Antiproliferative function of glia maturation factor beta.

Recombinant human glia maturation factor beta (GMF-beta) reversibly inhibits the proliferation of neoplastic cells in culture by arresting the cells in the G0/G1 phase. This phenomenon is not target-cell specific, as neural and nonneural cells are equally inhibited. When tested simultaneously, GMF-beta suppresses the mitogenic effect of acidic fibroblasts growth factor (aFGF), but the two are synergistic in promoting the morphologic differentiation of cultured astrocytes. GMF-beta also counteracts the growth-stimulating effect of pituitary extract and cholera toxin on Schwann cells. The results underscore the regulatory role of GMF-beta and its intricate interaction with the mitogenic growth factors.     

DNA fingerprinting of human isolates of Salmonella enterica serotype Paratyphi B in Malaysia

AIMS: DNA fingerprinting of Salmonella enterica serotype Paratyphi B isolated in Malaysia during 1982-83, 1992 and 1996-2002 was carried out by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility tests and D-tartrate utilization tests to assess the extent of genetic diversity of these isolates in Malaysia. METHODS AND RESULTS: Eighty-six human isolates and one food isolate of Salm. Paratyphi B were analysed by PFGE, antimicrobial susceptibility tests and D-tartrate utilization tests. Sixty-five strains were D-tartrate-negative (dT-) while 22 strains were D-tartrate-positive (dT+). Thirty-seven per cent of the Salm. Paratyphi B strains were resistant to one or more antimicrobial agents. PFGE analysis clearly distinguished the dT- and dT+ strains into two clusters based on the unweighted pair group average method (UPGMA). Twenty-two XbaI-pulsotypes were observed among the 65 dT- strains while 17 XbaI-pulsotypes were observed among the 22 isolates of Salm. Paratyphi B dT+. CONCLUSIONS: The present study showed that PFGE was very discriminative with 33.7% of the strains yielding distinct fingerprints. Paratyphoid fever in Malaysia is probably caused by one predominant, endemic clone of Salm. Paratyphi B dT- with various subtypes. There was no association between the pulsotypes and the severity of the disease indicating that the severity of the disease is probably multifactorial. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing strains of Salm. Paratyphi B. This is the first report on the application of PFGE on a large collection of Salm. Paratyphi B in Malaysia.     

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.     

Identification by families of pediatric adverse events and near misses overlooked by health care providers

Background:

Identifying adverse events and near misses is essential to improving safety in the health care system. Patients are capable of reliably identifying and reporting adverse events. The effect of a patient safety reporting system used by families of pediatric inpatients on reporting of adverse events by health care providers has not previously been investigated.

Methods:

Between Nov. 1, 2008, and Nov. 30, 2009, families of children discharged from a single ward of British Columbia’s Children’s Hospital were asked to respond to a questionnaire about adverse events and near misses during the hospital stay. Rates of reporting by health care providers for this period were compared with rates for the previous year. Family reports for specific incidents were matched with reports by health care providers to determine overlap.

Results:

A total of 544 familes responded to the questionnaire. The estimated absolute increase in reports by health care providers per 100 admissions was 0.5% (95% confidence interval −1.8% to 2.7%). A total of 321 events were identified in 201 of the 544 family reports. Of these, 153 (48%) were determined to represent legitimate patient safety concerns. Only 8 (2.5%) of the adverse events reported by families were also reported by health care providers.

Interpretation:

The introduction of a family-based system for reporting adverse events involving pediatric inpatients, administered at the time of discharge, did not change rates of reporting of adverse events and near misses by health care providers. Most reports submitted by families were not duplicated in the reporting system for health care providers, which suggests that families and staff members view safety-related events differently. However, almost half of the family reports represented legitimate patient safety concerns. Families appeared capable of providing valuable information for improving the safety of pediatric inpatients.It has been estimated that adverse events occur in about 1% of children treated in hospital and that, on average, 60% of these events are preventable.¹ To increase institutional awareness of adverse events, hospitals have implemented systems to encourage health care providers to report adverse events.² The reporting of adverse events can be improved by making electronic systems for reporting readily accessible³ and by ensuring a “just culture,” which includes nonpunitive reporting policies.⁴ However, adverse events reported by health care providers account for only a small fraction of total adverse events as determined by chart review.⁵ Time pressures to treat patients, fear of punishment, lack of belief in the benefit of reporting and differing opinions of what defines a reportable event contribute to low reporting rates.⁶ However, patients and their families are readily available, keen and motivated observers who may not be subject to these reporting barriers. Family members are capable of observing and reporting adverse events in a variety of clinical settings.⁷ It is known that the interpretation of safety events and the threshold for reporting differ among health care disciplines and individual health care providers.⁶ However, it is not clear how families of pediatric patients interpret safety-related events or what their threshold would be for reporting events.The purpose of this study was to test whether the introduction of an adverse event reporting system for use by families of pediatric patients at the time of discharge from a surgical ward would significantly change the rate of reporting of adverse events by health care providers. We also evaluated the types of events that families reported, the relevance of these events with respect to patient safety, families’ desires for anonymous reporting and families’ assessments of institutional responses to reported events. We anticipated that health care providers’ reporting rates would rise with the introduction of the family reporting system, on the assumption that greater attention would be paid to reporting safety-related events on the ward. We also anticipated that families would provide useful information about safety-related events, at least some of the time. In particular, we thought that facilitating communication from the patient’s family directly to the study institution’s Quality, Safety and Outcome Improvement Department would allow more opportunities to improve safety through changes in practice.     

Development of species-specific quantitative real-time PCR primers for detecting anginosus group streptococci based on the rpoB

In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene for detecting anginosus group streptococci (AGS), Streptococcus anginosus, S. constellatus, and S. intermedius. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 76 strains regarding 44 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of these species-specific qPCR primers was 40 fg at below a cycle threshold (C_T) value of 35. These results suggest that AGS species-specific qPCR primers are suitable for applications in epidemiological studies associated with infectious diseases related to AGS.     

Biochemical analysis of a Chinese cabbage phytocystatin-1

The phytocystatins are inhibitors of papain-like cysteine proteinases that are implicated in defense mechanisms and the regulation of protein turnover. BCPI-1, a Brassica rapa (Chinese cabbage) phytocystatin isolated from flower buds, contains an extended C-terminal region that contains a single Cys residue at position 102. In an effort to investigate the role of the C-terminus and this Cys residue in BCPI-1 activity, purified recombinant proteins of BCPI-1, including wild-type BCPI-1 (wtBCPI-1), N-terminus BCPI-1 (BCPI-1??C), C-terminus BCPI-1 (BCPI-1??N), and BCPI-1 with a single Cys residue exchange to Ser (BCPI-1C102S), were generated and their inhibitory activities against papain were investigated. Kinetic analysis revealed that the monomeric forms of wtBCPI-1 (K _i = 6.84 ± 0.3 × 10^?8 M) inhibited papain more efficiently than the dimeric forms of wtBCPI-1 (K _i = 1.01 ± 0.5 × 10^?7 M). Experiments with recombinant BCPI-1C102S demonstrated that the dimerization of wtBCPI-1 caused by the formation of an intermolecular disulfide bond at the cysteine residue. The inhibitory activity of the recombinant proteins, except BCPI-1??N, was reduced in the pH range of 7.0?C11.5 and was highly stable over a wide range of temperatures. Thus, dimerization mediated by the cysteine residue in the extended C-terminal region and alkaline conditions reduced the inhibitory activity of BCPI-1.     

Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics

Abstract

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein–ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC₅₀ was measured (IC₅₀ = 1.738?±?0.041?mM), and the dissociation constant was determined (K_{i Slope} = 0.46?±?0.04?mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. Abbreviations AKG alpha-ketoglutaric acid

pNPG 4-nitrophenyl-α-d-glucopyranoside

ANS 1-anilinonaphthalene-8-sulfonate

MD molecular dynamics.

Communicated by Ramaswamy H. Sarma