Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Expression of cDNAs encoding the precursor and the mature form of chicken mitochondrial aspartate aminotransferase in Escherichia coli

Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli. The precursor was found to sediment quantitatively together with insoluble cell material. In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria. Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing.     

人体单臂间歇运动对发汗调定点的影响

本工作系在微小气候相对恒定条件下,对10名健康男青年每人进行四项实验。实验 Ⅰ 为测定双侧腿足浸入43℃水中,诱发左前臂屈侧显现定量汗点时的口腔温度(舌下)阈值,作为发汗调定点参考值(ToSSP);实验 Ⅱ 为 Ⅰ 附加右臂间歇轻负荷运动(77W)时测定 ToSSP,部分对象还记录了皮肤电反应;实验 Ⅲ、Ⅳ 为 Ⅰ、Ⅱ 均附加4.5m/s 气流(22—25℃)直吹头面部,再分别测定 ToSSP。实验 Ⅰ 与 Ⅱ 同体对照22人次,Ⅲ 与 Ⅳ 同体对照24人次。结果表明,实验 Ⅱ、Ⅳ 的 ToSSP 均值及其潜伏期均值分别较 Ⅰ、Ⅲ 者降低(P<0.01)或缩短(P<0.001);Ⅰ、Ⅱ间的 ToSSP 均值差、潜伏期均值差,分别与 Ⅲ、Ⅳ 之间者无显著差异(P>0.2);Ⅱ、Ⅳ 的ToSSP 均值各与其实验开始前的口温均值亦无明显差异(P>0.5)。此结果支持运动时体温调定点下降的论点,并提示在研究体温调定点活动时,以 ToSSP 为指标较用发汗速率为优越,因 ToSSP 不为许多干扰因素所影响。     

Evolutionary relationships among aminotransferases. Tyrosine aminotransferase, histidinol-phosphate aminotransferase, and aspartate aminotransferase are homologous proteins

A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases. A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase. However, no significant similarity between these proteins and any of the other aminotransferases was detected. A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol-phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases. For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work-sheet. The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment. Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E. coli aspartate aminotransferase, rat and E. coli tyrosine aminotransferase, and S. cerevisiae and E. coli histidinol-phosphate aminotransferase are homologous proteins. Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5'-phosphate and the substrate, or appear to be essential for the conformation of the enzymes.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

The covalent structure of mitochondrial aspartate aminotransferase from chicken. Identification of segments of the polypeptide chain invariant specifically in the mitochondrial isoenzyme

The primary structure of mitochondrial aspartate aminotransferase from chicken is reported. The enzyme is a dimer of identical subunits. Each subunit contains 401 amino acid residues; the calculated subunit molecular weight of the apoform is 44,866. The degree of sequence identity with the homologous cytosolic isoenzyme from chicken is 46%. A comparison of the primary structures of the mitochondrial and the cytosolic isoenzyme from pig and chicken shows that 40% of all residues are invariant. The degree of interspecies sequence identity both of the mitochondrial and the cytosolic isoenzyme from chicken and pig (86% and 83%, respectively) markedly exceeds that of the intraspecies identity between mitochondrial and cytosolic aspartate aminotransferase in chicken (46%) or in pig (48%). Based on these values, the duplication of the aspartate aminotransferase ancestral gene is estimated to have occurred approximately 1000 million years ago, i.e. at the time of the emergence of eukaryotic cells. By sequence comparison it is possible to identify amino acid residues and segments of the polypeptide chain that have been conserved specifically in the mitochondrial isoenzyme during phylogenetic evolution. These segments comprise about a third of the total polypeptide chain and appear to cluster in a certain surface region. The cluster carries an excess of positively charged residues which exceeds the overall charge difference between the cytosolic (pI approximately 6) and the mitochondrial isoenzyme (pI approximately 9).     

31P-NMR analysis of sea urchin sperm activation: Reversible formation of high energy phosphate compounds by changes in intracellular pH

³¹P-NMR has been used to estimate the internal pH (pH_i) of sperm from the sea urchin Strongylocentrotus purpuratus. The values for pH_i obtained from the chemical shift of inorganic phosphate agree well with those obtained from amine accumulation. At low pH_i, when sperm are quiescent (immotile and non-respiring), they accumulate phosphocreatine (PCr), but have a low level of inorganic phosphate (P_i). Conversely, when the pH_i is elevated, sperm respiration and motility are activated, PCr is decreased and P_i is increased. This change is reversible upon decrease of the pH_i, whereupon respiration and motility are arrested, P_i disappears and PCr increases. We conclude that the overall balance of energy metabolism, and thus the phosphate potential, of sea urchin sperm are under the control of the pH_i.     

我国恙虫蚴的三个新种(蜱螨目, 恙虫科)

近几年来,我国科学家们在恙虫方面的研究工作巳经取得了显著的成就。然而,这对于拥有辽阔疆土的我国来说,实在只不过是一个开始。我们在这方面的工作也做得很少,虽然从1953年开始收集标本,由于各种条件所限制,未能及时整理,以致大量标本散失或霉烂,十分可惜。本文内容,仅仅是将我站恙虫病调研工作组1957年在本省各地所采集的材料中的三种恙虫蚴加以描述,该三种恙虫蚴均系文献上未曾记载过的种类。