The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

Ion-pair reversed-phase high-performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion

An isocratic HPLC method for the simple and selective determination of adenine nucleoside and nucleotides has been developed. The separation is achieved at room temperature by reversed-phase chromatography (Shiseido, Capcell Pak C18). A mixture of 0.1 M triethylamine (TEA) phosphate buffer and methanol (95:5, v/v) is used as a standard eluent. Influence of pH and concentrations of organic modifiers and TEA ion on capacity factors of adenine compounds has been investigated. It has been also found that the TEA ion in the eluent is adsorbed onto the reversed-phase surface. The results clearly demonstrate that ion-pair formation with TEA ion occurs probably both in the mobile phase and on the stationary phase and governs the retention of adenine and nucleotides in the present system. The HPLC system is applied to the analysis of adenine nucleotides formed as intermediates in the synthesis of 3′-phosphoadenosine 5′-phosphosulphate (PAPS) and to the assays of ATPases and 5′-nucleotidase activities in rat liver plasma membrane. This method is a new type of ion-pair reversed-phase HPLC system and is suitable for the separation of highly polar organic anions, especially for adenine nucleotides.     

Causal relationship between the loss of RUNX3 expression and gastric cancer

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.     

Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.     

Construction of a Pseudozyma antarctica strain without foreign DNA sequences (self-cloning strain) for high yield production of a biodegradable plastic-degrading enzyme

ABSTRACT

The basidiomycetous yeast Pseudozyma antarctica GB-4(0) esterase (PaE) is a promising candidate for accelerating degradation of used biodegradable plastics (BPs). To increase safety and reduce costs associated with the use of PaE, we constructed a self-cloning strain with high-PaE productivity. A Lys12 gene (PaLYS12)-deleted lysine auxotroph strain GB4-(0)-L1 was obtained from GB-4(0) by ultraviolet mutagenesis and nystatin enrichment. Subsequently, the PaE gene (PaCLE1) expression cassette consisting of GB-4(0)-derived PaCLE1, under the control of a xylose-inducible xylanase promoter with PaLYS12, was randomly introduced into the GB4-(0)-L1 genome. A PaE high-producing strain, PGB474, was selected from among the transformants by high throughput double-screening based on its ability to degrade emulsified polybutylene succinate-co-adipate. Quantitative PCR revealed that four copies of the PaE gene expression cassette were introduced into the PGB474 genome. PGB474 produced 2.0 g/L of PaE by xylose-fed-batch cultivation using a 3-L jar fermentor for 72 h.     

Disruption of adiponectin causes insulin resistance and neointimal formation

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.     

Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses

Immunoglobulin (Ig)-free light chains IgLC are present in serum and their production is augmented under pathological conditions such as multiple sclerosis, rheumatoid arthritis and neurological disorders. Until now, no (patho)physiological function has been ascribed to circulating Ig light chains. Here we show that IgLCs can confer mast cell dependent hypersensitivity in mice. Antigenic stimulation results in plasma extravasation, cutaneous swelling and mast-cell degranulation. We show that IgLCs have a crucial role in development of contact sensitivity, which could be completely prevented by a novel IgLC antagonist. Although IgE and IgG(1) are central to the induction of immediate hypersensitivity reactions, our results show that IgLCs have similar activity. IgLCs may therefore be a novel factor in the humoral immune response to antigen exposure. Our findings open new avenues in investigating the pathogenesis of autoimmune diseases and their treatments.     

Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity

Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity.