Purification and characterization of a cold-adapted isocitrate lyase and expression analysis of the cold-inducible isocitrate lyase gene from the psychrophilic bacterium<Emphasis Type="Italic"> Colwellia psychrerythraea</Emphasis>

Isocitrate lyase (ICL) from Colwellia psychrerythraea, a psychrophilic bacterium, was purified and characterized. The subunit molecular mass was 64 kDa, which is larger than that of other bacterial ICLs. The optimal temperature for its activity was 25 degrees C, the value of K(m) for the substrate ( DL-isocitrate) was minimum at 15 degrees C, and the catalytic efficiency ( k(cat)/ K(m)) value was maximum at 20 degrees C. Furthermore, the enzyme was remarkably thermolabile and completely inactivated by incubation for 2 min at 30 degrees C. These features indicate that ICL from this bacterium is a typical cold-adapted enzyme. A partial amino acid sequence of the C. psychrerythraea ICL was very similar to that of the closely related psychrophile Colwellia maris. Expression of the gene encoding the C. psychrerythraea ICL was found to be induced by low temperatures and by acetate in the medium. The cold adaptation of the catalytic properties of ICL and the stimulated expression of its gene at low temperatures strongly suggest that this enzyme is important for the growth of this bacterium in a cold environment.     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

Endonucleases that may recognize the ssDNA-backbone structure: purification from Sf9 cells and characterization

Endonucleases that cleave ssDNA under conditions that minimize the formation of secondary structures were detected in Spodoptera frugiperda Sf9 cells. One endonuclease was purified from Sf9 cells and another from Sf9 cells infected by baculoviruses. Polynucleotides containing an A- or T-tract, or a CAP binding region were digested in the presence of ATP at low Mg ions with these two nucleases. ATP could be replaced by citrate but not by EDTA. The cleavage patterns were different from those obtained with endonuclease I and exonuclease VII of Escherichia coli. The cleavages were dependent on the sequence of the polynucleotides but not associated with specific bases. EndoSfV cleaved mainly AT-rich regions.     

Regulation of proteinase-activated receptor 1 by inflammatory mediators in human vascular endothelial cells

Thrombin plays a critical role in haemostasis, inflammation, and cell proliferation, mediated by proteinase-activated receptor 1 (PAR-1; thrombin receptor). The physiological and pathological regulation of PAR-1 by inflammatory mediators has not yet been fully elucidated. The aim of this study is to investigate the effects of inflammatory mediators on mRNA and protein expression of PAR-1 in early passage human vascular endothelial cells. Endothelial cells were activated by inflammatory mediators, such as tumour necrosis factor alpha (TNFalpha), interferon gamma (IFN gamma), and bacterial substance lipopolysaccharide (LPS), and the PAR-1 expression was verified by flow cytometry or RT-PCR. By stimulating endothelial cells with TNFalpha, IFN gamma, and LPS, the PAR-1 expression on the cell surface remained almost unchanged for 48 h. After stimulation with 20-300 U/ml TNFalpha, the total cellular PAR-1 expression (both on cell surface and in the cytoplasm) significantly decreased at 24h and thereafter recovered to the basal level at 48 h. The stimulation with 100 U/ml TNFalpha transiently down-regulated the PAR-1 mRNA expression to approximately 0.3-fold of the basal level at 30 min, but it rebounded 3-fold above the basal level at 6h, and again decreased to 0.5-fold of the basal level at 12h, and finally returned to the basal level at 24h. In contrast, IFN gamma or LPS did not affect the PAR-1 mRNA expression.     

A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry

Recently, cDNAs encoding novel RFamide-related peptides (RFRPs) have been reported in the mammalian brains by a gene database search and the deduced RFRPs have been suggested to participate in neuroendocrine and pain mechanisms in the rat. Two peptides have been predicted to be encoded in the cDNA of rodent RFRPs. To assess precise functions of rodent RFRPs in the brain, in the present study we identified a naturally occurring RFRP in the rat hypothalamus by immunoaffinity purification combined with mass spectrometry (MS). The affinity chromatography showed that the rat hypothalamus contained RFRP-like immunoreactivity. The immunoreactive material was analyzed by a nanoflow electrospray ionization time-of-flight MS followed by tandem MS analysis. The mass peak corresponding to octadecapeptide was detected at 1010.54 m/z ([M+2H](2+)) and its sequence, ANMEAGTMSHFPSLPQRF-NH(2), was revealed by the fragmentation, showing a mature form encoded in the cDNA sequence of RFRPs. The identified endogenous RFRP will aid not only in defining its physiological roles but also facilitate the development of its agonists and antagonists in the rodent brain.     

Characterization of FAM10A4, a member of the ST13 tumor suppressor gene family that maps to the 13q14.3 region associated with B-Cell leukemia,multiple myeloma,and prostate cancer

Using the 650-kb DNA sequence from the minimally deleted region in B-cell chronic lymphocytic leukemia (BCLL), we have identified a new gene, FAM10A4, that maps to the proximal end of the region. This gene has been shown to be part of a now six-member family of genes with high homology to the ST13 tumor suppressor gene. We have established conditions to specifically undertake mutation studies of the chromosome 13 member of this family and have identified a Ser71Leu change in BCLL samples, which is apparently a polymorphism. The characterization of this gene will permit mutation studies in other tumor cell types such as multiple myeloma and prostate cancer, which also show genetic loss in the 13q14 region.     

Cloning,sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen-fixing bacterium,Azotobacter vinelandii

Isocitrate dehydrogenase (IDH: EC 1.1.1.42) of Azotobacter vinelandii was purified to an electrophoretically homogeneous state, and a gene (icd) encoding this enzyme was cloned and sequenced. The N-terminal amino acid sequence of the purified enzyme was consistent with that deduced from the nucleotide sequence of the icd gene. The deduced amino acid sequence of this gene showed high identity (62-66%) to those of the other bacterial monomeric IDHs. Expression of the icd gene in Escherichia coli was examined by measuring the enzyme activity and mRNA level. Primer extension analyses revealed that two species of mRNAs with different lengths of 5'-untranslated regions (TS-1 and TS-2) were present, of which the 5'-terminals (TS-1 and TS-2 sites) were cytosines located at 244 bp and 101 bp upstream of translational initiation codon, respectively. Conserved promoter elements were present at -35 and -10 regions from the TS-1 site, whereas no such a common motif was found in the upstream region of the TS-2 site. Deletion of the promoter elements upstream of the TS-1 site resulted in complete loss of IDH activity in the E. coli transformant. When the promoter elements upstream of the TS-1 site were intact, the levels of TS-1 and TS-2 were varied greatly by altering exogenous nutrients for growth. The cells grown in a nutrient-rich medium produced large amounts of TS-1 and had a low level of IDH activity. In a nutrient-poor medium, the cells contained large amounts of TS-2 and high levels of IDH activity.     

Milk basic protein (MBP) increases radial bone mineral density in healthy adult women

We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.     

Embryonic shoot apical meristem formation in higher plants

The shoot apical meristem (SAM) is essential for organ formation in higher plants. How the SAM is formed during plant development is poorly understood, however. In this review, we focus on several recent studies that provide new insights into the mechanism of SAM formation during embryogenesis. Recently, positive and negative regulators of the class I KNOX genes, which are thought to be necessary for SAM formation, have been identified; the Arabidopsis CUP-SHAPED COTYLEDON (CUC) genes are required for the expression of a class I KNOX gene, SHOOT MERISSTEMLES (STM) during embryogenesis, and the Arabidopsis ASYMMETRIC LEAVES1 (AS1), AS2, and several other genes negatively regulate KNOX gene expression in cotyledon primordia. Also, several genes that are involved in the formation of the adaxial–abaxial axis of cotyledons seem to regulate embryonic SAM formation. Electronic Publication