Pentagalloyl glucose (PGG) partially prevents arterial mechanical changes due to elastin degradation.

BACKGROUND: Elastic fibers are composed primarily of the protein elastin and they provide reversible elasticity to the large arteries. Degradation of elastic fibers is a common histopathology in aortic aneurysms. Pentagalloyl glucose (PGG) has been shown to bind elastin and stabilize elastic fibers in some in vitro studies and in vivo models of abdominal aortic aneurysms, however its effects on native arteries are not well described. OBJECTIVE: Perform detailed studies of the biomechanical effects of PGG on native arteries and the preventative capabilities of PGG for elastin degraded arteries. METHODS: We treated mouse carotid arteries with PGG, elastase (ELA), and PGG+ELA and compared the wall structure, solid mechanics, and fluid transport properties to untreated (UNT) arteries. RESULTS: We found that PGG alone decreased compliance compared to UNT arteries, but did not affect any other structural or biomechanical measures. Mild (30 sec) ELA treatment caused collapse and fragmentation of the elastic lamellae, plastic deformation, decreased compliance, increased modulus, and increased hydraulic conductance of the arterial wall compared to UNT. PGG+ELA treatment partially protected from all of these changes, in particular the plastic deformation. PGG mechanical protection varied considerably across PGG+ELA samples and appeared to correlate with the structural changes. CONCLUSIONS: Our results provide important considerations for the effects of PGG on native arteries and a baseline for further biomechanical studies on preventative elastic fiber stabilization.

An updated meta-analysis to understand the variable efficacy of drotrecogin alfa (activated) in severe sepsis and septic shock.

BACKGROUND: Significant debate continues over the efficacy of drotrecogin alpha activated (DAA) in sepsis. This updated meta-analysis provides an updated summary effect estimate and explores the reasons for outcome heterogeneity in placebo-controlled randomized clinical trials of DAA on 28-day all-cause mortality in patients with severe sepsis or septic shock. METHODS: Computer searches of MEDLINE, EMBASE, the Cochrane Library, ClinicalTrials.gov, published abstracts from major intensive care meetings and examination of reference lists were used to identify five placebo-controlled randomized clinical trials with 7260 patients. The primary endpoint was 28-day all-cause mortality. Secondary outcomes were 28-day incidence of severe bleeding and intracranial hemorrhage. RESULTS: DAA was not associated with improved 28-day all-cause mortality in patients with severe sepsis or septic shock (pooled relative risk (RR) of 0.97 [95% CI 0.83-1.14]), and is associated with an increase in serious bleeding. The significant heterogeneity in the pooled RR for 28-day mortality (I2 value of 59.4%, χ2 P-value 0.043) is no longer present with exclusion of the post-study amendment portion of PROWESS (I2 value of 0%, χ2 P-value 0.44 without PROWESS post-amendment). Using meta-regression, the best ranked predictor of outcome heterogeneity was baseline mortality in the placebo arm, which was among the highest in PROWESS. CONCLUSION: DAA is not associated with improved survival in patients with severe sepsis or septic shock. Further studies should be done to determine whether changes in supportive therapy for sepsis explain the variable efficacy of DAA in randomized controlled clinical trials observed over time.

Quantifying the biodiversity value of tropical primary, secondary, and plantation forests.

Biodiversity loss from deforestation may be partly offset by the expansion of secondary forests and plantation forestry in the tropics. However, our current knowledge of the value of these habitats for biodiversity conservation is limited to very few taxa, and many studies are severely confounded by methodological shortcomings. We examined the conservation value of tropical primary, secondary, and plantation forests for 15 taxonomic groups using a robust and replicated sample design that minimized edge effects. Different taxa varied markedly in their response to patterns of land use in terms of species richness and the percentage of species restricted to primary forest (varying from 5% to 57%), yet almost all between-forest comparisons showed marked differences in community structure and composition. Cross-taxon congruence in response patterns was very weak when evaluated using abundance or species richness data, but much stronger when using metrics based upon community similarity. Our results show that, whereas the biodiversity indicator group concept may hold some validity for several taxa that are frequently sampled (such as birds and fruit-feeding butterflies), it fails for those exhibiting highly idiosyncratic responses to tropical land-use change (including highly vagile species groups such as bats and orchid bees), highlighting the problems associated with quantifying the biodiversity value of anthropogenic habitats. Finally, although we show that areas of native regeneration and exotic tree plantations can provide complementary conservation services, we also provide clear empirical evidence demonstrating the irreplaceable value of primary forests.

Renal injury: similarities and differences in male and female rats with the metabolic syndrome.

The metabolic syndrome is complicated by nephropathy in humans and rats, and males are more affected than females. We hypothesized that female rats had reduced expression of glomerular oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1), attendant glomerular oxidant injury, and renal inflammation. Three groups, obese males (OM), obese females (OF), and lean males (LM) of first-generation (F(1)) hybrid rats derived from the Zucker fatty diabetic (ZDF) strain and the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) were studied from 6 to 41 weeks of age. OM had severe renal oxidant injury and renal failure. Their glomeruli expressed the LOX-1, and exhibited heavier accumulation of the lipid peroxide 4-hydroxynonenal (4-HNE). OM had compromised mitochondrial enzyme function, more renal fibrosis, and vascular leakage. Younger LM, OM, and OF ZS (ZDF/SHHF F(1) hybrid rat) rats, studied from 6 to 16 weeks of age, showed that unutilized renal lipids were comparable in OM and OF, although young OM had worse nephropathy and inflammation. In conclusion, glomerular LOX-1 expression is coupled to deposits of 4-HNE and glomerulosclerosis in OM. We presume that LOX-1 enhances glomerular uptake of oxidized lipids and renal inflammation, causing greater oxidant stress and severe glomerulosclerosis. In OF, renal protection from lipid oxidants appears to be conferred by blunted glomerular LOX-1 expression and renal inflammation.

Fate and effects of enrofloxacin in aquatic systems under different light conditions.

The fate and effects of fluoroquinolone antibacterials (FQ) in the environment is of significance because of apparent increased FQ resistance in environmental and clinical organisms. Here we simultaneously assessed the fate and effects of enrofloxacin (enro), an FQ often used in agriculture, on the chemistry and in situ microbial communities in receiving waters. We added enro to 25 microg/L in nine outdoor mesocosms maintained under three light conditions (in triplicate): full sunlight typical of the upper epilimnion (100% full-light exposure, FLE), partial shading typical of the lower epilimnion (28% FLE), and near-complete shading typical of the hypolimnion (0.5% FLE). Enro disappearance and ciprofloxacin (cipro) formation were monitored over time using LC/MS, and water chemistry and ambient microbial communities (using denaturing gradient gel electrophoresis; DGGE) were characterized. Enro half-lives were 0.8, 3.7, and 72 days for the 100%, 28%, and 0.5% FLE treatments, respectively, creating three distinct FQ exposure scenarios. Although FQ exposures ranged from approximately 6 microg/L for 24 h to approximately 21 microg/L for 30 days, no statistically significant exposure effects were noted in water quality or microbial communities (as indicated by whole-community 16S rDNA DGGE analysis and specific amplification of the QRDR region of gyrase A). Small changes in water chemistry were noted over time; however, changes could not be specifically attributed to FQs. In general, enro addition had minimal effect on water column conditions at the levels and durations used here; however, further investigation is needed to assess effects in aquatic sediments.

Nasal versus oral route for placing feeding tubes in preterm or low birth weight infants.

BACKGROUND: Enteral feeding tubes for preterm or low birth weight infants may be placed via either the nose or mouth. Nasal placement may compromise respiration. However, orally placed tubes may be more prone to displacement, local irritation, and vagal stimulation. OBJECTIVES: To assess the available evidence from randomised controlled trials concerning the effects of nasally placed compared with orally placed feeding tubes on growth and development, and the incidence of adverse consequences in preterm or low birth weight infants. SEARCH STRATEGY: We used the standard search strategy of the Cochrane Neonatal Review Group, including electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 2, 2004), MEDLINE (1966 - April 2004), EMBASE (1988 - April 2004), and CINAHL (1982- April 2004), conference proceedings, and previous reviews. SELECTION CRITERIA: Randomised or quasi-randomised controlled trials that compared the use of the nasal versus oral route for placing feeding tubes in preterm or low birth weight infants. DATA COLLECTION AND ANALYSIS: We extracted data using the standard methods of the Cochrane Neonatal Review Group, with separate evaluation of trial quality and data extraction by each author, and analysis of data using relative risk, risk difference and mean difference. MAIN RESULTS: We found only one eligible trial. Forty-two infants participated in the study. This primary aim of the trial was to assess the effect of oral versus nasal placement of feeding tubes on the incidence of apnea and periodic breathing in preterm infants. The trial did not report data on the pre-specified primary outcomes for this review (growth and development). REVIEWERS' CONCLUSIONS: There are insufficient data available to inform practice. A large randomised controlled trial is required to determine if the use of nasally placed feeding tubes compared with orally placed feeding tubes improves growth and development, without increasing adverse consequences in preterm or low birth weight infants.

chy1, an Arabidopsis mutant with impaired beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-CoA hydrolase.

The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal beta-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes beta-hydroxyisobutyryl-CoA. We demonstrated that a human beta-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes beta-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered beta-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.

New developments in our understanding of the beta-hydroxyacid dehydrogenases.

The beta-hydroxyacid dehydrogenases are a structurally conserved family of enzymes that catalyze the NAD(+) or NADP(+)-dependent oxidation of specific beta-hydroxyacid substrates like beta-hydroxyisobutyrate. These enzymes share distinct domains of amino acid sequence homology, most of which now have assigned putative functions. 6-phosphogluconate dehydrogenase and beta-hydroxyisobutyrate dehydrogenase, the most well-characterized members, both appear to be readily inactivated by chemical modifiers of lysine residues, such as 2,4,6-trinitrobenzene sulfonate (TNBS). Peptide mapping by ESI-LCMS showed that inactivation of beta-hydroxyisobutyrate dehydrogenase with TNBS occurs with the labeling of a single lysine residue, K248. This lysine residue is completely conserved in all family members and may have structural importance relating to cofactor binding. The structural framework of the beta-hydroxyacid dehydrogenase family is shared by many bacterial homologues. One such homologue from E. coli has been cloned and expressed as recombinant protein. This protein was found to have enzymatic activity characteristic of tartronate semialdehyde reductase, an enzyme required for bacterial biosynthesis of D-glycerate. A homologue from H. influenzae was also cloned and expressed as recombinant protein. This protein was active in the oxidation of D-glycerate, but showed approximately ten-fold higher activity with four carbon substrates like beta-D-hydroxybutyrate and D-threonine. This enzyme might function in H. influenzae, and other species, in the utilization of polyhydroxybutyrates, an energy storage form specific to bacteria. Cloning and characterization of these bacterial beta-hydroxyacid dehydrogenases extends our knowledge of this enzyme family.

Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T cell recognition.

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.

Novel beta -hydroxyacid dehydrogenases in Escherichia coli and Haemophilus influenzae.

Our laboratory has previously reported a structurally and mechanistically related family of beta-hydroxyacid dehydrogenases with significant homology to beta-hydroxyisobutyrate dehydrogenase. A large number of the members of this family are hypothetical proteins of bacterial origin with unknown identity in terms of their substrate specificities and metabolic roles. The Escherichia coli beta-hydroxyacid dehydrogenase homologue corresponding to the locus was cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant protein very specifically catalyzed the NAD(+)-dependent oxidation of d-glycerate and the NADH-dependent reduction of tartronate semialdehyde, identifying this protein as a tartronate semialdehyde reductase. Further evidence for identification as tartronate semialdehyde reductase is the observation that the coding region for this protein is directly preceded by genes coding for hydroxypyruvate isomerase and glyoxylate carboligase, two enzymes that synthesize tartronate semialdehyde, producing an operon clearly designed for d-glycerate biosynthesis from tartronate semialdehyde. The single beta-hydroxyacid dehydrogenase homologue from Haemophilus influenzae was also cloned, expressed, and purified with a 6-histidine tag. This protein also catalyzed the NAD(+)-dependent oxidation of d-glycerate but was significantly more efficient in the oxidation of four-carbon beta-hydroxyacids like d-hydroxybutyrate and d-threonine. This enzyme differs from all the presently known beta-hydroxybutyrate dehydrogenases which are well established members of the short chain dehydrogenase/reductase superfamily.

A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.

15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions.

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.

Prospective audit of cytomegalovirus-negative blood product utilization in haematology/oncology patients.

The transfusion management of immunocompromised patients often requires special blood product use such as cytomegalovirus (CMV)-negative cellular products, which are more costly than standard blood products and occasionally in short supply. We audited the use of CMV-negative products in haematology/oncology patients to determine the appropriateness of their use. A concurrent-prospective audit was conducted of all orders for CMV-negative packed red blood cell (PRBC) and platelet products in 201 haematology/ oncology patients. Once CMV serostatus was determined, orders for inappropriate CMV-negative units were cancelled, and filled as CMV untested units. During the 21-month period of this audit, the rates of inappropriate transfusions decreased for PRBC from 73.2% to 14.3% (chi2 = 68.4, P<0.001) and for platelets from 68.1% to 10.6% (chi2 = 65.6, P<0.001). The median time to cancellation of inappropriate CMV-negative orders was 11 days. This audit resulted in estimated cost savings of $16500 over the 21-month duration. Inappropriate requests for scarce and expensive blood products are substantially reduced by concurrent-prospective auditing of transfusion practice, in a manner that is both simple and cost effective.