The isolation and partial characterization of precursor forms of ostrich carboxypeptidase

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.     

不受欢迎的生物多样性：香港的外来植物物种

香港早在19世纪中叶开始就有外来植物入侵的记录,迄今为止,已发现多达238种已归化的外来或怀疑为外来的植物,其中又以薇甘菊（Mikania micrantha)、五爪金龙（Ipomoea cairica)、假臭草（Eupatorium catarium)、大黍（Panicum maximum)等最常见,外来植物最常见于受人为干扰的生境,例如荒废农田及路旁等,而较少在天然林地生境及贫瘠的灌草丛中发现,外来植物的对本地生态系统的影响主要局限于低地生境,它们常形成单优种群,减少了生境及贫瘠的灌草丛中发现,外来植物对本地生态系统的影响主要局限于低地生境,它们常形成单优种群,减少少了生境及动植物的多样性,外来动物对香港原生植物影响最大的是于20世纪70年代入侵的松树线虫（Bur-saphelenchus xylophilus)。外来的脊椎动物也有可能对香港的植物被演替产生影响,目前香港的外来植物当中,有些在大陆较少分布或没有记录,作为华南最大的港口,香港对外来物种的引入扮演着重要的角色,因此制定控制外来种在香港及华南地区的引入及传播的政策及措施非常重要。     

The purification and characterisation of m-calpain from ostrich brain

Calpains are intracellular cysteine proteases activated in a Ca(2+)-dependent manner. The purpose of the present study was to investigate the physico-chemical and kinetic properties of ostrich brain m-calpain. m-Calpain was purified by successive chromatographic steps on Toyopearl-Super Q 650s and Pharmacia Mono Q HR 5/5 columns. A Ca(2+) concentration of 5mM and a casein concentration of 5mg/ml were found to be necessary for optimum calpain activity. Ostrich m-calpain exhibited a M(r) of 84K using SDS-PAGE and a M(min) of 79.3K from amino acid analysis. The pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. The amino acid composition of m-calpain revealed 700 residues. The N-terminal sequence of m-calpain showed sequence identity with chicken (27%), human (23%) and rabbit (18%) and Schistoma mansoni (9%).     

Nucleotide polymorphism in the acidic chitinase locus (ChiA) region of the wild plant Arabidopsis thaliana

To investigate DNA variation in natural plant populations, a 1.8-kb region of the acidic chitinase locus (ChiA)was analyzed for 17 ecotypes of Arabidopsis thaliana sampled worldwide and 3 Arabis species in Japan. As in the Adh region, dimorphism was detected throughout the investigated ChiA region, suggesting the possibility that dimorphic DNA variation exists in the entire nuclear genome of A. thaliana. The ChiA region was divided into two blocks by an intragenic recombination between two parental sequence types, which diverged 7.4 MYA under the assumption that nucleotide mutation rate per site per year is mu = 10(- 9). Nucleotide diversity in the entire ChiA region was 0.0104. Tajima's test was significantly negative for both nucleotide and indel variations, which was manifested as an excess of unique polymorphisms. However, the level and pattern of polymorphism in the ChiA region were inconsistent with simple theoretical explanations. The HKA test detected no difference in the levels of intra- and interspecific variations between the ChiA and Adh regions. In the ChiA coding region, no difference in the patterns of synonymous and replacement variation was found in intra- and interspecific comparisons by the MK test. Although it was difficult to determine the exact genetic mechanism acting on the ChA locus, these results suggested that the ChA locus region was under the same genetic mechanism before and after the establishment of A. thaliana as a species.      

The purification and characterization of mu-calpain and calpastatin from ostrich brain

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.     

人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.