A particulate DNA polymerase activity in adult rat brain

A particulate fraction of adult rat brain (sucrose buoyant density 1.24 gm/ml) catalyzed the incorporation of [³H]dTTP into an acid-insoluble product in an endogenously templated reaction sensitive to ribonuclease pretreatment. Upon fractionation, this activity was identified in the cerebellum, pons, frontal lobes and base. The DNA polymerase present in these brain fractions exhibited a strong preference for the synthetic template dT_12–18·poly rA rather than dT_12–18·poly dA; dT₁₀ was completely inactive. Purification and equilibrium Cs₂SO₄ gradient centrifugation of the [³H]DNA product-endogenous template complex suggested that RNA was serving as primer for endogenous DNA synthesis.     

E-box元件修饰端粒酶核心启动子序列及体外转录活性分析

人类端粒酶启动子(hTERT启动子)在肿瘤基因治疗中的有效性已经得到了证实. 然而,hTERT启动子有限的肿瘤靶向转录活性困扰着它的临床应用.早期研究已经揭示,核心hTERT启动子上的-34位E-box元件与该启动子的肿瘤靶向转录活性有关.为进一步探索核心hTERT启动子序列3′端富余E-box元件是否能提高启动子的肿瘤靶向转录能力,用化学合成方法在野生型hTERT(WT-hTERT)核心启动子片段(编码蛋白起始子ATG上游-268 bp～-10 bp)的3′端接入3个E-box序列, 构建成修饰型hTERT(Mod-hTERT)启动子. 然后,分别用WT-hTERT和Mod-hTERT启动子去调控增强型绿色荧光蛋白(EGFP)及荧光素酶报告基因在293FT、HepGⅡ、SGC7901、U2OS、以及原代培养人成纤维细胞(PHF)中表达. 结果表明, 在Mod-hTERT启动子的各实验组细胞中,能够在端粒酶阳性的293FT、HepGⅡ及 SGC7901细胞组中观测到EGFP的表达,而在端粒酶阴性的U2OS及PHF细胞组中没有观测到EGFP的表达;在端粒酶阳性的293FT、HepGⅡ和SGC7901细胞株中,Mod-hTERT启动子调控下的荧光素酶活性要高于WT-hTERT启动子组（P＜0.01）; 而在端粒酶阴性的U2OS细胞组中,Mod-hTERT启动子调控下的荧光素酶活性则低于WT-hTERT启动子组（P＜0.01); 在PHF细胞组中,Mod-hTERT启动子组与WT-hTERT启动子组的荧光素酶活性差异不显著(P＞0.05).研究提示,在3′端增加E-box元件可以提高核心hTERT启动子序列的肿瘤靶向转录活性.     

Induction of morphogenesis by methionine starvation in Myxococcus xanthus: polyamine control

The induction of mycrocyst formation by methionine starvation was demonstrated in Myxococcus xanthus by several methods. Growing in a defined medium (M(1)), M. xanthus had a doubling time of 6.5 hr. Four amino acids-leucine, isoleucine, valine, and glycine-were required for growth under these conditions. When the concentration of several amino acids in the medium was reduced (M(2)), the doubling time increased to 10 to 12 hr, and a requirement for methionine was observed. Methionine starvation led to a slow conversion of the population to microcysts. Under conditions of methionine prototrophy (M(1)), microcyst formation could still be triggered in exponentially growing cells by the addition of either 5 mm ethionine or 0.1 m isoleucine plus 0.1 m threonine, feedback inhibitors of methionine biosynthesis. Vegetative growth in the absence of methionine was obtained in medium M(2) if the leucine concentration was raised to its level in medium M(1). Thus, methionine biosynthesis is controlled by the exogenous concentration of the required amino acid, leucine. During an examination of the effects of methionine metabolites on microcyst formation, the involvement of polyamines in morphogenesis was uncovered. Putrescine (0.05 m) induced the formation of microcysts; spermidine (2 to 5 mm) inhibited induction by methionine starvation, ethionine, or high isoleucine-threonine. Spermidine was the only polyamine detected in M. xanthus (16.0 mug/10(9) cells). Its concentration decreased by more than 50% shortly after microcyst induction by high isoleucine-threonine. It is postulated that spermidine is an inhibitor of microcyst induction; when spermidine formation is blocked by methionine starvation, morphogenesis is induced.     

Changes in Ribonucleic Acid Turnover During Aerobic and Anaerobic Growth in Rhodopseudomonas spheroides

Differences in the rate and extent of degradation of ribonucleic acid (RNA) labeled by a 30-sec pulse in aerobically or anaerobically grown Rhodopseudomonas spheroides have been studied by using rifampin to block RNA synthesis. In anaerobic cultures, unstable RNA is degraded with a half-life of 1.25 to 2.0 min, and about 40% of the pulse-labeled RNA is stable. In aerobic cultures, the half-life of unstable RNA is increased to 2.5 to 4.0 min, and 50% of the RNA is stable. When aerobic cultures are transferred to anaerobic conditions, there is a rapid drop in half-life and in the proportion of stable RNA. When anaerobic cultures are made aerobic, the reverse changes occur after a lag of about 30 min. Addition of puromycin to either aerobic or anaerobic cultures caused the pulse-labeled RNA to be degraded at the same rate and to the same extent as the RNA in an anaerobic control culture. In contrast, addition of chloramphenicol enhanced the difference in RNA half-life and increased the proportion of stable RNA by about 10% in each case. It is concluded that there is a difference in the stability of an RNA component under aerobic and anaerobic conditions.     

Ribonucleic Acid from Aerobically and Anaerobically Grown Rhodopseudomonas spheroides: Comparison by Hybridization to Chromosomal and Satellite Deoxyribonucleic Acid

Ribonucleic acid (RNA) species from aerobically and anaerobically grown Rhodopseudomonas spheroides were compared via hybridization to deoxyribonucleic acid (DNA). Both long-labeled and stable RNA bound to chromosomal DNA to the same extent, regardless of derivation. About 4% of the chromosomal DNA hybridized with total cell RNA and about 0.08% with stable RNA. About 4% of the mixed satellite DNA could be hybridized to total cell RNA from aerobic or anaerobic cultures, whereas essentially no stable RNA formed a hybrid with this DNA. Hybridization competition experiments with aerobic and anaerobic pulse-labeled RNA and chromosomal or satellite DNA demonstrated that no qualitative differences existed between the RNA species. It is concluded that identical species of RNA in the same relative amounts are synthesized by R. spheroides during aerobic or anaerobic growth on the same medium.     

Evaluation of Toll-Like Receptor 2 and 4 RNA Expression and the Cytokine Profile in Postmenopausal Women with Metabolic Syndrome

Objective

To evaluate the gene expression of Toll-Like (TLR-2 and TLR-4) receptors and cytokine profile in postmenopausal women with or without metabolic syndrome (MetS).

Methods

In this cross-sectional study, 311 Brazilian women (age≥45 years and amenorrhea≥12 months) were included. Women showing three or more of the following diagnostic criteria were diagnosed as positive for MetS: waist circumference>88 cm, triglycerides≥150 mg/dL, HDL cholesterol<50 mg/dL, blood pressure≥130/85 mmHg, and fasting glucose≥100 mg/dL. The expression of TLR-2 and TLR-4 in peripheral blood was evaluated by RNA extraction and subsequent real time PCR analysis. The cytokine profile, tumor necrosis factor alpha (TNF-α) and interleukins 1β, 6, and 10, were measured by ELISA.

Results

The expression of TLR-2 RNA was demonstrated in 32.5% and TLR-4 in 20.6% of the subjects. There was no association between the expression of TLR-2 and TLR-4 and the presence or absence of MetS (P>0.05). A greater production of IL-6 was associated with TLR-2 and TLR-4 expressions and greater production of TNF-α was associated only with TLR-2 expression (P>0.05). Only the lower quartile of IL-10 was associated with the presence of the MetS (P>0.05).

Conclusions

TLR-2 and TLR-4 expressions were associated with increased pro-inflammatory cytokines, IL-6 and TNF-α, with no association with biomarkers of MetS. The low concentrations of IL-10 may suggest an anti-inflammatory modulation in postmenopausal women with MetS.