Determination of methylation site of DNA-methyltransferase NlaX by a hybrid method

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.     

Importance of a Small N-Terminal Region in Mammalian Peptide Transporters for Substrate Affinity and Function

The two closely related, proton-coupled, electrogenic mammalian peptide transporters PEPT1 and PEPT2 differ substantially in substrate affinity and mode of function. The intestinal carrier PEPT1 has a lower affinity for most substrates than the isoform PEPT2 that is expressed in kidney, lung, brain and other tissues. A previous analysis of PEPT1-PEPT2 chimeras has suggested that the N-terminal half of the carrier proteins is important for substrate affinity. We constructed and analyzed new PEPT1-PEPT2 chimeras for identifying smaller segments within the N-terminal region of the transporter proteins that contribute to the kinetic properties. The first 59 or 91 amino-acid residues of PEPT1 were used to replace the corresponding region in PEPT2 leading to the chimeras CH3 and CH4, which could be analyzed when expressed in Xenopus laevis oocytes. Substrate affinities of both chimeras for the zwitterionic substrate D-Phe-Ala ranged between those that are characteristic for either PEPT1 or PEPT2, but when charged dipeptide substrates were employed, both chimeras possessed PEPT1-like affinities. The chimera CH3 carrying the N-terminal 59 amino-acid residues of PEPT1 exhibited a PEPT2-like phenotype with respect to pHout-dependency as well as to the current-voltage relationship of inward currents. In the chimera CH4 possessing the 91 amino-terminal residues of PEPT1, a pronounced alteration in the pHout-dependence was observed, with highest transport rates occurring at pH values as low as pH 4.0. Based on this analysis, we propose that the two identified aminoterminal regions in mammalian peptide carriers play an important role in determining the substrate affinity and also other characteristic features of the two transporter subtypes.     

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK,calpain and caspase 3

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.     

Isoflavone glycosides from Derris scandens

Five isoflavone glycosides, named derriscandenosides A–E (1–5), were isolated from the stems of Derris scandens, together with ten known compounds comprising one isoflavone, two benzoic acid derivatives, three glucosyl isoflavones and four rhamnosyl-(1→6)-glucosyl isoflavones. The structures of the glycosides were assigned on the basis of spectroscopic data, especially of the acetate derivatives. Three known rhamnosyl-(1→6)-glucosyl isoflavones isolated from a crude fraction were retested for hypotensive activity with varying results.     

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.