Enhanced expression of the LDH-A gene after gravity-changing stress in human RSa cells.

A major issue in radiation and space biology is whether gene expression levels are altered in cells exposed to gravity-changing stress. In the present study, genes up- or down-regulated in radiation-sensitive human RSa cells cultured under gravity-changing conditions, were identified using a PCR-based mRNA differential display method. Exposure of cells to gravity-changing stress was performed by free-fall with a drop-shaft facility or by an airplane-conducted parabolic flight. Among the candidates for gravity-changing stress-responsive genes obtained by the differential display analysis, the lactate dehydrogenase A gene (LDH-A) was confirmed by Northern blotting analysis to exhibit increased expression levels. The gravity-changing stress consisted of a combination of microgravity and hypergravity. However, exposure of the cells to hypergravity produced by centrifuge only slightly affected the LDH-A mRNA expression. Thus, LDH-A was found to be a candidate for the genes which play a role in the cellular response to gravity-changing stress, and mainly to microgravity.     

Genetic mechanisms of age regulation of protein C and blood coagulation.

Blood coagulation activity in humans increases with age. We previously identified two genetic elements, age-related stability element (ASE; GAGGAAG) and age-related increase element (AIE; unique stretch of dinucleotide repeats), which were responsible for age-related stable and increasing expression patterns, respectively, and together recapitulated normal age regulation of the human factor IX (hFIX) gene. Here we report the age-regulatory mechanisms of human anticoagulant protein C (hPC), which shows an age-stable pattern of circulatory levels. The murine protein C gene showed an age-related stable expression pattern in general agreement with that of the hPC. Through longitudinal analyses of transgenic mice carrying hPC minigenes, the hPC gene was found to have a functional age-related stability element (hPC ASE; CAGGAAG) in the 5'-upstream proximal region but was found to lack any age-related increase element. Three other ASE-like sequences present in the hPC gene, GAGGAAA and (G/C)AGGATG, also bound nuclear proteins but were not active in the age regulation of the hPC gene. Functional hPC ASE and hFIX ASE were apparently generated through convergent evolution, and hFIX ASE can fully substitute for the hPC ASE in conferring age-related stable expression pattern of the hPC gene. In the presence of the hPC ASE, hFIX AIE can convert the age-stable expression pattern of the hPC gene to a hFIX-like age-related increase pattern. These results support the universality of ASE and AIE functions across different genes. Clearance of hPC protein from the circulation was not significantly affected by age. We now have established the basic mechanisms responsible for the age-related increase of blood coagulation activity.     

Dynamin Isoforms Decode Action Potential Firing for Synaptic Vesicle Recycling

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Identification of Osteopontin as a Novel Ligand for the Integrin α8β1 and Potential Roles for This Integrin–Ligand Interaction in Kidney Morphogenesis

Epithelio–mesenchymal interactions during kidney organogenesis are disrupted in integrin α8β1-deficient mice. However, the known ligands for integrin α8β1—fibronectin, vitronectin, and tenascin-C—are not appropriately localized to mediate all α8β1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for α8β1. We have coexpressed the extracellular domains of the mouse α8 and β1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the α8β1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with α8β1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In “far Western” blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to α8β1-AP. Cell adhesion assays using K562 cells expressing α8β1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin α8β1 may help regulate kidney development and other morphogenetic processes.     

Development and polymorphism of simple sequence repeat DNA markers for Kandelia candel (L.) Druce

Microsatellite markers of Kandelia candel were developed. Forty‐nine clones yielded strong positive signals among 331 clones hybridized to repetitive sequence probes. Primer pairs were designed for 19 of these positive clones according to their sequence data. Five of the primer pairs showed polymorphism for 16 individuals from Amami‐O‐Shima Island, Japan. Three to nine alleles per locus were detected, and the observed heterozygosities ranged from 0.250 to 0.938. Because our previous study reported very low level of genetic diversity for allozymes in the same Amami population, these microsatellite markers should be powerful tools for the analysis of genetic structure.     

Microbial production of cis,cis-muconic acid from benzoic acid

Summary The authors isolated numerous microorganisms with the capacity to assimilate large amounts of benzoate from many soil samples. Several of them were selected and subjected to mutation mainly by ultraviolet irradiation. One mutant lacking active muconate-lactonizing enzyme, the parent strain of which was identified as belonging to the genus Arthrobacter, was isolated and found to be capable of producing cis, cis-muconic acid with a quantitative yield of 44.1 g/l over 48 h in a 30 1 jar fermentor by successive feeding of small amounts of benzoate. This mutant, however, was more sensitive to high concentrations of the substrate than the parent strain. As few intermediates and isomers other than cis, cis-muconic acid were accumulated in the large fermentor, a large amount of pure cis, cis-muconic acid was easily obtained from the broth by salting out and recrystallization at a high recovery rate.     

Development of formulae for estimating amylose content and resistant starch content based on the pasting properties measured by RVA of Japonica polished rice and starch

We searched for the easy and simple method to measure the novel indicators which reflect not only AAC, but also (RS) based on pasting properties using RVA. Novel indexes such as SB/Con and Max/Fin (Maximum viscosity/Minimum viscosity) ratios had a very high correlation with proportion of intermediate and long chains of amylopectin; Fb₁₊₂₊₃ (DP ≧ 13). In Japonica polished rice, estimation formulae for AAC and RS content were developed using novel indexes based on pasting properties by RVA, and these equations showed determination coefficients of 0.89 and 0.80 for calibration and 0.71 and 0.75 for validation test. We developed the estimation formulae for AAC and RS content for Japonica starch samples. These equations showed determination coefficients of 0.86 and 1.00 for calibration and 0.76 and 0.83 for validation test, which showed that these equations can be applied to the unknown rice samples.