Structure of P-protein of the glycine cleavage system: implications for nonketotic hyperglycinemia

The crystal structure of the P-protein of the glycine cleavage system from Thermus thermophilus HB8 has been determined. This is the first reported crystal structure of a P-protein, and it reveals that P-proteins do not involve the alpha(2)-type active dimer universally observed in the evolutionarily related pyridoxal 5'-phosphate (PLP)-dependent enzymes. Instead, novel alphabeta-type dimers associate to form an alpha(2)beta(2) tetramer, where the alpha- and beta-subunits are structurally similar and appear to have arisen by gene duplication and subsequent divergence with a loss of one active site. The binding of PLP to the apoenzyme induces large open-closed conformational changes, with residues moving up to 13.5 A. The structure of the complex formed by the holoenzyme bound to an inhibitor, (aminooxy)acetate, suggests residues that may be responsible for substrate recognition. The molecular surface around the lipoamide-binding channel shows conservation of positively charged residues, which are possibly involved in complex formation with the H-protein. These results provide insights into the molecular basis of nonketotic hyperglycinemia.     

Vitellogenin-immunohistochemistry in the liver and the testis of the medaka, Oryzias latipes, exposed to 17beta-estradiol and p-nonylphenol

Vitellogenin (VTG) produced in male fish has been used for a biomarker to study endocrine disrupters. However, the characteristics of VTG produced in male fish have not been studied well. In this study, we investigated the localization of VTG in the liver and the testis of male medaka (Oryzias latipes) treated with 17beta-estradiol (E2) and p-nonylphenol (NP). The male fish were exposed to 1 microg/L E2 and 500 microg/L NP for 1-12 days. Control groups were kept in water including only vehicle. The frozen sections of the liver and the testis were stained with immunohistochemical methods using an antiserum against medaka VTG as the first antibody. In the E2 and NP treated liver, the hepatocytes showed immunoreactivity. In particular, the cytoplasm close to the cell membrane surrounding the sinusoids was strongly immunopositive. In the testis of both treatments, the interstitial tissues and the cells (spermatocytes) in the seminiferous tubules were immunopositive. The concentration of VTG became gradually higher in both tissues with longer treatments. These results suggest that germ cells in the testis treated with E2 and NP are able to incorporate and accumulate VTG.     

Development of non-radioisotopic immunoassay systems for measuring flounder IGF-I

A time-resolved fluoroimmunoassay system (TR-FIA) for measuring flounder insulin-like growth factor-I (IGF-I) was developed using biotinylated flounder IGF-I, anti-fish IGF-I antiserum and europium-avidin conjugate. The detection limit per well was <5 pg/well corresponding to <0.5 ng/ml in a basic procedure for sample of 10 microl/well and to <0.08 ng/ml in a procedure modified for high volume samples (up to 70 microl/well). Specificity of the assay was validated using various IGF-Is and insulins. All IGFs except seabream IGF-I showed very low or no crossreactivity. Binding inhibition curves for flounder and seabream IGF-Is were completely identical to each other. Intra- and interassay variations ranged from coefficients of variations of 3.9% to 7.2%. Recovery tests using barfin flounder plasma varied from 82.7 to 101.6% in the added range from 20 to 160 ng/ml. This assay system was applied for measuring total plasma IGF-I in barfin flounder injected porcine growth hormone (GH). A group injected with GH at the dose of 0.05 IU/gBW showed a significant increase of total plasma IGF-I compared with those of albumin-injected (control) and initial groups. In addition, I was able to substitute time-resolved fluorometric detection in this assay system with enzymatic fluorometric detection (FIA). Binding inhibition curve for flounder IGF-I in this substituted assay system showed equal performance with that of the TR-FIA system. Correlation of IGF-I levels between TR-FIA and FIA was high (r(2)=0.957) in plasma samples from barfin flounders in various physiological conditions. Thus, the present study shows precision and efficiency of two non-radioisotopic immunoassay systems for measuring flounder IGF-I.     

Light effects on cell development and secondary metabolism in Monascus

In nature, light is one of most crucial environmental signals for developmental and physiological processes in various organisms, including filamentous fungi. We have found that both red light and blue light affect development in Monascus, influencing the processes of mycelium and spore formation, and the production of secondary metabolites such as -aminobutyric acid, red pigments, monacolin K and citrinin. Additionally, we observed that the wavelength of light affects these developmental and physiological processes in different ways. These findings suggest that Monascus possesses a system for differential light response and regulation.     

CIN85 associates with TNF receptor 1 via Src and modulates TNF-alpha-induced apoptosis

CIN85 is a multidomain protein that associates with receptors carrying tyrosine kinase domains. Here we report that it is also a component of the signaling complex associated with tumor necrosis factor receptor 1 (TNFR1), which lacks a tyrosine kinase domain. This was established by showing that CIN85 was co-precipitated with TNFR1, TRADD, cIAP-1 and TARF1/2, but not with FADD, RIP, caspase-8 or TRAF6. However, CIN85 did not bind directly to the cytoplasmic domain of TNFR1 (TNFR1-CYT) but to Src family kinases, Cbl and the p85alpha subunit of phosphatidylinositol 3-kinase (PI3-K p85alpha). Src bound directly to TNFR1-CYT, but Cbl and PI3-K p85alpha did not. A human cell line ectopically expressing CIN85 was 10 times more susceptible to TNF-alpha-induced apoptosis than control cells, which expressed identical levels of TNFR1 on their surface. However, the susceptibility of these two cell lines to CD95-induced apoptosis was the same. The three SH3 domains of CIN85 were essential for this increased susceptibility to apoptosis and its proline-rich regions were also required for maximal effect. TNF-alpha treatment recruited CIN85 to the TNFR1 signaling complex. Taken together, these results indicate that CIN85 associates with TNFR1 via Src and modulates TNF-alpha-induced apoptosis.     

Studies on the reactivity of CDDO, a promising new chemopreventive and chemotherapeutic agent: implications for a molecular mechanism of action

CDDO, a semi-synthetic triterpenoid derived from oleanolic acid, has the potential to be used as a chemopreventive and chemotherapeutic agent. The structure of CDDO contains two alpha,beta-unsaturated carbonyl moieties, suggesting a mechanism of action involving a conjugate nucleophilic addition. Spectroscopic evaluation with thiol nucleophiles illustrates that an addition does indeed occur, but this addition is selective and reversible.     

Long-term ecological dynamics: reciprocal insights from natural and anthropogenic gradients

Many ecological dynamics occur over time-scales that are well beyond the duration of conventional experiments or observations. One useful approach to overcome this problem is extrapolation of temporal dynamics from spatial variation. We review two complementary variants of this approach that have been of late increasingly employed: the use of natural gradients to infer anthropogenic effects and the use of anthropogenic gradients to infer natural dynamics. Recent studies have considered a variety of naturally occurring gradients associated with climate, CO2, disturbance and biodiversity gradients, as well as anthropogenic gradients such as those created by biological invasions, habitat fragmentation and land abandonment. These studies show that natural gradients are useful in predicting long-term consequences of human-induced environmental changes, whereas anthropogenic gradients are helpful in inferring the mechanisms behind natural dynamics because covarying factors are often more clearly understood in anthropogenic gradients than in natural gradients. We classify these studies into several categories, each with different strengths and weaknesses, and outline how the limitations can be overcome by combining the gradient-based approach with other approaches. Overall, studies reviewed here demonstrate that the development of basic ecological concepts and the application of these concepts to environmental problems can be more effective when conducted complementarily than when pursued separately.     

Chimeric structure of omp2 of Brucella from Pacific common minke whales (Balaenoptera acutorostrata)

In the Pacific common minke whale (Balaenoptera acutorostrata ), a new variant of Brucella has been detected using the polymerase chain reaction. Detailed analysis of the porin protein genes, omp2a and omp2b from the whale Brucella showed that these two genes have some motifs in common with Atlantic marine strains in the 5'-terminal one-third region. On the other hand, the nucleotide sequences in the 3'-terminal two-thirds region of the two genes were almost identical to the respective genes of terrestrial strains. Thus, Pacific whale Brucella omp2 genes are chimeras between marine and terrestrial strains.     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.