Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.     

Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.     

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phylogeny of mitochondrial DNA clones in tassel-eared squirrels Sciurus aberti

The tassel-eared squirrel, Sciurus aberti , includes six subspecies which occupy restrictive and apparently identical habitats in Ponderosa pine forests in the south-western United States and Mexico; the strict habitat requirement of this species is based on dietary requirements which are only fulfilled in these forests. To examine evolutionary relationships among certain subspecies of S. aberti , we obtained estimates of nucleotide diversity within subspecies as well as nucleotide divergence between subspecies using mitochondrial DNA (mtDNA) analysis. Restriction site polymorphisms were identified in samples of the four US subspecies: S. a. aberti (Abert), S. a. kaibabensis (Kaibab), S. a. ferreus (Ferreus), and S. a. chuscensis (Chuska) Fourteen mtDNA clones were resolved that were, with one exception, uniquely subspecific. Dendrograms constructed by neighbour-joining and maximum parsimony methods revealed two major assemblages: (1) an Abert/Kaibab group; and (2) a Ferreus/Chuska group. The Abert vs. Ferreus clones exhibited the greatest net nucleotide divergence, with a lineage separation estimate approximating 572 000 years ago assuming a nucleotide substitution rate of 7.15 × 10^-9/year/site. Five out of ten Chuska squirrels shared a clone with one Abert sample; the relative sizes of these two populations and their respective ranges as well as their close proximity support the proposal for relatively recent intermixing of Abert and Chuska populations resulting in what appears to be Abert → Chuska migration. Nucleotide diversity within subspecies ranked as Kaibab < Ferreus < Abert < Chuska; the relatively high diversity for the Chuska sample is based on the apparent introgression of Abert mtDNA. The relative diversity exhibited by Kaibab, Ferreus and Aberti samples corresponds to the range size of the respective subspecies.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.     

水库对投饵网箱养鱼的负荷力

本文用１８个１４．３ｍ￣３的围隔组成的围隔群,以鲤为材料,研究了水库对投饵网箱养鱼的负荷力。实验中观测了水温、透明度、ｐＨ值、溶氧、化学耗氧量、生化需氧量与非离子氨等环境因素的变化。结束时将这些变化与我国渔业水质标准相比较,查明该负荷力约为３０００ｋｇ·ｈａ￣（－１）,建议增加２５－３５％的安全储备,以最大载鱼量１８００－２３００ｋｇ·ｈａ￣（－１）作为推荐值。     

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.     

卡特拉鱼形态,食性和生长的研究

观测卡特拉鱼的形态特征和1—3龄鱼主要可量性状及其比例的变动。1—3龄鱼除主食商品饲料外,同时也食较大比例的浮游生物。着重对浮游生物的种类和比例进行了分析。肠长与体长呈直线相关。Y=6.38X-89.95。1—4龄鱼体长和体重相对生长率和生长指标都随年龄增加而逐渐下降。体长与体重呈曲线相关。W=0.00002261L~(3.0569)。采用 Von.Bertalanffy 生长方程分析其生长特性。方程参数 L_∞=821.2mm,W_∞=19616.6g,t_0=0.6年,K=0.3。体长生长不具拐点,生长速度随年龄增加而递减;体重生长具拐点,其值4.3年。此时体重生长速度达最大值。4、3龄前是生长旺盛期,此后进入生长缓慢期。     

Analysis of feeding mechanism in a pitcher ofNepenthes hybrida

The process of digestion of captured feeds in a pitcher, an insect-trapping organ, ofNepenthes was studied. Changes in bacterial population, pH and NH₄ ⁺ concentrations in pitcher juice were examined. Strong activities of both acid- and alkaline phosphatase, phosphoamidase, esterase C4 and esterase C8 were found in the pitcher juice. Optimum pH of proteases in the juice and those secreted from bacteria showed pH 3.0 and pH 8.0–9.0, respectively. Twenty six strains of bacteria were isolated from 4 pitchers: 10 strains were gram positive, 16 strains were gram negative (10 strains had casein hydrolase activity). A proton excretion was induced by NH₄ ⁺ released from the added solutions, and accordingly, the pH of the solutions fell. As a simulation model of the digestion process of feeds in pitcher juice and polypeptone solution was added into the washed pitcher. A good correlation was found among the NH₄ ⁺ concentration, pH and bacterial cell titer.