Suppression of the accumulation of triosephosphates and increased formation of methylglyoxal in human red blood cells during hyperglycaemia by thiamine in vitro

The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications. A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate. To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions. In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal. The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate. Biochemical changes implicated in the development of diabetic complications were thereby prevented. This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.     

Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy

The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.     

Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.     

A novel osteoblast-derived C-type lectin that inhibits osteoclast formation

We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.     

Arginine biosynthesis in Campylobacter jejuni TGH9011: determination of the argCOBD cluster

Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. Group V is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.     

The molecular basis of multiple vector insertion by gene targeting in mammalian cells

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.     

Natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities

This article reviews compounds of botanical origin which are capable of lowering plasma levels of glucose and cholesterol and blood pressure, as well as compounds inhibiting atherosclerosis and thrombosis. Hypoglycemic natural products comprise flavonoids, xanthones, triterpenoids, alkaloids, glycosides, alkyldisulfides, aminobutyric acid derivatives, guanidine, polysaccharides and peptides. Hypotensive compounds include flavonoids, diterpenes, alkaloids, glycosides, polysaccharides and proteins. Among natural products with hypocholesterolemic activity are beta-carotene, lycopene, cycloartenol, beta-sitosterol, sitostanol, saponin, soybean protein, indoles, dietary fiber, propionate, mevinolin (beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitor) and polysaccharides. Heparins, flavonoids, tocotrienols, beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins), garlic compounds and fungal proteases exert antithrombotic action. Statins and garlic compounds also possess antiatherosclerotic activity.     

Hypercholesterolemia and its correlates in Taiwanese elderly people

BACKGROUND: Our study used data collected in Chung-Shing-Shin-Tseun community in Taiwan in May 1998 to evaluate the relationship between hypercholesterolemia and the cardiovascular and sociodemographic risk factors in elderly people. METHODS: Individuals aged 65 and over were recruited as study subjects. A total of 1,093 persons, out of 1,774 registered residents, were contacted in face-to-face interview. The response rate was 61.6 percent. However, only 586 respondents took blood tests and completed questionnaires. Analysis in this study was based on these 586 subjects. The t-test, chi-square analysis, and multivariate logistic regression were used to study the significant correlates of hypercholesterolemia. RESULTS: Our results showed that 66 percent were men and 34 percent were women. The mean age was 73.1 +/- 5.3 years. The mean total cholesterol value was 5.1 +/- 1 mmol/l in elderly men and 5.5 +/- 1.3 mmol/l in elderly women. The proportions of hypercholesterolemia were 43.7 percent in elderly men and 59.6 percent in elderly women. After controlling the other covariates, the multivariate logistic regression analysis showed that the significant related factors of hypercholesterolemia were age, hypertriglyceridemia, and hyperuricemia. No significant association was found between hypercholesterolemia and gender, obesity, high systolic pressure, high diastolic pressure, hyperglycemia, educational level, retirement status, or marital status. CONCLUSION: Hypercholesterolemia is significantly associated with hypertriglyceridemia and hyperuricemia in elderly people. It is important to determine other metabolic disorders if one metabolic disorder is disclosed.