in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Tau protein kinase I/GSK-3Β/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain

Previously, tau protein kinase I/glycogen synthase kinase-3/kinase F_A(TPKI/GSK-3/F_A) was identified as a brain microtubule-associated tau kinase possibly involved in the Alzheimer disease-like phosphorylation of tau. In this report, we find that the TPKI/GSK-3/F_A can be stimulated to phosphorylate brain tau up to 8.5 mol of phosphates per mol of protein by heparin, a polyanion compound. Tryptic digestion of³²P-labeled tau followed by high-performance liquid chromatography and high-voltage electrophoresis/thin-layer chromatography reveals 12 phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and peptide sequence analysis further reveals that TPKI/GSK-3//F_A after heparin potentiation phosphorylates tau on sites of Ser¹⁹⁹, Thr²³¹, Ser²³⁵, Ser²⁶², Ser³⁹⁶, and Ser⁴⁰⁰, which are potential sites abnormally phosphorylated in Alzheimer tau and potent sites responsible for reducing microtubule binding possibly involved in neuronal degeneration. The results provide initial evidence that TPKI/GSK-3/F_A after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau and neuronal degeneration in Alzheimer disease brains.Abbreviations F_A the activating factor of type 1 protein phosphatase - GSK-3 glycogen synthase kinase-3 - TPKI tau protein kinase I - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - PHF paired helical filaments - HPLC high-performance liquid chromatography     

人微小纤溶酶原基因的克隆和表达

用PCR方法扩增人微小纤溶酶原(Microplasmingen,mPlg)基因,再与表达载体重组．构造mPlg原核表达质粒并转化大肠杆菌。阳性克隆pSSE-mPlg经温度诱导表达,SDS-PAGE等方法证明表达产物的分子量约为29kDa。占全菌总蛋白的24％左右,并在菌内形成包涵体。经半胱氨酸再氧化法和空气氧化法复性。表达产物r-mPlg经SK作用后显示纤溶活性。同时对蛋白质浓度、复性时间等因素对复性的影响进行了初步探讨。     

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.     

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.     

Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem.

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.     

phbB,phbC基因克隆,序列分析及植物表达载体的构建

利用聚合酶链式反应技术,从真养产碱杆菌Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ Ｈ１６染色体ＤＮＡ中的主增并克隆了调控聚－β－羧基丁酸生物合成的两个关键酶基因;依赖ＮＡＤＰＨ的乙酰乙酰ＣｏＡ还原酶基因和ＰＨＢ合成酶基因。限制性内切酶图谱和核苷酸序列分析证实了克隆结果,并表明所克隆的基因与国外报道的有很高的同源性。     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products

Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library. The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli. The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies. The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS). The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS. Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS. This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS. The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo. The third α-helix and three adjacent amino acids in the third repeat (R3) of c-Myb were replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein. This chimeric protein bound to PBS with a low affinity but failed to bind to MBS. Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6. This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding.