Significance of platelet-derived endothelial cell growth factor in the angiogenesis of human gastric cancer

Most patients with primary IgA nephropathy (IgAN) have a significantly higher memory repertoire of IgA1-producing B lymphocytes in their bone marrow together with high plasma levels of IgA1. The connection between the mucosal immune system and the bone marrow compartment is probably based on traffic of either antigen-presenting cells (APC) or antigen-specific lymphocytes. Cytokines play an important role in the proliferation and differentiation of lymphoid cells. In order to mimic the in vivo situation as much as possible, we assessed cytokine production profiles ex vivo in 23 IgAN patients and matched controls, using lipopolysaccharide (LPS)- or phytohaemagglutinin (PHA)-stimulated whole blood (WB) cultures. Interferon-gamma (IFN-gamma), IL-2, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-alpha) production in culture supernatants were determined by cytokine-specific ELISAs. Compared with controls, PHA-stimulated cultures resulted in significantly higher IL-10 (P<0.001), IL-2 (P<0.005) and IFN-gamma (P<0.001) levels in IgAN patients, but no significant differences in TNF-alpha or IL-6 levels were found. In LPS-stimulated cultures, the only significant difference (P<0.001) between the two groups was the increased IL-10 production in IgAN patients. The enhanced cytokine production in stimulated WB cultures suggests altered monocyte-related T cell responses in patients with IgAN. Increased IL-10 production may eventually result in an increased number of IgA-producing B lymphocytes in the bone marrow. In addition, high levels of endogenous IL-10 may down-regulate the effector functions of monocytes, or possibly APC in general, and consequently the IgA response at the mucosal level.     

Sulfotransferase gene expression in primary cultures of rat hepatocytes

Hepatocyte cultures have been used in pharmacotoxicological studies, and sulfotransferases (ST) are important drug-metabolizing enzymes in liver. The expression of sulfotransferases in hepatocyte cultures has not been examined systematically. In the present study, the mRNA levels of different sulfotransferases in male and female rat hepatocytes were examined by northern-blot analyses. Various culture conditions such as different matrices (collagen, matrigel, collagen sandwich, or co-culture with epithelial cells), medium (Way-mouth's MB 752/1 and Modified Chee's Medium) and glucocorticoid supplementation (dexamethasone, 0.1 microM) were compared. Phenol ST (ST1A1) mRNA levels decreased to about 50% of initial mRNA levels within 10 hr of culture. At 96 hr, ST1A1 mRNA levels were approximately 20% of initial values when cultured on collagen, matrigel or co-culture. The two media did not differ in ability to maintain ST1A1 mRNA levels in the absence of dexamethasone (DEX); however, DEX addition to either medium resulted in ST1A1 mRNA levels greater than 100% of the initial mRNA levels at 96 hr, with the greatest increase observed using the matrigel substratum and Chee's medium. In the absence of DEX, the mRNA levels of N-hydroxy-2-acetylaminoflurene sulfortransferase (ST1C1), estrogen sulfotransferase (ST1E2) and hydroxysteroid sulfotransferase (ST-20/21, ST-40/41, ST-60) fell to approximately 20% of their initial levels within 24 hr, and to less than 5% at 96 hr. The loss of expression of these sulfotransferases was observed with all culture conditions. Addition of DEX to the media resulted in ST-40/41 and ST-60 mRNA expression at 20 and 35% of their initial values, respectively, in cultures maintained on matrigel and Chee's medium at 96 hr. These data suggest that sulfotransferases lose their constitutive expression in hepatocyte culture, but retain their inducibility.     

Quinolinic acid and lymphocyte subsets in the intrathecal compartment as biomarkers of SIV infection and simian AIDS

Cerebrospinal fluid (CSF) samples were collected from monkeys infected with SIVmac251 (SIV) or HIV-1/SIVmac chimeric viruses (SHIV(HXBc2) and SHIV(89.6P)) to investigate quinolinic acid (QUIN) levels in the intrathecal compartment. CSF levels of QUIN were elevated in the SIV-infected monkeys, especially in animals with end-stage disease, and in those infected with pathogenic SHIV(89.6P), but not after infection with the nonpathogenic construct SHIV(HXBc2). QUIN elevations occurred in association with reduced CD4+ and increased CD8+ lymphocytes, cellular alterations that were more pronounced in CSF than in the blood. These findings support the view that the intrathecal compartment provides a unique window on viral infection, and are in keeping with the a priori prediction that QUIN increases primarily in response to more pathogenic viral strains.     

Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.     

Expression of the brain creatine kinase gene in rat RT4 peripheral neurotumor cell lines and its modulation by cell confluence

Creatine kinases (CK) catalyze the reversible transfer of a high energy phosphate group between creatine phosphate and ADP to regenerate ATP in cell types where the requirements for ATP are extensive and/or sudden. Previously, we have shown in primary rat brain cell cultures that brain CK (CKB) mRNA levels are highest in astrocytes and oligodendrocytes and much lower in neuronal cells. However, little is known of the factors which regulate CKB expression in the central nervous system and peripheral nervous system. To begin to investigate these factors, we asked in this report (1) if this pattern of CKB expression was also characteristic of some established glial and neuronal cell lines derived from the PNS; (2) whether CKB expression could be rapidly modulated by culture conditions, and (3) if CKB is expressed in cells with characteristics of glial cell progenitors. In subconfluent cells, CKB mRNA and enzyme activity were found to be high in both the rat RT4 peripheral neurotumor stem cell RT4-AC36A and its glial cell derivative RT4-D6. Conversely, CKB mRNA and activity were 5- and 8-fold lower, respectively, in the neuronal derivative RT4-E5 and, more dramatically, CKB was undetectable in neuronal RT4-B8 cells. Maintaining RT4-D6 glial cells at confluence rapidly increased CKB enzyme activity by 7-fold, such that D6 cells contained about 25% of the CKB level in lysates prepared from either whole adult rat brain or primary cultures of rat brain astrocytes. The levels of CKB mRNA and immunoreactive protein were also correspondingly increased in confluent D6 cells. These confluence-mediated increases in CKB appeared to be due to cell-cell contact and not the depletion of serum growth factors or an increase in intracellular cAMP. This study indicates that CKB expression is highest in cells displaying glial properties and can be rapidly modulated by appropriate culture conditions. The results are discussed in relation to the factors which may regulate CKB expression in vivo.