65Zn2+ Transport by lobster hepatopancreatic lysosomal membrane vesicles

In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.     

Mapping data elements to terminological resources for integrating biomedical data sources

Background

Data integration is a crucial task in the biomedical domain and integrating data sources is one approach to integrating data. Data elements (DEs) in particular play an important role in data integration. We combine schema- and instance-based approaches to mapping DEs to terminological resources in order to facilitate data sources integration.

Methods

We extracted DEs from eleven disparate biomedical sources. We compared these DEs to concepts and/or terms in biomedical controlled vocabularies and to reference DEs. We also exploited DE values to disambiguate underspecified DEs and to identify additional mappings.

Results

82.5% of the 474 DEs studied are mapped to entries of a terminological resource and 74.7% of the whole set can be associated with reference DEs. Only 6.6% of the DEs had values that could be semantically typed.

Conclusion

Our study suggests that the integration of biomedical sources can be achieved automatically with limited precision and largely facilitated by mapping DEs to terminological resources.

     

Evidence of self-sustained motoneuron firing in young and older adults.

Motoneurons demonstrate a type of self-sustained firing behavior that seems to be produced by a prolonged period of depolarization caused by intrinsic long-term changes in the motoneuron. Such self-sustained firing behavior has previously been reported in human motor units. The purpose of the present study was to investigate the occurrence of self-sustained firing behavior in older adults. Eight young (mean age 24 yrs) and eight older (mean age 73 yrs) individuals participated in the investigation. While subjects produced light dorsiflexion contractions, a brief vibration stimulus was applied to the tibialis anterior muscle. Motor unit recordings were also obtained from the tibialis anterior muscle. Self-sustained firing behavior was evidenced by the appearance of new motor unit recruitment following vibration, even as the motor units that fired before the vibratory stimulus maintained a steady firing rate. The proportion of motor units exhibiting self-sustained firing activity was similar in both young and older adults (approx. 23% of trials). We conclude that self-sustained firing behavior is a ubiquitous phenomenon that does not seem to be affected by the aging process.     

Characteristics of rabbit muscle adenylate kinase inhibition by ascorbate

Earlier studies [1-3] showed that of the glycolytic enzymes, the muscle isozymes PFK-1, LDH, and AK were inhibited by ascorbic acid. These studies on the characteristics of the inhibition of RMAK by ascorbate are part of a hypothesis [3] that ascorbate facilitates the storage of skeletal muscle glycogen by inhibiting glycolysis when the muscle is at rest. These studies examine conditions for RMAK inhibition, prevention of inhibition, and reversal of ascorbate inhibition. We found that the concentration of RMAK was an important condition for inhibition. Above 200 nM RMAK, inhibition by ascorbate could not be demonstrated and below that concentration RMAK became increasingly sensitive to ascorbate inhibition. Associated with increased sensitivity to inhibition by ascorbate is a deviation from a linear to a concave relationship between low RMAK concentrations and enzyme activity. At low RMAK concentrations, the concave relationship becomes convex in the presence of muscle aldolase. In addition, aldolase reverses inhibitions by ascorbate. A comparison of inhibition of RMAK byascorbate and inhibition of LDH-m4 [3] is discussed. Other proteins prevent RMAK inhibition but do not reverse inhibition by ascorbate. The role of RMAK as a factor in the control of the rate of glycolysis is presented as is the role of compartmentalization with respect to the proposed role for ascorbate inhibition.     

Role of deficient type III interferon-lambda production in asthma exacerbations

Rhinoviruses are the major cause of asthma exacerbations, and asthmatics have increased susceptibility to rhinovirus and risk of invasive bacterial infections. Here we show deficient induction of interferon-lambdas by rhinovirus in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of rhinovirus-induced asthma exacerbation and virus load in experimentally infected human volunteers. Induction by lipopolysaccharide in asthmatic macrophages was also deficient and correlated with exacerbation severity. These results identify previously unknown mechanisms of susceptibility to infection in asthma and suggest new approaches to prevention and/or treatment of asthma exacerbations.     

Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori

BACKGROUND: Amoxicillin-based therapies are highly effective for the treatment of Helicobacter pylori infections, but the efficacy may decrease as the incidence of amoxicillin resistance is increasing. So far, the molecular mechanism underlying stable amoxicillin resistance has only been identified for a few naturally occurring amoxicillin-resistant (Amx) H. pylori isolates, and is mediated by mutations in penicillin-binding protein 1A (PBP1A). In this study the molecular mechanism underlying amoxicillin resistance of seven additional Amx H. pylori isolates has been established. METHODS: H. pylori strain 26695 (minimal inhibitory concentration (MIC) 0.125 mg/l) was naturally transformed with total DNA and pbp1A polymerase chain reaction (PCR) products from the seven Amx H. pylori isolates, and the MIC of amoxicillin and pbp1A gene sequence of the obtained Amx transformants were determined. RESULTS: Replacement of the wild-type pbp1A gene of H. pylori reference strain 26695 by the pbp1A gene of the Amx H. pylori isolates resulted in an increased MIC (0.5-1.0 mg/l). Sequence analysis of the smallest PBP1A fragments able to transfer the resistance indicated that several amino acid substitutions in or adjacent to the second (SKN402-404) and third (KTG555-557) conserved penicillin-binding protein motifs (PBP-motifs) mediate amoxicillin resistance in H. pylori. This was confirmed by site-directed mutagenesis using oligonucleotides that contained defined mutations in or adjacent to these PBP-motifs. CONCLUSION: In naturally occurring Amx H. pylori isolates, amoxicillin resistance is mediated by various mutational changes located in or adjacent to the second and third PBP-motifs of the PBP1A. Although we cannot exclude the role of the other genes in amoxicillin resistance, it is likely that multiple mutational changes in the PBP1A gene are the predominant cause of amoxicillin resistance in H. pylori. The findings of this study currently preclude the rapid detection of amoxicillin resistance in H. pylori by molecular tests.     

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.     

A rapid functional assay for the human trace amine-associated receptor 1 based on the mobilization of internal calcium

The molecular targets for trace amines (TAs) such as p-tyramine and beta-phenylethylamine have been recently discovered and have been shown to comprise a family of G-protein-coupled receptors based on DNA sequence homologies. These have been termed trace amine-associated receptors (TAARs) because TAs do not activate all of the identified receptors. Because TA may be involved in modulating a variety of behaviors including mood, cognition, and addiction, it is of interest to discover novel ligands for TAARs to probe the role TAs play in brain function. Pharmacophore development for the G(s)-coupled human TAAR1 (hTAAR1) would be aided by a rapid functional assay amenable to screening libraries of compounds. Accordingly, the authors used RD-HGA16 CHO-1 cells from Molecular Devices, which stably express the promiscuous G(q), G(alpha16), to create a cell line stably expressing hTAAR1, thereby coupling receptor activation to the mobilization of internal calcium. They used this cell line to develop a homogenous fluorometric imaging plate reader-based assay using the Calcium 3 fluorescent dye. The EC50 and Emax data obtained for known TAs are in close agreement with previous work using transient hTAAR1 expression systems or a chimeric receptor. These data indicate that the hTAAR1 retains its reported pharmacological characteristics when coupled to G(alpha16).