The location of a closed channel gate in the GABAA receptor channel

Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABAA receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-A resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9' to 14' but the M1-M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABAA receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of alpha1Val257 (2') near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location.     

Channel opening by anesthetics and GABA induces similar changes in the GABAA receptor M2 segment

For many general anesthetics, their molecular basis of action involves interactions with GABA(A) receptors. Anesthetics produce concentration-dependent effects on GABA(A) receptors. Low concentrations potentiate submaximal GABA-induced currents. Higher concentrations directly activate the receptors. Functional effects of anesthetics have been characterized, but little is known about the conformational changes they induce. We probed anesthetic-induced conformational changes in the M2 membrane-spanning, channel-lining segment using disulfide trapping between engineered cysteines. Previously, we showed that oxidation by copper phenanthroline in the presence of GABA of the M2 6' cysteine mutants, alpha(1)T261Cbeta(1)T256C and alpha(1)beta(1)T256C resulted in formation of an intersubunit disulfide bond between the adjacent beta-subunits that significantly increased the channels' spontaneous open probability. Oxidation in GABA's absence had no effect. We examined the effect on alpha(1)T261Cbeta(1)T256C and on alpha(1)beta(1)T256C of oxidation by copper phenanthroline in the presence of potentiating and directly activating concentrations of the general anesthetics propofol, pentobarbital, and isoflurane. Oxidation in the presence of potentiating concentration of anesthetics had little effect. Oxidation in the presence of directly activating anesthetic concentrations significantly increased the channels' spontaneous open probability. We infer that activation by anesthetics and GABA induces a similar conformational change at the M2 segment 6' position that is related to channel opening.     

Bacterial L-arabinose isomerases: industrial application for D-tagatose production

D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.     

Plant genome modification by homologous recombination

The mechanisms and frequencies of various types of homologous recombination (HR) have been studied in plants for several years. However, the application of techniques involving HR for precise genome modification is still not routine. The low frequency of HR remains the major obstacle but recent progress in gene targeting in Arabidopsis and rice, as well as accumulating knowledge on the regulation of recombination levels, is an encouraging sign of the further development of HR-based approaches for genome engineering in plants.     

Plant dehydrins and stress tolerance: Versatile proteins for complex mechanisms

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.Key words: abiotic stress, dehydration stress, drought, cold acclimation, freezing tolerance, LEA proteins, dehydrins     

Nasal carriage of methicillin resistant Staphylococcus aureus and their antibiotic susceptibility patterns in children attending day-care centers

Nasal colonization with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is being increasingly reported, especially in places where people are in close contact and in reduced hygiene, such as day-care centers. In this study we investigated the frequency of MRSA colonization and their antibiotic susceptibility patterns in 1-6 years old children of day-care centers in Hamadan, West of Iran.Five hundred nasal swabs were collected from children of 27 day-care centers that had no risk factors for colonization by S. aureus. The specimens were cultured for isolation of S. aureus by standard methods. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. For evaluation of the frequency of erythromycin induced clindamycin resistance, disk approximation test (D-test) was applied.Totally, 148 (29.6%) children were colonized by S. aureus. Out of 260 male, 94 (36.2%) and of 240 female, 54 (22.5%) cases were nasal carriers of S. aureus (P value = 0.001). Six (4.1%) of the 148 S. aureus isolated from children were MRSA strains. None of MRSA and methicillin susceptible S. aureus (MSSA) was resistant to vancomycin and clindamycin. Three of the 6 strains of MRSA and 7 (4.9%) of the 142 MSSA strains were resistant to erythromycin, and D-test was positive in all of them.We conclude that the rate of colonization by S. aureus is high in children attending day-care centers but colonization with MRSA is not common in our areas. Clindamycin or trimethoprim-sulfamethoxazol could be used in mild to moderataly severe diseases caused by CA-MRSA. However, if the CA-MRSA isolates are erythromycin resistant, D-test should be carried out for detection of inducible clindamycin resistance.     

Bacterial structure and spatiotemporal distribution in a horizontal subsurface flow constructed wetland

In this study, bacterial community structure in a horizontal subsurface flow constructed wetland (HSF-CW) planted with Phragmites australis was investigated using the 16S rRNA cloning–sequencing technique. Two layer depths were considered: the rhizosphere zone (RH) and the deep-layer zone (DL) in different sampling periods. Bacteria-specific primers 008F and 1492R were used to amplify the 16S rRNA genes and construct six clone libraries. A total of 1,284 individual sequences were used to assess the HSF-CW diversity. Phylogenetic analysis of RH and DL clone libraries shows that 41.57 and 42.17 % of the 16S rRNA sequences are affiliated with the Proteobacteria in the RH and the DL, respectively. The remaining major phylogenetic groups are Bacteroidetes, Planctomycetes, and Chloroflexi with 11.78, 9.36, and 7.6 %, respectively, in the RH and 11.38, 6.48, and 7.65 % in the DL, respectively. Minor divisions such as Verrucomicrobia, TM7, Nitrospira, and Gemmatimonadetes represented <6 % of the total sequences, while 14.2 % were unidentified Bacteria. Among the Proteobacteria, the Alphaproteobacteria subclass is represented in both locations, while the Deltaproteobacteria and Gammaproteobacteria subclasses were predominant in the RH and the DL, respectively. Results suggest that Archaea and Bacteria in the HSF-CW are the essential actors in the nitrogen cycle and that the established microbial community is efficient in nitrogen removal from wastewater.