Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Type II secretory pathway for surface secretion of DraD invasin from the uropathogenic Escherichia coli Dr+ strain

The virulence of the uropathogenic Escherichia coli Dr(+) IH11128 strain is associated with the presence of Dr fimbrial structures and a DraD invasin which can act as a fimbrial capping domain at the bacterial cell surface. However, a recent study suggests that the DraD protein is surface exposed in two forms: fimbria associated and fimbria nonassociated (prone to interaction with the N-terminal extension of the DraE protein located on the fimbrial tip). The actual mechanism of DraD surface secretion is presently unknown. We identified a previously unrecognized type II secretory pathway (secreton) in the uropathogenic E. coli Dr(+) strain which is well conserved among gram-negative bacteria and used mainly for secretion of virulence determinants. An active secreton is composed of 12 to 15 different proteins, among which GspD functions as an outer-membrane channel to permit extrusion of proteins in a folded state. Therefore, we inactivated the pathway by inserting the group II intron into a gspD gene of the type II secretion machinery by site-specific recombination. DraD secretion by the E. coli Dr(+) and gspD mutant strains was determined by immunofluorescence microscopy (with antibodies raised against DraD) and an assay of cell binding between bacteria and HeLa cells. The specificity of DraD-mediated bacterial binding for the integrin receptor was confirmed by examination of the adhesion of DraD-coated beads to HeLa cells in the presence and absence of alpha(5)beta(1) monoclonal antibodies. The investigations that we performed showed that type II secretion in E. coli Dr(+) strains leads to DraD translocation at the bacterial cell surfaces.     

Identification of Ruta graveolens L. metabolites accumulated in the presence of abiotic elicitors

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, gamma-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.     

Neurochemical dementia diagnostics: State of the art and research perspectives

The aim of this review is to present current state of the art on the field of routine neurochemical dementia diagnostics (NDD) with a focus on cerebrospinal fluid (CSF) biomarkers: amyloid beta peptides, tau protein, and its phosphorylated form (pTau). After several years of experience, it is reasonably to postulate that CSF biomarkers analysis is an increasingly important tool within the early and differential diagnosis of dementia syndromes. Actual research activities are briefly discussed, too, including: (i) possibilities and limitations of the diagnosis of incipient Alzheimer's disease in preclinical stages (e.g., mild cognitive impairment), (ii) the role of multiplexing technologies in dementia biomarkers research, (iii) the role of biomarkers in differential diagnosis of dementia syndromes, (iv) approaches to improve analytical performance of available methods, and (v) research activities to identify dementia biomarkers in blood.     

Pioglitazone induces lipid accumulation in the rat heart despite concomitant reduction in plasma free fatty acid availability

Thiazolidinediones are insulin-sensitizing drugs which have been proved to be effective in the treatment of type 2 diabetes. However, the action of thiazolidinediones on myocardial metabolism is only poorly recognized. Therefore, the aim of our study was to investigate the effects of two-week pioglitazone treatment (3 mg/kg/d) on lipid and carbohydrate metabolism in the heart of rats fed on a standard chow or on a high-fat diet (HFD) for three weeks. High-fat feeding increased myocardial protein expression of all peroxisome proliferator-activated receptor (PPAR) isoforms. The greatest response was, however, noted in the case of PPARγ. Surprisingly, administration of pioglitazone induced accumulation of free fatty acids (FFA) and diacylglycerol in the heart in both groups, despite concomitant reduction in plasma FFA concentration. The content of triacylglycerol was increased only in the HFD group. Pioglitazone treatment also shifted myocardial substrate utilization towards greater contribution of glucose in both groups, as evidenced by decreased rate of palmitate oxidation and higher 2-deoxyglucose uptake and elevated glycogen content. This could induce a mismatch between the rate of myocardial fatty acid uptake and oxidation leading to increased intracellular availability of fatty acids for non-oxidative metabolic pathways like synthesis of acylglycerols. Our data suggests that thiazolidinediones improve cardiac insulin sensitivity by mechanisms other than reduction in intramyocardial lipid content.     

Structures of β-hairpin antimicrobial protegrin peptides in lipopolysaccharide membranes: mechanism of gram selectivity obtained from solid-state nuclear magnetic resonance

The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both gram-positive and gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains gram-positive activity but is 30-fold less active against gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of gram-positive bacteria. (31)P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. (13)C chemical shifts and (13)C-(1)H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. Indeed, (1)H spin diffusion from lipid chains to the peptides shows that PG-1 fully inserts into the LPS-rich membrane whereas the mutant does not. The (13)C-(31)P distances between the most hydrophobically embedded Arg of PG-1 and the lipid (31)P are significantly longer in the LPS membrane than in the POPE/POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against gram-negative bacteria. The data provide a rational basis for controlling gram selectivity of AMPs by adjusting the charge densities.     

Differences and similarities in binding of pyruvate and L-lactate in the active site of M4 and H4 isoforms of human lactate dehydrogenase

We present QM/MM calculations that show differences in geometries of active sites of M(4) and H(4) isoforms of human LDH ligated with oxamate, pyruvate or L-lactate. As the consequence of these differences, binding isotope effects of the methyl hydrogen atoms of pyruvate and l-lactate may be used to experimentally distinguish these isoforms. Based on the FEP calculations we argue that L-lactate is a better candidate for the experimental studies. Our calculations of energies of interactions of ligands with the active site residues provide explanation for the observed experimentally sensitivity to inhibition of the M(4) isoenzyme isoform and pinpoint the differences to interactions of the ligand with the histidine residue. We conclude that pyruvate interacts much stronger in the active site of H(4) than M(4) isoform and that the latter interactions are weaker than with water molecules in the aqueous solution.     

Carboxylate-induced degradation of poly(3-hydroxybutyrate)s

This communication shows that thermal degradation of poly(3-hydroxybutyrate)s (PHBs) is induced by carboxylate groups via a newly proposed E1cB mechanism. In PHBs with end groups in the form of carboxylic acid salts with Na+, K+, and Bu4N+ counterions, the proposed mechanism explains the dependence of thermal stability on the size of the counterion. The degradation via intermolecular alpha-deprotonation by carboxylate is suggested to be the main PHB decomposition pathway at moderate temperatures. The results of the present study show the ability to control the degradation and stability of poly(3-hydroxybutyrate)s as well as of their blends via chemical structure and concentration of the carboxylate polymer end groups.