Kinetic β-deuterium isotope effects suggest a covalent mechanism for the protein folding enzyme peptidylprolyl cis/trans-isomerase

The cis/trans interconversion of Glt-Ala-Ala-Pro-Phe-4-nitroanilide and Glt-Ala-Gly-Pro-Phe-4-nitroanilide was studied both enzymatically and nonenzymatically by measuring kinetic β-deuterium isotope effects. The hydrogen atom at the α-carbon atom of the Xaa residue within the Xaa-Pro moiety was substituted by deuterium. In the nonenzymatic case the transition state of rotation is reflected by k_H/k_D > 1. When catalysed by 17 kDa PPIase the same bond rotation is characterized by k_H/k_D < 1. This suggests a covalent mechanism of catalysis which involves an approximately tetravalent carbon of the prolyl imidic bond for the transition state of reaction.     

Synthesis and biological properties of 4-norleucine-neuropeptide Y; secondary structure of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide amide isolated from porcine brain. The NPY analog, 4-norleucine-NPY was synthesized by a solid-phase method and purified to homogeneity in 20% yield by reverse-phase chromatography. Investigation of the biological properties indicated that the analog is an agonist of NPY. Secondary structural analyses revealed that NPY and the analog exhibited predominantly alpha-helical and beta-sheet structures, respectively; however, experiments in trifluoroethanol indicated that the analog has the potential of assuming an alpha-helical structure. Based on circular dichroism (CD), Raman spectroscopy and Chou-Fasman analyses, a model has been proposed for the secondary structure of NPY.     

Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure,and quantitative determinations of catecholamines and somatostatin

Summary The ultrastructural and transmitter development of lumbar sympathetic ganglia was studied in embryonic day-6 through-18 chick embryos. At embryonic day 6, ganglia are populated by two morphologically distinct types of neuronal cells and Schwann cell precursors. The neuronal populations basically comprise a granule-containing cell and a developing principal neuron. Granule-containing cells have, an irregularly shaped or oval nucleus with small clumps of chromatin attached to the inner nuclear membrane and numerous large (up to 300 nm) membrane-limited granules. Developing principal neurons display a more rounded vesicular nucleus with evenly distributed chromatin, prominent nucleoli, more developed areas of Golgi complexes, and rough endoplasmic reticulum and large dense-core vesicles up to 120 nm in diameter. There are granule-containing cells with fewer and smaller granules which still display the nucleus typical for granule-containing cells. These granule-containing cells may develop toward developing principal neurons or the resting state of granule-containing cells found in older ganglia. Both granule-containing cells and developing principal neurons proliferate and can undergo degeneration. At embryonic day 9 there are far more developing principal neurons than granule-containing cells. Most granule-containing cells have very few granules. Mitotic figures and signs of cell degeneration are still apparent. Synapse-like terminals are found on both developing principal neurons and granule-containing cells. Ganglionic development from embryonic day 11 through 18 comprises extensive maturation of developing principal neurons and a numerical decline of granule-containing cells. Some granule-containing cells with very few and small granules still persist at embryonic day 18. The mean catecholamine content per neuron increases from 0.044 femtomol at embryonic day 7 to 0.22 femtomol at embryonic day 15. Concomitantly, there is a more than 6-fold increase in tyrosine hydroxylase activity. Adrenaline has a 14% share in total catecholamines at embryonic day 15. Somatostatin levels are relatively high at embryonic day 7 (1.82 attomol per neuron) and are 10-fold reduced by embryonic day 15. Our results suggest the presence of two morphologically distinct sympathetic neuronal precursors at embryonic day 6: one with a binary choice to become a principal neuron or to die, the other one, a granule-containing cell, which alternatively may develop into a principal neuron, acquire a resting state or die.     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Ultrastructure of neutrophilic granulopoiesis in the bone marrow of patients with chronic myeloid leukemia (CML). A morphometric study with special emphasis on azurophil (primary) and specific (secondary) granules

In seven patients with chronic myeloid leukemia (CML) and ultrastructural and morphometric study was performed on neutrophilic granulopoiesis in bone marrow trephine biopsies. Bone marrow specimens from five patients without hematological abnormalities served as controls. In stable phases of CML, abnormalities of the maturing granulocytic lineage were most conspicuously expressed by an infrequently occurring nuclear disfiguration (blebs and disturbed bridging of segments). Morphometric evaluation included the numbers of azurphil (primary) and specific (secondary) granules, the cisternal length of the endoplasmic reticulum and the area of the mitochondrial profiles. These variables could be determined in early and late myeloblasts, promyelocytes, metamyelocytes, band cells and mature polymorphonuclear granulocytes. Statistical analysis with regard to control specimens demonstrated no significant differences in the total amount of neutrophil granules or of the other cell organelles.     

A histomorphometric analysis of trephine biopsies of bone marrow from 65 patients with chronic myeloid leukemia. Classification of patients into subgroups with different survival patterns

A histomorphometric (planimetric) study was performed on trephine biopsies of the bone marrow taken at presentation from 65 patients (31 males and 34 females, with a median age of 48 years) with chronic myeloid leukemia (CML). Specimens from 20 patients (9 males and 11 females, with a median age of 53 years) without any hematologic disorders served as controls. Of the various histologic variables tested, only the counts of neutrophilic granulocytes per 1 sq mm, the ratio of granulocytopoiesis to megakaryopoiesis and the density of reticulin (argyrophilic) fibers revealed a significant correlation with the prognosis. The CML patients were separated into two groups with different survival patterns. Group I (34 patients with a median survival of 24 months) mostly contained cases with the so-called "megakaryocytic subtype" of CML, which is accompanied by variable degrees of fibrosis; group II (31 patients with a median survival of 36 months) mainly contained cases with the "granulocytic subtype," which is not accompanied by myelofibrosis. Among the morphometric parameters, a positive correlation existed between the megakaryocyte count and the reticulin fiber density, which underlines the important role of that cell lineage in fibrillogenesis. There were multiple interrelationships between the histomorphometric variables and the laboratory data. Consequently, multivariate regression methods (using Cox's proportional hazards model) were applied to assess the relative predictive value of the patient characteristics for survival. The derived prognostic model divided the patients into two risk groups, with median survivals of 14 and 41 months, respectively. In order of their entry into the regression model, these variables were percentage of neutrophils in the differential blood count, amount of granulopoiesis, liver size, percentage of peripheral myeloblasts and density of reticulin fibers in the bone marrow. In comparing the two patient groups, based on bone marrow histomorphometric parameters, this model revealed that two of those factors (amount of granulopoiesis and density of reticulin fibers) had a significant correlation with the prognosis.     

The lipopolysaccharide of the phototrophic bacterium Ectothiorhodospira vacuolata

The lipopolysaccharide of Ectothiorhodospira vacuolata was obtained by the phenol-water procedure. It contained a 3-O-methyl-hexose, glucose, galacturonic and glucuronic acids. The finding of d-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate (tentatively identified) suggested a core-structure. The lipid fraction of the lipopolysaccharide contained phosphate and both, 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The major fatty acids were amine-bound 3-OH-10:0 and 3-OH-12:0 and esterbound 14:0 and 16:0 Sodium deoxycholate gel-electrophoresis, showing a single band only, indicated R-type character of the lipopolysaccharide of Ectothiorhodospira vacuolata.Abbreviations DOC sodium deoxycholate - GC/MS combined gasliquid chromatography - PAGE polyacrylamide gel-electrophoresis     

Practical application of the protein C activator Protac from Agkistrodon contortrix venom

The protein C activator Protac from A. contortrix venom is being investigated as a potential antithrombotic agent and as a tool for the preparation of activated protein C. Its established major application is the zymogen activation in functional protein C determinations based on either a clotting assay or a chromogenic substrate technique. The sensitivity of the activated partial thromboplastin time as an indicator reaction for Protac activated protein C depends on the contact activator component of the reagent. Protein C dose-response increased in the following order: kaolin greater than ellagic acid greater than sulfatide. This phenomenon is due to a competition of molecular affinities between Protac, plasma components and the different activating surfaces.     

Homologous and heterologous β-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331–336) and this heterologous β-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous β-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.