An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe.

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

红豆草组织培养物的超低温保存及其超微结构的观察

红豆草(Onobrychis viciaefolia Scop.)组织培养物在5%DMSO+10%甘油+8%蔗糖的冰冻保护剂及以1℃/分钟的速度降温到-35—-40℃,停留2小时后,投入液氮,40℃水浴快速化冻等条件下,存活率达60—70%,并保持了高的分化能力。电子显微镜的观察结果表明,快速冰冻和1℃/分钟慢速冰冻至-35℃—40℃不停留,对细胞结构造成严重的致死性破坏;-35℃停留30分钟对细胞结构的损伤是可逆性的;停留2小时的其超微结构基本上与对照材料无明显差别。     

3H]prostaglandin uptake in vivo by rabbit uterine tissues and blastocysts

Day-6 pregnant rabbits were anesthetized and subjected to a mid-ventral laparotomy. [3H] Prostaglandin F2alpha) (PGF2alpha) [3H]PGE2, [14C]Urea or [14C]Sucrose were instilled into the uterine lumen via the uterotubal junction. The amounts instilled/uterine horn were respectively 3.7 +/- 0.3, 3.5 +/- 0.3, 5.7 +/- 1.3 and 2.7 +/- 1.6 muCi in 20mul of buffer. Animals were killed at 1, 2, 9, 19 or 21 h after radioactive instillation, and the amounts of radioactivity in blastocysts, uterine tissue, peritoneal cavity washings and urine evaluated by liquid scintillation spectrometry. A gradient of radioactivity was observed from the uterotubal junction to the cervical end of the uterus. Large amounts of [3H]PG were found in the injected horn and associated blastocysts with a considerable crossover to the non-injected horn, but little in the associated blastocysts. Much of the blastocysts associated- [3H]PG remained unmetabolized. Large amounts of metabolized [3 H] were found in urine. [14C]Urea was taken up by uterine tissue in the injected horn, but there was little cross over to the non-injected horn. Urea was also found in urine. Much of the [14C]Sucrose remained in the injected horn, and little was recovered from the urine. It was found that at 9 h, but not at 19 h, after [3 H]PG instillation, the PG was localized at the site of the blastocysts in the injected but not in the contralateral horn. Significantly more [3H]PGF2alpha than [3H]PGE2 was localized in this situation. [14C]Urea was not localized at the site of the blastocysts in urea injected horns. (ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of tumor growth following treatment with a glutamine antimetabolite

Assessment of arterial-venous differences across transplanted methylcholanthrene-induced sarcomas in rats revealed significant decreases in plasma concentrations of glutamine, serine and glucose. Treatment with the glutamine antimetabolite, acivicin, significantly reduced tumor weights by 65% at the conclusion of the experiment 34 days after tumor induction. These results suggest that glutamine is an essential metabolic substrate for tumor growth and that blockade of glutamine utilization can inhibit the growth of these transplantable sarcomas.     

Temporal comparison of recombination and synaptonemal complex formation during meiosis in S. cerevisiae

In synchronous cultures of S. cerevisiae undergoing meiosis, an early event in the meiotic recombination pathway, site-specific double strand breaks (DSBs), occurs early in prophase, in some instances well before tripartite synaptonemal complex (SC) begins to form. This observation, together with previous results, supports the view that events involving DSBs are required for SC formation. We discuss the possibility that the mitotic pathway for recombinational repair of DSBs served as the primordial mechanism for connecting homologous chromosomes during the evolution of meiosis. DSBs disappear during the period when tripartite SC structure is forming and elongating (zygotene); presumably, they are converted to another type of recombination intermediate. Neither DSBs nor mature recombinant molecules are present when SCs are full length (pachytene). Mature reciprocally recombinant molecules arise at the end of or just after pachytene. We suggest that the SC might coordinate recombinant maturation with other events of meiosis.     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

Some observations on the localization of mitomycin C-induced aberrations in human lymphocytes

Actively cycling human lymphocytes were treated with mitomycin C for 1 h (1.4 μg/ml) and then grown in medium containing 10 μg/ml bromodeoxyuridine. Serial 5-h colcemid accumulation samples were taken up to 35 h and the air-dried methaphase spreads stained for replication banding. A complete cell-cycle subphasing analysis was made, and classified cells scored for all categories of chromatid-type aberrations and their location.

Inspite of the high dose which produced massive delay and cycle perturbation, there was no evidence for selectively lethality of early-S cells, in fact such cells were in excess. Extreme localization of aberrations to late-replicating (mostly centromeric) regions was found at all subphases and in pre-S cells. This rules out ‘localization by default’ as an explanation for the observed preferential occurence of ‘break points’ in these regions.

The frequency of incomplete intrachanges, low in late S, rises dramaticallyin early S to become maximal in pre-S cells.