In vivo and in vitro function of human UDP-galactose 4 '-epimerase variants

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4'-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coil resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three alpha-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia. (C) 2011 Elsevier Masson SAS. All rights reserved.

Phosphonoacetate biosynthesis: In vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of a marine Roseobacter

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.

Electrophysiological, pharmacological and molecular profile of the transient outward rectifying conductance in neonatal rat symapthetic preganglionic neurons in vitro.

Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K + channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1-10 µM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Ka (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Ka and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 µM). Single-cell RT-PCR revealed mRNA expression for the a-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory ß-subunits was detected for Kvß2 in all SPN with differential expression of mRNA for KChIP1, Kvß1 and Kvß3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the ß-subunit Kvß2. Differential expression of the accessory ß subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons. © 2011 IBRO.

Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory-secretory candidate vaccine antigens

Excretory secretory products (ESP) of Schistosoma mansoni developing larvae are ideal potential vaccines as such molecules may readily induce host primary immune responses, and local memory immune response effectors that would target, surround, and pursue the larvae while negotiating the lung blood capillaries. We herein characterized the cytokines response ESP, e.g., SG3PDH, 14-3-3-like protein, TPX, and calpain induce in the natural context of infection, and defined the global cytokine profile conducive to effective schistosome larvae killing. Accordingly, spleen cells (SC) taken from naive, and 7-, or 9-day S. mansoni-infected mice were stimulated in vitro with the selected ESP, in a recombinant or multiple antigen peptide (MAP) form, and examined for production of T helper type (Th) 1, Th2, and Th17 cytokines, and the ability to mediate in vitro attrition of lung-stage schistosomula. The study indicated that larval ESP principally elicit Th1 and Th17 type cytokines. Recombinant SG3PDH was the only test ESP to additionally activate SC from S. mansoni-infected BALB/c mice to release higher IL-4 levels than unstimulated SC and mediate significant (P

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.