A novel mouse model of hepatocarcinogenesis triggered by AID causing deleterious p53 mutations.

Activation-induced cytidine deaminase (AID), the only enzyme that is known to be able to induce mutations in the human genome, is required for somatic hypermutation and class-switch recombination in B lymphocytes. Recently, we showed that AID is implicated in the pathogenesis of human cancers including hepatitis C virus (HCV)-induced human hepatocellular carcinoma (HCC). In this study, we established a new AID transgenic mouse model (TNAP-AID) in which AID is expressed in cells producing tissue-nonspecific alkaline phosphatase (TNAP), which is a marker of primordial germ cells and immature stem cells, including ES cells. High expression of TNAP was found in the liver of the embryos and adults of TNAP-AID mice. HCC developed in 27% of these mice at the age of approximately 90 weeks. The HCC that developed in TNAP-AID mice expressed alpha-fetoprotein and had deleterious mutations in the tumour suppressor gene Trp53, some of which corresponded to those found in human cancer. In conclusion, TNAP-AID is a mouse model that spontaneously develops HCC, sharing genetic and phenotypic features with human HCC, which develops in the inflamed liver as a result of the accumulation of genetic changes.

Expression of activation-induced cytidine deaminase in human hepatocytes via NF-kappaB signaling.

Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. The fact that constitutive expression of AID in mice causes tumors in various organs, including lymphoid tissues and lungs, suggests the important role of the aberrant editing activity of AID on various tumor-related genes for carcinogenesis. AID expression, however, is restricted to activated B cells under physiological conditions. We demonstrate here that ectopic AID expression is induced in response to tumor necrosis factor-alpha stimulation in cultured human hepatocytes. The proinflammatory cytokine-mediated expression of AID is achieved by IkappaB kinase-dependent nuclear factor (NF)-kappaB signaling pathways. Hepatitis C virus, one of the leading causes of hepatocellular carcinoma (HCC), enhanced AID expression via NF-kappaB activation through expression of viral core protein. The aberrant expression of AID in hepatoma-derived cells resulted in accumulation of genetic alterations in the c-myc and pim1 genes, suggesting that inappropriate expression of AID acts as a DNA mutator that enhances the genetic susceptibility to mutagenesis in human hepatocytes. Our current findings indicate that the inappropriate expression of AID is induced by proinflammatory cytokine stimulation and may provide the link between hepatic inflammation and the development of HCC.

Identification of homologous biologically active sites on the N-terminal domain of laminin alpha chains.

Laminin, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha, beta, and gamma chains. To date, five different alpha chains have been identified. N-terminal domain VI in the alpha1 chain has various biological activities. Here we screened biologically active sequences on domain VI of the laminin alpha2, alpha3, and alpha5 chains using a large number of overlapping peptides. HT-1080 human fibrosarcoma cell attachment to the peptides was evaluated using peptide-coated plastic plates and peptide-conjugated Sepharose beads. We identified four cell adhesive sequences from laminin alpha2 chain domain VI, two sequences from the alpha3 chain, and two sequences from the laminin alpha5 chain. Sequences homologous to A13 (RQVFQVAYIIIKA, alpha1 chain 121-133) on all the alpha chains (FQIAYVIVKA, alpha2 chain 130-139; GQLFHVAYILIKF, alpha3 chain 96-108; FHVAYVLIKA, alpha5 chain 74-83) showed strong cell attachment activity. A5-16 (LENGEIVVSLVNGR, alpha5 chain 147-160) showed the strongest cell attachment activity in the plate assay, and the homologous peptide in the alpha3 chain promoted similar strong cell attachment activity. A5-16 and its homologous peptide in the alpha2 chain promoted moderate cell attachment, while the homologous peptide to A5-16 in the alpha1 chain did not show activity. A2-7 (SPSIKNGVEYHYV, alpha2 chain 108-120) showed cell attachment activity only in the plate assay, but homologous sequences in the alpha1, alpha3, and alpha5 chains did not promote activity. A2-7 promoted endothelial cell sprouting from aortic rings in vitro and melanoma colonization to murine lungs in vivo. However, none of the homologous peptides of A2-7 promoted experimental pulmonary metastasis by B16-BL6 melanoma cells. These results indicate that there are chain-specific active sites in domain VI of the laminin alpha chains, some of which contain conserved activities.

Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat.

BACKGROUND/AIMS: The aim of this study was to investigate whether both matrix metalloproteinase-2 (MMP-2) and membrane type 1 MMP (MT1-MMP) participate in the spontaneous resolution of liver fibrosis. METHODS: Transcription of both genes was examined by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Gelatinase activity was investigated by zymography. RESULTS: Gene expression by RT-PCR showed that both genes increased in the process of liver fibrosis, then decreased gradually in the recovery phase. ISH revealed that distribution of positive cells changed quickly in the recovery phase. Positive cells were widely seen in the liver, mainly around fibrous septa, in the aggressive phase, but were exclusively observed at the interface between the resolving fibrous band and the parenchyma, then were diffusely located in the lobules in the recovery phase. Main cells expressing both mRNAs seemed to be stellate cells for their morphology, though they did not express characteristic cell markers. Some hepatocytes and Kupffer cells expressed both mRNAs in the recovery phase. Gelatinase activity of MMP-2 increased in the recovery phase of 8-week-treated rat liver by gelatin zymography. CONCLUSIONS: The results of present study suggest that both enzymes participate in the destruction of extracellular matrix in coordination with MMP-13.

Reversibility of hepatic fibrosis: from the first report of collagenase in the liver to the possibility of gene therapy for recovery.

Since the authors reported the presence of collagenase in the liver as well as its increased activity in the early stage of hepatic fibrosis and its reduced activity in advanced fibrosis in rats induced by chronic CCl4 intoxication, in baboons fed alcohol chronically and in patients with alcoholic fibrosis, other investigators have demonstrated the same tendency of collagenase activity biologically and histochemically. Very recently, the authors demonstrated definite gene expression of collagenase during the recovery from experimental hepatic fibrosis using Northern blotting and in situ hybridization. The findings of in situ hybridization not only demonstrated the cells expressing collagenase, but also suggested much information on the mechanism of the recovery from fibrosis. Hepatic stellate cells play a key role not only in fibrogenesis but also in fibrolysis. The authors' recent observation revealed that collagenase (matrix metalloproteinase-13 (MMP-13)) gene expression appears very early in the process of recovery from liver fibrosis, and that both stellate cells and hepatocytes express MMP-13. Recovery from liver cirrhosis requires the gene expression of collagenase, increased production of the collagenase enzyme, and activation of the enzyme balanced with the specific inhibitors of collagenase. The understanding of molecular mechanisms of MMP-1 gene expression which is under investigation in our laboratory may provide us a new strategy for the treatment of liver fibrosis including the possibility of gene therapy.

[Coronary risk appraisal for primary prevention of coronary heart disease in a community].

PURPOSE: In discussion on application of "National Health Promotion Toward 21st Century in Japan" in Kanagawa prefecture, it was noted that the age-adjusted mortality rate of death from ischemic heart disease in this part of Japan was higher than that for the whole nation in 1996. To facilitate development of a strategy for primary prevention of coronary heart disease (CHD), the present study was conducted to predict 2-yr incidence of CHD and decrease with simulations assuming improvement in CHD risk factors. METHODS: Using CHD risk prediction algorithm; the Weibull accelerated failure regression model based on the Framingham Heart Study, a 2-yr incidence of CHD was predicted for 1652 residents (515 male, 1137 female) on the basis of results of a health check up in 1998. We then estimated the probable decrease in CHD recalculated assuming decrease in total cholesterol (TC), increase in HDL-cholesterol (HDL-C), decrease in systolic blood pressure (SBP), or quitting the smoking habit. RESULTS: 1. The 2-yr probability of developing CHD for men free of heart disease was 2.79 +/- 2.17%, and that for men who had heart disease was 10.25 +/- 2.17%. The 2-yr probability for women free of heart disease was 16.80 +/- 14.40%, and that for women who had heart disease was 3.66 +/- 1.09%. As the reported probability of developing CHD in the U.S.A. is remarkably higher than in Japan, the fact that the present model was based on American data explains why these predicted probabilities are higher than values reported from Japanese cohort studies. 2. For men free of heart disease, a strategy for high risk case such as a decrease in TC and an increase in HDL-C, or quitting the smoking habit, was more effective than a population-based strategy. For women free of heart disease, the population-based strategy was more effective. 3. Women more than 60-yrs old who had a high 2-yr probability of developing CHD were divided into three groups; high, middle, and low risk. The mean body weight, mean body mass index, mean diastolic blood pressure, and mean blood glucose in the high risk group were significantly higher than the values in the other groups. Decrease in systolic blood pressure was a more effective strategy for decrease in CHD incidence in the high risk group than in the other groups. CONCLUSIONS: CHD risk prediction of this type may be considered useful for setting target CHD risk factors and for focusing interventions to prevent CHD effectively.

Amino acids and peptides. Part 39: a bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin.

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.

Neuronal cell adhesion, mediated by the heparin-binding neuroregulatory factor midkine, is specifically inhibited by chondroitin sulfate E. Structural ans functional implications of the over-sulfated chondroitin sulfate.

The heparin-binding neurotrophic factor midkine (MK) has been proposed to mediate neuronal cell adhesion and neurite outgrowth promotion by interacting with cell-surface heparan sulfate. We have observed that over-sulfated chondroitin sulfate (CS) D and CS-E show neurite outgrowth-promoting activity in embryonic day (E) 18 rat hippocampal neurons (Nadanaka, S., Clement, A., Masayama, K., Faissner, A., and Sugahara, K. (1998) J. Biol. Chem. 273, 3296-3307). In the present study, various CS isoforms were examined for their ability to inhibit the MK-mediated cell adhesion of cortical neuronal cells in comparison with heparin from porcine intestine and heparan sulfate from bovine kidney. E17-18 rat cortical neuronal cells were cultured on plates coated with recombinant MK in a grid pattern. The cells attached to and extended their neurites along the MK substratum. Cell adhesion was inhibited by squid cartilage over-sulfated CS-E as well as by heparin, but not by heparan sulfate or other CS isoforms. Direct interactions of MK with various glycosaminoglycans were then evaluated using surface plasmon resonance, showing that CS-E bound MK as strongly as heparin, followed by other over-sulfated CS isoforms, CS-H and CS-K. Furthermore, E18 rat brain extracts showed an E disaccharide unit, GlcUAbeta1-3GalNAc(4,6-O-disulfate). These findings indicate that CS chains containing the E unit as well as heparin-like glycosaminoglycans may be involved in the expression and/or modulation of the multiple neuroregulatory functions of MK such as neuronal adhesion and migration and promotion of neurite outgrowth.

Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride.

BACKGROUND/AIMS: Liver fibrosis is a dynamic state between matrix production and degradation. Since our report in 1974, many studies have examined collagenase and liver fibrosis, but not the identification of cells responsible for collagenase production in vivo. The aim of this study was to investigate the gene expression of interstitial collagenase in the progressive and recovery phases of experimental rat liver fibrosis by in situ hybridization. METHODS: We examined the gene expression of interstitial collagenase (MMP-13) in the progressive and recovery phase of experimental rat liver fibrosis induced by chronic CCl4 intoxication by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. In order to identify the cells expressing MMP-13 mRNA by in situ hybridization, immunohistochemistry was performed using serial sections. RESULTS: In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR, but not by in situ hybridization. The livers of rats treated with CCl4 for 4 weeks showed fatty metamorphosis but no definite fibrosis. Positive signals for MMP-13 mRNA were observed in scattered mesenchymal cells, within lobules which seem to be stellate cells from immunohistochemical staining. Once the fibrosis became prominent, the faint band for MMP-13 mRNA was detected only by RT-PCR and very few signals, if any, by in situ hybridization. On the other hand, in the recovery phase of liver fibrosis, gene expression of MMP-13 was markedly enhanced. Strong positive cells by in situ hybridization were observed mainly at the interface between the resolving fibrous septa and the parenchyma. Overlapping both images of in situ hybridization and immunohistochemical staining with the help of a computer revealed that some positive cells, but not all cells, were stellate cells stained with a-smooth muscle actin antibody. CONCLUSIONS: MMP-13 participates in the degradation of newly-formed matrix in the recovery from rat liver fibrosis more than in the remodeling of extracellular matrix for the formation of fibrosis. Hepatic stellate cells play a crucial role in MMP-13 production in the recovery from fibrosis, though not all stellate cells were positive for MMP-13 mRNA. Further investigation into gene expression of MMP-13 in recovery will lead to new strategies for the treatment of liver cirrhosis.

High and low affinity heparin-binding sites in the G domain of the mouse laminin alpha 4 chain.

G domains of the mouse laminin alpha 1 and alpha 4 chains consisting of its five subdomains LG1-LG5 were overexpressed in Chinese hamster ovary cells and purified by heparin chromatography. alpha 1LG1-LG5 and alpha 4LG1-LG5 eluted at NaCl concentrations of 0.30 and 0.47 m, respectively. In solid phase binding assays with immobilized heparin, half-maximal concentrations of 14 (alpha 1LG1-LG5) and 1.4 nm (alpha 4LG1-LG5) were observed. N-Glycan cleavage of alpha 4LG1-LG5 did not affect affinity to heparin. The affinity of alpha 4LG1-LG5 was significantly reduced upon denaturation with 8 m urea but could be recovered by removing urea. Chymotrypsin digestion of alpha 4LG1-LG5 yielded high and low heparin affinity fragments containing either the alpha 4LG4-LG5 or alpha 4LG2-LG3 modules, respectively. Trypsin digestion of heparin-bound alpha 4LG1-LG5 yielded a high affinity fragment of about 190 residues corresponding to the alpha 4LG4 module indicating that the high affinity binding site is contained within alpha 4LG4. Competition for heparin binding of synthetic peptides covering the alpha 4LG4 region with complete alpha 4LG1-LG5 suggests that the sequence AHGRL1521 is crucial for high affinity binding. Introduction of mutation of H1518A or R1520A in glutathione S-transferase fusion protein of the alpha 4LG4 module produced in Escherichia coli markedly reduced heparin binding activity of the wild type. When compared with the known structure of alpha 2LG5, this sequence corresponds to the turn connecting strands E and F of the 14-stranded beta-sheet sandwich, which is opposite to the proposed binding sites for calcium ion, alpha-dystroglycan, and heparan sulfate.

Inactive aldehyde dehydrogenase 2 worsens glycemic control in patients with type 2 diabetes mellitus who drink low to moderate amounts of alcohol.

BACKGROUND: Alcohol intake can have hypoglycemic or hyperglycemic effects in patients with type 2 diabetes mellitus. The present study was designed to investigate the glycemic control of male patients with diabetes mellitus from the aspect of the genetic status of alcohol metabolism. METHODS: One hundred sixty-three men with type 2 diabetes mellitus were enrolled in the present study. They were all outpatients at the Diabetes Center of Saiseikai Central Hospital. The genotype of the aldehyde dehydrogenase 2 (ALDH2) gene of each patient was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the patients were divided into those with active or inactive ALDH2 phenotype. We compared the amount of habitual alcohol intake and clinical data that included physical findings and blood chemistry of the patients in the active and inactive ALDH2 groups. The glycemic control of each patient was evaluated by the serum level of HbAlc. RESULTS: Of the 163 patients with type 2 diabetes mellitus, 90 patients had the active ALDH2 phenotype and 73 patients had the inactive ALDH2 phenotype. The mean HbA1c level of the active ALDH2 group was nearly the same as that of the inactive ALDH2 group. However, the HbA1c level of the light-to-moderate drinkers (1-400 g/week) in the inactive ALDH2 group was highest and was significantly higher than the HbA1c level of the light-to-moderate drinkers of the active ALDH2 group. The HbA1c of the patients with diabetic complications was higher than the HbAlc of those without diabetic complications in both the active and inactive ALDH2 groups. However, the HbA1c level of the light-to-moderate drinkers without diabetic complications in the inactive ALDH2 group was significantly higher and the incidence of 24 hr urinary C-peptide was higher than the respective level of the light-to-moderate drinkers without diabetic complications in the active ALDH2 group. CONCLUSIONS: Habitual light-to-moderate alcohol intake worsens glycemic control in diabetic patients who have the inactive ALDH2 phenotype. The data on 24 hr urinary C-peptide level suggested that increased acetaldehyde after light-to-moderate drinking by inactive ALDH2 diabetic patients may increase the HbA1c value by the insulin-resistant condition that resulted in hyperinsulinemia.

Molecular mechanism of the reversibility of hepatic fibrosis: with special reference to the role of matrix metalloproteinases.

The participation of matrix metalloproteinases (MMP) and their specific inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMP), in both the formation and degradative recovery processes of liver fibrosis were mainly reviewed from the molecular biological aspect. Since authors first reported increased activity of interstitial collagenase in the early stage of hepatic fibrosis in rats induced by chronic CCl4 intoxication, in baboons fed alcohol chronically and in patients with alcoholic fibrosis, other investigators have also demonstrated increased activity biologically and histochemically. However, species-specific differences in response have been found and gene-level research on the rat model has not demonstrated increased mRNA transcription of collagenase. It has also been clarified that activated stellate cells can also produce matrix components. Very recently, authors observed the participation of interstitial collagenase in the recovery from experimental hepatic fibrosis by using polymerase chain reaction northern blotting and in situ hybridization. The in situ hybridization findings not only demonstrated the cells responsible for interstitial collagenase, but also suggested a great deal about the mechanism of recovery from fibrosis. Hepatic stellate cells are activated via the expression of c-myb and nuclear factor-kappaB (NFkappaB) which is induced by oxidative stress, and inhibited by antioxidant (1-alpha-tocopherol) and butylated hydroxytoluene. The activation mechanism is now being revealed. The relationship between the activation mechanism of stellate cells and the production and secretion of MMP and TIMP in the formation and recovery process of hepatic fibrosis should be investigated from the promoter gene level. This approach might help develop a new strategy for the treatment of liver fibrosis.

The use of docosahexaenoic acid supplementation to ameliorate the hyperactivity of rat pups induced by in utero ethanol exposure.

It has been demonstrated thatin utero ethanol (EtOH) exposure induces hyperactive behavior and learning disturbances in offspring. In order to investigate the effects of docosahexaenoic acid (DHA) on these neurobehavioral dysfunctions of rat pups induced byin utero EtOH exposure, pregnant Wistar rats were divided into four treatment groups depending on the type of oil added to the diet and drinking water as follows; (a) 5% safflower oil with tap water (TW/n-6), (b) 3% safflower oil and 2% DHA with tap water (TW/n-3), (c) 5% safflower oil with 10%-EtOH (ET/n-6), (d) 3% safflower oil and 2% DHA with 10%-EtOH (ET/n-3) at gestational day (GD) 7.10%-EtOH was administered to dams in ET/n-6 and ET/n-3 groups from GD 7 to the pups' weaning (postnatal week 4), and all pups were fed with the same diet that was given to their dams during the entire examination period. The open-field test and the water E-maze test were conducted for all pups, and a spontaneous motor activity test and the Sidman electric shock avoidance test were performed for some of male pups. Amounts of monoamine metabolites in striatum were then determined, and fatty acid analyses of total brain lipids were performed.The male pups in the ET/n-6 group showed significandy more rearing and square-crossing movements in the open-field test, and significandy higher spontaneous motor activity during the dark period in the daily cycle compared to the males in the TW/n-6 group. The male pups in the ET/n-3 group showed fewer of these behaviors in the open-field test compared to the ET/n-6 group males, and a normal pattern of spontaneous motor activity.Learning disturbance induced byin utero EtOH exposure was not observed in the E-shaped water maze, but was observed in the avoidance rates in the Sidman electric shock avoidance test. However, there was no significant modifying effect of DHA on the avoidance rates in EtOH exposed pups.The analysis of the fatty acid composition of total lipids in the brains of the pups revealed high levels of DHA in the diet reflected an increased level of brain DHA and caused a decreased level of the brain arachidonic acid. Retroco nversion from DHA to eicosapentaenoic acid was also observed. However, there was no significant effect of DHA on the levels of monoamine metabolites.These results support the hypothesis that DHA can counteract the attention deficit hyperactivity disorder.

Characterization of glycosylphosphatidylinositiol-anchored, secreted, and intracellular vertebrate mono-ADP-ribosyltransferases.

Mono-ADP-ribosylation is a posttranslational modification of proteins in which the ADP-ribose moiety of nicotinamide adenine dinucleotide is transferred to an acceptor amino acid. Five mammalian ADP-ribosyltransferases (ART1--ART5) have been cloned and expression is restricted to tissues such as cardiac and skeletal muscle, leukocytes, brain, and testis. ART1 and ART2 are glycosylphosphatidylinositol (GPI)-anchored ectoenzymes. ART5 appears not to be GPI-linked and may be secreted. In skeletal muscle and lymphocytes, ART1 modifies specific members of the integrin family of adhesion molecules, suggesting that ADP-ribosylation affects cell-matrix or cell-cell interactions. In lymphocytes, ADP-ribosylation of surface proteins is associated with changes in p56lck tyrosine kinase-mediated signaling. The catalytic sites of bacterial toxins and vertebrate transferases have conserved structural features, consistent with a common reaction mechanism. ADP-ribosylation can be reversed by ADP-ribosylarginine hydrolases, resulting in the regeneration of free arginine. Thus, an ADP-ribosylation cycle may play a regulatory role in vertebrate tissues.

Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase is a major protein of the cerebrospinal fluid and was originally known as beta-trace. We investigated the binding ability of prostaglandin D synthase toward bile pigments, thyroid hormones, steroid hormones, and fatty acids in this present study. We found that the recombinant enzyme binds bile pigments and thyroid hormones, resulting in quenching of the intrinsic tryptophan fluorescence, the appearance of induced circular dichroism of the lipophilic ligands, and a red shift of the absorption spectra of bilirubin and biliverdin. The binding of prostaglandin D synthase to lipophilic ligands was also demonstrated by the resonant mirror technique and surface plasmon resonance detection. The dissociation constants were calculated to be 33 nM, 37 nM, 660 nM, 820 nM, and 2.08 microM for biliverdin, bilirubin, L-thyroxine, 3,3',5'-triiodo-L-thyronine, and 3,3', 5-triiodo-L-thyronine, respectively. Biliverdin and bilirubin underwent a shift in their absorption peaks from 375 to 380 nm and from 439 to 446 nm, respectively, after binding to prostaglandin D synthase. Bilirubin bound to the enzyme showed a bisignate CD spectrum with a (-) Cotton effect at 422 nm and a (+) Cotton effect at 472 nm, indicating a right-handed chirality. The ligands also inhibited prostaglandin D synthase activity noncompetitively in a concentration-dependent manner, with IC50 values between 3.9 and 10. 9 microM. Epididymal retinoic acid-binding protein and beta-lactoglobulin, two other lipocalin proteins that bind retinoids such as prostaglandin D synthase, did not show any significant interaction with bile pigments or thyroid hormones. These results show that prostaglandin D synthase binds small lipophilic ligands with a specificity distinct from that of other lipocalins.

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.

Characterization of NAD:arginine ADP-ribosyltransferases.

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.

Collagen biosynthesis in gastric cancer: immunohistochemical analysis of prolyl 4-hydroxylase.

BACKGROUND AND OBJECTIVES: The mechanism of the desmoplastic response in gastric carcinoma tissues is largely unknown. The objective of this study is to determine the localization of prolyl 4-hydroxylase (PH), an enzyme that plays a crucial role in collagen biosynthesis. METHODS: Freshly prepared gastric carcinoma tissues from 51 cases, including 13 of the scirrhous type (diffusely infiltrative type), were immunostained by using monoclonal antibodies to human placental PH. RESULTS: Although cytoplasmic staining for PH was observed in both fibroblasts and carcinoma cells, there was increased expression of the alpha-subunit in fibroblasts and no difference in expression between the scirrhous and non-scirrhous type gastric carcinomas. In scirrhous type samples, there was increased PH expression in fibroblasts located in the tumor periphery when compared with fibroblasts in the tumor center. These findings suggested that maintenance of a balance between production and degradation of collagen in gastric carcinoma tissues might be important for stroma formation. CONCLUSIONS: It is speculated that activated fibroblasts participate in collagen biosynthesis at the tumor periphery rather than in the tumor center and that increased collagen biosynthesis at the tumor periphery in scirrhous gastric carcinoma may assist further invasion of tumor cells.