Transforming growth factor-beta1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-beta1 (TGF-beta1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-beta1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-beta1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-beta1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.     

Effects of static stress on the mechanical properties of cultured collagen fascicles from the rabbit patellar tendon

In-vitro tissue culture experiments were performed to study the effects of static stress on the mechanical properties of collagen fascicles obtained from the rabbit patellar tendon. After collagen fascicles having the diameter of approximately 300 microm were cultured for 1 and 2 wk under static stress between 0 and 3 MPa, their mechanical properties and crimp morphology were determined using a micro-tensile tester and a light microscope, respectively. The tensile strength and tangent modulus of the fascicles were significantly decreased by culture under no load compared to control fascicles. A statistically significant correlation, which was described by a quadratic curve, was observed between applied stress and tensile strength. The maximum tensile strength (16.7 MPa) was obtained at the applied stress of 1.2 MPa; the strength was within a range of control values. There was a similar correlation between applied stress and tangent modulus, and the modulus was maintained at control level under 1.3 MPa stress. The stress of 1.2 to 1.3 MPa is equivalent to approximately 50 percent of the peak stress developed in the intact rabbit patellar tendon by running. Strain at failure of cultured collagen fascicles was negatively correlated with applied stress, and that at 1.2 to 1.3 MPa stress was almost the same as the control value. Crimp morphology in the fascicles cultured under about 1.2 MPa stress was similar to that in control fascicles. These results indicate that cultured collagen fascicles change the mechanical properties and structure in response to static tensile stress. In addition, their mechanical properties and structure are maintained at control level if the static stress of 50 percent of in-vivo peak stress is applied.     

Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54

The RAD52 epistasis group genes are involved in homologous recombination, and they are conserved from yeast to humans. We have cloned a novel human gene, RAD54B, which is homologous to yeast and human RAD54. Human Rad54B (hRad54B) shares high homology with human Rad54 (hRad54) in the central region containing the helicase motifs characteristic of the SNF2/SWI2 family of proteins, but the N-terminal domain is less conserved. In yeast, another RAD54 homolog, TID1/RDH54, plays a role in recombination. Tid1/Rdh54 interacts with yeast Rad51 and a meiosis-specific Rad51 homolog, Dmc1. The N-terminal domain of hRad54B shares homology with that of Tid1/Rdh54, suggesting that Rad54B may be the human counterpart of Tid1/Rdh54. We purified the hRad54 and hRad54B proteins from baculovirus-infected insect cells and examined their biochemical properties. hRad54B, like hRad54, is a DNA-binding protein and hydrolyzes ATP in the presence of double-stranded DNA, though its rate of ATP hydrolysis is lower than that of hRad54. Human Rad51 interacts with hRad54 and enhances its ATPase activity. In contrast, neither human Rad51 nor Dmc1 directly interacts with hRad54B. Although hRad54B is the putative counterpart of Tid1/Rdh54, our findings suggest that hRad54B behaves differently from Tid1/Rdh54.     

Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation

Integrin-mediated adhesion induces the formation of focal adhesions that link the extracellular matrix and intracellular actin cytoskeletal networks. We previously showed that integrin-linked kinase (ILK), which can interact with beta1 and beta3 integrins, and its interacting protein, affixin, play an essential role in the initial assembly of focal adhesion structures and actin stress fibers. Although the relevant structures are also observed in integrin alphaIIbbeta3 in platelets, the precise underlying molecular mechanism remains unclarified. Here, we found that ILK stably forms a complex with ss-affixin in platelets. Thrombin stimulation induces their association with integrin beta3, which is followed by their incorporation into the Triton-insoluble membrane-cytoskeletal fraction. During the course of thrombin-induced platelet aggregation, ILK activity was enhanced within 90s to 2.1-fold of the basal level, independent of phosphatidylinositol 3-kinase. Taken together with the observation that the treatment with an anti-integrin beta3 antibody stimulates ILK activity without inducing platelet aggregation, these results suggest that the outside-in signaling induced by fibrinogen binding to integrin enhances ILK activity and results in the initial phase to reorganize the actin cytoskeleton.     

Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana

Leaf variegation has long been known as a recessive genetic trait in higher plants. Unlike albino mutants, leaf-variegated mutants are non-lethal and thus enable us to study a novel mechanism of plastid development and maintenance. Variegation results from a defect that makes chloroplast development unstable, since at least part of the tissues gives rise to normal chloroplasts. Despite the fact that leaf-variegated mutants have contributed to the findings of maternal inheritance or have been used as genetic markers, these mutations and the responsible loci have been poorly understood at the molecular level. A comprehensive study of the leaf-variegated mutants is possible in Arabidopsis, since such mutants have been known and the cloning can be at relative ease as a model plant. Here I summarize recent progress on characterization of the Arabidopsis leaf-variegated mutants. Detailed analysis of the responsible loci revealed that variegation is caused by a defect in various metabolic pathways related to organelle functions. Thus, studies on these genes provide us with novel redundant mechanisms by which heteroplasmic organelles such as plastids and mitochondria can survive from an environmental stress.     

Glycosidase-catalyzed deoxy oligosaccharide synthesis. Practical synthesis of monodeoxy analogs of ethyl beta-thioisomaltoside using Aspergillus niger alpha-glucosidase

Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl alpha-D-glucopyranoside (Glc alpha-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glc alpha-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl beta-D-thioglucopyranoside (Glc beta-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger alpha-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH3CN (1:1 v/v) at 37 degrees C. High activity of the enzyme was observed in the reaction between 2D-Glc alpha-O-pNP and Glc beta-S-Et to afford the monodeoxy analogs of ethyl beta-thiomaltoside and ethyl beta-thioisomaltoside that contain a 2-deoxy alpha-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-alpha-D-arabino-hexopyranosyl-(1,4)-beta-D-thioglucopyranoside and ethyl 2-deoxy-alpha-D-arabino-hexopyranosyl-(1,6)-beta-D-thioglucopyranoside, in 6.72% and 46.6% isolated yields (based on 2D-Glc alpha-O-pNP), respectively. Moreover, from 3D-Glc alpha-O-pNP and Glc beta-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl beta-thioisomaltoside that was modified at the glycon alpha-D-glucopyranose moiety, namely ethyl 3-deoxy-alpha-D-ribo-hexopyranosyl-(1,6)-beta-D-thioglucopyranoside, in 23.0% isolated yield (based on 3D-Glc alpha-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glc alpha-O-pNP and Glc beta-S-Et.     

Induced Response of Tomato Plants to Injury by Green and Red Strains of Tetranychus Urticae

We studied the induced response of tomato plants to the green strain and the red strain of the spider mite Tetranychus urticae. We focused on the olfactory response of the predatory mite Phytoseiulus persimilis to volatiles from T. urticae-infested tomato leaves in a Y-tube olfactometer. Tomato leaves attracted the predatory mites when slightly infested with the red strain, or moderately or heavily infested with the green strain. In contrast, neither leaves that were slightly infested with green-strain mites, nor leaves that were moderately or heavily infested with the red strain attracted the predators. We discuss the specific defensive responses of tomato plants to each of the two strains.     

Selection of Orthologous Genes for Construction of a Highly Resolved Phylogenetic Tree and Clarification of the Phylogeny of Trichosporonales Species

The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have clinical, agricultural and biotechnological value. Thus, understanding why and how evolutionary diversification occurred within this order is extremely important. This study clarified the phylogenetic relationships among Tricosporonales species. To select genes suitable for phylogenetic analysis, we determined the draft genomes of 17 Trichosporonales species and extracted 30 protein-coding DNA sequences (CDSs) from genomic data. The CDS regions of Trichosporon asahii and T. faecale were identified by referring to mRNA sequence data since the intron positions of the respective genes differed from those of Cryptococcus neoformans (outgroup) and are not conserved within this order. A multiple alignment of the respective gene was first constructed using the CDSs of T. asahii, T. faecale and C. neoformans, and those of other species were added and aligned based on codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment. Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both nucleotide (72,531) and amino acid (24,173) sequences were greater than in previous reports. In addition, we found that several genes, such as phosphatidylinositol 3-kinase TOR1 and glutamate synthase (NADH), had good resolution in this group (even when used alone). Our study proposes a set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary diversification in Trichosporonales. These can also be used for epidemiological and biogeographical studies, and may also serve as the basis for a comprehensive reclassification of pleomorphic fungi.