estimated to be 237,500 by gradient gel electrophoresis. The native enzyme is probably composed of four subunits, each with a molecular weight of not more than 60,000. Amino acid analyses of the purified enzyme revealed the presence of 1.2 residues of glucosamine/mol of enzyme, in addition to all of the common amino acids. The presence of enzyme-bound NAD was confirmed, probably 1 NAD molecule bound/enzyme subunit. In addition to adenosine, 3-deazaadenosine was found to be an effective substrate as well in the direction of synthesis.


Purification of mRNA guanylyltransferase from vaccinia virions. GTP:RNA guanylyltransferase, the enzyme which catalyzes the guanylylation of the 5' termini of viral mRNAs, has been isolated and purified approximately 10,000-fold from cores of vaccinia virus. S-adenosyl-methionine:mRNA (guanine-7)-methyltransferase copurified with guanylyltransferase activity through chromatography on DNA agarose, phosphocellulose, and centrifugation in glycerol gradients, suggesting that the two activities are closely associated. The molecular weight of native guanylyltransferase- and 7-methyltransferase-associated activities was approximately 120,000 as determined by glycerol gradient centrifugation. Guanylytransferase purified by electrophoresis on polyacrylamide gels at pH 4.5 lacked 7-methyltransferase activity. Analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels of electrophoretically purified native guanylyltransferase showed the presence of one major band of polypeptide which had a molecular weight of approximately 59,000.


Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H. Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.


Modification of rat liver RNA polymerase I after in vivo stimulation by hydrocortisone or methylisobutylxanthine. Following in vivo administration of hydrocortisone or methylisobutylxanthine to rats, higher levels (1.5- to 2.3-fold) of RNA polymerase I activity are present in liver nuclei and nucleoli of the treated animals as compared to control animals. The elevated specific activity is retained after purification of the enzyme under conditions where the enzyme is dependent on exogenous template for activity. The elevated polymerase activity in nuclei, nucleoli, and soluble enzyme can be destroyed by mild trypsin treatment which results in a rapid decay of the specific activity to the control level. Under these conditions, the control polymerase I activity is stable. The results indicate that in vivo stimulation by hydrocortisone or methylisobutylxanthine results in a conversion of the enzyme to a form that is catalytically more active but has an increased sensitivity to proteolysis.


Rapid accumulation of vitellogenin messenger RNA during secondary estrogen stimulation of Xenopus laevis. Accurate quantitation of low concentrations of vitellogenin mRNA by hybridization to vitellogenin cDNA allows analysis of the accumulation of new vitellogenin mRNA sequences throughout secondary estrogen stimulation. Administration of a secondary injection of estradiol-17 beta to male Xenopus laevis which have been withdrawn from estrogen for 60 days results in synthesis of complete vitellogenin mRNA molecules in as little as 1 h after restimulation. Vitellogenin mRNA accumulates at a rate of 13 molecules/cell/min--at least four times the rate observed in primary estrogen stimulation and peaks at a level twice that observed in primary stimulation. Administration of estrogen to male Xenopus laevis evokes stable long lived changes in the pattern of vitellogenin gene expression and constitutes a type of cellular "memory effect."


A pyruvate-containing peptide of proline reductase in Clostridium sticklandii. Proline reductase in Clostridium sticklandii is composed of 10 apparently identical subunits. Each subunit contains a pyruvate residue that became labeled when the cell culture was supplemented with [14C]serine. No NH2-terminal amino acid was detected either by dansylation, by Edman degradation, or by aminopeptidase M digestion. The results suggest that the NH2 terminus may be blocked by pyruvate. A pyruvate-containing peptide, also blocked at the NH2 terminus, was isolated from the NH2-terminal portion of proline reductase. From amino acid analysis the peptide was found to be rich in basic amino acids and to have a molecular weight of 4621. Its COOH-terminal amino acid was found to be serine and since the peptide was released from proline reductase by very mild alkali hydrolysis, it is suspected that an ester bond is involved.


Separation and characterization of two activities from HeLa cell nuclei that degrade double-stranded RNA. Two enzymatic activities that degrade double-stranded RNA have been partially purified from HeLa cell nuclei using reoviral [3H]RNA as the substrate. The two active fractions, separated by chromatography on phosphocellulose, are designated PC I and PC II. Both fractions degrade a variety of double-stranded RNAs with an absolute requirement for a divalent cation. However, they are distinct by at least five criteria. 1)PC I degrades a variety of single- and double-stranded RNAs, single- and double-stranded DNAs, and DNA.RNA hybrids, in addition to double-stranded reoviral RNA. In contrast, PC II has maximal activity with reoviral RNA, some activity with rRNA, and much less activity with the other substrates. 2) Analyses of reaction products by sucrose gradient centrifugation and chromatography on Sephadex G-100 and DEAE-cellulose indicate that, PC I cleaves reoviral RNA endonucleolytically to a final mixture of mono- and oligonucleotides, whereas the only acid- or alcohol-soluble products of PC II are 5'-XMPs produced exonucleolytically. 3) PC I activity  is stimulated 2-fold more by MnCl2 than by MgCl2, whereas PC II activity is stimulated 3-fold more by MgCl2 than by MnCl2. 4) PC I activity is inhibited by NaCl concentrations as low as 10 mM, whereas PC II requires 50 to 80 mM NaCl for optimal activity. 5)Estimated by their sedimentation rates in glycerol gradients, PC I and PC II have apparent molecular weights of 55,000 and 20,000, respectively.


The effect of colchicine and dibucaine on the morphogenesis of Semliki Forest virus. The effect of colchicine, Nocodazole, and dibucaine on the assembly of Semliki Forest virus was investigated. Colchicine, Nocodazole, and dibucaine reduced the production of extracellular virus by 75 to 90%. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [3H]leucine into material precipitated by trichloroacetic acid. Colchicine (100 micron) disrupted the microtubles of the baby hamster kidney cells (BHK-21), whereas dibucaine did not alter microtubule polymerization. The stage of virus assembly inhibited by colchicine and dibucaine was studied by experiments with [3H]-leucine or [35S]methionine. At various times after addition of one of these drugs, the incorporation of the labeled precursors into viral proteins associated with fractions enriched for endoplasmic reticulum or plasma membrane from the cell was evaluated. The results clearly show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the cell where they accumulate. The studies strongly suggest that the cytoskeletal system is involved in the final stages of morphogenesis of Semliki Forest virus from the plasma membrane.


Kinetic studies on the activation of human factor X. The role of metal ions on the reaction catalyzed by the venom coagulant protein of Viper russelli. The effect of Ca2+, Mg2+, and Mn2+ on the initial rate of activation of human Factor X by the venom coagulant protein of Vipera russelli has been investigated. Neither Mg2+ nor Mn2+ alone support the reaction. Ca2+ is an essential activator and exhibits cooperative kinetics. Both Mg2+ and Mn2+ enhance the reaction cooperatively when Ca2+ is present at suboptimal concentrations. Similarly, Ca2+ quenches the intrinsic fluorescence of human Factor X in a cooperative manner. While neither Mg2+ nor Mn2+ by themselves affect the fluorescence of human Factor X, they decrease the cooperativity of the Ca2+ binding to the protein as judged by Hill plots of the Ca2+ -induced fluoresence quenching. EPR measurements indicate that there are three high affinity Mn2+ binding sites on human Factor X which can also bind Ca2+. Positive cooperativity was not observed for Mn2+ binding. These data indicate that Ca2+ can cause a conformational change of the Factor X molecule which allows the activation reaction to proceed. We propose that Mn2+ does not support the activation of human Factor X because it cannot induce a necessary conformational change in the absence of Ca2+.


Formation of 20-hydroxyprostaglandins by lungs of pregnant rabbits. Homogenates or particulate fractions (1,000 to 100,000 X g) from lungs of pregnant rabbits were incubated with prostaglandins or prostaglandin metabolites and the products were purified by chromatography and identified by gas chromatography-mass spectrometry. In the presence of NADPH, particulate fractions from pregnant rabbit lungs converted prostaglandins E1, E2, and F2alpha as well as 13,14-dihydro-15-oxoprostaglandin E2 and 13, 14-dihydro-15-oxoprostaglandin F2alpha to their 20-hydroxy derivatives. In the cases of the 3 primary prostaglandins, the corresponding omega-carboxylic acids were also isolated. The omega-hydroxylation reaction occurred in the presence of the microsomal fraction. The mitochondrial fraction was much less active whereas the cytosol fraction converted prostaglandins to their 13, 14-dihydro-15-oxo derivatives. When prostaglandin F2alpha was incubated with homogenates of lungs from pregnant rabbits, omega-oxidation was combined with oxidation of the 15-hydroxyl group and reduction of the 13, 14-double bond to give 13, 14-dihydro-20-hydroxy-15-oxoprostaglandin F2alpha as well as the corresponding derivative with an omega-carboxylic acid group. Lungs from nonpregnant rabbits were much less active than lungs from pregnant rabbits in the omega-oxidation of prostaglandins.


Human adenosine deaminase binding protein. Assay, purification, and properties. In many human tissues adenosine deaminase exists as a complex composed of two proteins; one protein has adenosine deaminase activity while the other represents a binding protein with no other known binding activity. A rapid, quantitative assay for human adenosine deaminase binding protein has been developed utilizing 125I-labeled calf adenosine deaminase. In addition this binding protein has been purified 1,690-fold from human kidney using adenosine deaminase affinity chromatography and appears to be homogenous by sedimentation equilibrium, sodium dodecyl sulfate, and native polyacrylamide gel electrophoresis. This highly purified binding protein exists as a dimer of native molecular weight 190,000, complexes with calf adenosine deaminase in a ratio of 1:2, respectively, and contains carbohydrate which reacts specifically with phytohemagglutinin and ricin lectins. A second form of this adenosine deaminase binding protein may exist, resulting from degradation of its carbohydrate moiety.


Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis. The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.


Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly. A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.


Chemical synthesis of 5beta-cholestane-3alpha, 7alpha, 24, 25-tetrol and its metabolism in the perfused rabbit liver. 5beta-[G-3H]Cholestane-3alpha, 7alpha, 24xi, 25-tetrol (IV) was synthesized via dehydration and peroxidation of 5beta-[G-3H]cholestane-3alpha, 7alpha, 25-triol. Following perfusion of the labeled compound in the isolated rabbit liver, the bile alcohol and bile acid metabolites secreted into the bile were identified by a combination of thin layer chromatography, gas-liquid chromatography, and gas-liquid chromatography/mass spectrometry. The following bile alcohols were tentatively identified: 5beta-cholest-23-ene-3alpha, 7alpha, 25-triol, 5beta-cholest-25-ene-3alpha, 7alpha, 12alpha, 24xi-tetrol, and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol. The amount of administered tetrol recovered unchanged ranged from 1 to 88%. Cholic acid was the major product, but limited amounts of chemodeoxycholic acid were also formed. The 24-hydroxyl group in the steroid side chain did not prevent 12alpha-hydroxylation.


Differentiation of 3T3-L1 fibroblasts to adipocytes. Effect of insulin and indomethacin on the levels of insulin receptors. Differentiating (3T3-L1) and nondifferentiating (3T3-C2) fibroblastic cell lines possess two classes of insulin receptors, high affinity (KD = 1.0 to 3.7 X 10(-9) M) and low affinity (KD = 2.0 to 3.6 X 10(-8) M). Confluent cultures of 3T3-L1 cells induced to differentiate by insulin (1.74 x 10(-6) M) or indomethacin (1.25 x 10(-4) M) exhibit a 3-fold increase in the number of high affinity and low affinity receptors per cell or a 1.5- to 1.8-fold increase in the number of receptors per micron2 of surface area. In contrast, nondifferentiating 3T3-C2 cells treated with insulin or indomethacin lose almost completely the high affinity insulin receptors while retaining the same levels of low affinity receptors. The loss of high affinity receptors of the 3T3-C2 cells is accompanied by the disappearance of the stimulatory effect of insulin on the production of CO2 from glucose and on the uptake of aminoisobutyrate. The levels of high affinity insulin receptors appear to be regulated by different mechanisms in the differentiating (3T3-L1) and nondifferentiating (3T3-C2) cell lines. The mode of this regulation may have a bearing on the ability of a particular cell line to differentiate.


Reconstitution of the erythrocyte anion channel. Band 3, the membrane protein which mediates erythrocyte anion exchange, was purified on a concanavalin A column. Triglycerides, diglycerides, cholesteryl esters, cholesterol, and phosphatidylcholine were found to copurify. The column product gave at least two and probably three bands by isoelectric focusing. Antibodies prepared against the purified Band 3 appeared to react only with the cytoplasmic face of Band 3. Vesicles prepared with Band 3 had an accelerated uptake of SO4(2-) which could be inhibited by 2-(j'-aminophenyl)-6-methyl benzene thiazo-3', 7-disulfonic acid, a potent inhibitor of anion transport in the intact system. The possible source of this difference is discussed. Band 3 was spin labeled, probably at one specific site. The spectra showed that the spin label was highly immobilized, but no dipole-dipole interactions between spin labels on adjacent Band 3 subunits were apparent.


Stimulation of prostaglandin synthesis by tumor-promoting phorbol-12, 13-diesters in canine kidney (MDCK) cells. Cycloheximide inhibits the stimulated prostaglandin synthesis, deacylation of lipids, and morphological changes. Tumor-promoting 12-O-tetradecanoyl-phorbol-13-acetate and phorbol-12, 13-di-decanoate, but not the non-tumor-promoting 4alpha-phorbol-12, 13-di-decanoate, stimulated deacylation of cellular lipids, prostaglandin biosynthesis, and morphological changes in cultured MDCK cells. The increased prostaglandin biosynthesis and morphological changes required at least 24 h for expression. Cycloheximide inhibited the stimulated prostaglandin biosynthesis, the changes in morphology, and the increased lipid deacylation, but hydrocortisone (1.0 microgram/ml) did not. Indomethacin (0.5 microgram/ml) completely inhibited the stimulated prostaglandin biosynthesis and also inhibited some deacylation of cellular lipids. Indomethacin, however, did not effect the 12-O-tetradecanoyl-phorbol-13-acetate-stimulated changes in morphology.


Acute haemodilutional autotransfusion in the surgical management of scoliosis. Homologous blood transfusions are not without risks, especially in young women and girls. In patients undergoing certain elective surgical procedures autologous blood transfusion is a good alternative. Normovolaemic haemodilution in association with autotransfusion offers the additional advantages of reduced loss of red cell mass during the operation and an increase in tissue blood flow. In this study twenty-seven adolescent patients undergoing Harrington instrumentation for idiopathic scoliosis had haemodilutional autotransfusions during their operations, none requiring homologous blood. Eighteen were given an average of 750 millilitres of homologous blood in the postoperative period which compared favourably with controls who required 1280 millilitres.


The characteristics of juvenile idiopathic scoliosis and results of its treatment. Fifty-nine children with juvenile idiopathic scoliosis have been reviewed. In six of eighteen who had no treatment the curvatures did not increase over eighteen months or more, while in twelve they increased at a median annual rate of six degrees. The pattern and magnitude of the curvature and the age at diagnosis could not be used accurately to predict progression. In forty-two patients progression occurred and Milwaukee brace treatment was begun, thirty being managed with periods of part-time wear. The median correction was 13 per cent. Serial measurements of the rib-vertebra angle difference (RVAD) were useful to predict a tendency to progress. Part-time wear of the brace was successful in cases where the RVAD values fell towards zero or became negative with treatment. Sixteen patients required surgical treatment; all had high positive RVAD values. However, with the plan of brace treatment outlined, part-time wear controlled many of the curvatures.


The assessment of prognosis in Perthes' disease. Seventy children who had suffered from Perthes' disease were reviewed clinically and radiologically three to eight years from the onset of the condition in order to determine retrospectively the most satisfactory method of assessing the prognosis and the correlation between the clinical and radiological result. In younger children the femoral head was more likely to be spherical at the conclusion of the pathological process but not necessarily of normal proportions nor normally covered by the acetabulum. The prognosis was significantly poorer for girls than for boys. Clinical factors were not an aid to prognosis in the individual cases, but overall there was a close correlation between the clinical and the radiological end-results. The most reliable radiological factors indicating the prognosis were the extent of uncovering of the femoral head, the Catterall grouping, the presence of calcification lateral to the outer limit of the acetabulum and lateral displacement of the femoral head, as measured by comparing the head to tear-drop distances on each side.


Tennis elbow--a radial tunnel syndrome?

Tennis elbow--a radial tunnel syndrome? The hypothesis that the chronic tennis-elbow syndrome is caused by an entrapment neuropathy of the radial nerve (Roles and Maudsley 1972) was put to the test prospectively in ten patients. Detailed orthopaedic, neurological and neurophysiological examinations did not reveal any involvement of the radial nerve. It is concluded that this syndrome cannot be explained by an entrapment neuropathy.


Osteochondral fractures of the lateral femoral condyle: a result of indirect violence to the knee. Twenty patients with an average age of eighteen and a half years sustained osteochondral fractures of the lateral femoral condyle as the result of a sudden twist and valgus strain to the straight or almost straight knee. All the patients felt sudden pain at the moment of injury, all had a haemarthrosis, and yet the fracture escaped early diagnosis in one-third of the cases. Internal fixation of the fragment with early mobilisation is recommended if the diagnosis is made within two weeks of injury, and excision of the fragment if it is only later identified.


Is chondromalacia patellae a separate clinical entity? The articular cartilage of the patella was studied in 100 knees at necropsy. In twenty-one of these knees the cartilage changes were related to the trabecular architecture of the underlying bone. It would appear that the initiation and location of cartilage damage and its rate and degree of progression are related to the relative stiffness of the underlying cancellous bone. On the basis of our observations we suggest that the diagnosis "chondromalacia of the patella" should be reserved for patients with asymptomatic or transiently symptomatic fibrillation of the articular cartilage of the central medial patellar facet. Those patients with persistent patellofemoral pain should be considered to have some other syndrome.


Where ankle fusion stands today.

Where ankle fusion stands today. A retrospective study involving thirty-six patients with thirty-seven ankle fusions was undertaken to assess the rate of fusion, the subjective and objective results, the residual subtalar and midtarsal movement, and the functional disability. Fusion occurred in thirty-one ankles (84 per cent). Twenty-four patients were reviewed, on average 7.5 years after fusion, and eighteen had good or excellent results; only four had been unable to return to their previous employment. The conclusion is that fusion is still a good treatment for the painful post-traumatic arthritis ankle, the resulting functional disability being minimal.


The surface ultrastructure of ganglia.

The surface ultrastructure of ganglia. The surface ultrastructure of ganglia has been studied using the scanning electron microscope. This study showed that the ganglion wall consists of multidirectional strata of collagen fibres and has no cellular lining. The wall has a sponge-like appearance and does not appear degenerate or necrotic. Comparison with synovial membrane and adventitious bursa confirmed that these are distinct structures which have a cellular lining. Ganglia probably arise from the multifunctional mesenchymal cells which are found within their walls. The ganglion fluid may also originate from these cells.


Experimental production of forward dislocation in the human cervical spine. Entire human cadaveric cervical spines with the basiocciput were subjected to load in a compression apparatus to simulate the clinical situation of forward dislocation. The movements were recorded by lateral cineradiography. Vertical load was measured by a potentiometric transmitter synchronised with each frame of the cineradiograph. The lower part of the spine was flexed and fixed, and the upper extended and free to move forward. Vertical compression then produced bilateral dislocation of the facets without fracture. If lateral tilt or axial rotation occurred as well, a unilateral dislocation was produced. The maximum vertical load was only 145 kilograms, and coincided with the rupture of the posterior ligament and capsule and the stripping of the anterior longitudinal ligament, but this occurred before dislocation. The low vertical load indicates a peculiar vulnerabiity of the cervical spine in this position and correlates well with the minor trauma often seen in association with forward dislocation.


The distribution of surface strain in the cadaveric lumbar spine. The fourth lumbar vertebrae and L4-5 discs from six cadaveric lumbar spines were subjected to detailed strain gauge analysis under conditions of controlled loading. With central compression loads, maximal compressive strain was found to occur near the bases of the pedicles and on both superficial and deep surfaces of the pars interarticularis, which emphasises the importance of the posterior elements of lumbar vertebrae in transmitting load. Radial bulge and tangential strain of the disc wall were maximal at the posterolateral surface, in agreement with the fact that disc degeneration and prolapse commonly occur there. Under posterior offset loads simulating extension, both compressive and tensile strains were found to be increased on both surfaces of the pars interarticularis, which suggests that hyperextension may lead to stress fractures and spondylolisthesis. Posterior offset loads also increased the radial bulge of the posterior disc wall and tangential strain at the anterior surface of the disc. Anterior offset loads simulating flexion increased the radial bulge of the anterior disc wall and tangential strain at the posterior surface of the disc. These findings are compatible with movement of the nucleus pulposus within the disc during flexion and extension. This hypothesis was supported by post-mortem discography.


Mechanical tests on the tibial components of non-hinged knee prostheses. Cadaveric knees replaced with the Geomedic, ICLH, Marmor and Total Condylar prostheses were tested in axial compression, in rotation and in hyperextension in order to observe the strength of fixation of the tibial components. In axial compression the strengths at failure varied widely, both with any one prosthesis and between prostheses. This is attributed largely to the strength of the cancellous bone of the tibia, which was measured in each case and also varied widely. Three natural knees failed at loads of 7300, 7600 and 8300 newtons respectively, whereas the strengths of replaced knees ranged from 3000 to 15750 newtons. At least one example of each design failed at less than 7300 newtons, suggesting little or no reserve of strength. The strength of fixation was greater when the tibial prosthesis was large enough to rest on the whole cross-section of the tibia. In rotation the three prostheses embodying rollers in troughs were stiffer than the Marmor which had a nearly flat tibial-bearing surface. The presence or absence of the cruciate ligaments had a negligible effect on torsional stiffness. In hyperextension, knees replaced with the ICLH, Marmor and Total Condylar prostheses failed by rupture of the posterior capsule at moments of about 60 newton-metres, compared with about 100 for natural knees. With the Marmor prosthesis the anterior cruciate ligament was avulsed at about 20 newton-metres compared with about 75 in natural knees, suggesting that in this respect the retention of the cruciate ligaments contributes little. None of the four knees tested after inserting a Geomedic prosthesis showed strengths as high as those replaced with the other three designs.


The microsurgical revascularisation of resected segments of tibia in the dog. A segment of tibia 4.5 centimetres long was removed from one hind limb of fifteen dogs. It was then replaced and the main vasculature was restored by a microsurgical technique. In eight controls the segment was replaced without such restoration. In two-thirds of the former cases the microvascular reconstruction was successful; the rate of infection was found to be reduced, bone union was guaranteed and the rate of union accelerated. Success or failure of the reconstruction was clearly demonstrated in five cases by early bone scanning using technetium-labelled polyphosphate.


The release of gentamicin from polymethylmethacrylate beads. An experimental and pharmacokinetic study. Gentamicin incorporated in beads of polymethylmethacrylate has been shown capable of being released over a period of several months in concentrations sufficiently high to control most pathogens. The therapeutic efficacy of such beads has been demonstrated in a model of osteomyelitis of the femur in the dog. Good tolerance has been shown, both in the animal model and in tissue cultures. In forty-one patients with infection of either bone or soft tissue, mainly of the lower limb, the findings were similar. The concentrations in serum and urine were low, which excludes side-effects. The insertion of gentamicin-PMMA beads may prove to be a valuable new form of local antibiotic therapy.


Intra-abdominal sepsis--ultrasound evaluation. The advent of grey-scale ultrasound has made this facility an irreplaceable tool for diagnostic imaging techniques. Although well established in obstetric management, its application to abdominal "body" scanning is not yet fully realized and accepted. These case presentations demonstrate the role of ultrasound in the diagnosis, localization and clinical management of patients with intra-abdominal sepsis. It is emphasized that ultrasound should be used early in the work-up of such problems and is rapidly becoming the investigation of choice. The diagnostic information provided facilitates management, reduces the need for complex investigations and occasionally makes surgery unnecessary.


Mature placenta simulating fetus papyraceus on ultrasonic examination. A case of a heavily calcified, mature placenta simulating the appearance of a fetus papyraceus is presented. Calcification surrounding the more sonolucent cotyledons simulated the body and limbs of a dead fetus. Awareness of the morphologic alterations which occur as the placenta matures should prevent confusion of normal placental changes with a pathologic entity.


Renal pseudotumor due to gas-filled hepatic flexure. Renal pseudotumors--both intrinsic and extrinsic--are numerous. On a recent urogram, performed with abdominal pressure and tomography, a mass lesion was simulated by the gas-filled colon. The problem was resolved with a combination of ultrasonography, nephrotomography, and barium enema examinations.


Cerebrospinal fluid ascites.

Cerebrospinal fluid ascites. One new and one previously reported case of cerebrospinal fluid ascites following ventricular-peritoneal shungting are presented. Ultrasonic investigation is now most helpful.


The angiography of intrarenal leiomyoma. A patient with a leiomyoma of the kidney was studied by ultrasound, intravenous pyelography and angiography. Only three renal leiomyomas have been investigated by angiography. The angiographic features of our patient's tumor differed from these others, being much more vascular with multiple tortuous vessels and an inhomogeneous capillary phase.


Interstitial gastric and duodenal emphysema. A 31-year-old woman developed combined interstitial gastric and duodenal emphysema associated with acute dilatation of the stomach and duodenum following an asthmatic attack. The literature is reviewed.


A needle-catheter kit in knee arthrography. Use of a sheathed Teflon needle in knee arthrography facilitates removal of synovial effusion and eliminates trauma due to a rigid needle.


Narcotic smuggling: the double condom sign. The "double condom sign," on a plain film of the abdomen, can be used to identify smugglers who are concealing non-opaque narcotics within the gastrointestinal tract.


133Xe uptake by fatty livers.

133Xe uptake by fatty livers. Of 19 studies on 15 patients with morbid obesity and fatty infiltration of the liver, 16 had increased uptake and retention of 133Xe in the liver following 5-minute rebreathing of 133Xe and oxygen. This observation shows a high correlation with the histologically proven presence of fat in the hepatocytes, and offers a simple, accurate and precise indication of the excess fat in the liver.


Reversible renal failure secondary to severe compensated aortic regurgitation. Severe renal failure associated with proteinuria occurred in a 21-year patient, who had massive rheumatic aortic regurgitation. There was no sign of congestive heart failure or extra-cellular dehydration. Subacute bacterial endocarditis was ruled out by appropriate laboratory investigations. Prosthetic aortic valve replacement resulted in normalization of the renal function and marked reduction of proteinuria. Renal histology showed severe sclerotic endarteritis involving predominantly the large arteries, and membrano-proliferative-like glomerulopathy without immune deposits. The role of the massive aortic regurgitation in the production of renal failure and histologic alterations is suggested.


Venous thrombectomies and partial interruption of the vena cava in 125 cases of thrombophlebitis. In 125 cases surgically treated for thrombophlebitis of the lower limbs, thrombectomy was done in 96, while caval clipping alone was done in 31. Beginning in 1965, technical improvements and systematic control of the 3 major cross points of the lower limb venous system resulted in gradually improved results as regards patency rate and clinical status. When the return flow was increased by arteriovenous fistula, the patency rate and the clinical results were significantly improved. Comparison of a first group of 23 patients submitted to venous thrombectomy alone with 102 patients protected by filters or serrated clips shows a drastic decrease in lethal pulmonary embolism postoperatively. Mortality  in the unprotected group was 8.7% while in the protected series it fell to .98%: the latter death was in fact due to forced selection of an  operation of second choice. In the protected series, 69 preoperative pulmonary embolisms were recorded in  55 patients. Only  one minor postoperative embolism occurred, this originating from an axillary phlebitis. We find  caval clipping to be formally indicated whether or not a venous disobstruction procedure is feasible.


Effect of increased oxygen on peripheral circulation in acute, temporary limb hypoxia. This study was intended to investigate the effect of high arterial oxygen tension on the peripheral circulation between normal and hypoxic area by using twelve dogs. For this purpose, acute temporary ischemia was produced at the right hindlimb by an occlusion of the right external iliac artery for 1 and 2 hours, and the left hind-limb was let alone as a control. The peripheral vascular resistance and the arterio-venous difference in oxygen tension between the right and the left hind-limb were measured continuously during 1 hour from immediately after reopening of the blood flow of the right external iliac artery. They were compared between at the condition of breathing room air at 1 ATA and pure oxygen at 2 ATA. The high arterial oxygen tension causes vasoconstriction. When there exists hypoxia in the peripheral tissue, however, it does not act on as a vasoconstrictive factor until the oxygen deficit is improved.


Left coronary artery originating from the pulmonary artery correction and total myocardial blood flow measurements. In a boy aged 12 years correction of an anomalous coronary artery system was performed by suture of the left coronary artery origin at the pulmonary artery trunk and a vein by-pass to the proximal LAD. Peroperative blood flow gives values for myocardial perfusion before and after by-pass with relation to patency of the vein graft. Before correction pure coronary blood flow was 540 ml/min, shunt flow 540 ml/min; in total 1,080 ml/min through the right coronary artery. After correction the vein by-pass conducted 310 ml/min and the right coronary artery 180 ml/min. At follow-up 2 months later coronary angiography showed a normal right coronary artery without collateral connections to the left coronary artery system which was well perfused from the by-pass. Left heart hypertrophy was reduced from 568 ml/m2. A weak systolic murmur at the apex could be a slight unimportant mitral insufficiency.


Apparent rupture of the ventricular septum after myocardial infarction in an infant with congenital heart disease. Clinical and angiocardiographic simulation of rupture of the interventricular septum, in an infant with an endocardial cushion defect and documented myocardial infarction is reported. Close straddling of a proven congenital muscular apical ventricular septal defect by trabeculae carne is believed to have led to the angiocardiographic simulation of a post-infarction apical ventricular septal defect. In the surgical approach to post-infarction ventricular septal defects, the implication of such a simulation could be important.


A proposed technique for treatment of hypoplastic left heart syndrome. The Hypoplastic Left Heart Syndrome includes a variety of anomalies, all characterized by a hypoplastic or absent left ventricle. A technique is described for producing a normal course of circulation in these cases. Its ultimate success will depend on the patients having a low pulmonary vascular resistence.


Superiority of aortic over femoral cannulation for cardiopulmonary bypass, with specific attention to lower extremity neuropathy. The advantage of aortic cannulation over femoral cannulation in cardiopulmonary bypass has been well established in our experience. Over a four year period, we compared the incidence of complications of these two modalities in a large group of patients. Specific emphasis is placed on the lack of lower extremity neurological deficit and retrograde dissection in patients who underwent aortic cannulation.


Open mitral commissurotomies. Analysis of 100 cases. Routine use of open mitral commissurotomy in all cases of rheumatic mistral stenosis is the recommended treatment of choice in recent years. Effective division of the subvalvular apparatus (chordae tendineae and papillary muscles) can be accomplished easily during open commissurotomy and therefore recurrences after the open approach is less than the recurrences after closed commissurotomy. These subvalvular elements can not be freed easily during closed mitral commissurotomy. Low mortality rates after open approach suggests that this operation can be performed safely in cases of MS. Open approach has several advantages versus closed commissurotomy. We believe that closed commissurotomy should be replaced by open commissurotomy in all cases of MS.


Sinoatrial disorders, the "sick sinus" syndrome. Experience with implanted cardiac pacemakers. The long term results of permanent pacemaker implantation in patients with disorders of the sinoatrial node and atrial conduction pathways were studied. In a consecutive series of 300 patients treated by pacemaker implantation, 68 ((22.3%) had sinoatrial disorders. Mean age of the group was 54 years (33-82 years); 37 were male, 31 female. Diagnosis was confirmed by serial electrocardiography with overdrive suppression of sinoatrial node function as indicated. Twenty-two patients presented with sinus bradycardia alone (Type I disorder) and 46 with bradycardia-tachycardia syndrome (Type II disorder) were completely controlled. of the 46 Type II patients, in 30 (65.2%) tachyarrhythmias were controlled by pacing alone and in 14 (30.4%) by pacing and antiarrhythmic drug therapy. Congestive cardiac failure was relieved in 80% and anginal pain in 70% of patients with rate control alone. Systemic embolisation did not recur after pacemaker implantation. The average duration of pacemaker therapy was 52 months (12-96 months). In this series, long term ventricular demand pacing proved beneficial in both types of sinoatrial disorders.


High molecular weight MAPs are part of the mitotic spindle. We have found that the microtubule-associated proteins of high molecular weight are located in the mitotic spindle. Indirect immunofluorescence studies reveal that the pattern of distribution of these proteins is similar to that described for tubulin and corresponds to the known phases of mitosis.


Protective effect of betamethasone on the subendocardial ischemia after the cardiopulmonary bypass. The experimental and clinical studies on the protective effect of Betamethasone upon the myocardium particularly on the prevention of the subendocardial ischemia after the cardiopulmonary bypass was investigated on the ultrastructural alterations of the epicardium and the endocardium obtained by the transmural left ventricular myocardial biopsy. Pretreatment of Betamethasone, 5 mg/Kg, with single intravenous injection a half to one hour before cardiopulmonary bypass was effective to preserve the myocardial ultrastructure after the anoxic arrest and in the recovery period after restoring the coronary circulation. The intramyocardial gradient of the ultrastructure was diminished by Betamethasone hardly demonstrating the no-reflow phenomenon. Hemodynamic and metabolic changes did not show the significant effectiveness of Betamethasone. It was suspected that Betamethasone stabilized the cell membrane and lysosome and suppressed the interstitial edema in the myocardium during and after anoxic arrest.


Selective binding, uptake, and retrograde transport of tetanus toxin by nerve terminals in the rat iris. An electron microscope study using colloidal gold as a tracer. A series of specific macromolecules (tetanus toxin, cholera toxin, nerve growth factor [NGF], and several lectins) have been shown to be transported retrogradely with high selectivity from terminals to cell bodies in various types of neurons. Under identical experimental conditions (low protein concentrations injected), most other macromolecules, e.g. horseradish peroxidase (HRP), albumin, ferritin, are not transported in detectable amounts. In the present EM study, we demonstrate selective binding of tetanus toxin to the surface membrane of nerve terminals, followed by uptake and subsequent retorgrade axonal transport. Tetanus toxin or albumin was adsorbed to colloidal gold particles (diam 200 A). The complex was shown to be stable and well suited as an EM tracer. 1-4 h after injection into the anterior eye chamber of adult rats, tetanus toxin-gold particles were found to be selectively associated with membranes of nerve terminals and preterminal axons. Inside terminals and axons, the tracer was localized mainly in smooth endoplasmic reticulum (SER)-like membrane compartments. In contrast, association of albumin-gold complexes with nervous structures was never observed, in spite of extensive uptake into fibroblasts. Electron microscope and biochemical experiments showed selective retrograde transport of tetanus toxin-gold complexes to the superior cervical ganglion. Specific binding to membrane components at nerve terminals and subsequent internalization and retrograde transport may represent an important pathway for macromolecules carrying information from target organs to the perikarya of their innervating neurons.


Cell wall structure and deposition in Glaucocystis. Events leading to cell wall formation in the ellipsoidal unicellular alga Glaucocystis are described. The wall is deposited in three phases: (a) a thin nonfibrillar layer, (b) cellulosic microfibrils arranged in helically crossed polylamellate fashion, and (c) matrix substances. At poles of cells, microfibrils do not terminate but pass around three equilaterally arranged points, resulting in microfibril continuity between the twelve helically wound wall layers. These findings were demonstrated in walls of both mother cells and freeze-fractured growing cells, and models of the wall structure are presented. Cellular extension results in spreading apart, and in rupture, of microfibrils. On freeze-fractured plasma membranes, there were 35 nm X 550 nm structures associated with the ends of microfibrils. These are interpreted as representing microfibril-synthesizing centers (terminal complexes) in transit upon the membrane. These terminal complexes are localized in a zone, or zones. The plasma membrane is subtended by flattened sacs, termed shields, which become cross-linked to the plasma membrane after completion of wall deposition. During wall deposition, microtubules lie beneath the shields, and polarized filaments lie between shields and plasma membrane. The significance of these findings in relation to understanding the process of cellulose deposition is discussed, and comparisons are made with the alga Oocystis.