Detection of chromosomally located and plasmid-borne genes on 20 kb DNA fragments in parasporal crystals from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains CryˉB and HD73, producing recombinant strains CryˉB(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant CryˉB(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than CryˉB(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins.     

Knockout of the gene (<Emphasis Type="Italic">ste15</Emphasis>) encoding a glycosyltransferase and its function in biosynthesis of exopolysaccharide in <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15 ⁻) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis.     

A novel pro-apoptosis protein PNAS-4 from <Emphasis Type="Italic">Xenopus laevis</Emphasis>: Cloning,expression, purification,and polyclonal antibody production

Human PNAS-4 was identified as a novel pro-apoptotic protein in mammalian cells. Here we report the cloning, expression, purification, and antibody production of a PNAS-4 homolog (named xPNAS-4) from Xenopus laevis, an extensively used model organism in exploring gene functions during embryonic development. Recombinant histidine-tagged xPNAS-4 protein was expressed in Escherichia coli as insoluble inclusion bodies. The inclusion bodies were subsequently dissolved in 8 M urea and purified to near homogeneity by Ni2+ affinity chromatography. The resulting denatured protein was refolded by stepwise dilution of urea concentration via dialysis. This procedure yielded about 4 mg refolded protein per liter of E. coli culture with a purity of 95%. The purified protein was identified by liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) and used to raise anti-xPNAS-4 polyclonal antibodies that were suitable for detecting the expression of PNAS-4 in X. laevis embryos by Western blotting. The availability of recombinant protein and specific polyclonal antibodies will provide a valuable tool in studying apoptotic mechanisms of this protein. To our knowledge, this is the first report to demonstrate the presence of PNAS-4 in X. laevis.     

The <Emphasis Type="Italic">Endo-β-Mannanase</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis</Emphasis>, rice,and poplar

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal β-1,4-d-mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-β-mannanase. Plant endo-β-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-β-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-β-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-β-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-β-mannanase genes. The phylogenetic analysis that included the endo-β-mannanases from plants and other organisms implied that plant endo-β-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-β-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

Establishment,Characterization, and Virus Susceptibility of a New Marine Cell Line from Red Spotted Grouper (<Emphasis Type="Italic">Epinephelus akaara</Emphasis>)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10^8.5 TCID₅₀ ml⁻¹), while the viral titer of frog Rana grylio virus 9807 (RGV₉₈₀₇) reached 10^3.5 TCID₅₀ ml⁻¹. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.     

Hippocampal AMPA receptor gating controlled by both TARP and cornichon proteins

Transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon proteins (CNIH-2/3) independently modulate AMPA receptor trafficking and gating. However, the potential for interactions of these subunits within an AMPA receptor complex is unknown. Here, we find that TARPs γ-4, γ-7, and γ-8, but not γ-2, γ-3, or γ-5, cause AMPA receptors to "resensitize" upon continued glutamate application. With γ-8, resensitization occurs with all GluA subunit combinations; however, γ-8-containing hippocampal neurons do not display resensitization. In recombinant systems, CNIH-2 abrogates γ-8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match that of hippocampal neurons. In hippocampus, γ-8 and CNIH-2 associate in postsynaptic densities and CNIH-2 protein levels are markedly diminished in γ-8 knockout mice. Manipulating neuronal CNIH-2 levels modulates the electrophysiological properties of extrasynaptic and synaptic γ-8-containing AMPA receptors. Thus, γ-8 and CNIH-2 functionally interact with common hippocampal AMPA receptor complexes to modulate synergistically kinetics and pharmacology.     

Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides.

Rhesus theta-defensin 1 (RTD-1) is a unique tridisulfide, cyclic antimicrobial peptide formed by the ligation of two 9-residue sequences derived from heterodimeric splicing of similar 76-amino acid, alpha-defensin-related precursors, termed RTD1a and RTD1b (Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., and Selsted, M. E. (1999) Science 286, 498-502). The structures of RTD-2 and RTD-3 were predicted to exist if homodimeric splicing of the RTD1a and RTD1b occurs in vivo. Western blotting disclosed the presence of putative theta-defensins, distinct from RTD-1, in leukocyte extracts. Two new theta-defensins, RTD-2 and RTD-3, were purified by reverse-phase high performance liquid chromatography and characterized by amino acid analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and comparison to the synthetic standards. RTD-2 and RTD-3 are the predicted homodimeric splicing products of RTD1b and RTD1a, respectively. The cellular abundances of RTD-1, -2, and -3 were 29:1:2, indicating that there is a preference for the heterodimeric ligation that generates RTD-1. RTD-1, -2, and -3 had similar antimicrobial activities against Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans, whereas the activity of RTD-2 against Escherichia coli was 2-3-fold less than those of RTD-1 and RTD-3. Equal amounts of each theta-defensin bound to E. coli cells, indicating that the differences in antibacterial activities are the result of post-binding processes.     

Apoptotic macrophage-derived foam cells of human atheromas are rich in iron and ferritin, suggesting iron-catalysed reactions to be involved in apoptosis

We investigated the presence of low-molecular-weight iron and ferritin in human atheromas, and their possible relation to the apoptotic process. Arterial wall segments with fatty streaks were collected from coronary arteries and thoracic aortas of 12 clinical autopsy cases with general atherosclerosis. Normal appearing regions from the same cases together with normal coronary arteries from seven young forensic autopsy cases, without any sign of atherosclerosis, were used for comparison. Anti-CD68 (macrophage marker) and anti-ferritin antibodies were applied to serial sections of the arterial wall segments, fixed in formadehyde and embedded in paraffin wax, using an avidin-biotin complex (ABC) technique. Similarly, apoptotic cells were assayed by the TUNEL technique, while low-molecular-weight iron was cytochemically detected by autometallography. Cell counting and computerised image analysis were performed to compare the distribution of macrophages, ferritin- and iron-rich cells, and apoptotic cells in the intima, media, and adventitia of the arteries.

Pronounced ferritin accumulation, occurrence of lysosomal low-molecular-weight iron, and apoptosis mainly concerned CD68-positive cells (macrophages) in the atherosclerotic lesions. No ferritin- or CD68-positivity was found in normal coronary arteries from the young forensic-autopsy cases, while a moderate number of such cells were observed in the intima of normal looking vessel areas from the control cases. In the intima, cytosolic ferritin and low-molecular-weight iron with a lysosomal type distribution were found in many CD68-positive macrophages which frequently were surrounded by erythrocytes. A substantial number of apoptotic cells within the intima, media, and adventitia were registered in all atherosclerotic lesions examined, although mainly in the vulnerable macrophage-enriched areas of the atheroma shoulder.

We suggest that iron may occur within the cytosol, mainly bound in ferritin, but also in low-molecular weight, redox-active form within the acidic vacuolar apparatus of macrophages and macrophage-derived foam cells following erythrophagocytosis or phagocytosis of apoptotic cells. Low-molecular-weight iron within lysosomes, present due to degradation of iron-containing structures, such as ferritin, may partially become exocytosed and contribute to cell-mediated LDL-oxidation. Moreover, such lysosomal iron may also sensitise lysosomes to oxidative stress and induce apoptosis of macrophage/foam-cells that may result in instability and rupture of atherosclerotic plaques.     

局灶性脑缺血再灌损伤时神经细胞内Ca~(2+)时空动态变化的实验研究

本文用插线法制作局灶性脑缺血／再灌损伤模型,利用激光共聚焦扫描显微镜观察活体脑片细胞内Ｃａ２＋的分布及动态变化,结果表明：（１）缺血／再灌时间不同,梗塞面积不同,缺血４小时梗塞面积占同侧半球的１６．３％,缺血４小时再灌２０小时梗塞面积增加到２５．９％,缺血２４小时梗塞面积占同侧半球的６０．４％。（２）本文首次观察到在缺血４小时纹状体区域的Ｃａ２＋变化明显高于皮层,并且再灌后皮层及纹状体区域Ｃａ２＋的含量明显增加