Cytokine deprivation of naive CD8+ T cells promotes minimal cell cycling but maximal cytokine synthesis and autonomous proliferation subsequently: a mechanism of self-regulation.

Naive CD8+ T cells differentiate into effectors secreting various cytokines that aid their function. IL-2, but not IL-15, promoted this differentiation of naive CD8+ T cells into effectors. However, the amount of IL-2 present during differentiation had a dichotomous effect on their subsequent function. High concentrations of IL-2 enhanced proliferation and cell cycling initially, but the effectors subsequently failed to produce cytokines and proliferate autonomously, although CD28 expression was maintained. In contrast, suboptimal amounts of IL-2 during priming promoted apoptosis, little proliferation and cell cycling, yet the CD8+ effectors generated produced high levels of cytokines and proliferated autonomously. Interestingly, the effects of IL-2 on naive CD8+ T cells were totally opposite those on naive CD4+ T cells. Although IL-2 impaired cytokine synthesis by CD8+ T cells, the expression of LFA1 and CD44 as well as Fas-dependent cytotoxicity were enhanced. However, loss of cytokine synthesis was not due to increased cytotoxicity, as inhibition occurred even in the absence of perforin/FasL. Interestingly, CD8+ effectors secreting reduced amounts of cytokines exhibited enhanced IL-2Ralpha, but reduced IL-2Rbeta, expression. Furthermore, sorted CD8+ IL-2Ralphahigh cells secreted less cytokines than IL-2Ralphalow cells. These results suggest that the presence of excessive IL-2 during the activation of naive CD8+ T cells, while promoting cell cycling initially, may compromise long-term immunity.     

The effect of Hoe-427 (an ACTH4-9 analog) on free-choice ethanol consumption in male and female rats.

Ethanol consummatory patterns of individual male and female rats and the effects of Hoe-427 (Ebiratide), an ACTH4-9 analog, thereon, were studied in a test system using 24 hour, two-bottle free choice consumption between 0.2% saccharin and 10% ethanol in 0.2% saccharin. Single, daily i.p. doses (0.03mg/rat) of either ACTH4-10 or its analog resulted in a significant reduction of daily ethanol consumption with no effects on saccharin consumption. After 4 days of treatment, male rats consistently exhibited a rebound increase in ethanol consumption; this effect was not seen in females. The daily ethanol consummatory patterns of the female animals seemed to exhibit a 4-6 day cyclic rhythymicity, suggesting an interaction with estrous cycles. These results support a role for ACTH4-10 in the initiation of ethanol consummatory behavior in rats and suggests the existence of sex differences in this phenomenon.     

Studies on pachytene and somatic chromosomes ofPhaseolus mungo L.

The pachytene and somatic chromosomes ofPhaseolus mungo L. (2n=22), were identified and classified on the basis of their relative length, arm ratio, chromomere pattern and nucleolar association. A comparison of the karyotypes during the somatic and pachytene stages, revealed that some chromosomes are of different relative lengths during the two phases. Thus, the two nucleolar organizer chromosomes are the 2nd and 3rd longest at somatic metaphase, but only 7th and 9th longest during pachytene. The pachytene chromosomes belong to the differentiated group.     

Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM ^+ve/let-7b^{down-regulated}), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM ^−ve/let-7b^up-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM ^+ve/let-7b^up-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.     

Isoenzymicity in mouse liver guanine deaminase demonstrable under substrate stress

Two isoenzymes of guanine deaminase could be demonstrated in the liver of mice subjected to guanine stress while the salinetreated controls showed only one. The one appearing under stress was a regulatory protein showing a sigmoidal substrate saturation curve, but was not influenced by GTP, allantoin or Mg²⁺     

A Stereoselective Synthesis of α - and β-2′-Deoxy-2-thiouridine

Abstract

A stereoselective glycosylation procedure is described for the synthesis of protected α- and β-2′-deoxy-2-thiouridine (dS²U) in 68% and 94% yield, respectively. Evidence is presented that suggests the reaction proceeds through a silylated thio-glycoside intermediate. This intermediate undergoes an efficient S² → N¹ rearrangement mediated by SnCl₄. The phosphoramidite and phosphodiester synthons and a dS²U dinucleotide are also synthesized and the X-ray structure of β-dS²U is presented.