Influence of insulin sensitivity and the TaqIB cholesteryl ester transfer protein gene polymorphism on plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities and their response to hyperinsulinemia in non-diabetic men.

Lecithin:cholesteryl acyl transferase (LCAT), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP), and lipoprotein lipases are involved in high density lipoprotein (HDL) metabolism. We evaluated the influence of insulin sensitivity and of the TaqIB CETP gene polymorphism (B1B2) on plasma LCAT, CETP, and PLTP activities (measured with exogenous substrates) and their responses to hyperinsulinemia. Thirty-two non-diabetic men without hyperlipidemia were divided in quartiles of high (Q(1)) to low (Q(4)) insulin sensitivity. Plasma total cholesterol, very low + low density lipoprotein cholesterol, triglycerides, and apolipoprotein (apo) B were higher in Q(4) compared to Q(1) (P < 0.05 for all), whereas HDL cholesterol and apoA-I were lowest in Q(4) (P < 0.05 for both). Plasma LCAT activity was higher in Q(4) than in Q(1) (P < 0. 05) and PLTP activity was higher in Q(4) than in Q(2) (P < 0.05). Insulin sensitivity did not influence plasma CETP activity. Postheparin plasma lipoprotein lipase activity was highest and hepatic lipase activity was lowest in Q(1). Insulin infusion decreased PLTP activity (P < 0.05), irrespective of the degree of insulin sensitivity. The CETP genotype exerted no consistent effects on baseline plasma lipoproteins and LCAT, CETP, and PLTP activities. The decrease in plasma PLTP activity after insulin was larger in B1B1 than in B2B2 homozygotes (P < 0.05). These data suggest that insulin sensitivity influences plasma LCAT, PLTP, lipoprotein lipase, and hepatic lipase activities in men. As PLTP, LCAT, and hepatic lipase may enhance reverse cholesterol transport, it is tempting to speculate that high levels of these factors in association with insulin resistance could be involved in an antiatherogenic mechanism. A possible relationship between the CETP genotype and PLTP lowering by insulin warrants further study.

High-density lipoprotein cholesterol is related to the TaqIB cholesteryl ester transfer protein gene polymorphism and smoking, but not to moderate alcohol consumption in insulin-dependent diabetic men.

In non-diabetic subjects, the high-density lipoprotein (HDL) cholesterol level is increased by alcohol and decreased by smoking. The biallelic B1B2 polymorphism of the cholesteryl ester transfer protein (CETP) gene is a genetic determinant of HDL cholesterol. We evaluated the effect of moderate alcohol consumption, the CETP gene polymorphism and clinical variables on HDL cholesterol and other lipoprotein parameters in insulin-dependent diabetic (IDDM) men. Thirteen moderate alcohol using IDDM men (median alcohol consumption 17 g/d) and 13 abstainers, individually matched for the CETP gene polymorphism and clinical factors including smoking, were studied. HDL cholesterol, serum apo AI and serum CETP activity levels were very similar in alcohol users compared to abstainers (1.36 +/- 0.28 vs 1.36 +/- 0.36 mmol l-1, 1.71 +/- 0.31 vs 1.75 +/- 0.33 g l-1 and 134 +/- 27 vs 138 +/- 53 nmol l-1h-1, respectively, n.s. for all). No significant differences in apo B-containing lipoproteins were observed. Multiple regression analysis (multiple r = 0.68) showed that HDL cholesterol was positively associated with the presence of the B2 allele (0.23 mmol l-1 higher for each B2 allele present, p = 0.004) and negatively with smoking (0.15 mmol l-1 lower per 10 cigarettes smoked daily, p = 0.011), but not with alcohol consumption (p = 0.66). This study suggests that moderate alcohol consumption has no beneficial effect on the lipoprotein profile in IDDM men. HDL cholesterol is adversely influenced by smoking, whereas considerable variation in its level appears to be explained by the CETP gene polymorphism.

Cholesteryl ester transfer protein gene polymorphism is a determinant of HDL cholesterol and of the lipoprotein response to a lipid-lowering diet in type 1 diabetes.

The TaqIB cholesteryl ester transfer protein (CETP) gene polymorphism (B1B2) is a determinant of HDL cholesterol in nondiabetic populations. Remarkably, this gene effect appears to be modified by environmental factors. We evaluated the effect of this polymorphism on HDL cholesterol levels and on the lipoprotein response to a linoleic acid-enriched, low-cholesterol diet in patients with type 1 diabetes. In 44 consecutive type 1 diabetic patients (35 men), CETP polymorphism, apolipoprotein (apo) E genotype, serum lipoproteins, serum CETP activity (measured with an exogenous substrate assay, n = 30), clinical variables, and a diet history were documented. The 1-year response to diet was assessed in 14 type 1 diabetic patients, including 6 B1B1 and 6 B1B2 individuals. HDL cholesterol was higher in 10 B2B2 than in 14 B1B1 homozygotes (1.63 +/- 0.38 vs. 1.24 +/- 0.23 mmol/l, P < 0.01). HDL cholesterol, adjusted for triglycerides and smoking, was 0.19 mmol/l higher for each B2 allele present. CETP activity levels were not significantly different between CETP genotypes. Multiple regression analysis showed that VLDL + LDL cholesterol was associated with dietary polyunsaturated:saturated fatty acids ratio (P < 0.02) and total fat intake (P < 0.05) in the B1B1 homozygotes only and tended to be related to the presence of the apo E4 allele (P < 0.10). In response to diet, VLDL + LDL cholesterol fell (P < 0.05) and HDL cholesterol remained unchanged in 6 B1B1 homozygotes. In contrast, VLDL + LDL cholesterol was unaltered and HDL cholesterol decreased (P < 0.05) in 6 B1B2 heterozygotes (P < 0.05 for difference in change in VLDL + LDL/HDL cholesterol ratio). This difference in response was unrelated to the apo E genotype. Thus, the TaqIB CETP gene polymorphism is a strong determinant of HDL cholesterol in type 1 diabetes. This gene effect is unlikely to be explained by a major influence on the serum level of CETP activity, as an indirect measure of CETP mass. Our preliminary data suggest that this polymorphism may be a marker of the lipoprotein response to dietary intervention.

G(AnH)MTetra, a naturally occurring 1,6-anhydro muramyl dipeptide, induces granulocyte colony-stimulating factor expression in human monocytes: a molecular analysis.

N-Acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutam yl-m- diaminopimelyl-D-alanine [G (Anh)MTetra], a naturally occurring breakdown product of peptidoglycan from bacterial cell walls, was studied for its ability to induce granulocyte colony-stimulating factor (G-CSF) mRNA and protein expression in human adherent monocytes. Resting monocytes did not express G-CSF mRNA or secrete G-CSF protein. In contrast, monocytes exposed to G(Anh)MTetra showed a dose-dependent increase in G-CSF mRNA accumulation, which correlates with the secretion of G-CSF protein. Maximal levels of G-CSF mRNA were reached within 2 h of activation. Expression of G-CSF was mediated by an increase in the stability of G-CSF transcripts rather than by an increase in the transcription rate of the G-CSF gene. Experiments with the protein synthesis inhibitor cycloheximide revealed that G(Anh)MTetra-induced G-CSF mRNA expression was independent of new protein synthesis. Furthermore, it was shown that the effect of G(Anh)MTetra was regulated by a protein kinase C-dependent pathway, whereas protein kinase A and tyrosine kinases were not involved. Finally, it was shown that G(Anh)MTetra also induced G-CSF mRNA expression in human endothelial cells. The data indicate that, besides lipopolysaccharide, other naturally occurring bacterial cell wall components are able to induce G-CSF expression in different hematopoietic cells.

The regulation of interleukin 5 and interleukin 3 gene expression in human T cells.

Interleukin 5 (IL-5) and Interleukin 3 (IL-3) mRNA levels in human peripheral blood T cells were compared by semi-quantitative polymerase chain reaction (PCR) analysis. Unstimulated T Cells did not express IL-5 and IL-3 mRNA. IL-5 and IL-3 mRNA expression were similarly induced by the lectin concanavalin A (Con A). The protein kinase C (PKC) activator phorbol myristate acetate (PMA) triggered both IL-3 and IL-5 mRNA expression, whereby IL-5 and IL-3 mRNA expression was observed after 9 and 3 h treatment, respectively. Stimulation with calcium ionophore A23187 induced IL-3 mRNA expression, whereas it failed to induce IL-5 mRNA. In contrast to IL-3 mRNA, the expression of IL-5 mRNA was dependent on de novo protein synthesis, since cycloheximide (CHX) blocked the Con A plus PMA induced IL-5 mRNA expression. In contrast, cyclosporin A (CsA) inhibited but failed to completely block the expression of IL-3 and IL-5 mRNA. mRNA studies in T cell subsets revealed that the expression of IL-5 mRNA was restricted to the CD4 positive T cell subset in response to Con A plus PMA stimulation. On the other hand, IL-3 mRNA expression was noticed in both the CD4 and the CD8 positive T cell subset. These data indicate that the selective expression of IL-5 by human T cells can either be explained by activation of a selective intracellular signalling pathway or by selective activation of a T cell subset. Alternatively, both processes could be involved.

G(Anh)MTetra, a natural bacterial cell wall breakdown product, induces interleukin-1 beta and interleukin-6 expression in human monocytes. A study of the molecular mechanisms involved in inflammatory cytokine expression.

It is believed that induction of cytokine expression by bacterial cell wall components plays a role in the development and course of sepsis. However, most attention has been focused on lipopolysaccharide (LPS). We studied the ability of N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D- isoglutamyl-m-diaminopimelyl-D-alanine (G(Anh)MTetra), a naturally occurring breakdown product of peptidoglycan that is produced by soluble lytic transglycosylase of Escherichia coli, to induce cytokine expression in human monocytes. G(Anh)MTetra was found to strongly induce interleukin (IL)-1 beta and IL-6 mRNA expression after 2 h and IL-1 beta and IL-6 protein secretion after 48 h of activation. The increase in mRNA accumulation was at least partly due to an increase in the transcription rates of the respective genes and was accompanied by a strong induction of nuclear factor-kappa B and activator protein-1 transcription factor expression. Experiments using inhibitors of protein kinase C, protein kinase A, and tyrosine kinase-dependent pathways revealed that G(Anh)MTetra-induced IL-1 beta and IL-6 mRNA expression involves activation of an H7-inhibitable pathway. By using the protein synthesis inhibitor cycloheximide, it was shown that G(Anh)MTetra-induced IL-6 mRNA expression depends on the synthesis of new protein, whereas G(Anh)MTetra-induced IL-1 beta mRNA accumulation does not. When responses to G(Anh)MTetra were compared with those to LPS and muramyldipeptide (MDP), it was found that the optimal response to G(Anh)MTetra induction was similar to that of LPS but significantly higher than the response to MDP. Furthermore, maximal G(Anh)MTetra-induced IL-1 beta and IL-6 mRNA expression could be enhanced by co-stimulation with LPS or MDP, suggesting that different receptors and/or transduction pathways were involved. These results indicate that G(Anh)MTetra induces IL-1 beta and IL-6 expression in human monocytes suggesting a possible role for G(Anh)MTetra in the release of cytokines during sepsis.

Introduction of disulfide bonds into Bacillus subtilis neutral protease.

The effects of engineered disulfide bonds on autodigestion and thermostability of Bacillus subtilis neutral protease (NP-sub) were studied using site-directed mutagenesis. After modelling studies two locations that might be capable of forming disulfide bonds, both near previously determined autodigestion sites in NP-sub, were selected for the introduction of cysteines. Analysis of mutant enzymes showed that disulfide bonds were indeed formed in vivo, and that the mutant enzymes were fully active. The introduced disulfides did not alter the autodigestion pattern of the NP-sub. All mutant NP-subs exhibited decreased thermostability, which, by using reducing agents, was shown to be caused by the introduction of the cysteines and not by the formation of the disulfides. Mutants containing one cysteine exhibited intermolecular disulfide formation at elevated temperatures, which, however, was shown not to be the cause of the decreased thermostability. Combining the present data with literature data, it would seem that the introduction of disulfide bridges is unsuitable for the stabilization of proteases. Possible explanations for this phenomenon are discussed.

The supportive effects of IL-7 on eosinophil progenitors from human bone marrow cells can be blocked by anti-IL-5.

Human rIL-7 was studied for its effects on myeloid and erythroid progenitors from human bone marrow cells. IL-7 did not support the granulocytic/monocytic or erythroid lineage but exclusively stimulated eosinophil colony formation (CFU-Eo) (4 +/- 3 vs 48 +/- 17 CFU-Eo/10(5) nonadherent fraction-non-T cell (NAF-NT) cells). This supportive effect was not mediated by T cells or monocytes because similar results were obtained with or without T cell or adherent depleted cell fractions. In addition, it was shown that CD34+ sorted cells could be stimulated by IL-7 (0 vs 15 +/- 9 CFU-Eo/3 x 10(3) CD34+ cells) Furthermore studies with IL-3 or granulocyte-macrophage CSF (GM-CSF) demonstrated an additive effect on the IL-7 supported colony formation. Finally, experiments were performed with anti-IL-3, anti-GM-CSF, anti-IL-1, and anti-IL-5 to exclude the possibility that IL-7 indirectly stimulated the eosinophil progenitor cell. Anti-GM-CSF, anti-IL-1, or anti-IL-3 did not influence the supportive effects of IL-7. However, anti-IL-5 did abolish the effects of IL-7 on the eosinophil colony formation (69 +/- 15 vs 3 +/- 2 CFU-Eo/10(5) NAF-NT, n = 3). Similar results were obtained with CD34+ sorted cells. Moreover, IL-5 mRNA expression could be demonstrated in IL-7-stimulated NAF-NT cells. These data suggest that the supportive effects of IL-7 on eosinophil precursors are mediated by the endogenous release of IL-5.

Introduction of a stabilizing 10 residue beta-hairpin in Bacillus subtilis neutral protease.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product.

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. sterothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8 degrees C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostability was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B. caldolyticus.

Identification of autodigestion target sites in Bacillus subtilis neutral proteinase.

Autocatalytic degradation of purified Bacillus subtilis neutral proteinase was examined under various conditions. At elevated temperatures, under non-inhibitory conditions, mature protein was rapidly degraded, but no accumulation of specific breakdown products occurred. However, by incubating purified neutral proteinase on ice during extended periods of time, specific peptides accumulated. These peptides were analysed by SDS/PAGE and Western blotting, and the N-terminal sequences were determined for the four major peptides, which had sizes of 30, 22, 20 and 15 kDa. Sequence data identified five fission sites in the neutral proteinase, three of which were identical with autodigestion target sites in thermolysin, a thermostable neutral proteinase. Comparison of the identified fission sites of the B. subtilis neutral proteinase with the known substrate-specificity of the enzyme indicated that they were in agreement, showing a preference for the generation of fissions at the N-terminal side of large hydrophobic residues, such as leucine, isoleucine and methionine. These results suggest a high degree of similarity in the three-dimensional structures of B. subtilis neutral proteinase and thermolysin.

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease.

The role of the C-terminal Leu300 in maintaining thermal stability of the neutral protease of Bacillus subtilis was investigated. From model building studies based on the three-dimensional structure of thermolysin, the neutral protease of B. thermoproteolyticus, it was concluded that this residue is located in a hydrophobic pocket composed of residues located in the C-terminal and the middle domain. To test the hypothesis that Leu300, by contributing to a stabilizing interaction between these domains, is important for enzyme stability, several neutral protease mutants were constructed and characterized. The thermostability of the enzyme was lowered by deleting Leu300 or by replacing this residue by a smaller (Ala), a polar (Asn) or a sterically unfavourable (Ile) amino acid. Thermostability was increased upon replacing Leu300 by Phe. These results are in agreement with model-building studies. The effects on thermostability observed after mutating the corresponding Val318 in the thermostable neutral protease of B.stearothermophilus were less pronounced.

One-step affinity purification of Bacillus neutral proteases using bacitracin-silica.

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.

An economic water-jacketed vacuum-enforced mini-column system for quick separation of double and single stranded DNA.

A jacketed column system that can be applied in separation of double and single stranded DNA by hydroxylapatite chromatography is described. The construction of the column allows high flow rates and a short manipulation time. The system is simple, inexpensive, and easy to construct.