Development of cell-expressed and virion-incorporated CCR5-targeted vaccine

Our previous study demonstrated that the immunization with a cycloimmunogen derived from extracellular loop-2 (ECL-2) of CCR5 (cDDR5) attenuated acute phase of CCR5-tropic simian-human immunodeficiency virus (SHIV)_SF162P3 replication in vivo. Although the study showed that the antisera raised against cDDR5 reacted with cell-expressed CCR5, we have not yet demonstrated whether the antisera can react with virion-incorporated CCR5. Here, we show that rhesus cDDR5 (rcDDR5)-specific antibodies react with not only cell-expressed but also virion-incorporated simian CCR5s (siCCR5s), but may predominantly exert their inhibitory effects on simian immunodeficiency virus (SIV) infection by the binding of cell-expressed rather than virion-incorporated CCR5s. These results suggest that the virion-incorporated CCR5 may contribute to the reactivation of the anti-rcDDR5 antibody-producing B-cells by SIV particles after rcDDR5 immunization, although the binding of anti-rcDDR5 antibody to virion-incorporated CCR5 results in a partial inhibitory effect on SIV infection.     

The 4′-hydroxy group is responsible for the in vitro cytogenetic activity of resveratrol

We previously reported that 3,5,4′-trihydroxy-trans-stilbene (resveratrol), a polyphenolic phytoalexin found in grapes, induces a high frequency of sister chromatid exchanges (SCEs) in vitro. In this study, to investigate structure activity relationships, we synthesized six analogues of resveratrol differing in number and position of hydroxy groups, and we investigated their activity in chromosomal aberration (CA), micronucleus (MN) and sister chromatid exchange (SCE) tests in a Chinese hamster cell line (CHL). Two of the six analogues (3,4′-dihydroxy-trans-stilbene and 4-hydroxy-trans-stilbene) showed clear positive responses in a concentration-dependent manner in all three tests. Both were equal to or stronger than resveratrol in genotoxicity. The 4′-hydroxy (OH) analogue had the simplest chemical structure and was the most genotoxic. The other analogues did not have a 4′-hydroxy group. These results suggested that a 4′-hydroxy group is essential to the genotoxicity of stilbenes.     

Bioconversion of dethiobiotin to biotin by a cell-free system of a bioYB transformant of Bacillus sphaericus

Abstract Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Effect of biodegradable chelating ligands on Fe uptake in and growth of marine microalgae

Iron (Fe) is an important nutrient for phytoplankton. The low solubility of Fe in oxic waters can be a growth-limiting factor for phytoplankton. Synthetic aminopolycarboxylates (APCs) such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are widely used as Fe complexing agents for microalgae culture. However, the presence of these non-ready biodegradable APC_S in aquatic systems may have serious environmental consequences. In the present study, the effects of biodegradable chelating ligands (hydroxyiminodisuccinic acid (HIDS), methylglycinediacetic acid (MGDA), and iminodisuccinate (IDS)) on Fe uptake in and growth of three coastal microalgae (Heterosigma akashiwo, Prymnesium parvum, and Skeletonema marinoi-dohrnii complex) were investigated, and the results were compared with those of non-ready biodegradable APCs (EDTA, ethylenediamine tetra-methylene phosphonic acid (EDTMP), and DTPA). The biodegradable chelating ligands did not have significant growth inhibition effect on the phytoplankton. Although the growth of the algae (except S. marinoi-dohrnii complex) was not affected substantially by 1.5 and 7.5 μM of DTPA, growth inhibition occurred by 7.5 μM of EDTMP and 150 μM of EDTA, DTPA, and EDTMP. The effect of chelating ligands on microalgal growth was likely to be associated with the intracellular Fe uptake influenced by the chelating ligands. On average, intracellular Fe concentrations for biodegradable chelating ligands were substantially higher than those for non-ready biodegradable APCs. Except H. akashiwo, the ratio of intra/extracellular Fe concentrations was highest for MGDA followed by IDS and HIDS. The results indicate that biodegradable chelating ligands are more efficient than non-ready biodegradable APCs in intracellular Fe uptake and algal growth.     

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase.

Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, ch1-1, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels.     

Drebrin attenuates the interaction between actin and myosin-V

Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.     

Astrocytes with previous chronic exposure to amyloid β‐peptide fragment 1–40 suppress excitatory synaptic transmission

Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long‐term exposure of amyloid β‐peptide (Aβ) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long‐term Aβ exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal‐naïve neurons co‐cultured with astrocytes that have previously experienced chronic Aβ_1‐40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post‐synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aβ1–40 is attributable to a small number of synapses with higher release probability.