POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells

The genomes of eukaryotic cells predict the existence of multiple DNA polymerases, which are proposed to serve specialized roles in DNA replication and repair. We report here the isolation of the full-length human DNA POLQ gene, and an initial characterization of its gene product, DNA polymerase θ. POLQ is of particular interest as it is orthologous to Drosophila Mus308, a gene implicated in cellular resistance to interstrand DNA cross-linking agents. The POLQ cDNA encodes a polypeptide of 2592 amino acids with an ATPase-helicase domain in the N-terminal part of the protein, a central spacer domain, and a DNA polymerase domain in the C-terminal portion. This arrangement is conserved with Mus308. Expression of an mRNA of ~8.5 kb was detected in human cell lines. In a survey of human and mouse tissues, expression was highest in testis. Immunoblotting with POLQ antibodies detected a protein of >250 kDa in extracts from HeLa cells. Prominent fragments of ~100 kDa suggest that POLQ is readily proteolyzed. Full-length human POLQ was expressed from a baculovirus system. Purified POLQ showed DNA polymerase activity on nicked double-stranded DNA and on a singly primed DNA template. The enzyme activity was resistant to aphidicolin, consistent with its membership of the A family of DNA polymerases, and inhibited by dideoxynucleotides. POLQ further exhibited a single-stranded DNA-dependent ATPase activity.     

Localization of Na+-HCO-3 cotransporter (NBC-1) variants in rat and human pancreas

Mutations inNa⁺-HCO cotransporter (NBC-1) causeproximal renal tubular acidosis (pRTA) associated with ocularabnormalities. One pRTA patient had increased serum amylase, suggestingpossible evidence of pancreatitis. To further delineate a link betweenNBC-1 inactivation and pancreatic dysfunction, immunohistochemicalanalysis was performed on rat and human pancreas using antibodiesagainst kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters.In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and bothapical and basolateral membranes of medium and large duct cells. Inhuman pancreas, on the other hand, the anti-pNBC-1 antibody did notlabel acinar cells, although it did label the basolateral membranes ofthe entire duct system. The labeling by anti-kNBC-1 antibody wasdetected in only a limited number of rat pancreatic duct cells. Toexamine the effects of pRTA-related mutations, R342S and R554H, onpNBC-1 function, we performed functional analysis and found that bothmutants had reduced transport activities compared with the wild-typepNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO uptake into duct cellsin both rat and human pancreas. The loss of pNBC-1 function ispredicted to have significant impact on overall ductal HCO secretion, which could potentially lead topancreatic dysfunction.

     

Guaiacylglycerol-7'-O-methyl 8'-vanillic acid ether and related compounds from Boreava orientalis

The threo and erythro forms of guaiacylglycerol-7'-O-methyl 8'-vanillic acid ethers, threo and erythro guaiacylglycerol 8'-vanillin ethers, and threo guaiacylglycerol 8'-(4-hydroxymethyl-2-methoxyphenyl) ether have been isolated from fruits of Boreava orientalis. Structural determinations were made on the basis of UV, MS, 1H- and 13C-NMR spectral data, including two-dimensional shift correlation. The relative configurations were assigned on the basis of 1H-NMR chemical shifts.     

Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes

Adipose tissue expresses a variety of genes including tumor necrosis factor alpha and type-1 plasminogen activator inhibitor (PAI-1); and these factors, produced by adipocytes, may be associated with the risk of coronary events in obesity. In this study, we characterized the production of fibrinolytic factors including tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PAI-1 in the differentiation of preadipocytes, and examined the hormonal regulation of these fibrinolytic factors in mature adipocytes. Mouse 3T3-L1 preadipocytes were employed as a model of adipocytes. Adipocyte differentiation was induced by insulin, dexamethasone, and 3-isobutyl-1-methyl xanthine (IBMX). alpha-Glycerophosphate dehydrogenase (GPDH) activity and glucose transporter 4 (GLUT4) mRNA, indices for adipocyte maturation, were induced on Day 4, and gradually increased. GPDH activity reached its maximum level on Day 14. The level of tPA, a major PA in preadipocytes, dramatically decreased with differentiation. On the other hand, that of uPA reciprocally increased. PAI-1 production was also dramatically induced concomitant with differentiation. In mature adipocytes, uPA production was dominant (25 microg/ml/24 h vs. 0.8 microg/ml/24 h for tPA). Total PA activity in the mature adipocytes was reduced by insulin or dexamethasone, but not by glucagon. Insulin, IBMX, and dexamethasone significantly decreased both uPA and tPA production, and increased PAI-1 production. Glucagon had no effect on the production of these fibrinolytic factors. Our results reveal that uPA is one of the markers for the differentiation of 3T3-L1 cells and that insulin, IBMX, and dexamethasone are potent regulators of the fibrinolytic activity in differentiated 3T3-L1 cells, reciprocally affecting PA and PAI-1 levels in them.     

IL-1beta down-regulates tissue-type plasminogen activator by up-regulating low-density lipoprotein receptor-related protein in AML 12 cells

Interleukin-1 (IL-1) regulation of tPA in hepatocytes was studied in mouse hepatocyte line AML12. IL-1 induced transient accumulation of tPA mRNA as high as threefold by 2 h after the start of treatment. The cytokine also induced the mRNA for serum amyloid A, a typical acute-phase protein in mice, with more sustained kinetics in a time-dependent manner. In contrast to the induction of mRNA, tPA activity and protein levels in the harvested medium were dramatically diminished by IL-1. IL-1 stimulated the uptake of (125)I-tPA by AML 12. This uptake was inhibited by 39-kDa receptor-associated protein (RAP), but not by the sugar mannan. These results revealed that low-density lipoprotein receptor-related protein (LRP), which is known to be a receptor for tPA and to be blocked by RAP, was up-regulated by IL-1. We also demonstrated, for the first time, that IL-1 transiently increased the mRNA level of LRP threefold by 30 min after the start of IL-1 treatment of AML 12. The receptor-mediated endocytosis of tPA by hepatocytes may thus play a crucial role in the down-regulation of fibrinolysis during the acute-phase response.     

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.     

cDNA microarray analysis of Helicobacter pylori-mediated alteration of gene expression in gastric cancer cells

Helicobacter pylori infection stimulates several intracellular signaling pathways and is accompanied by increased gene expression in gastric epithelial cells. High-density cDNA microarray was used to characterize the mRNA expression profile of genes in human gastric cancer cells (MKN45, AGS) cocultured with H. pylori. Coculture with cag pathogenicity island (PAI)-positive H. pylori (wild-type) significantly up-regulated mRNA expression in 8 of 2304 genes tested. In 6 (interleukin-8, I(kappaB)alpha, A20, ERF-1, keratin K7, glutathione peroxidase) of the 8 genes, up-regulation was confirmed by RT-PCR. In coculture with isogenic cagE-negative mutant ((Delta)cagE), which encodes a type IV secretion system with other genes in the cag PAI, no significant up-regulation was found. We further analyzed the role of A20. Transfection of expression vector encoding A20 resulted in an inhibition of H. pylori-mediated NF-kappaB activation, indicating that H. pylori-mediated A20 expression could be a negative regulator of NF-kappaB activation. Taken together, these results indicate the importance of microarray technology as a tool for analyzing the complex interplay between H. pylori and the host.     

Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1

The gene encoding a ferredoxin (Fd) from Haloarcula japonica strain TR-1 was cloned and sequenced. Sequence analysis of the cloned Ha. japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids. The deduced amino acid sequence of Ha. japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea. The Ha. japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. Ha. japonica Fd could then be produced as a fusion with HisTag (6xHis) in Ha. japonica host cells. The absorption and ESR spectra of the Fd/HisTag fusion protein revealed the presence of a [2Fe-2S] cluster which is characteristic of native Ha. japonica Fd.     

Microfabricated polymer chip for capillary gel electrophoresis

A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length.