alpha 1-Acid Glycoprotein Decreases Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice

The mechanisms underlying immune deficiency in diabetes are largely unknown. In the present study, we demonstrate that diabetic mice are highly susceptible to polymicrobial sepsis due to reduction in rolling, adhesion, and migration of leukocytes to the focus of infection. In addition, after sepsis induction, CXCR2 was strongly downregulated in neutrophils from diabetic mice compared with nondiabetic mice. Furthermore, CXCR2 downregulation was associated with increased G-protein coupled receptor kinase 2 (GRK2) expression in these cells. Different from nondiabetic mice, diabetic animals submitted to mild sepsis displayed a significant augment in alpha 1-acid glycoprotein (AGP) hepatic mRNA expression and serum protein levels. Administration of AGP in nondiabetic mice subjected to mild sepsis inhibited the neutrophil migration to the focus of infection, as well as induced t-selectin shedding and rise in CD11b of blood neutrophils. Insulin treatment of diabetic mice reduced mortality rate, prevented the failure of neutrophil migration, impaired GRK2-mediated CXCR2 downregulation, and decreased the generation of AGP. Finally, administration of AGP abolished the effect of insulin treatment in diabetic mice. Together, these data suggest that AGP may be involved in reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. Diabetes 61:1584-1591, 2012

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

Genetic studies of the human plasma orosomucoid (alpha-1 acid glycoprotein)

Orosomucoid (ORM) or alpha-1 acid glycoprotein is an acute phase protein of human plasma whose function is suggested to be the competitive inhibition of cellular recognition by infective agents. Isoelectric focusing (IEF) and immunoblotting have been combined and optimum conditions have been determined for reliable classification of different ORM phenotypes. Addition of 6 M urea in an IEF gel revealed additional microheterogeneity in the ORM system which has not been previously reported. 1,667 individuals from different native ethnic groups of North and South America, Africa and New Guinea have been screened to determine the distribution of ORM alleles. Two common alleles, ORM1*1 and ORM1*2 have been observed and their frequencies were determined. Genetically independent variation consistent with expression of the ORM2 locus was observed in American and African blacks but was not observed in other sampled populations. The population allele frequencies for this new locus were 0.958, 0.025, 0.006, 0.011, for alleles ORM2*1, ORM2*2, ORM2*3, ORM2*4, respectively. Family studies confirm the autosomal codominant inheritance of the phenotypes observed at both ORM loci. ^

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.

Alpha-lipoic acid does not acutely affect resistance and conduit artery function or oxidative stress in healthy men

Aims Alpha-lipoic acid (ALA) is a thiol compound with antioxidant properties used in the treatment of diabetic polyneuropathy. ALA may also improve arterial function, but there have been scant human trials examining this notion. This project aimed to investigate the effects of oral and intra-arterial ALA on changes in systemic and regional haemodynamics, respectively. Methods In study 1, 16 healthy older men aged 58 +/- 7 years (mean +/- SD) received 600 mg of ALA or placebo, on two occasions 1 week apart, in a randomized cross-over design. Repeated measures of peripheral and central haemodynamics were then obtained for 90 min. Central blood pressure and indices of arterial stiffness [augmentation index (AIx) and estimated aortic pulse wave velocity] were recorded non-invasively using pulse wave analysis. Blood samples obtained pre- and post-treatments were analysed for erythrocyte antioxidant enzyme activity, plasma nitrite and malondialdehyde. In study 2 the effects of incremental cumulative doses (0.5, 1.0, 1.5 and 2.0 mg ml(-1) min(-1)) of intra-arterial ALA on forearm blood flow (FBF) were assessed in eight healthy subjects (aged 31 +/- 5 years) by conventional venous occlusion plethysmography. Results There were no significant changes on any of the central or peripheral haemodynamic measures after either oral or direct arterial administration of ALA. Plasma ALA was detected after oral supplementation (95% confidence intervals 463, 761 ng ml(-1)), but did not alter cellular or plasma measures of oxidative stress. Conclusions Neither oral nor intra-arterial ALA had any effect on regional and systemic haemodynamics or measures of oxidative stress in healthy men.

Characterization of the human cytochrome P450 forms involved in metabolism of tamoxifen to its alpha-hydroxy and alpha,4-dihydroxy derivatives

Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial. cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing a-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250,mu M) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100,mu M) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of a,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.

Evidence for a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as a-lipoic acid and alpha-tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha-lipoic acid and alpha-tocopherol, alone or in combination, prior to incubation with H2O2 or staurosporine. alpha-lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H2O2 and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with a-lipoic acid and/or a-tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of BcI-2, Bax, HSP70 or pERK1/2 or pJNK. alpha-lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha-lipoic acid and alpha-tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha-lipoic acid as an antioxidant.

alpha-tocopherol and alpha-lipoic acid enhance the erythrocyte antioxidant defence in cyclosporine A-treated rats

The aim of this study was to determine the effects of dietary antioxidant supplementation with alpha-tocopherol and alpha-lipoic acid on cyclosporine A (cyclosporine)-induced alterations to erythrocyte and plasma redox balance. Rats were randomly assigned to either control, antioxidant (alpha-tocopherol 1000 IU/kg diet and alpha-lipoic acid 1.6 g/kg diet), cyclosporine (25 mg/kg/day), or cyclosporine + antioxidant treatments. Cyclosporine was administered for 7 days after an 8 week feeding period. Plasma was analysed for alpha-tocopherol, total antioxidant capacity, malondialdehyde, and creatinine. Erythrocytes were analysed for glutathione, methaemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Cyclosporine administration caused a significant decrease in superoxide dismutase activity (P < 0.05 control versus cyclosporine) and this was improved by antioxidant supplementation (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P < 0.05 control versus cyclosporine + antioxidant). Animals receiving cyclosporine and antioxidants showed significantly increased (P < 0.05) catalase activity compared to both groups not receiving cyclosporine. Cyclosporine administration induced significant increases in plasma malondialdehyde and creatinine concentration (P < 0.05 control versus cyclosporine). Antioxidant supplementation prevented the cyclosporine induced increase in plasma creatinine (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P > 0.05 control versus cyclosporine + antioxidant), however, supplementation did not alter the cyclosporine induced increase in plasma malondialdehyde concentration (P > 0.05 cyclosporine versus cyclosporine + antioxidant). Antioxidant supplementation resulted in significant increases (P < 0.05) in plasma and erythrocyte alpha-tocopherol in both of the supplemented groups compared to non-supplemented groups. In conclusion, dietary supplementation with alpha-tocopherol and alpha-lipoic acid enhanced the erythrocyte antioxidant defence and reduced nephrotoxicity in cyclosporine treated animals.

Vitamin E and alpha-lipoic acid supplementation increase bleeding tendency via an intrinsic coagulation pathway

Vitamin E and a-lipoic acid are potent nutritional antioxidants, and when used together, their antioxidant capabilities are improved as a-lipoic acid recycles vitamin E. Supplementation of vitamin E has been shown to prolong platelet aggregation but the effects of vitamin E and alpha-lipoic acid supplementation on bleeding tendency have yet to be reported. Young, male rats consumed either control diet (n=5) or vitamin E and a-lipoic acid-supplemented diet (n=5) for 14 weeks. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured as markers of intrinsic and extrinsic coagulation pathways respectively in addition to lipid peroxidation (malondialdehyde). Supplementation significantly prolonged APTT (23.8 +/- 1.5 vs 31.4 +/- 1.2s, p < 0.05) compared to the con-trol diet; however, there was no significant difference in PT (27.8 +/- 1.5 vs 26.6 +/- 0.9s, p > 0.05). While vitamin E was increased (p < 0.05), there was no significant difference in plasma levels of malondialdehyde (p > 0.05). Dietary supplementation of vitamin E and alpha-lipoic acid increases bleeding tendency via inhibition of the intrinsic coagulation pathway with no change in markers of lipid peroxidation. Such supplementation could benefit patients with cardiovascular disease who exhibit elevated levels of coagulation and oxidative stress.

Design, Synthesis, Potency and Cytoselectivity of Anticancer Agents Derived by Parallel Synthesis from alpha-Aminosuberic Acid

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 mu M), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 mu M). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.

A preliminary evaluation of a novel method to monitor a triple antioxidant combination (vitamins E, C and alpha-lipoic acid) in diabetic volunteers using in vitro methaemoglobin formation

Eight otherwise healthy diabetic volunteers took a daily antioxidant supplement consisting of vitamin E (200 IU), vitamin C (250 mg) and α-lipoic acid (90 mg) for a period of 6 weeks. Diabetic dapsone hydroxylamine-mediated methaemoglobin formation and resistance to erythrocytic thiol depletion was compared with age and sex-matched non-diabetic subjects. At time zero, methaemoglobin formation in the non-diabetic subjects was greater at all four time points compared with that of the diabetic subjects. Resistance to glutathione depletion was initially greater in non-diabetic compared with diabetic samples. Half-way through the study (3 weeks), there were no differences between the two groups in methaemoglobin formation and thiol depletion in the diabetic samples was now lower than the non-diabetic samples at 10 and 20 min. At 6 weeks, diabetic erythrocytic thiol levels remained greater than those of non-diabetics. HbA1c values were significantly reduced in the diabetic subjects at 6 weeks compared with time zero values. At 10 weeks, 4 weeks after the end of supplementation, the diabetic HbA1c values significantly increased to the point where they were not significantly different from the time zero values. Total antioxidant status measurement (TAS) indicated that diabetic plasma antioxidant capacity was significantly improved during antioxidant supplementation. Conversion of α-lipoic acid to dihydrolipoic acid (DHLA) in vivo led to potent interference in a standard fructosamine assay kit, negating its use in this study. This report suggests that triple antioxidant therapy in diabetic volunteers attenuates the in vitro experimental oxidative stress of methaemoglobin formation and reduces haemoglobin glycation in vivo. © 2003 Elsevier Science B.V. All rights reserved.

Polímeros biorreabsorvíveis como substrato para cultura de células e engenharia tecidual

Biomateriais poliméricos são desenvolvidos para uso como substitutos de tecidos danificados e/ou estimular sua regeneração. Uma classe de biomateriais poliméricos são os biorreabsorvíveis, compostos que se decompõem tanto in vitro quanto in vivo. São empregados em tecidos que necessitam de um suporte temporário para sua recomposição tecidual. Dentre os vários polímeros biorreabsorvíveis, destacam-se os alfa-hidróxi ácidos, entre eles, diferentes composições do poli(ácido lático) (PLA), como o poli(L-ácido lático) (PLLA), poli(D-ácido lático) (PDLA), poli(DL-ácido lático) (PDLLA), além do poli(ácido glicólico) (PGA) e da policaprolactona (PCL). Estes polímeros são considerados biorreabsorvíveis por apresentarem boa biocompatibilidade e os produtos de sua decomposição serem eliminados do corpo por vias metabólicas. Diversas linhas de pesquisa mostram que os diferentes substratos à base de PLA estudados não apresentam toxicidade, uma vez que as células são capazes de crescer e proliferar sobre eles. Além disso, diversos tipos de células cultivadas sobre diferentes formas de PLA são capazes de se diferenciarem sobre os diferentes polímeros e passar a produzir componentes de matriz extracelular. Neste trabalho, é revisada a utilização de substratos à base de alfa-hidróxi ácidos, com destaque para diferentes formas de PLA, utilizados como substratos para cultura de células, bem como suas aplicações.