Dysmyelinating and demyelinating Charcot–Marie–Tooth disease associated with two myelin protein zero gene mutations

Mutations in the myelin protein zero (MPZ) gene are the third most frequent cause of hereditary motor and sensory neuropathies (HMSN), also called Charcot–Marie–Tooth disorders (CMT). Only in case of recurrent mutations occurring in the MPZ gene is it possible to draw phenotype–genotype correlations essential for establishing the prognosis and outcomes of CMT1. We have surveyed a cohort of 67 Polish patients from CMT families with demyelinating neuropathy for mutations in the MPZ gene. In this study, we report two CMT families in which the Ile135Thr and Pro132Leu mutations have been identified for the MPZ gene. These MPZ gene mutations had not been identified hitherto in the Polish population. The Pro132Leu mutation segregates with a severe early-onset dysmyelinating–hypomyelinating neuropathy, whereas the Ile135Thr substitution is associated with the classical phenotype of CMT1. To the best of our knowledge, we present here, for the first time, morphological data obtained in two sural nerve biopsies pointing to a hypomyelination–dysmyelination process in a family harboring the Pro132Leu mutation in the MPZ gene.     

Evaluation and optimization of ethanol production from carob pod extract by <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> using response surface methodology

In this research, ethanol production from carob pod extract (extract) using Zymomonas mobilis with medium optimized by Plackett–Burman (P–B) and response surface methodologies (RSM) was studied. Z. mobilis was recognized as useful for ethanol production from carob pod extract. The effects of initial concentrations of sugar, peptone, and yeast extract as well as agitation rate (rpm), pH, and culture time in nonhydrolyzed carob pod extract were investigated. Significantly affecting variables (P = 0.05) in the model obtained from RSM studies were: weights of bacterial inoculum, initial sugar, peptone, and yeast extract. Acid hydrolysis was useful to complete conversion of sugars to glucose and fructose. Nonhydrolyzed extract showed higher ethanol yield and residual sugar compared with hydrolyzed extract. Ethanol produced (g g⁻¹ initial sugar, as the response) was not significantly different (P = 0.05) when Z. mobilis performance was compared in hydrolyzed and nonhydrolyzed extract. The maximum ethanol of 0.34 ± 0.02 g g⁻¹ initial sugar was obtained at 30°C, initial pH 5.2, and 80 rpm, using concentrations (g per 50 mL culture media) of: inoculum bacterial dry weight, 0.017; initial sugar, 5.78; peptone, 0.43; yeast extract, 0.43; and culture time of 36 h.     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Bacterial diversity of soil in the vicinity of Pindari glacier,Himalayan mountain ranges,India, using culturable bacteria and soil 16S rRNA gene clones

Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers

Mutations in fibroblast growth factor receptors are known as the genetic basis of skeletal growth disorders. The mechanism of pathogenesis, as determined by mutation-induced changes in receptor structure, interactions, and function, is elusive. Here we study three pathogenic Cys mutations, associated with either thanatophoric dysplasia or achondroplasia, in the TM domain of fibroblast growth factor receptors 3 (FGFR3). We characterize the dimerization propensities of the mutant TM domains in detergents and in lipid bilayers, in the presence and absence of reducing agents, and compare them to previous measurements of wild-type. We find that the Cys mutations increase the propensity for dimerization in detergent, with the Cys370 mutant exhibiting the highest propensity for disulfide bond formation, the Cys371 mutant having an intermediate propensity, and Cys375 the lowest. Thus, disulfide bonds readily form in detergents, with efficiency that correlates with the severity of the phenotype. In lipid bilayers, however, the Cys370 mutant, which dimerizes strongly in detergent, behaves as the wild-type, suggesting that Cys370-mediated disulfide bonds do not form between the isolated TM domains in bilayers. Thus, the nature of the hydrophobic environment plays an important role in defining the structure and flexibility of transmembrane dimers. These results and previous findings from cellular studies lead us to propose a conformational flexibility mechanism of receptor stabilization as a basis for disregulated FGFR3 signaling in thanatophoric dysplasia and achondroplasia.     

An in vitro comparative study on the reactivation of nerve agent-inhibited guinea pig and human acetylcholinesterases by oximes

The reactivation of nerve agent-inhibited acetylcholinesterase (AChE) by oxime is the most important step in the treatment of nerve agent poisoning. Since the evaluation of nerve agent antidotes cannot be conducted in humans, results from animal experiments are extrapolated to humans. Guinea pig is one of the animal models that is frequently used for conducting nerve agent antidote evaluations. Several investigations have demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited AChE. If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the reactivation of guinea pig and human AChEs inhibited by six different G and V type nerve agents. Reactivation kinetic studies with five mono- and bis-pyridinium oximes showed that oxime reactivation of nerve agent-inhibited human AChE in most cases was faster than guinea pig AChE. The most significant enhancement was observed in the reactivation of human AChE inhibited by nerve agents containing bulky side chains GF, GD, and VR, by H-series oximes HLo-7, HI-6, and ICD-585. In these cases, species-related differences observed between the two AChEs, based on the second-order reactivation rate constants, were 90- to over 400-fold. On the other hand, less than 3-fold differences were observed in the rates of aging of nerve agent-inhibited guinea pig and human AChEs. These results suggest that the remarkable species-related differences observed in the reactivation of nerve agent-inhibited guinea pig and human AChEs were not due to differences in the rates of aging. These results also suggest that guinea pig may not be an appropriate animal model for the in vivo evaluation of oxime therapy.     

Chemiluminescence of the peroxomonosulphate-cobalt(II)-aliphatic monocarboxylic acids system.

A weak chemiluminescence (CL) emission was observed due to the production of singlet oxygen ((1)O(2)) during the decomposition of peroxomonosulphate (HSO(5)(-)) catalysed by cobalt(II). Low molecular mass aliphatic monocarboxylic acids, such as formic, acetic, propionic, butyric and valeric acids, influenced the CL emission, and the reaction of aliphatic monocarboxylic acids with HSO(5)(-)/Co(2+) solution was further investigated using a flow injection analysis (FIA) CL method. The results indicated that the CL intensities of aliphatic monocarboxylic acids were improved with increase in the carbon chain length in the potassium peroxomonosulphate-cobalt(II) sulphate system. Generation of singlet oxygen was confirmed by the fact that the CL emission of aliphatic monocarboxylic acids with the HSO(5)(-)/Co(2+) solution was quenched by NaN(3), and from the CL spectrum of the reaction system. Additionally, a possible mechanism of aliphatic monocarboxylic acids CL emission enhancement was proposed.