Balamuthia mandrillaris: The multiple nuclei of Balamuthia amebas; their location, activity, and site of development

Multiple nuclei were first noted in the pseudopodia of Balamuthia mandrillaris amebas feeding on mammalian cells. Phase microscope observations of live amebas in vitro reveal that while many amebas have a single nucleus, others have multiple nuclear-like structures, now confirmed as nuclei with hematoxylin and Feulgen stains. In the live cultures, two nuclei located near the tip of an extended pseudopodium were seen to fuse resulting in one larger morphologic unit. Such merging of nuclei has not been previously reported. Other nuclei were located at positions that subsequently became the site for the outgrowth of an additional pseudopod branch. A newly discovered large structure, a polyploid nucleus, was located in the mid-part of the ameba. Nucleoli of uniform size were seen to develop from the central mass of chromatin and each became surrounded by a vesicular component as they moved into the protoplasm as morphologically complete nuclei.     

Nest site selection in leatherbacks, Dermochelys coriacea: individual patterns and their consequences

We determined individual nest placement patterns for female leatherbacks nesting at Awa:la-Ya:lima:po, French Guiana, by measuring distance from the nest to several landscape features, such as the highest spring tide line (HSTL) and the vegetation line. Distance from the nest to the HSTL differed significantly between females, indicating the existence of individual nesting patterns. There was a significant repeatability of nest site choice relative to the HSTL, indicating that females showed within-individual consistency in their nest placement. Despite individual preferences, there was much within-individual variation and a lack of predictability in the nesting patterns; that is, the locations of subsequent nests could not be predicted based on knowledge of previous nest choices, indicating a certain degree of scatter. The significant repeatability suggests that nest choice behaviour in female leatherbacks is heritable and may show the potential for further evolution. We tested sea-finding ability of hatchlings, a potential consequence of nest site choice, in Matapica, Suriname, by using orientation arenas to quantify the strength and direction of travel after emergence. The orientation tests showed that hatchlings were unable to move seaward in vegetated arenas, providing evidence that vegetation acts as a strong selective pressure driving nest placement seaward. It appears that leatherbacks have adopted a regional rather than a local optimum for nest placement patterns, possibly resulting from their weak beach fidelity and the frequent erosion and destruction of their nesting beaches. We discuss the evolutionary and conservation implications for this species in the context of current environmental changes.     

Different site, different splice

The MKK3/6-p38 pathway has been found to induce the relocalization of premessenger-RNA splicing factors from the nucleus to the cytoplasm. This represents the first physiological mechanism that alters the nuclear ratios of splicing factors and modulates alternative splice-site choice in vivo.     

Active site of α1-antitrypsin: Homologous site in antithrombin-III

Examination of peptides resulting from reaction of bovine trypsin and human α₁-antitrypsin in near-equimolar amounts showed anomalous cleavage of antitrypsin at a Met-Ser bond 37 residues from the C-terminus, giving evidence that this is the active site for trypsin inhibition. Alignment of the C-terminal 141 residues of α₁-antitrypsin with the C-terminal 147 residues of human antithrombin-III showed homology with 30% identity and allowed the identification of a homologous active site in antithrombin.     

WWOX,large common fragile site genes,and cancer

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.     

The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes

The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in [32P]orthophosphoric acid for about four generations. The cells were harvested in the logarithmic phase, resuspended in a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees C in the presence of 3H-labelled amino acids. This procedure preferentially labels growing peptidyl chains. Polysomes were isolated, the fraction in the post-translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis. The data revealed that at least 75% of post-translocational ribosomes in isolated native polysomes carry a tRNA in their E site. The results are consistent with the allosteric three-site model for the elongation cycle but disagree with the two-site model.     

ADAM10 overexpression shifts lympho- and myelopoiesis by dysregulating site 2/site 3 cleavage products of Notch

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b(+)Gr-1(+) cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage(-)Sca-1(+)c-Kit(+) cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage(-)Sca-1(+)c-Kit(+) cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.     

Nucleotide binding site/leucine-rich repeats,Pto-like and receptor-like kinases related to disease resistance in grapevine

Nucleotide Binding Site/Leucine-Rich Repeat (NBS-LRR) and Serine/Threonine Kinase (STK) genes are two of the known classes of resistance (R-) genes in plants, and occur in large multigene families. Systematic identification of genes for NBS-LRRs and STKs provides a means of access to genomic regions that may be involved in disease resistance. Here we present a picture of these two families of R-gene analogs (RGAs) in grape with the aim of developing a set of resistance-related sequence-tagged-site (STS) markers. One hundred and three NBS-LRR sequences were isolated. They included members of the CC (coiled-coil) and TIR (Toll-interleukin receptor) sub-classes. A comparative analysis with other angiosperm NBSs is provided. Fifty-three genes for receptor-like kinases (RLKs) with serine/threonine specificity were identified. RLK sequences formed a putative monophyletic group within the kinase superfamily. They were similar to both cytoplasmic RLKs, such as Pto, and RLKs with LRR, S-locus, lectin-like and thaumatin-like extracellular binding-domains. The latter resembled the products of the R-related genes Xa21, FLS2, Rlk10, SFR2, and PR5K. Forty-five reference RGAs were converted into STSs by using appropriately designed specific primers. RGA-STSs were present in diverse grape genotypes, and >85% of the primers were capable of amplifying the STSs across the taxa Vitis and Muscadinia. DNA sequence polymorphism among these RGAs was assessed by SSCP (single-strand conformation polymorphism) analysis in over 20 Vitis spp. Finally, 45 universal primers for grape RGAs are proposed that should permit tagging of R-related regions in any grape genome.Communicated by R. Hagemann     

Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification

The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.     

Pupation site and emergence time influence the mating success of bagworm females,Oiketicus kirbyi

In commercial oil palm plantations in Costa Rica, we tested the hypotheses that pupation site and emergence time affect the mating success of protogynous female bagworms,Oiketicus kirbyi (Guilding) (Lepidoptera: Psychidae). Greater proportions of female than male pupae on upper leaves of oil palms and greater proportions of mated females in the upper rather than lower crown strata support the hypothesis that selection of pupation site by female larvae influences the mating success of adults. Increasing captures of males with increasing trap height further suggest that enhanced mating success of females in tree tops may be attributed either to most effective dissemination of sex pheromone on higher sites, or to males foraging predominantly in the upper strata of oil palms. As the majority of females pupated in the middle rather than upper crown of oil palms, selection of pupation site by females may be affected by additional as yet unknown factors. Emergence of females significntly preceded emergence of males. Increasing proportions of mated females throughout the emergence seasons probably resulted from an increased ‘availability’ of males. In tropical rainforests with local variations inO. kirbyi developmental time and stage, protogyny may represent an evolutionary strategy that furthers outbreeding.     

Development of the cell-division site in FtsA-filaments

Early changes at cell-division sites were studied in non-septate filaments induced by growth of ftsATs mutant cells under non-permissive conditions. The positions of localized regions of plasmolysis were used as markers for the locations of partial and complete annular structures that are thought to be precursors of the periseptal annuli that flank the septum during cytokinesis. The results confirmed that these structures were localized at potential division sites and suggested a model in which older division sites play a role in the generation of new sites for future use, with each older site being used only once for this purpose. The results also suggest that the details of division-site development can profitably be studied in cells in which early events in the differentiation process are uncoupled from the septation event.     

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.     

A Rangwapithecus gordoni mandible from the early Miocene site of Songhor,Kenya

A mandible of Rangwapithecus gordoni from the early Miocene site of Songhor, Kenya, provides additional information about this relatively poorly known taxon. The R. gordoni sample is small, being composed of dental and a few gnathic parts. The fossil described here provides examples of previously unknown dental and mandibular anatomy, and confirms former reassignments of isolated anterior teeth based on less certain evidence. The phylogenetic status of Rangwapithecus, its distribution, and paleobiology are briefly reviewed. Rangwapithecus shows a suite of dental and gnathic features that warrants its generic distinction from Proconsul. Derived features shared with Nyanzapithecus and Turkanapithecus indicate that it is an early member of the subfamily Nyanzapithecinae. Its molar morphology suggests a considerable component of folivory in its diet. A review of the hypodigm shows Rangwapithecus to be restricted to Songhor. This distribution parallels that of Limnopithecus evansi, and is mirrored by Limnopithecus legetet and Micropithecus clarki suggesting that Songhor may have differed ecologically from other more or less contemporary sites in the region.     

广聚萤叶甲成虫产卵行为及其产卵部位的选择性

【目的】明确广聚萤叶甲成虫产卵行为及其产卵部位的选择性。【方法】在室内条件下,对广聚萤叶甲成虫交配及产卵的系列行为、产卵场所选择、不同部位豚草植株叶片叶绿素b的含量进行了观察和测定:(1)将1对成虫放到养虫笼内的一株豚草上,观察交配时间,记录产卵数量、前后2粒卵之间的产卵时间间隔;(2)在均匀分为5部分(0～10、11～20、21～30、31～40和41～50 cm)的豚草植株上,随机放置10对成虫,观察雌虫对于产卵场所的选择。(3)将上述5个部位的豚草叶片通过丙酮匀浆法处理,用紫外分光光度计测定其在645和663 nm的吸光值,计算叶绿素b含量。【结果】广聚萤叶甲成虫完成一次成功交配平均需96.09 min。雌虫一般需45 min的时间来寻找其适应的产卵场所,在产卵过程中,成虫习惯将卵产于叶片背面,雌虫喜欢用口器来清理刚产下的卵粒。在一株50 cm高的豚草植株上,雌虫喜欢将卵产在植株中部21～30 cm和中上部31～40cm的叶片上(从下往上划分)。卵块数量和豚草不同部位叶片叶绿素b含量呈显著的正相关性。【结论】广聚萤叶甲成虫喜欢产卵在叶绿素b含量较高的叶片背面,可能以视觉识别叶片颜色来选择和定位产卵场所。     

Roles of active site and novel K+ ion-binding site residues in human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase

The human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric (alpha(2)beta(2)) thiamine diphosphate (TDP)-dependent enzyme. The recently solved human BCKD structure at 2.7 A showed that the two TDP-binding pockets are located at the interfaces between alpha and beta' subunits and between alpha' and beta subunits. In the present study, we show that the E76A-beta' mutation results in complete inactivation of BCKD. The result supports the catalytic role of the invariant Glu-76-beta' residue in increasing basicity of the N-4' amino group during the proton abstraction from the C-2 atom on the thiazolium ring. A substitution of His-146-beta' with Ala also renders the enzyme completely inactive. The data are consistent with binding of the alpha-ketoacid substrate by this residue based on the Pseudomonas BCKD structure. Alterations in Asn-222-alpha, Tyr-224-alpha, or Glu-193-alpha, which coordinates to the Mg(2+) ion, result in an inactive enzyme (E193A-alpha) or a mutant BCKD with markedly higher K(m) for TDP and a reduced level of the bound cofactor (Y224A-alpha and N222S-alpha). Arg-114-alpha, Arg-220-alpha, and His-291-alpha interact with TDP by directly binding to phosphate oxygens of the cofactor. We show that natural mutations of these residues in maple syrup urine disease (MSUD) patients (R114W-alpha and R220W-alpha) or site-directed mutagenesis (H291A-alpha) also result in an inactive or partially active enzyme, respectively. Another MSUD mutation (T166M-alpha), which affects one of the residues that coordinate to the K(+) ion on the alpha subunit, also causes inactivation of the enzyme and an attenuated ability to bind TDP. In addition, fluorescence measurements establish that Trp-136-beta in human BCKD is the residue quenched by TDP binding. Thus, our results define the functional roles of key amino acid residues in human BCKD and provide a structural basis for MSUD.