Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae

Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.     

Elementary steps in the formation of horseradish peroxidase compound I: direct observation of compound 0, a new intermediate with a hyperporphyrin spectrum

The reaction of horseradish peroxidase (HRP) with H2O2 has been studied in 50% v/v methanol/water over the 25.0 to -36.0 degrees C temperature range by using the low-temperature stopped-flow technique. All reactions were carried out under pseudo-first-order conditions with [H2O2] much greater than [HRP]. Arrhenius plots for the pseudo-first-order rate constant kobs were linear over the 17.6 to -36.0 degrees C temperature range studied with an activation energy of 4.8 +/- 0.5 kcal/mol. Above 0 degrees C, kobs varies linearly with peroxide concentration. However, saturation kinetics are observed below -16.0 degrees C, indicating that there is at least one reversible elementary step in this reaction. Double-reciprocal plots at -26.0 degrees C at pH* 7.3 for the reaction give kappa max(obs) = 163 s-1 and KM = 0.190 mM. Rapid-scan optical studies carried out at -35.0 degrees C with [H2O2] much greater than KM reveal the presence of a transient intermediate referred to as compound 0 whose conversion to compound I is rate limiting. The Soret region of the optical spectrum of compound 0 resembles that of a "hyperporphyrin" with prominent bands near 330 and 410 nm. The temperature dependencies of kappa max(obs) and KM have been measured over the -16.0 to -26.0 degrees C range and give an activation energy for kappa max(obs) of 1.6 +/- 0.7 kcal/mol and an enthalpy of formation for compound 0 of 4.0 +/- 0.7 kcal/mol.     

Autoantibodies from patients with systemic lupus erythematosus can affect DNA and RNA synthesis in cultured cells

Sera reacting positively for anti-DNA antibodies from systemic lupus erythematosus (SLE) patients were tested for their effect on DNA and RNA synthesis in permeabilized cultured cells and isolated nuclei. The immunoglobulin fraction obtained by ammonium sulfate precipitation of serum was shown to exert considerable influence on DNA and RNA synthesis in cultured cells and nuclei. A component of this antibody population is anti-DNA. These antibodies exert different effects on DNA template activity which is a function of their conformational specificity. Intracellular penetration of autoantibodies as noted in SLE may be one of the reasons for clinical manifestations of disease in these patients.     

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.     

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.     

In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus

The reaction velocity of glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) was quantified with a cytophotometer by continuous monitoring of the reaction product as it was formed in liver cryostat sections from normal, young mature female rats at 37 degrees C. Control incubations were performed in media lacking both substrate and coenzyme for G6PDH activity and lacking substrate for PGDH activity. All reaction rates were non-linear but test minus control reactions showed linearity with incubation time up to 5 min using Nitro BT as final electron acceptor. End point measurements after incubation for 5 min at 37 degrees C revealed that the highest specific activity of G6PDH was present in the intermediate area (Vmax = 7.79 +/- 1.76 mumol H2 cm-3 min-1) and of PGDH in the pericentral and intermediate areas (Vmax = 17.19 +/- 1.73 mumol H2 cm-3 min-1). In periportal and pericentral areas, Vmax values for G6PDH activity were 4.48 +/- 1.03 mumol H2 cm-3 min-1) and 3.47 +/- 0.78 mumol H2 cm-3 min-1), respectively. PGDH activity in periportal areas showed a Vmax of 10.84 +/- 0.33 mumol H2 cm3 min-1. Variation of the substrate concentration for G6PDH activity yielded similar KM values of 0.17 +/- 0.07 mM, 0.15 +/- 0.13 mM and 0.22 +/- 0.11 mM in periportal, pericentral and intermediate areas, respectively. KM values of 0.87 +/- 0.12 mM in periportal and of 1.36 +/- 0.10 mM in pericentral and intermediate areas were found for PGDH activity. The significant difference between KM values for PGDH in areas within the acinus support the hypothesis that PGDH is present in the cytoplasmic matrix and in the microsomes. A discrepancy existed between KM and Vmax values determined in cytochemical assays using cryostat sections and values calculated from biochemical assays using diluted homogenates. In cytochemical assays, the natural microenvironment for enzymes is kept for the demonstration of their activity and thus may give more accurate information on enzyme reactions as they take place in vivo.     

Benthic algal biomass and productivity in high subarctic streams,Alaska

Year-round measurements of the standing crop of epilithic algae (as chlorophyll a concentration) in two streams — one second and one fourth order (map scale 1:63 360) — in interior Alaska (64°–65° N) were only about one tenth that reported from streams of temperate North America. Cell densities in these streams, however, were similar to those in comparable temperate streams. Year-round domination of the benthic flora by very tiny diatoms (Achnanthes spp.) may explain the apparent disparity between low chlorophyll a content and nearly average cell densities. Chlorophyll a standing crop in a more alkaline groundwater-fed stream, however, was higher and within the range of similarly sized temperate streams. Maximum chlorophyll a standing crop varied positively with alkalinity in 5 clear-water streams where standing crop was measured on natural or artificial substrates. Seasonal mean concentrations of sestonic chlorophyll a (used as estimates of benthic algal chlorophyll a standing crop) varied directly and significantly with alkalinity among ten clear-water streams; and, with total phosphorus among 8 of 10 clear-water and 5 brown-water streams studied. During the summer, when there is little darkness, gross primary productivity (as estimated by the diurnal dissolved-oxygen method) was similar to that of northern temperate streams. Gross primary productivity was also seen to vary directly with alkalinity in 5 clear-water streams of this region.U.S. Fish and Wildlife Service     

Different preparations of natural and recombinant human interleukin-6 (IFN-beta 2, BSF-2) similarly stimulate acute phase protein synthesis and uptake of alpha-aminoisobutyric acid by cultured rat hepatocytes

1. Rat hepatocytes were cultured for 2 days in Williams E medium containing 1 microM insulin and dexamethasone. 2. Production of five plasma proteins was determined by electroimmunoassay in the media, and amino acid uptake was measured by [alpha-14C]aminoisobutyric acid accumulation in hepatocytes. 3. Supernatants from rat peritoneal macrophages and IL-6/IFN-beta 2/BSF-2 obtained from four different laboratories similarly stimulated synthesis of fibrinogen, alpha 1-cysteine proteinase inhibitor and alpha 2-macroglobulin, as well as [14C]-accumulation in cultured hepatocytes. 4. It is concluded that IL-6 is the principal hepatocyte stimulating factor responsible for typical features of the acute phase response of liver cells.     

On the occurrence of macroorchidism and mental handicap in the Aarskog syndrome

In this report we present follow-up on two moderately mentally retarded boys with Aarskog syndrome. As 22 other mentally normal Aarskog patients these two boys presented a catch-up after a delayed puberty with a final adult height of 160 cm. A remarkable finding was the development of macroorchidism in two mentally retarded Aarskog patients. The pathogenesis of macroorchidism in the fragile X syndrome and in other X-linked mental retardation syndromes is discussed.     

The primary structure of cytochrome c from the nematode Caenorhabditis elegans.

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an 'invariant' phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.