BglⅠ限制性核酸内切酶与环状pBR322-DNA专一性和非专一性相互作用的动力学及热力学

 本文选择限制性核酸内切酶BglⅠ和pBR322-DNA为试验系统,用酶促反应的动力学和热力学方法来研究内切酶对环状DNA分子的专一性和非专一性结合及切割过程,求得了各限制位点的切割速度k及活化能E,各限制位点催化速度常数k_c,酶同限制位点专一性结合的平衡常数k_S非专一性结合的平衡常数k_N及其热力学参数△H,△S。研究表明:不论底物的构型如何(线状还是环状),内切酶都以相似的动力学和热力过程对其进行结合与切割。     

Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen

Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+ signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3-like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI.     

Sulfated modification can enhance the immune-enhancing activity of lycium barbarum polysaccharides

In test in vitro, four sulfated lycium barbarum polysaccharides (sLBPSs) with different degrees of sulfation (DS), sLBPS_0.7, sLBPS_1.1, sLBPS_1.5 and sLBPS_1.9, were added into cultured chicken peripheral lymphocytes and the changes of lymphocytes proliferation were compared by MTT assay taking the non-modified LBPS as control. Two sLBPSs with better efficacy, sLBPS_1.5 and sLBPS_1.9 were selected. In test in vivo, one hundred 14-day-old chickens were averagely divided into five groups randomly. The chickens except blank control group were vaccinated with Newcastle disease vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in three experimental groups were injected with 0.5 mL of sLBPS_1.5, sLBPS_1.9 and LBPS at 4 mg mL⁻¹, in vaccination control group, with 0.5 mL of physiological saline, once a day for three successive days. On days 7, 14, 21 and 28 after the first vaccination, the changes of peripheral lymphocytes proliferation and serum HI antibody titer were determined. The result showed that two sLBPSs could significantly promote lymphocytes proliferation and enhance serum antibody titer. These results indicated that sulfated modification could enhance the immune-enhancing activity of LBPS, which there was a certain relativity with the DS of sulfated polysaccharide. sLBPS_1.5 possessed the best efficacy and would be expected as the component drug of a new-type immunopotentiator.     

NO, but not CO, attenuates anaphylaxis-induced postsinusoidal contraction and congestion in guinea pig liver

The pathophysiology of the hepatic vascular response to anaphylaxis in guinea pig is not known. We studied effects of anaphylaxis on hepatic vascular resistances and liver weight in isolated perfused livers derived from guinea pigs sensitized with ovalbumin. We also determined whether nitric oxide (NO) or carbon monoxide (CO) modulates the hepatic anaphylaxis. The livers were perfused portally and recirculatingly at constant flow with diluted blood. With the use of the double-occlusion technique to estimate the hepatic sinusoidal pressure (Pdo), portal venous resistance (Rpv) and hepatic venous resistance (Rhv) were calculated. An antigen injection caused venoconstriction characterized by an increase in Rpv greater than Rhv and was accompanied by a large liver weight gain. Pretreatment with the NO synthase inhibitor NG-nitro-l-arginine methyl ester, but not the heme oxygenase inhibitor zinc protoporphyrin IX, potentiated the antigen-induced venoconstriction by increasing both Rpv and Rhv (2.2- and 1.2-fold increase, respectively). In conclusion, anaphylaxis causes both pre- and postsinusoidal constriction in isolated guinea pig livers. However, the increases in postsinusoidal resistance and Pdo cause hepatic congestion. Endogenously produced NO, but not CO, modulates these responses.     

A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.     

Identification of a Toll-Like Receptor 1 in Guinea Fowl (Agelastes niger)

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, thus playing important roles in host defense. This study determined the first sequence of a TLR1 type 1 in the guinea fowl (GFTLR1). The open reading frame of GFTLR1 type 1 contains 2,115 nucleotides and encodes 705 amino acids. Amino acid analysis indicated that GFTLR1 type 1 shares 92.3?% homology with the green jungle fowl, 92.1?% with the chicken, 90.4?% with the turkey, and 84.4?% with Cooper's hawk. Genetic patterns were identified within the TLR1 type 1 of the chicken and the guinea fowl. GFTLR1 type 1 was found to have 92 polymorphic amino acid sites, of which 16 were in the leucine-rich repeat (LRR) domain, 3 in a C-terminal LRR domain, and 6 in a Toll/interleukin-1 receptor domain. The data showed that avian TLR1 type 1 genes are under purifying selection and highly conserved, because dN/dS was less than 1.     

Colorectal Cancer with Residual Polyp of Origin: A Model of Malignant Transformation

The majority of colorectal cancers (CRCs) arise from adenomatous polyps. In this study, we sought to present the underrecognized CRC with the residual polyp of origin (CRC RPO +) as an entity to be utilized as a model to study colorectal carcinogenesis. We identified all subjects with biopsy-proven CRC RPO + that were evaluated over 10 years at Mayo Clinic, Rochester, MN, and compared their clinical and pathologic characteristics to CRC without remnant polyps (CRC RPO −). Overall survival and disease-free survival overlap with an equivalent hazard ratio between CRC RPO + and RPO − cases when age, stage, and grade are adjusted. The somatic genomic profile obtained by whole genome sequencing and the gene expression profiles by RNA-seq for CRC RPO + tumors were compared with that of age -and gender-matched CRC RPO − evaluated by The Cancer Genome Atlas. CRC RPO + cases were more commonly found with lower-grade, earlier-stage disease than CRC RPO −. However, within the same disease stage and grade, their clinical course is very similar to that of CRC RPO −. The mutation frequencies of commonly mutated genes in CRC are similar between CRC RPO + and RPO − cases. Likewise, gene expression patterns are indistinguishable between the RPO + and RPO − cases. We have confirmed that CRC RPO + is clinically and biologically similar to CRC RPO − and may be utilized as a model of the adenoma to carcinoma transition.     

中国红豆杉细胞经重复诱导和蔗糖饲喂后云南紫杉烷C生产的相应基因表达变化

红豆杉悬浮培养细胞具有可持续生产抗癌药物紫杉醇及其他紫杉烷的潜力。在中国红豆杉悬浮培养细胞中,云南紫杉烷 C(Tc) 是主要的次生代谢产物。为促使代谢前体由生成其他紫杉烷的代谢支路转到生产紫杉醇,实验采用实时定量PCR技术 (RQ-PCR) 揭示细胞培养过程中紫杉醇及紫杉烷合成关键基因的动态变化。在细胞培养的第7天和第12天,以100 μmol/L 2,3-二羟丙基茉莉酸 (DHPJA) 进行诱导,同时在第7天饲喂20 g/L的蔗糖,在此过程中考察6个关键基因 (TASY,TDAT,T5αH,TαH,T10βH和T14βH) 的表达变化。上述联合调控手段使得Tc产量在第1次诱导8 d后达 (554.46±21.28) mg/L,第2次诱导9 d后高达 (997.72±1.51) mg/L。代谢早期基因TASY和TDAT在第1次诱导后表达量分别提高了182和98倍,在第2次诱导后表达量分别提高了208和131倍。在每次诱导后基因表达量提高约持续24 h,之后下降。其他4个基因 (T5αH、TαH、T10βH和T14βH) 的情况有所不同。基因TαH在2次诱导后表达量分别提高了3 061和1 016倍。其他3个基因T5αH、T10βH、T14βH在第1次诱导后表达量分别提高13、38、20倍,在第2次诱导后分别提高7、16、6倍。RQ-PCR结果表明基因表达和Tc积累之间存在紧密相关性：基因表达的变化与Tc产量的变化相一致,诱导可提高6个基因的表达量。基因的高表达随着培养过程逐渐衰减,再次诱导可再次促使基因的高表达。