Circulating pathogen-associated molecular pattern - binding proteins and High Mobility Group Box protein 1 in nascent metabolic syndrome: implications for cellular Toll-like receptor activity.

OBJECTIVE: The Metabolic Syndrome, (MetS) a global epidemic, is a state of low grade chronic inflammation and confers an increased risk for diabetes and CVD. We have previously reported increased activity of the pathogen recognition receptors, Toll-like receptors (TLRs), TLR2 and TLR4 in MetS. We hypothesized that increased TLR activity in MetS is due in part to increased levels of circulating PAMP-binding proteins, soluble CD14 (sCD14), lipopolysaccharide binding protein (LBP) and the damage associated molecular pattern (DAMP), High Mobility Group Box protein 1 (HMGB-1). METHODS: We measured sCD14, LBP and HMGB-1 in fasting plasma from nascent MetS (n = 37) and healthy control subjects (n = 32) by ELISA. We also investigated the effects of sCD14 and LBP on TLR4 activity in human aortic endothelial cells (HAECs). RESULTS: Following adjustment for body mass index and waist circumference, sCD14, LBP and HMGB-1 levels remained significantly increased in MetS. Also their levels increased with increasing numbers of MetS risk factors. Only sCD14 correlated significantly with monocyte TLR4 protein and activity. None of these soluble biomarkers correlated with TLR2 protein. Both sCD14 and HMGB-1 correlated significantly with HOMA-IR. In LPS primed HAECs, sCD14 compared to LBP, resulted in a greater increase in both TLR4 abundance and inflammatory biomediators (NF-κB, IL-1ß, IL-8 and TNF-α). CONCLUSION: Thus, we make the novel observation that sCD14 reflects increased monocyte TLR4 protein and activity in nascent MetS and by contributing to increased cellular inflammation could explain, in part, the increased risk for diabetes and CVD.

The hexosamine biosynthetic pathway can mediate myocardial apoptosis in a rat model of diet-induced insulin resistance.

AIMS: Type 2 diabetes is characterized by deranged metabolic pathways that may result in cardiovascular complications. For example, hyperglycaemia promotes flux through the hexosamine biosynthetic pathway (HBP) leading to greater O-GlcNAcylation of target proteins, with pathophysiological outcomes. This study investigated mechanisms whereby increased HBP flux elicits myocardial apoptosis in a rat model of diet-induced hyperglycaemia/insulin resistance. METHODS: Four-week-old male Wistar rats were fed a high-fat diet (86 days) after which insulin resistance was assessed vs. matched controls. Oxidative stress was evaluated, and apoptotic peptide levels, BAD phosphorylation and overall O-GlcNAcylation assessed by immunoblotting. Protein-specific O-GlcNAcylation and BAD-Bcl-2 dimerization were determined by immunoprecipitation and Western blotting. RESULTS: Rats consuming the high-fat diet exhibited a moderate elevation in body weight, higher fasting insulin and glucose levels, and insulin resistance vs. controls. Overall protein O-GlcNAcylation was increased in hyperglycaemic/insulin-resistant hearts. In parallel, myocardial peptide levels of apoptotic markers (caspase-3, cytochrome-c, BAD) were significantly higher with insulin resistance. To gain mechanistic insight into our findings, we evaluated O-GlcNAcylation of BAD, a pro-apoptotic Bcl-2 homolog. Here we found increased BAD O-GlcNAcylation and decreased BAD phosphorylation (Ser136) in hyperglycaemic/insulin-resistant rat hearts. These data are in agreement with competition by phosphorylation and O-GlcNAcylation for the same or neighbouring site(s) on target proteins. Moreover, we observed increased BAD-Bcl-2 dimerization in hyperglycaemic/insulin-resistant hearts. CONCLUSION: The main finding of this study is that increased apoptosis in hyperglycaemic/insulin-resistant hearts can also be mediated through HBP-induced BAD O-GlcNAcylation and greater formation of BAD-Bcl-2 dimers (pro-apoptotic).

Lack of anticonvulsant tolerance to the neuroactive steroid pregnanolone in mice.

GABA-potentiating neuroactive steroids such as pregnanolone have potent protective effects in the pentylenetetrazol seizure test. We sought to determine if tolerance develops to the anticonvulsant activity of pregnanolone with chronic administration. Mice were treated with two daily injections of a 2 x ED50 dose of pregnanolone (25 mg/kg, i.p.) for 7 days. On the day after the chronic treatment protocol, the dose-response relationship for protection in the pentylenetetrazol seizure test was obtained. The ED50 value after the chronic treatment protocol was not significantly different from that in naive mice (12 mg/kg), indicating that tolerance does not develop to the anticonvulsant activity of pregnanolone. In subsequent experiments, we extended the chronic treatment protocol to 14 days with three daily injections of pregnanolone (25 mg/kg, i.p.). Again, no tolerance was observed (ED50, 13 mg/kg). The anticonvulsant activity of pregnanolone was well correlated with plasma levels in both the naive and chronically (14 day) treated mice. The estimated plasma concentrations of pregnanolone representing threshold (10%) protection (125-150 ng/ml) and 50% protection (575-700 ng/ml) were similar in naive and chronically treated animals. In both chronically treated and naive animals, plasma levels of pregnanolone declined rapidly (t1/2, 16-19 min) and there was a corresponding reduction in the anticonvulsant activity. Our results with pregnanolone suggest that tolerance does not develop to the anticonvulsant activity of neuroactive steroids as it does with other GABA potentiating drugs such as benzodiazepines, supporting the potential clinical utility of neuroactive steroids in chronic seizure therapy.

Isolation, primary sequence determination, and disulfide bond structure of cyanovirin-N, an anti-HIV (human immunodeficiency virus) protein from the cyanobacterium Nostoc ellipsosporum.

A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga) Nostoc ellipsosporum, purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.

Detection of pathogenic and non-pathogenic bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The proteins isolated from the whole cells of bacterial pathogens and related non-pathogenic simulants were analyzed directly, with minimal sample preparation by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Inspection of mass spectrometric profiles obtained from direct MALDI-MS analysis of the protein extracts revealed specific biomarkers for individual bacterial cells. The observed biomarkers enabled us not only to detect pathogenic bacteria (Bacillus anthracis, Yersinia pestis and Brucella meliteusis), but also to distinguish them from the corresponding non-pathogenic species. By examining a series of strains of several Bacillus species (anthracis, thuringiensis, cereus and subtilis), it was possible to derive genus, species and strain-specific biomarkers from the measured molecular masses of the intact proteins. Additional series of biomarkers were obtained from direct mass spectrometric analysis of tryptic digests of the protein extracts. The application of this technique for rapid chemotaxonomic classification of microorganisms is demonstrated.