Ginsenosides regulate ligand-gated ion channels from the outside

Treatment with ginsenosides, the major active ingredients of Panax ginseng, produces a variety of physiological effects on the central and peripheral nervous systems. Ginsenosides inhibit various types of ligand-gated ion channel but it is not clear whether they act from within or outside the cell since they are somewhat membrane-permeable. In the present study, we used the Xenopus oocyte gene expression system to determine from which side of the cell membrane the ginsenoside Rg3 (Rg3), and M4, a ginsenoside metabolite, act to regulate ligand-gated ion channel activity. Ligand-gated ion currents were measured using the two-electrode voltage clamp technique. Rg3 and M4 inhibited 5-HT3A and a3b4 nACh receptor-mediated ion currents when present outside of the cell but not when injected intracellularly. We also examined the effect of these agents on oocytes expressing the gustatory cGMP-gated ion channel, which is known to have a cGMP binding site on the intracellular side of the plasma membrane and is only activated by cytosolic cGMP. Rg3 inhibited cGMP-gated ion currents when applied extracellularly or to an outside-out patch clamp, but not when injected into the cytosol or when using an excised inside-out patch clamp. These results indicate that Rg3 and M4 regulate ligand-gated ion channel activity from the extracellular side.     

A stable gene transfer system for hematopoietic progenitor cells from human bone marrow using pseudotyped retroviral vectors

Recombinant DNA technology has permitted tremendous progression in delivering genes into cells; however, further advances in gene replacement techniques are needed prior to application to hematological diseases. One of the greatest obstacles to gene therapy in human hematopoietic stem cells is the lack of a defined protocol in humans and low transduction efficiency. Currently, murine leukemia virus (MuLV) is the most popular choice as a gene transfer vehicle but it cannot infect non-dividing cells. In our study, vesicular stomatitis G protein pseudotyped MuLV and HIV-1 were produced by a split gene transfection method. Mononuclear cells were separated from healthy human bone marrow and pre-stimulated with cytokines to form myeloid cell lineages. The cells were infected at different MOls with highly concentrated virus and infection rates were analyzed by flow cytometry and progenitor cell assays. eGFP expression was much higher when using HIV-1 system than when using MuLV. Progenitor cell assays agreed with the results obtained by FACS, but the difference was less great. We conclude that the lentiviral system is more suitable for gene transfer to hematopoietic progenitor cells probably because it stably infects both dividing and non-dividing cells. In addition, fibronectin was shown to improve the rate of infection with HIV-1.     

Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking

Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.     

Ribosome-inactivating activity and cDNA cloning of antiviral protein isoforms of Chenopodium album

We have characterized a novel type I ribosome-inactivating protein (CAP30) from the leaves of Chenopodium album. Purified native CAP30 depurinated the ribosomes of Chenopodium, tomato, and tobacco leaves in vitro. To further characterize this protein, cDNA clones were isolated from a leaf cDNA library using a DNA probe derived from the N-terminal amino acid sequence. Two full-length cDNA clones, CAP30A and CAP30B, were isolated. The two clones were highly homologous (91.4% identity over 280 amino acids) at the deduced amino acid level. Both contain a putative signal peptide of 25 amino acid and a conserved domain commonly found in ribosome-inactivating proteins. This suggests that CAP30 is a single-chain ribosome-inactivating protein. Expression of CAP30 mRNA peaked twice, at 12 and 72 h, after tobacco mosaic virus (TMV) infection or wounding. Transformed Escherichia coli cells expressing pre- or mature CAP had greatly reduced growth rates. These results suggest that CAP30 functions as a broad-spectrum defense-related protein with both antiviral and anti-microbial activity.     

Role of thioredoxin peroxidase in aging of stationary cultures of Saccharomyces cerevisiae

A soluble protein from Saccharomyces cerevisiae acts as a peroxidase but requires a NADPH-dependent thioredoxin system and was named thioredoxin peroxidase (TPx). The role of TPx in aging of stationary cultures of S. cerevisiae was investigated in a wild-type strain and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. The occurrence of oxidative stress during aging of stationary cultures of the yeast has been proposed. Comparison of 5-day-old (young) stationary cultures of S. cerevisiae and of cultures aged for 3 months (old) revealed decreased viability, increased generation of reactive oxygen species, modulation of cellular redox status, and increased cellular oxidative damage reflected by increased protein carbonyl content and lipid peroxidation. The magnitude of this stress was augmented in yeast mutant lacking TPx. These results suggest that TPx may play a direct role in cellular defense against aging of stationary cultures presumably, functioning as an antioxidant enzyme.     

Parasites and phytoplankton, with special emphasis on dinoflagellate infections

Planktonic members of most algal groups are known to harbor intracellular symbionts, including viruses, bacteria, fungi, and protozoa. Among the dinoflagellates, viral and bacterial associations were recognized a quarter century ago, yet their impact on host populations remains largely unresolved. By contrast, fungal and protozoan infections of dinoflagellates are well documented and generally viewed as playing major roles in host population dynamics. Our understanding of fungal parasites is largely based on studies for freshwater diatoms and dinoflagellates, although fungal infections are known for some marine phytoplankton. In freshwater systems, fungal chytrids have been linked to mass mortalities of host organisms, suppression or retardation of phytoplankton blooms, and selective effects on species composition leading to successional changes in plankton communities. Parasitic dinoflagellates of the genus Amoebophrya and the newly described Perkinsozoa, Parvilucifera infectans, are widely distributed in coastal waters of the world where they commonly infect photosynthetic and heterotrophic dinoflagellates. Recent work indicates that these parasites can have significant impacts on host physiology, behavior, and bloom dynamics. Thus, parasitism needs to be carefully considered in developing concepts about plankton dynamics and the flow of material in marine food webs.     

Comparative analysis of GFP(UV)-beta1,3-N-acetylglucosaminyltransferase 2 production in two insect-cell-based expression systems

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.     

Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.     

Universal fast walking applied to cDNA

The elucidation of cDNA sequence remains problematic in cases such as genes possessing long coding regions, low expression levels, or poor library coverage. The recently described Universal Fast Walk (UFW) procedure offers a means of determining DNA sequence adjacent to characterised regions. To date, however, the approach has been applied only to genomic DNA. We demonstrate the first successful application of the UFW procedure to the elucidation of cDNA sequence, a previously unknown region of the large tammar wallaby ATRX gene in the theoretically more challenging 3' direction. To do this, we modified the previously published method by including an initial linear amplification and a final, fully nested PCR. We also exchanged buffers between preparative enzyme reactions to ensure optimal conditions for successive steps. These additional steps ensured a product not observed in their absence. UFW, therefore, represents a powerful alternative mechanism for the cloning and sequencing of cDNA, harnessing the exquisite sensitivity and specificity of fully nested PCR in challenging cloning scenarios where conventional 5' or 3' RACE may fail.     

3'-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

Tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a chemical carcinogen thought to be involved in the initiation of lung cancer in smokers. NNK is metabolically activated to methylating and pyridyloxobutylating species that form promutagenic adducts with DNA nucleobases, e.g. O⁶-[4-oxo-4-(3-pyridyl)butyl]guanine (O⁶-POB-dG). O⁶-POB-dG is a strongly mispairing DNA lesion capable of inducing both G→A and G→T base changes, suggesting its importance in NNK mutagenesis and carcinogenesis. Our earlier investigations have identified the ability of O⁶-POB-dG to hinder DNA digestion by snake venom phosphodiesterase (SVPDE), a 3′-exonuclease commonly used for DNA ladder sequencing and as a model enzyme to test nuclease sensitivity of anti-sense oligonucleotide drugs. We now extend our investigation to three other enzymes possessing 3′-exonuclease activity: bacteriophage T4 DNA polymerase, Escherichia coli DNA polymerase I, and E.coli exonuclease III. Our results indicate that, unlike SVPDE, 3′-exonuclease activities of these three enzymes are not blocked by O⁶-POB-dG lesion. Conformational analysis and molecular dynamics simulations of DNA containing O⁶-POB-dG suggest that the observed resistance of the O⁶-POB-dG lesion to SVPDE-catalyzed hydrolysis may result from the structural changes in the DNA strand induced by the O⁶-POB group, including C3′-endo sugar puckering and the loss of stacking interaction between the pyridyloxobutylated guanine and its flanking bases. In contrast, O⁶-methylguanine lesion used as a control does not induce similar structural changes in DNA and does not prevent its digestion by SVPDE.