A new d-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new d-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although α-ketoisoleucine was the most favourable of all α-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

Ubiquinone (coenzyme Q10) binding sites: low dielectric constant of the gate prevents the escape of the semiquinone

The photosynthetic reaction center (RC) from purple bacteria is frequently used as a model for the interaction of ubiquinones (coenzyme Q) with membrane proteins. Single-turnover flash activation of RC leads to formation of the semiquinone (SQ) of the secondary acceptor quinone after odd flashes and quinol after even flashes. The ubiquinol escapes the binding site in 1 ms, while the SQ does not leave the binding site for at least 5 min. Observed difference between these times suggests a large energetic barrier for the SQ. However, high apparent dielectric constant in the vicinity of the quinone ring (>or=25) results in a relatively small electrostatic energy of SQ stabilization. To resolve this apparent contradiction I suggest that a significant part of the kinetic stabilization of the SQ is achieved by the special topology of the binding site in which quinone can exit the binding site only by moving its headgroup toward the center of the membrane. The large energetic penalty of transferring the charged headgroup to the membrane dielectric can explain the observed kinetic stability of the SQ.     

The human beta-defensin-3, an antibacterial peptide with multiple biological functions

A group of interesting molecules called defensins exhibit multiple functions but have been primarily recognized to possess a broad spectrum of antimicrobial activities. Studies have reported two different types of defensins (α and β) from human and animals, a cyclic θ defensin from rhesus, and several defensin-like peptides from plants. There is no amino acid sequence homology between these peptides, but they all contain three Cys-Cys disulfide linkages while the connectivities are different. Human β-defensin-3 (HβD-3) is the most recently discovered member of the host-defense peptide family that has attracted much attention. This molecule is expressed either constitutively or induced upon a challenge, and a growing evidence indicates the involvement of such molecules in adaptive immunity as well. It has been shown to exhibit antibacterial activities towards Gram-negative and Gram-positive bacteria as well as an ability to act as a chemo-attractant. Analysis of NMR structural data suggested a symmetrical dimeric form of this peptide in solution, which consists of three β strands and a short helix in the N-terminal region. While the disulfide linkages are known to provide the structural stability and stability against proteases, the biological relevance of this dimeric form was contradicted by another biological study. Since there is considerable current interest in developing HβD-3 for possible pharmaceutical applications, studies to further our understanding on the determinants of antibacterial activities and immunomodulatory function of HβD-3 are considered to be highly significant. The knowledge of its biosynthetic regulation will also help in understanding the role of HβD-3 in immunity. This article presents an overview of the expression and regulation of HβD-3 in humans, and the structure-function correlations among HβD-3 and its modified peptides are discussed emphasizing the functional importance. The future scope for studies on HβD-3 and design of short potent antimicrobial peptides, based on the native HβD-3 molecule, that do not interfere in the immunomodulatory function is also outlined.     

Statistics for disinterested scientists.

This paper presents a brief introduction to some basic statistical tools and techniques with examples applied to biological data. Perhaps after becoming more familiar with statistics and more appreciative of their power in extracting information from data the previously fearful and skeptical researcher may be more inclined to make them an integral part of his research technique and also include them in his publications. For problems of greater difficulty, consultation at the early stages, preferably before undertaking the project, with a good biostatistician is suggested.     

Molecular modeling of hair keratin/peptide complex: Using MM-PBSA calculations to describe experimental binding results

Molecular dynamics simulations of a keratin/peptide complex have been conducted to predict the binding affinity of four different peptides toward human hair. Free energy calculations on the peptides' interaction with the keratin model demonstrated that electrostatic interactions are believed to be the main driving force stabilizing the complex. The molecular mechanics-Poisson-Boltzmann surface area methodology used for the free energy calculations demonstrated that the dielectric constant in the protein's interior plays a major role in the free energy calculations, and the only way to obtain accordance between the free energy calculations and the experimental binding results was to use the average dielectric constant.     

Coumarin-3-carboxylic acid derivatives as potentiators and inhibitors of recombinant and native N-methyl-d-aspartate receptors

N-Methyl-d-aspartate receptors (NMDARs) are known to be involved in a range of neurological and neurodegenerative disorders and consequently the development of compounds that modulate the function of these receptors has been the subject of intense interest. We have recently reported that 6-bromocoumarin-3-carboxylic acid (UBP608) is a negative allosteric modulator with weak selectivity for GluN2A-containing NMDARs. In the present study, a series of commercially available and newly synthesized coumarin derivatives have been evaluated in a structure-activity relationship (SAR) study as modulators of recombinant NMDAR activity. The main conclusions from this SAR study were that substituents as large as iodo were accommodated at the 6-position and that 6,8-dibromo or 6,8-diiodo substitution of the coumarin ring enhanced the inhibitory activity at NMDARs. These coumarin derivatives are therefore excellent starting points for the development of more potent and GluN2 subunit selective inhibitors, which may have application in the treatment of a range of neurological disorders such as neuropathic pain, epilepsy and depression. Surprisingly, 4-methyl substitution of UBP608 to give UBP714, led to conversion of the inhibitory activity of UBP608 into potentiating activity at recombinant GluN1/GluN2 receptors. UBP714 also enhanced NMDAR mediated field EPSPs in the CA1 region of the hippocampus. UBP714 is therefore a novel template for the development of potent and subunit selective NMDAR potentiators that may have therapeutic applicability in the treatment of patients with cognitive deficits or schizophrenia.     

Proteomic analysis and protein carbonylation profile in trained and untrained rat muscles

Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in trained and untrained animal models. We analyzed two muscles characterized by different metabolisms: tibialis anterior and soleus. Whilst tibialis anterior is mostly composed of fast-twitch fibers, the soleus muscle is mostly composed of slow-twitch fibers. By a proteomic approach we identified 15 protein spots whose expression is influenced by training. Among them in tibialis anterior we observed a down-regulation of several glycolitic enzymes. Concerning carbonylation, we observed the existence of a high basal level of protein carbonylation. Although this level shows some variation among individual animals, several proteins (mostly involved in energy metabolism, muscle contraction, and stress response) appear carbonylated in all animals and in both types of skeletal muscle. Moreover we identified 13 spots whose carbonylation increases after training.     

Starvation causes disturbance in amino acid and fatty acid metabolism in Diporeia

The benthic amphipod Diporeia spp. was once the predominant macroinvertebrate in deep, offshore regions of the Laurentian Great Lakes. However, since the early 1990s, Diporeia populations have steadily declined across the area. It has been hypothesized that this decline is due to starvation from increasing competition for food with invasive dreissenid mussels. In order to gain a better understanding of the changes in Diporeia physiology during starvation, we applied two-dimensional gas chromatography coupled with time of flight mass spectrometry (GCXGC/TOF-MS) for investigating the responses in Diporeia metabolome during starvation. We starved Diporeia for 60 days and collected five organisms every 12 days for metabolome analyses. Upon arrival to the laboratory, organisms were flash frozen and served as control (day 0). We observed an increase in lipid oxidation and protein catabolism with subsequent declines of essential amino acids (proline, glutamine, and phenylalanine), down-regulation of glycerophospholipid and sphingolipid metabolism, and decreased polyunsaturated fatty acid abundance in nutritionally stressed Diporeia. Abundance of 1-Iodo-2-methylundecane, a metabolite closely related to insect pheromones, also declined with starvation. This research has further substantiated the applicability of GCXGC/TOF-MS as a research tool in the field of environmental metabolomics. The next step is to apply this new knowledge for evaluating nutritional status of feral Diporeia to elucidate the underlying cause(s) responsible for their decline in the Great Lakes.     

鹅圆环病毒感染性克隆的构建及其转染试验

设计带BamHⅠ酶切标记位点的引物,PCR扩增鹅圆环病毒(Goose circovirus,GoCV)全长基因组,将2个基因组顺式连接插入到pGEM-T Easy载体中,获得GoCV全长基因组头尾串联二聚体感染性克隆质粒pGEMT-2GoCV。EcoRⅠ酶切线性化pGEMT-2GoCV,与脂质体混合转染GoCV阴性鹅胚和雏鹅,常规PCR检测发现GoCV在转染鹅体内增殖,鹅胚转染组于孵出第2周和第4周检出血清阳性,且其中一个个体于4周龄扑杀时检出法氏囊阳性,雏鹅转染组于转染后2周检出血清阳性。试验进一步对扩增片段进行了BamHⅠ标记位点的检测,并应用GoCV实时荧光定量PCR方法对转染阳性样品进行了定量,结果显示阳性法氏囊组织中病毒含量为1.57×106拷贝/mg,阳性血清含病毒拷贝数在3.52×104～5.92×105拷贝/μL。综上,本试验构建的GoCV全长基因组头尾串联二聚体感染性克隆DNA可以转染鹅胚和雏鹅并增殖出带标记的GoCV克隆。     

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1 year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.