Inosine induced mutations

Summary Two complementary 24 base single stranded oligonucleotides containing randomly located inosine residues were synthesized in vitro. Once annealed, the two oligonucleotides were cloned into derivatives of ColE1 and transformed into Escherichia coli. Sequence analysis of 157 clones yielded 305 mutations. The pattern of the mutations revealed the following: (1) The frequency of inosine induced mutations was significantly less than predicted from its content in the oligonucleotides; (2) Inosine incorporation resulted almost exclusively in base changes to guanine; (3) The mutation distribution is biased towards A/T to G/C substitutions; (4) There were reproducible position biases; and (5) There was a reproducible strand bias which was independent of the cassette orientation with respect to the plasmid origin of replication.     

Cell death induced by chemical homobifunctional cross-linkers. Cross-linker induced apoptosis

Physical association of proteins that underlies cytotoxic signal induction and transduction suggests a possibility of regulating cell response by modifying protein–protein interactions. For protein complexing, chemical cross-linking agents have been traditionally used. However, the ability of various cross-linkers to induce and modify cell responses, cell death in particular, is still obscure. We have undertaken the investigation to test the apoptosis-inducing and modifying properties of the homobifunctional cross-linkers-dimethyl suberimidate (DMS) and 1,5-bis(succinimido-oxycarbonyloxy)pentane (BSOCOP). The functional groups of these cross-linkers are different but both are able to interact with available amino groups. It was shown that bifunctional cross-linkers, unlike their monofunctional analogues, are capable of inducing cell death in transformed cells, thus indicating the crucial role of cross-linking in cell killing. DMS- and BSOCOP-treated cells were shown to undergo cell death by apoptosis, though the signaling pathways were distinct. DMS inhibited bcl-X_L and bak but not bax gene expression, while BSOCOP potentiated bax mRNA synthesis immediately after application. Cell pre-incubation with DMS, but not with BSOCOP, resulted in an increasing sensitivity to TNF, although activities of anti-Fas cytotoxic antibodies were then inhibited. Thus, this study has demonstrated for the first time that chemical cross-linkers are capable of inducing apoptosis by themselves and modifying the TNF-dependent and Fas-mediated cell death that may have potential therapeutic significance.     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

The counter regulatory response induced by CpG oligonucleotides prevents bleomycin induced pneumopathy

Bleomycin (BLM) induces life-threatening pneumonitis and pulmonary fibrosis in 20% of patients, limiting its use as a chemotherapeutic agent. Oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN) stimulate cells that express Toll-like receptor 9 to initiate an inflammatory response. This short-lived inflammation is physiologically suppressed by a counter-regulatory process that peaks five days later. Using a murine model of BLM-induced lung injury, the effect of CpG ODN treatment on pulmonary inflammation, fibrosis and mortality was examined. Administering CpG ODN 5 days before BLM (so that the peak of the counter-regulatory process induced by CpG ODN coincided with BLM delivery) resulted in a dose-dependent reduction in pulmonary toxicity (p < 0.005). Delaying the initiation of therapy until the day of or after BLM administration worsened the inflammatory process, consistent with the counter-regulatory process rather than initial pro-inflammatory response being critical to CpG induced protection. The protection afforded by CpG ODN correlated with reduced leukocyte accumulation and inflammatory cytokine/chemokine production in the lungs. These changes were associated with the increased production of IL-10, a critical element of the counter-regulatory process triggered by CpG ODN, and the concomitant down-regulation of BLM-induced IL-17A and TGF-β1 (which promote pulmonary toxicity). This work represents the first example of the physiologic counter-regulation of TLR induced immune activation being harnessed to block an unrelated inflammatory response.     

Stress induced experimental colitis

Stress induces chemical changes in the central nervous system which alters the biochemistry and physiology of the digestive tract. The present study determines arachidonic acid oxidation and damage in the colon following stress. Ten rats were stressed by the cold-restraint method; ten were controls. Stress induced 0.5 +/- 0.7 (S.D.) mucosal erosions whereas controls had none. Subepithelial hemorrhage and erosions occurred only in the proximal two-thirds of the colon. Prostaglandin E(2) synthesis was increased after stress compared to the control (381 +/- 130 vs. 1610 +/- 372 ng/g/min). Leukotriene C(4) synthesis also increased after stress (4217 +/- 994 vs. 11300 +/- 1662 ng/g/min). Synthesis of prostaglandin E(2) increased (r = 0.9381) with leukotriene C(4). The response of the colon to stress is less severe than that in the stomach and may be related to regional regulation of prostaglandin and leukotriene synthesis.     

Super-suppressor induced interallelic complementation

Summary In yeast the dominant super-suppressorS ₅ has a distinct expression in heterozygotes depending on the particular combination of alleles at thead ₁ orad ₂ loci. If thead ₁ combination is represented by two suppressible alleles, the phenotype of diploid is wild. If thead ₁ combination consists of a suppressible and a non-suppressible allele the phenotype of the diploid is partially mutant. Such a difference in the manifestation of suppressor depending on the combination of alleles is more pronounced in the case ofad ₂ mutations. In the case when bothad ₂ alleles are suppressible, the diploid is prototrophic, but when only one allele is suppressible, the diploid is an adenineless auxotroph as a rule.This type ofS ₅-effect gave us the possibility to study interallelic complementation atad ₂ locus in presence of the super-suppressor. It was shown that some combinations of noncomplementing alleles do complement as a result of suppression.Comparison of the two complementation maps with and without suppressor is made for thead ₂ locus. The mechanisms of the phenomena and of super-suppression are discussed.     

氧化与细胞凋亡

细胞凋亡(apoptosis)是细胞的主动死亡,它参与调节机体许多生理及病理过程．近年的研究表明细胞凋亡与氧化有密切关系．当细胞外诸如辐射、高氧、高温、感染、衰老等信息通过活性氧传入细胞,引起细胞脂质过氧化或与细胞凋亡有关的基因的表达,通过一系列生化反应,最终发生细胞凋亡．原癌基因bcl-2可能起着调节作用．