Studies on Mold Dextranases

Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28°C for 5 to 6 days in medium containing 1% dextran, 1% NaNO₃ or polypeptone, 0.2% yeast extracts, 0.4% K₂HPO₄ and small amounts of inorganic salts, pH 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus.     

Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degrees C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti--RP S19 rabbit antibodies and an anti--isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.     

Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.     

DNA sequence comparison considering both amino acid and nucleotide insertions/deletions because of evolution and experimental error

Amino acid similarity often needs to be considered in DNA sequence comparison to elucidate gene functions. We propose a Smith-Waterman-like algorithm which considers amino acid similarity and insertions/deletions in sequences at the DNA level and at the protein level in a hybrid manner. The algorithm is applied to cDNA sequences of Oryza sativa and those of Arabidopsis thaliana. The results are compared with the results of application of NCBI's tblastx program (which compares the sequences in the BLAST manner after translation). It is shown that the present algorithm is very helpful in discovering nucleotide insertions/deletions originating from experimental errors as well as amino acid insertions/deletions due to evolutionary reasons.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

Measurement of fasting serum apoB-48 levels in normolipidemic and hyperlipidemic subjects by ELISA

The present study was designed to evaluate the metabolism of chylomicron and chylomicron remnants by measuring serum apolipoprotein B-48 (apoB-48) levels in 335 normolipidemic and 253 hyperlipidemic subjects using a novel ELISA system. The distribution of fasting serum apoB-48 levels in normolipidemic subjects varied widely, ranging from <1 to >24 microgram/ml (mean, 5.2 +/- 3.8 microgram/ml; median, 3.9 microgram/ml). Serum apoB-48 levels correlated with serum triglyceride (TG) concentrations (r = 0.45, P < 0.001), but not with total cholesterol levels. Serum apoB-48 levels were 7 to 18 times higher in patients with Type I, Type V, and Type III hyperlipidemia, and only slightly higher in patients with Type IIa, Type IIb, and Type IV hyperlipidemia, compared with normolipidemic subjects. The calculated apoB-48/TG ratio was elevated only in patients with dysbetalipoproteinemia (apoE2/2 phenotype). In normolipidemic subjects, oral fat loading resulted in about 2-fold increase in serum apoB-48 levels, with a peak level recorded at 3-4 h postloading, and then returned to the baseline level within 6 h. On the other hand, in patients with dysbetalipoproteinemia, serum apoB-48 levels did not change considerably. Our results indicate that serum apoB-48 is a very useful parameter for evaluating lipoprotein metabolism in exogenous pathways.     

Ca2+ entry-dependent inactivation of L-type Ca current: a novel formulation for cardiac action potential models

Cardiac L-type Ca current (I(Ca,L)) is controlled not only by voltage but also by Ca(2+)-dependent mechanisms. Precise implementation of I(Ca,L) in cardiac action potential models therefore requires thorough understanding of intracellular Ca(2+) dynamics, which is not yet available. Here, we present a novel formulation of I(Ca,L) for action potential models that does not explicitly require the knowledge of local intracellular Ca(2+) concentration ([Ca(2+)](i)). In this model, whereas I(Ca,L) is obtained as the product of voltage-dependent gating parameters (d and f), Ca(2+)-dependent inactivation parameters (f(Ca): f(Ca-entry) and f(Ca-SR)), and Goldman-Hodgkin-Katz current equation as in previous studies, f(Ca) is not a instantaneous function of [Ca(2+)](i) but is determined by two terms: onset of inactivation proportional to the influx of Ca(2+) and time-dependent recovery (dissociation). We evaluated the new I(Ca,L) subsystem in the framework of the standard cardiac action potential model. The new formulation produced a similar temporal profile of I(Ca,L) as the standard, but with different gating mechanisms. Ca(2+)-dependent inactivation gradually proceeded throughout the plateau phase, replacing the voltage-dependent inactivation parameter in the LRd model. In typical computations, f declined to approximately 0.7 and f(Ca-entry) to approximately 0.1, whereas deactivation caused fading of I(Ca,L) during final repolarization. These results support experimental findings that Ca(2+) entering through I(Ca,L) is essential for inactivation. After responses to standard voltage-clamp protocols were examined, the new model was applied to analyze the behavior of I(Ca,L) when action potential was prolonged by several maneuvers. Our study provides a basis for theoretical analysis of I(Ca,L) during action potentials, including the cases encountered in long QT syndromes.     

Pioglitazone reduces monocyte adhesion to vascular endothelium under flow by modulating RhoA GTPase and focal adhesion kinase

Thiazolidinediones (TZDs), potent peroxisome proliferator-activated receptor gamma ligands, have been shown to improve endothelial function in vascular diseases. We investigated the effects of pioglitazone, a TZD, on monocyte-endothelial interaction under flow and found that pretreatment (20 mumol/l, 48 h) significantly reduced U937 adhesion to human umbilical vein endothelial cells. Integrin expression was not altered, however, the activation of RhoA GTPase was significantly reduced after treatment. Further, pioglitazone treatment significantly reduced phosphorylation of focal adhesion kinase (FAK) at 925Y, but not at 397Y, suggesting a specific role in FAK-dependent signaling. These results indicate a novel anti-inflammatory role for this compound.