PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.     

云南省角蝉科二新属二新种

最近我们从全国动物学科研单位和高等院校借调来大批的角蝉科标本,进行系统的分类研究,从中发现有二新属及二新种,均采自云南西双版纳,现在先把它们加以记载。     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.     

Does human attractin have DP4 activity?

Mutations in the mouse ATRN gene, which encodes attractin, offer links between this protein and pigmentation, metabolism, immune status and neurodegeneration. However, the mechanisms of attractin action are not understood. The protein was first identified in humans in a circulating form in serum. A protease activity was postulated similar to the membrane-bound ectoenzyme DP4/CD26. In the last decade, both DP4/CD26 and attractin were controversially described to be the major source of human serum DP4 activity. We purified attractin from human plasma, and found that the DP4-like activity of the preparation shows nearly identical kinetic properties to that of recombinant human DP4. In contrast, the native electrophoretic behavior of this activity is clearly different from human and porcine DP4, but co-migrates with the protein band identified as attractin by Western blotting and N-terminal sequencing. Nevertheless, a DP4 impurity could be demonstrated in purified plasma attractin and the activity could be removed by ADA affinity chromatography, resulting in a homogenous attractin preparation without DP4 activity. These results are substantiated by expression of different attractin isoforms, in which no DP4 activity was found either. This indicates that the multidomain protein attractin acts as a receptor or adhesion protein rather than a protease.     

Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu(1) conversion is favored at pH 6.0 while Gln(1) conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.     

Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.     

A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus

Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (～10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.     

A universal scaling law determines time reversibility and steady state of substitutions under selection

Monomorphic loci evolve through a series of substitutions on a fitness landscape. Understanding how mutation, selection, and genetic drift drive this process, and uncovering the structure of the fitness landscape from genomic data are two major goals of evolutionary theory. Population genetics models of the substitution process have traditionally focused on the weak-selection regime, which is accurately described by diffusion theory. Predictions in this regime can be considered universal in the sense that many population models exhibit equivalent behavior in the diffusion limit. However, a growing number of experimental studies suggest that strong selection plays a key role in some systems, and thus there is a need to understand universal properties of models without a priori assumptions about selection strength. Here we study time reversibility in a general substitution model of a monomorphic haploid population. We show that for any time-reversible population model, such as the Moran process, substitution rates obey an exact scaling law. For several other irreversible models, such as the simple Wright-Fisher process and its extensions, the scaling law is accurate up to selection strengths that are well outside the diffusion regime. Time reversibility gives rise to a power-law expression for the steady-state distribution of populations on an arbitrary fitness landscape. The steady-state behavior is dominated by weak selection and is thus adequately described by the diffusion approximation, which guarantees universality of the steady-state formula and its applicability to the problem of reconstructing fitness landscapes from DNA or protein sequence data.     

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.