Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha

The protocadherin-alpha (CNR/Pcdhalpha) and protocadherin-gamma (Pcdhgamma) proteins, members of the cadherin superfamily, are putative cell recognition/adhesion molecules in the brain. Overexpressed cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines, although hardly any overexpressed CNR/Pcdhalpha proteins are expressed on the cell surface, except on HEK293T cells, which show low expression. We analyzed the expression of CNR/Pcdhalpha and Pcdhgamma in HEK293T cells and found that they formed a protein complex and that Pcdhgamma enhanced the surface expression of CNR/Pcdhalpha. This enhanced surface expression was confirmed by flow cytometry analysis and by marking cell surface proteins with biotin. The enhancement was observed using different combinations of CNR/Pcdhalpha and Pcdhgamma proteins. The surface expression activity was enhanced by the extracellular domains of the proteins, which could bind each other. Their cytoplasmic domains also had binding activity and influenced their localization. Their protein-protein interaction was also detected in extracts of mouse brain and two neuroblastoma cell lines. Thus, interactions between CNR/Pcdhalpha and Pcdhgamma regulate their surface expression and contribute to the combinatorial diversity of cell recognition proteins in the brain.     

Development of image processing program for yeast cell morphology

Every living organism has its own species-specific morphology. Despite the relatively simple ellipsoidal shape of budding yeast cells, the global regulation of yeast morphology remains unclear. In the past, each mutated gene from many mutants with abnormal morphology had to be classified manually. To investigate the morphological characteristics of yeast in detail, we developed a novel image-processing program that extracts quantitative data from microscope images automatically. This program extracts data on cells that are often used by yeast morphology researchers, such as cell size, roundness, bud neck position angle, and bud growth direction, and fits an ellipse to the cell outline. We evaluated the ability of the program to extract quantitative parameters. The results suggest that our image-processing program can play a central objective role in yeast morphology studies.     

Comprehensive metabolite profiling of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> using gas chromatography-mass spectrometry

A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula. A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition. S. meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step. Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS. The different compounds were identified by comparison with the NIST 98 database and available standards. From about 200 peaks in each chromatogram 65 compounds have been identified so far. A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools. A principal component analysis (PCA) was able to distinguish S. meliloti cells grown on different carbon sources based on their metabolite profile. A comparison of the metabolite composition of a S. meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant. Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine. This finding further emphasises the importance of integrating metabolic data into post-genomic research.     

Regulation of gene expression of epithelial calcium channels in intestine and kidney of mice by 1alpha,25-dihydroxyvitamin D3

In wild-type (VDR(+/+)) mice, ECaC2 expression was confirmed in the intestine and kidney, while ECaC1 expression was exclusively confined to the kidney. Both mRNAs expression of ECaC1 and ECaC2 in the kidney and ECaC2 mRNA expression in the intestine increased time- and dose-dependently in response to 1alpha,25(OH)(2)D(3) injection in VDR(+/+) mice, but not in VDR(-/-) mice. The mRNA levels of ECaC2 in the intestine of VDR(-/-) mice were remarkably reduced when compared to VDR(+/+) mice, while no significant differences were observed in both mRNA levels of ECaC1 and ECaC2 in the kidney between VDR(+/+) mice and VDR(-/-) mice. In the primary renal tubular cells (PRTC) isolated from VDR(+/+) mice, both ECaCs mRNA expression increased in response to 1alpha,25(OH)(2)D(3) treatment, but not in the PRTC of VDR(-/-) mice. PTH increased both ECaCs mRNA expression in the PRTC of VDR(+/+) mice. These results suggest that 1alpha,25(OH)(2)D(3) directly modulates the gene expression of ECaC1 and ECaC2 together with PTH in the kidney of mice. 1alpha,25(OH)(2)D(3) also modulates the gene expression of ECaC2 in the intestine of mice, however, further studies are needed to elucidate the direct action of 1alpha,25(OH)(2)D(3) on the expression of ECaC2 in the intestine.     

Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression

3-Methylcrotonyl-CoA carboxylase (MCCase; EC 6.4.1.4) is a mitochondrial biotin enzyme and plays an essential role in the catabolism of leucine and isovalerate in animals, bacterial species, and plants. MCCase consists of two subunits, those that are biotin-containing and non-biotin-containing. The genes responsible for these subunits have been isolated in soybean, Arabidopsis thaliana, and tomatoes, but not in mammals. In humans, MCCase deficiency has been thought to be a rare metabolic disease, but the number of patients with MCCase deficiency appears to be increasing with a wide range of clinical presentations, some that result in a lethal condition and others that are asymptomatic. In this report, we have isolated and carried out chromosomal mapping of the gene for the biotin-containing subunit (A subunit) of the human MCCase gene, MCCA. The cDNA predicts an open reading frame coding for a 725-amino-acid protein with mitochondrial signal peptide, biotin carboxylase, and biotin-carrier domains. The gene is composed of at least 19 exons and covers more than 70 kb of sequence on band q27 of chromosome 3. MCCA was abundantly expressed in mitochondria-rich organs, such as the heart, skeletal muscles, kidney, and liver. In exon 13, we observed a His/Pro polymorphism at codon 464 (an A to C transition at nucleotide position 1391 in the cDNA sequence). Then, we determined the DNA sequences of the 5' untranslated region and entire coding regions in two patients with MCCase deficiency, but no sequence substitution was detected, suggesting that the gene mutations might be in the non-biotin-containing subunit (B subunit) gene, MCCB, in these patients.     

Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica

A brackish-water mollusc, Corbicula japonica, uses large quantities of D- and L-alanine as intracellular osmotically active solutes, osmolytes, for regulation of intracellular osmolarity. We purified alanine racemase from the mantle of C. japonica to characterize its enzymological properties. The molecular masses of the enzyme were estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 140 kDa by gel filtration on high-performance liquid chromatography, suggesting the trimeric or tetrameric nature of the enzyme. Neither dialysis nor chromatographic procedures in the absence of pyridoxal 5'-phosphate led to loss of enzyme activity, although carbonyl reagents, hydroxylamine and phenylhydrazine, inhibited the activity. These results suggest that alanine racemase of the animal may bind pyridoxal 5'-phosphate tightly as a cofactor. Kinetic experiments using the partially purified enzyme revealed that alanine was the sole substrate among 17 kinds of L-amino acids tested. The Lineweaver-Burk plot for L-alanine as substrate resulted in Km value of 22.6 mM, and the value for D-alanine was 9.2 mM. Together with the previous evidence that D- and L-alanine levels of this animal change with the external salinity maintaining the D-/L-alanine ratio at unity, the present results seem to indicate that the physiological role of alanine racemase in this animal is to supply D-alanine as a main intracellular osmolyte. J. Exp. Zool. 289:1-9, 2001.     

Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range

The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24+/-5% in slugs of 10(3) cells to 10+/-3% when 10(5) cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28+/-20%, well above the initial value of 10+/-3%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a approximately 10% lower threshold and a approximately 30% upper threshold. To explain this, we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant.     

G protein betagamma subunits induce stress fiber formation and focal adhesion assembly in a Rho-dependent manner in HeLa cells

In fibroblasts, the G protein alpha subunits Galpha(12) and Galpha(13) stimulate Rho-dependent stress fiber formation and focal adhesion assembly, whereas G protein betagamma subunits instead exert a disruptive influence. We show here that the latter can, however, stimulate the formation of stress fibers and focal adhesions in epithelial-like HeLa cells. Transient expression of beta(1) with gamma(2), gamma(5), gamma(7), and gamma(12) in quiescent HeLa cells induced stress fiber formation and focal adhesion assembly as did expression of the constitutively active Galpha(12). Co-expression of betagamma with Galpha(i2) and the C-terminal fragment of the beta-adrenergic receptor kinase, both of which are known to bind and sequester free betagamma, blocked betagamma-induced stress fiber and focal adhesion formation. Inhibition was also noted with co-expression of a dominant negative mutant of Rho. Botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, and a Rho-associated protein kinase inhibitor, Y-27632, similarly inhibited betagamma-induced stress fiber and focal adhesion assembly. These results indicate that G protein betagamma subunits regulate Rho-dependent actin polymerization in HeLa cells.     

Systemic administration of HVJ viral coat-liposome complex containing human insulin vector decreases glucose level in diabetic mouse: A model of gene therapy

In this study, we examined the feasibility of a systemic administration of HVJ-liposome complex containing human insulin construct into the blood in mice via the tail vein. Transfection of human insulin vector resulted in a transient decrease in serum glucose in streptozotocin (SZT)-induced diabetic mice, accompanied by the detection of human insulin in the liver and spleen. In accordance with the decreased glucose, plasma immunoreactive insulin could be detected up to 14 days after a single transfection in mice transfected with insulin vector. Repeated intravenous injection of human insulin vector every week resulted in a sustained decrease in serum glucose over a 4-week period, accompanied by the detection of C-peptide fragments and a significant decrease in BUN and creatinine. Here, we demonstrated the feasibility of intravenous systemic administration of an insulin vector that results in a sustained improvement of diabetic glucose metabolism.