Cloning, sequence analysis and expression of gene alyVI encoding alginate lyase from marine bacterium Vibrio sp. QY101.

The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp. QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli. Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa. AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography. AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl2. A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI. However, AlyVI could degrade both M block and G block. These results indicate that a novel alginate lyase-encoding gene has been cloned.     

人胸腺素α原cDNA的克隆及在大肠杆菌中的表达

采用基因工程方法制取人胸腺素α原获得成功。用２０ｕｇ／ｍｌ植物血球凝集素（ＰＨＡ）和５００Ｕ／ｍｌ重组人白细胞介素２（ＩＬ－２）联合刺激人胎儿胸腺细胞,从中提取总ＲＮＡ,经反转录ＰＣＲ获得了人胸腺素α原ｃＤＮＡ;将之克隆入ｐＵＣ１９中,序列测定表明与已报道序列一致,进一步将之亚克隆入原核表达载体ｐＢＶ２２０,转化大肠杆菌ＤＨ５ａ．观察到在不改变氨基酸编码的前提下,增加胸腺素ａ原上游引物中Ａ、Ｔ含量,可以明显提高胸腺素α原的表达量,同时,不同培养基对它的表达也有影响。胸腺素α原在大肠杆菌中以可溶形式表达,不需复性。初步活性测定显示,它可明显刺激人外周血淋巴细胞Ｅ－玫瑰花结形成率。重组人胸腺素α原在大肠杆菌中表达,为其临床应用及基础研究奠定了基础。     

Interferon-γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development

Interferon-γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF-γ addition was delayed after culture initiation. The effects of INFγ on BFU-E did not require the presence of interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1α, TNFα, or GM-CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL-1α- or TNFα-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INFγ antibody. In contrast, GM-CSF—induced suppression of BFU-E was negated by the simultaneous addition of anti-INFγ. We have previously shown that the addition of TNFα does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM-CSF—induced inhibition of BFU-E colony growth. © 1995 Wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

Fatty Acyl-CoA Inhibits 1-Alkyl-sn-Glycero-3-Phosphate Acetyltransferase in Microsomes of Immature Rabbit Cerebral Cortex: Control of the First Committed Step in the De Novo Pathway of Platelet-Activating Factor Synthesis

Abstract: Microsomal fractions of cerebral cortices of 15-day-old rabbits were used to study the 1-alkyl- sn -glycero-3-phosphate (AGP) acetyltransferase that generates 1-alkyl-2-acetyl- sn -glycero-3-phosphate in the de novo path of platelet-activating factor synthesis. The AGP acetyltransferase activity was inhibited by small concentrations of medium-long chain fatty acyl-CoA thioesters. In contrast, the AGP acyltransferase used oleoyl-CoA as substrate and was not inhibited by the presence of acetyl-CoA in high molar excess. The inhibition of AGP acetyltransferase was seen at concentrations of oleoyl-CoA as low as 0.5 µ M using 12.5 µ M AGP and 200 µ M acetyl-CoA. The inhibition by oleoyl-CoA was noncompetitive for the acetyl-CoA substrate. However, there was evidence that the oleoyl-CoA was competing with AGP in the acetyltransferase reaction, as the inhibition was lessened by increasing the AGP substrate concentration. Several acyl-CoA thioesters were effective as inhibitors of the AGP acetyltransferase, including oleoyl-, palmitoyl-, lauroyl-, and octanoyl-CoA. Propionyl- and butyryl-CoA were less effective as inhibitors, and propionyl-CoA was found to be a competitive inhibitor for acetyl-CoA. We have noted earlier that MgATP is an effective inhibitor of the AGP acetyltransferase and here we show that the inhibition by oleoyl-CoA can be increased by the presence of 0.1 m M MgATP. In brain ischemia, a decline in ATP levels would likely lead to a corresponding fall in acyl-CoA concentrations, thereby relieving the inhibition of AGP acetyltransferase and permitting the flow of AGP into the de novo pathway of platelet-activating factor synthesis.     

GTPγS restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells

Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finchet al., J Biol Chem 268, 5823–5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTPS, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTPS still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.     

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.     

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D 2,4-dichloro-phenoxyacetic acid - CaMV Cauliflower mosaic virus - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FDA fluorescein diacetate - HEPES (hydroxyethyl)piperazine(ethanesulfonic acid) - HR hypersensitive reaction - Tris tris (hydroxymethyl)amino-methane