Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.     

Structural significance of the acyl group at the C-10 position and the A ring of the taxane core of paclitaxel for inducing nitric oxide and tumor necrosis factor production by murine macrophages

The antitumor agent, paclitaxel (Taxol), mimics the actions of lipopolysaccharide (LPS) on murine macrophages (Mphi). Various synthetic analogs of paclitaxel were examined for their potencies to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by murine peritoneal Mphi, and by human peripheral blood cells. The benzoyl group at C-2, the hydroxy group at C-7 and the acetyl group at C-10 were found to be critically important sites to activate murine Mphi. Nor-seco-taxoid analogs lacking the A ring of the taxane core of paclitaxel were inactive, but inhibit paclitaxel- or LPS-induced NO production. All the compounds tested did not induce TNF production by human blood cells.     

Synthesis of highly potent second-generation taxoids through effective kinetic resolution coupling of racemic beta-lactams with baccatins

A series of highly potent second-generation taxoids bearing a 2-methylprop-1-enyl or a 2-methylpropyl group at C-3' with modifications at the C-2, C-10, and C-14 positions was synthesized through the coupling of racemic cis-beta-lactams with properly protected/modified baccatin and 14-OH-baccatin. A high level of kinetic resolution was observed for all cases examined. The observed highly efficient enantiomer differentiation is ascribed to the markedly different chiral environment between the (+)- and (-)-beta-lactams in their approach to the chiral framework of the enantiopure lithium alkoxide of a baccatin in the ring-opening coupling process. It was also observed that substantially higher selectivity was achieved when 14-OH-baccatin-1,14-carbonate was used. Analysis of the transition state models revealed that the repulsive interactions between the 3-TIPS group of a (-)-beta-lactam with 1, 14-carbonate group of the baccatin substantially increases the asymmetric bias in the kinetic resolution process, favoring the reaction of a (+)-beta-lactam, which leads to the observed excellent selectivity.     

NO Gas Loss from Biologically Crusted Soils in Canyonlands National Park,Utah

In this study, we examined N gas loss as nitric oxide (NO) from N-fixing biologically crusted soils in Canyonlands National Park, Utah. We hypothesized that NO gas loss would increase with increasing N fixation potential of the biologically crusted soil. NO fluxes were measured from biologically crusted soils with three levels of N fixation potential (Scytonema-Nostoc-Collema spp. (dark)>Scytonema-Nostoc-Microcoleus spp. (medium)>Microcoleus spp. (light)) from soil cores and field chambers. In both cores and field chambers there was a significant effect of crust type on NO fluxes, but this was highly dependent on season. NO fluxes from field chambers increased with increasing N fixation potential of the biologically crusted soils (dark>medium>light) in the summer months, with no differences in the spring and autumn. Soil chlorophyllasis Type a content (an index of N fixation potential), percent N, and temperature explained 40% of the variability in NO fluxes from our field sites. Estimates of annual NO loss from dark and light crusts was 0.04-0.16 and 0.02-0.11-N/ha/year. Overall, NO gas loss accounts for approximately 3-7% of the N inputs via N fixation in dark and light biologically crusted soils. Land use practices have drastically altered biological soil crusts communities over the past century. Livestock grazing and intensive recreational use of public lands has resulted in a large scale conversion of dark cyanolichen crusts to light cyanobacterial crusts. As a result, changes in biologically crusted soils in arid and semi-arid regions of the western US may subsequently impact regional NO loss.     

Modeling soil CO<Subscript>2</Subscript> emissions from ecosystems

We present a new soil respiration model, describe a formal model testing procedure, and compare our model with five alternative models using an extensive data set of observed soil respiration. Gas flux data from rangeland soils that included a large number of measurements at low temperatures were used to model soil CO₂ emissions as a function of soil temperature and water content. Our arctangent temperature function predicts that Q₁₀ values vary inversely with temperature and that CO₂ fluxes are significant below 0 °C. Independent data representing a broad range of ecosystems and temperature values were used for model testing. The effects of plant phenology, differences in substrate availability among sites, and water limitation were accounted for so that the temperature equations could be fairly evaluated. Four of the six tested models did equally well at simulating the observed soil CO₂ respiration rates. However, the arctangent variable Q₁₀ model agreed closely with observed Q₁₀ values over a wide range of temperatures (r² = 0.94) and was superior to published variable Q₁₀ equations using the Akaike information criterion (AIC). The arctangent temperature equation explained 16–85% of the observed intra-site variability in CO₂ flux rates. Including a water stress factor yielded a stronger correlation than temperature alone only in the dryland soils. The observed change in Q₁₀ with increasing temperature was the same for data sets that included only heterotrophic respiration and data sets that included both heterotrophic and autotrophic respiration.     

The production of xylitol by enzymatic hydrolysis of agricultural wastes

Agricultural waste products, beech wood and walnut shells, were hydrolyzed at 40°C using mixed crude enzymes produced byPenicillium sp. AHT-1 andRhizomucor pusillus HHT-1.d-xylose, 4.1 g and 15.1 g was produced from the hydrolysis of 100 g of beech wood and walnut shells, respectively. For xylitol production,Candida tropicalis IFO0618 and the waste product hydrolyzed solutions were used. The effects on xylitol production, of adding glucose as a NADPH source,d-xylose and yeast extract, were examined. Finally, a 50% yield of xylitol was obtained by using the beech wood hydrolyzed solution with the addition of 1% yeast extract and 1% glucose at an initial concentration.     

Geometric effect of matrix upon cell differentiation: BMP-induced osteogenesis using a new bioglass with a feasible structure

A new biocompatible glass, which is composed of CaO, P2O5, SiO2, and Al2O3 (abbreviated CPSA) and is characterized by higher elasticity than previous bioglass products, was molded into fibers with a diameter of 9 microm. With CPSA fibers, two geometrically different structures, balls and bundles (each 20 mg in weight), were prepared, combined with 2.2 microg of rhBMP-2 (a gift from Yamanouchi Co., Japan) and implanted subcutaneously into rats. The histology showed remarkably higher bone formation in the ball-CPSA/BMP at 2 and 4 weeks than in the bundle-CPSA/BMP. The ball-CPSA/BMP showed 10 times higher alkaline phosphatase (ALP) activity at the second week and 5 times higher osteocalcin content at the fourth week than the bundle-CSPA/BMP. Vascular development in the implants was evaluated by mRNA expression of Flt-1 and KDR, two receptors for vascular endothelial growth factor (VEGF). Both receptors showed higher expression in the case of the ball, while they were not detected in the bundle. It is concluded that the BMP-induced bone formation depends highly upon the porous vasculature-inducing geometry of the matrix, which can be constructed with the new CPSA fibers.     

3′-Fluorine-Substitution in 3-Fluorocarbocyclic Oxetanocin A Augments the Drug's Inhibition of HHV-6B Propagation in Chronically Infected Cultures

An infection of TaY cells, which originated from an adult T-cell leukemia, with an HHV-6B OK isolate resulted in a chronically infected culture, termed TaY(OK). Cell cloning analysis revealed that the TaY(OK) culture consisted of a mixture of cells permissive and refractory to the infection, and that the permissive cells were continuously produced from the refractory cell population. Since the chronically infected culture has been maintained for over 2 years without the addition of uninfected TaY cells, we used it for an evaluation of the antiviral potency of nucleoside analogs, especially carbocyclic oxetanocins (COXTs). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays showed a lack of toxicity of ganciclovir (GCV), COXTs, and their derivatives, to TaY(OK) cells at 1 μm . Therefore we compared the antiviral potencies of these drugs at 1 μm by monitoring the viral loads produced during a 1-day period during the course of the drug treatment. Among the drugs tested, 3′-fluorocarbocyclic oxetanocin A (3′-C.OXT-A) was the most effective for inhibiting the virus production, and at concentrations ranging from 0.5 μm to 10 μm , the inhibition of the viral production was dose-dependent. A comparison of the chemical structures of the derivatives with that of C.OXT-A, which is the parental molecule, suggested that the 3′-fluorine-modification might account for the higher anti-HHV-6 activity and lower cytotoxicity.     

Enzymatic properties and primary structures of two α-amylase isozymes from the Pacific abalone Haliotis discus hannai

Two α-amylase (EC 3.2.1.1) isozymes, HdAmy58 and HdAmy82, with approximate molecular masses of 58 kDa and 82 kDa, respectively, were isolated from the digestive fluid of the Pacific abalone Haliotis discus hannai. Optimal temperatures and pHs for HdAmy58 and HdAmy82 were at 30 °C and 6.7, and 30 °C and 6.1, respectively. Both enzymes similarly degraded starch, glycogen, and maltooligosaccharides larger than maltotriose producing maltose and maltotriose as the major degradation products. However, the activity toward maltotetraose was appreciably higher in HdAmy82 than HdAmy58. cDNAs encoding HdAmy58 and HdAmy82 were cloned and the amino-acid sequences of 511 and 694 residues for HdAmy58 and HdAmy82, respectively, were deduced. The putative catalytic domains of HdAmy58 and HdAmy82 were located in the 17–511th and 19–500th amino-acid regions, respectively, and they showed approximately 50% amino-acid identity to each other. These sequences also showed 62–99% amino-acid identity to the catalytic domains of known α-amylases that belong to glycoside-hydrolase-family 13. The difference in the molecular masses between HdAmy58 and HdAmy82 was ascribed to the extension of approximately 190 residues in the C-terminus of HdAmy82. This extended region showed 41–63% amino-acid identity with the ancillary domains of several α-amylases previously reported.     

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was ~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.