Characterization of the oriC region of Mycobacterium smegmatis.

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.     

K-77(2)雄性不育小麦花粉壁结构特征

K-77(2)不育花粉内壁比保持系厚将近一倍。在保持系花粉内壁中,有径向排列的、断断续续的、着色深的管状结构。而不育花粉内壁中则没有管状结构,且在不育花粉内壁中形成许多小泡。在花粉粒后期,往往在Z层与内壁连接处断开。推测,由于花粉内壁结构被破坏,影响了正常的营养运输和萌发所需酶的合成而抑制花粉发育。     

Extracellular action potentials recorded from the interior of the giant esophageal cell of Ascaris

Exploration of the cytoplasm of the giant esophageal cell of Ascaris with a recording microelectrode shows the existence of shallow spaces where the microelectrode tip becomes extracellular in spite of being in the interior of the cell. When the microelectrode penetrates into these spaces from the cytoplasm, the resting potential shifts to a different level or entirely disappears. At the same time the large intracellular spikes are replaced by small transients similar to extracellularly recorded action potentials. It is concluded that such spaces are in communication with the external solution, and separated from the cytoplasm by an electrically active membrane; i.e., able to generate action potentials. Measurement of the potential differences between the interior of the spaces and the external solution shows that although some are not polarized, many spaces have a resting potential of the same polarity as that of the cytoplasm. It is suggested that although they are of larger size these spaces may be equivalent to the tubular systems which in other muscle cells are known to be involved in the spread of excitation into the cytoplasm.     

Ablation of ghrelin receptor in leptin-deficient ob/ob mice has paradoxical effects on glucose homeostasis when compared with ablation of ghrelin in ob/ob mice

The orexigenic hormone ghrelin is important in diabetes because it has an inhibitory effect on insulin secretion. Ghrelin ablation in leptin-deficient ob/ob (Ghrelin(-/-):ob/ob) mice increases insulin secretion and improves hyperglycemia. The physiologically relevant ghrelin receptor is the growth hormone secretagogue receptor (GHS-R), and GHS-R antagonists are thought to be an effective strategy for treating diabetes. However, since some of ghrelin's effects are independent of GHS-R, we have utilized genetic approaches to determine whether ghrelin's effect on insulin secretion is mediated through GHS-R and whether GHS-R antagonism indeed inhibits insulin secretion. We investigated the effects of GHS-R on glucose homeostasis in Ghsr-ablated ob/ob mice (Ghsr(-/-):ob/ob). Ghsr ablation did not rescue the hyperphagia, obesity, or insulin resistance of ob/ob mice. Surprisingly, Ghsr ablation worsened the hyperglycemia, decreased insulin, and impaired glucose tolerance. Consistently, Ghsr ablation in ob/ob mice upregulated negative β-cell regulators (such as UCP-2, SREBP-1c, ChREBP, and MIF-1) and downregulated positive β-cell regulators (such as HIF-1α, FGF-21, and PDX-1) in whole pancreas; this suggests that Ghsr ablation impairs pancreatic β-cell function in leptin deficiency. Of note, Ghsr ablation in ob/ob mice did not affect the islet size; the average islet size of Ghsr(-/-):ob/ob mice is similar to that of ob/ob mice. In summary, because Ghsr ablation in leptin deficiency impairs insulin secretion and worsens hyperglycemia, this suggests that GHS-R antagonists may actually aggravate diabetes under certain conditions. The paradoxical effects of ghrelin ablation and Ghsr ablation in ob/ob mice highlight the complexity of the ghrelin-signaling pathway.     

Wistar大鼠心脏自发性病变的病理学观察

目的了解Wistar大鼠心脏自发性病变发病情况,为长期致癌性研究、老年病学研究及毒性病理学提供背景资料。方法采用160只清洁级Wistar大鼠,雌雄各半,常规饲养,分别在9月龄、12月龄、18月龄、24月龄时处死40只大鼠,HE及Masson三色法染色,观察心脏的病理改变。结果 9月龄Wistar大鼠心脏未见明显病理改变;12月龄Wistar大鼠月龄心脏病变的发病率为2.5%（1/40）,表现为少数心肌细胞变性坏死伴少量以单核细胞为主的炎细胞浸润;18月龄大鼠心脏病变的发病率为57.5%（23/40）,表现为轻至中度心肌病,雄性发病率高于雌性。24月龄大鼠100%（40/40）出现不同程度的心肌病,并有2.5%（1/40）发生心内膜下纤维组织增生。Masson染色显示9月龄大鼠心脏血管周围及心脏瓣膜环下有少量胶原纤维,随年龄增长,血管周围及心脏瓣膜环下胶原纤维逐渐增多,并延伸入心肌细胞间。结论随年龄增长,大鼠心脏自发病变比率升高,主要病变为心肌病,偶尔可发生心内膜下纤维组织增生;胶原纤维沉积首先发生于血管周围及心脏瓣膜环下,随年龄增长而增多,可能与大鼠心肌病的的发生密切相关。     

鱼类生殖细胞移植的研究进展

生殖细胞移植在生物学、畜牧学及兴起的鱼类生物工程上都有很多用途。目前,采用基于基因组的育种方法已选育出多种带有所需遗传性状的鱼类新品系;但由于缺乏鱼卵与胚胎的低温保存技术,尚未找到长期保存其种质资源的方法。就鱼类生殖细胞移植的最新研究进展进行综述,同时针对鱼类生殖细胞移植及其低温保存技术研究中存在的问题进行讨论,并提出了鱼类生殖细胞移植的应用前景,以期能积极推动鱼类生物工程的研究,加速鱼类生殖细胞移植在产业中的应用。     

广西巴马小型猪血清淀粉样P物质基因真核载体构建及细胞转染

血清淀粉样P物质(Serum Amyloid Pcomponent,SAP)是一种在进化上高度保守的血清糖蛋白,它可与各种类型的原纤维结合,在免疫应答和炎症反应等多种免疫疾病中发挥作用.以广西巴马小型猪肝组织总RNA为模板,利用RT-PCR技术扩增出相应cDNA片段,连接到克隆载体pMD18-T上进行检测,测序结果为675 bp,与GenBank所提供相关序列同源性为100％,并成功构建pEGFP-N1 -SAP重组真核表达载体,利用脂质体(Lipofectamine 2000)介导法将重组质粒导入到NIH-3T3细胞中培养,经转染24 h后,置于倒置荧光显微镜下观察,发现含有重组质粒的NIH-3T3细胞中表达出绿色荧光,为进一步研究SAP基因的功能特点及在试验动物相关疾病模型的应用提供务件.     

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.