Ginsenoside Rd Protects Neurons Against Glutamate-Induced Excitotoxicity by Inhibiting Ca2+ Influx

Our previous studies have demonstrated that ginsenoside Rd (GSRd), one of the principal ingredients of Pana notoginseng, has neuroprotective effects against ischemic stroke. However, the possible mechanism(s) underlying the neuroprotection of GSRd is/are still largely unknown. In this study, we treated glutamate-injured cultured rat hippocampal neurons with different concentrations of GSRd, and then examined the changes in neuronal apoptosis and intracellular free Ca²⁺ concentration. Our MTT assay showed that GSRd significantly increased the survival of neurons injured by glutamate in a dose-dependent manner. Consistently, TUNEL and Caspase-3 staining showed that GSRd attenuated glutamate-induced cell death. Furthermore, calcium imaging assay revealed that GSRd significantly attenuated the glutamate-induced increase of intracellular free Ca²⁺ and also inhibited NMDA-triggered Ca²⁺ influx. Thus, the present study demonstrates that GSRd protects the cultured hippocampal neurons against glutamate-induced excitotoxicity, and that this neuroprotective effect may result from the inhibitory effects of GSRd on Ca²⁺ influx.     

A new DNA-cloning vector forHaemophilus influenzae Rd.

A new, high-efficiency, DNA-cloning vector pJ1-8 was derived in two steps from the chimeric plasmid pD7 consisting of RSF 0885(amp ^r) andHaemophilus influenzae chromosomal DNA. pJl-8 has only oneEcoRI site and a molecular weight of only 2.5 × 10⁶. No detectableamp ^r transformation was obtained with pJl-8 DNA. However,amp ^r transformation increases markedly ifHaemophilus influenzae chromosomal DNA segments are spliced into it, providing a very facile assay for detecting inserts.     

Reexamination of phenotypic defects in rec-1 and rec-2 mutants of Haemophilus influenzae Rd.

Radiolabeled donor DNA is efficiently taken up into competent H. influenzae Rd rec-2 mutant cells but does not undergo the rapid degradation observed in wild-type cells. Furthermore, donor label is not recovered in the chromosome even after 1 h. The donor DNA appears to remain in a protected state in a compartment that can be separated from the rest of the cell. We interpret this as a failure of the donor DNA to be translocated out of the transformasome. In contrast, rec-1 cells translocate labeled donor DNA normally. The donor label accumulates in the recipient chromosome, but, as expected for cells with a recombination defect, there is no preferential localization of the label in sites homologous to the donor DNA. In addition, we have observed two enzymatic activities that act on transformasome-associated DNA of rec-2 cells, an endonuclease which may play a role in the translocation of closed circular DNA and a phosphatase.     

Systems properties of the Haemophilus influenzae Rd metabolic genotype.

Haemophilus influenzae Rd was the first free-living organism for which the complete genomic sequence was established. The annotated sequence and known biochemical information was used to define the H. influenzae Rd metabolic genotype. This genotype contains 488 metabolic reactions operating on 343 metabolites. The stoichiometric matrix was used to determine the systems characteristics of the metabolic genotype and to assess the metabolic capabilities of H. influenzae. The need to balance cofactor and biosynthetic precursor production during growth on mixed substrates led to the definition of six different optimal metabolic phenotypes arising from the same metabolic genotype, each with different constraining features. The effects of variations in the metabolic genotype were also studied, and it was shown that the H. influenzae Rd metabolic genotype contains redundant functions under defined conditions. We thus show that the synthesis of in silico metabolic genotypes from annotated genome sequences is possible and that systems analysis methods are available that can be used to analyze and interpret phenotypic behavior of such genotypes.     

人参皂甙Rd预处理可减轻谷氨酸所致的PC12细胞损伤

目的:研究人参皂甙Rd(Ginsenoside Rd)预处理对谷氨酸所致PC12细胞损伤的影响。方法:将体外培养的PC12细胞分为3组,分别为对照组(Control)、谷氨酸损伤组(Glu)和人参皂甙Rd预处理组(Rd)。Control组细胞正常培养;Glu组细胞暴露于含10mM谷氨酸的DMEM培养基中损伤24 h;Rd组细胞经50μM的人参皂甙Rd预处理30 min后,在谷氨酸浓度为10 mM的DMEM培养基中损伤24 h。采用MTT检测细胞活力和乳酸脱氢酶(LDH)检测试剂盒检测LDH释放量;流式细胞仪检测胞内活性氧(ROS)水平;Western blot检测还原型谷胱甘肽蛋白(GSH)表达;专用试剂盒检测细胞内过氧化氢酶(CAT)和超氧化物歧化酶(SOD)含量,相差显微镜观测细胞形态。结果:50μM的人参皂甙Rd预处理30 min,可明显提高谷氨酸诱导的PC12细胞的活力,降低其LDH释放量、胞内ROS含量,并提高胞内GSH蛋白表达,增加CAT、SOD含量并改善细胞形态。结论:人参皂甙Rd预处理可减轻谷氨酸引起的PC12细胞损伤。     

The genome of Haemophilus influenzae Rd has a unique NotI site

Previous analysis of physical maps of Haemophilus influenzae, which is circular and 1.9 Mb in length [Lee and Smith, J. Bacteriol. 170 (1988) 4402-4405; Kauc et al., J. Bacteriol. 171 (1989) 2474-2479], did not detect any NotI (GCGGCCGC) restriction sites. A transposon, Tn916, was constructed to contain a NotI linker cloned into its NciI site and introduced into the H. influenzae chromosome. NotI digestion of chromosomes containing a Tn916-associated NotI site followed by separation of fragments by field-inversion gel electrophoresis revealed the presence of two fragments obtained by two NotI cuts, one in Tn916 and the other, a unique, 'natural' NotI site in the original chromosomal DNA. The examination of other Haemophilus strains demonstrated the presence of one or more NotI sites in all of those tested.     

Initial proteome analysis of model microorganism Haemophilus influenzae strain Rd KW20

The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.     

Modification of DNA in chromatin with methyltransferase from Haemophilus influenzae Rd.

The accessibility of DNA in nucleosome dimers (as a model of the chromosomal chain of nucleosomes) was determined by means of modification methylases from Haemophilus influenzae Rd. Using these enzymes, the rate of modification of nucleosome dimers is about one fifth the rate observed with protein-free DNA from chromatin subunit dimers. Methylated DNA sites in nucleosome dimers are readily accessible to micrococcal nuclease. The analysis of the fragment pattern of nucleosomes after methylation and mild nuclease treatment reveals that the methylated sites are predominantly located in the internucleosomal linker DNA. Polylysine binding experiments further support this interpretation. This compound preferentially interacts with the nucleosomal core DNA and protects it against internal cleavage. It neither affects the degradation of methylated sites drastically nor does it inhibit the methylation of nucleosome dimers. Thus, a combination of protection, cleavage and modification is proposed as a useful tool for the analysis of the structure of chromatin.     

DNA methylases of Hemophilus influenzae Rd. I. Purification and properties

Hemophilus influenzae strain Rd DNA contains small amounts of 5-methylcytosine (0.012%) and significantly greater amounts of N-6-methyladenine (0.34%). Four DNA adenine methylases have been identified and purified from crude extracts of H. influenzae Rd by means of phosphocellulose chromatography. Each of the four enzymes requires (S-adenosyl-l-methionine as a methyl group donor and each differs in its ability to methylate various DNAs in vitro. DNA methylase I is related to the genetically described modification-restriction system in H. influenzae Rd, and is presumably the modification enzyme for that system. DNA methylase II introduces approximately 130 methyl groups into a phage T7 DNA molecule and protects T7 DNA from the H. influenzae Rd restriction enzyme, endonuclease R, described by Smith and Wilcox (1970). These findings indicate that DNA methylase II is the modification enzyme corresponding to endonuclease R. A third modification-restriction system, which does not affect T7 DNA, has been detected in H. influenzae Rd. DNA methylase III is apparently the modification enzyme for this system. The biological function of DNA methylase IV remains unknown.     

Physiological characterization of Haemophilus influenzae Rd deficient in its glutathione-dependent peroxidase PGdx

The chimeric peroxidase PGdx of Haemophilus influenzae Rd belongs to a recently identified family of thiol peroxidases capable of reducing hydrogen peroxide as well as alkylhydroperoxides by means of glutathione redox cycling. In the present study, we constructed a H. influenzae Rd strain, deficient in its PGdx encoding gene (open reading frame HI0572). The mutant was shown by disk inhibition and liquid culture growth assays to exhibit increased susceptibility to organic hydroperoxides. The hampered growth was restored by complementing the interrupted gene on the genome with a replicating plasmid bearing an intact copy of the gene, hereby rejecting the possible influences of polar effects. Elevated levels of hydrogen peroxide scavenging activity, due to the catalase HktE, were measured in the absence of a functional pgdx gene rendering the mutant more resilient against hydrogen peroxide. On the other hand, after initiation of the stationary phase, aerobic cultures of the pgdx mutant were practically devoid of living cells, whereas wild-type counterparts retained viability. This observed feature was alleviated by complementation with the functional gene or with the addition of catalase.     

A New Transformation-Deficient Mutant of Haemophilus influenzae Rd with Normal DNA Uptake

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM, is induced during competence development in wild-type H. influenzae but not in RJ248.     

结缕草胁迫诱导型启动子Rd29A的克隆及功能鉴定

以结缕草基因组DNA为模板,根据已报道Rd29A启动子序列设计了一对特异引物,在优化的PCR反应条件下扩增出了Rd29A启动子的基因片段,通过序列分析与文献报道的基因序列有41.65％的同源性.在GenBank上的登录号为EU346948.利用GFP基因作为报告基因,构建了用于比较鉴定所克隆启动子活性的pBIG (35S-GFP)和pBIRG (Rd29A-GFP)两个植物表达载体,进而采用基因枪法对洋葱表皮细胞进行遗传转化,检测Rd29A启动子在受体细胞中调控基因表达的活性.结果表明,克隆到的Rd29A启动子活性强于组成型的35S启动子,利用GFP瞬时表达的分析方法有效的筛选到用于进行草坪草抗逆育种的启动子.     

Functional Annotation of Conserved Hypothetical Proteins from Haemophilus influenzae Rd KW20

Haemophilus influenzae is a Gram negative bacterium that belongs to the family Pasteurellaceae, causes bacteremia, pneumonia and acute bacterial meningitis in infants. The emergence of multi-drug resistance H. influenzae strain in clinical isolates demands the development of better/new drugs against this pathogen. Our study combines a number of bioinformatics tools for function predictions of previously not assigned proteins in the genome of H. influenzae. This genome was extensively analyzed and found 1,657 functional proteins in which function of 429 proteins are unknown, termed as hypothetical proteins (HPs). Amino acid sequences of all 429 HPs were extensively annotated and we successfully assigned the function to 296 HPs with high confidence. We also characterized the function of 124 HPs precisely, but with less confidence. We believed that sequence of a protein can be used as a framework to explain known functional properties. Here we have combined the latest versions of protein family databases, protein motifs, intrinsic features from the amino acid sequence, pathway and genome context methods to assign a precise function to hypothetical proteins for which no experimental information is available. We found these HPs belong to various classes of proteins such as enzymes, transporters, carriers, receptors, signal transducers, binding proteins, virulence and other proteins. The outcome of this work will be helpful for a better understanding of the mechanism of pathogenesis and in finding novel therapeutic targets for H. influenzae.     

The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp

Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus.     

Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene.

The Haemophilus influenzae Rd rec-1+ gene was cloned from a partial chromosomal digest into a plasmid vector as a 20-kilobase-pair (kbp) BstEII fragment and then subcloned. The smallest subclone with rec-1+ activity carried a 3.1-kbp EcoRI fragment. The identity of the rec-I gene in these clones was confirmed by transforming an Rd strain carrying a leaky rec-1 mutation (recA4) to resistance to methyl methanesulfonate (MMS) by using whole or digested plasmids. It was demonstrated that the Rec+ phenotype of the MMSr transformants was linked to the strA, novAB, and mmsA loci, as expected if the recA4 allele had been replaced by rec-1+. In growing cultures (rec-1 or rec+), all rec-1+-carrying plasmids induced near-maximal levels of transformability when their hosts reached stationary phase; these levels are 100 to 1,000 times higher than the values seen with strains not carrying a Rec plasmid. Transfer of the 3.1-kbp subclone was greatly reduced compared with transfer of similarly sized vector plasmids, and the resulting transformants grew slowly; this suggests an explanation of my failure to directly clone this fragment from chromosomal DNA digests. Transfer of a rec-1+ plasmid to a very poorly genetically transformable H. influenzae Rb strain resulted in greatly increased transformability. Transfer of such plasmids to a noncompetent H. influenzae Rc strain did not render this strain competent. It is suggested that transformability of Rd and Rb strains is limited by rec-1 expression but that the noncompetence of Rc has some other basis.