First Report of Grey Mould Disease on Abelmoschus esculentus Caused by Botrytis cinerea in China

Okra (Abelmoschus esculentus), or Lady's finger, is a plant originating from Africa and is grown as a multipurpose crop in Asia, America and Europe nowadays. Grey mould disease was observed on okras in several okra fields in Ledong County, Hainan Province, and Shunyi District, Beijing City, during September to December in 2014 with the incidence varying from 20 to 55%. To identify the pathogen, the detailed morphological, cultural characteristics, pathogenicity test along with the DNA sequences for four gene regions ITS, G3PDH, HSP60 and RPB2 of our isolate were compared with those of some previously reported Botrytis cinerea and other similar species of the genus Botrytis. Results suggested this is the first report of grey mould disease on Abelmoschus esculentus caused by Botrytis cinerea in China.     

A monoclonal antibody that inhibits translation in Sf21 cell lysates is specific for glyceraldehyde-3-phosphate dehydrogenase

Monoclonal antibody (Mab) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to be specific for a 60-kDa form of elongation factor-1 alpha (EF-1alpha), primarily because the antigen immunoprecipitated by Mab 8B7 cross-reacted with Mab CBP-KK1, an antibody generated to EF-1alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded an additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mab 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pI 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mab 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation.     

Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis

Thirty-seven kilodalton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.     

Ionic strength dependence of F-actin and glycolytic enzyme associations: a Brownian dynamics simulations approach

The association of glycolytic enzymes with F-actin is proposed to be one mechanism by which these enzymes are compartmentalized, and, as a result, may possibly play important roles for: regulation of the glycolytic pathway, potential substrate channeling, and increasing glycolytic flux. Historically, in vitro experiments have shown that many enzyme/actin interactions are dependent on ionic strength. Herein, Brownian dynamics (BD) examines how ionic strength impacts the energetics of the association of F-actin with the glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase (aldolase), and triose phosphate isomerase (TPI). The BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases but also suggest that these interactions are significant, at physiological ionic strengths. Furthermore, BD agrees with experiments that muscle LDH, aldolase, and GAPDH interact significantly with F-actin whereas TPI does not. BD indicates similarities in binding regions for aldolase and LDH among the different species investigated. Furthermore, the residues responsible for salt bridge formation in stable complexes persist as ionic strength increases. This suggests the importance of the residues determined for these binary complexes and specificity of the interactions. That these interactions are conserved across species, and there appears to be a general trend among the enzymes, support the importance of these enzyme-F-actin interactions in creating initial complexes critical for compartmentation.     

The critical role of the loops of triosephosphate isomerase for its oligomerization,dynamics, and functionality

Triosephosphate isomerase (TIM) catalyzes the reaction to convert dihydroxyacetone phosphate into glyceraldehyde 3‐phosphate, and vice versa. In most organisms, its functional oligomeric state is a homodimer; however, tetramer formation in hyperthermophiles is required for functional activity. The tetrameric TIM structure also provides added stability to the structure, enabling it to function at more extreme temperatures. We apply Principal Component Analysis to find that the TIM structure space is clearly divided into two groups—the open and the closed TIM structures. The distribution of the structures in the open set is much sparser than that in the closed set, showing a greater conformational diversity of the open structures. We also apply the Elastic Network Model to four different TIM structures—an engineered monomeric structure, a dimeric structure from a mesophile—Trypanosoma brucei, and two tetrameric structures from hyperthermophiles Thermotoga maritima and Pyrococcus woesei. We find that dimerization not only stabilizes the structures, it also enhances their functional dynamics. Moreover, tetramerization of the hyperthermophilic structures increases their functional loop dynamics, enabling them to function in the destabilizing environment of extreme temperatures. Computations also show that the functional loop motions, especially loops 6 and 7, are highly coordinated. In summary, our computations reveal the underlying mechanism of the allosteric regulation of the functional loops of the TIM structures, and show that tetramerization of the structure as found in the hyperthermophilic organisms is required to maintain the coordination of the functional loops at a level similar to that in the dimeric mesophilic structure.     

Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus

To gain insight into the molecular determinants of thermoadaptation within the family of archaeal glyceraldehyde-3-phosphate dehydrogenases (GAPDH), a homology-based 3-D model of the mesophilic GAPDH from Methanobacterium bryantii was built and compared with the crystal structure of the thermophilic GAPDH from Methanothermus fervidus. The homotetrameric model of the holoenzyme was initially assembled from identical subunits completed with NADP molecules. The structure was then refined by energy minimization and simulated-annealing procedures. PROCHECK and the 3-D profile method were used to appraise the model reliability. Striking molecular features underlying the difference in stability between the enzymes were deduced from their structural comparison. First, both the increase in hydrophobic contacts and the decrease in accessibility to the protein core were shown to discriminate in favor of the thermophilic enzyme. Besides, but to a lesser degree, the number of ion pairs involved in cooperative clusters appeared to correlate with thermostability. Finally, the decreased stability of the mesophilic enzyme was also predicted to proceed from both the lack of charge-dipole interactions within alpha-helices and the enhanced entropy of unfolding due to an increase in chain flexibility. Thus, archaeal GAPDHs appear to be governed by thermoadaptation rules that differ in some aspects from those previously observed within their eubacterial counterparts.     

Engineering glyceraldehyde-3-phosphate dehydrogenase for switching control of glycolysis in Escherichia coli

Glycolysis has evolved to be a highly robust mechanism for maintaining the cellular metabolism of living organisms. However, relevant modifications of glycolytic activity are required to intentionally modulate cellular phenotypes. Here, we designed a platform that allows switching control of glycolysis in Escherichia coli in response to an environmental signal, in this case, temperature. This system functions by regulating the expression of gapA, which encodes glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes. Because a very low level of gapA expression is capable of maintaining cellular physiology, we also modified GAPDH through directed evolution to provide sensitive regulation of glycolytic activity. The switching control of glycolysis was successfully demonstrated by regulating the expression of engineered gapA through changes in temperature. This system offers potential control over the cell's central carbon‐metabolism switch, providing the ability to perform reprogrammed tasks with desired timing depending on environmental signals. Biotechnol. Bioeng. 2012; 109: 2612–2619. © 2012 Wiley Periodicals, Inc.     

Differential synthesis of glyceraldehyde-3-phosphate dehydrogenase polypeptides in stressed yeast cells

Abstract Three unlinked genes, TDH1, TDH2 and TDH3 , encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (triose-phosphate dehydrogenase; TDK) in the yeast Saccharomyces cerevisiae . We demonstrate that the synthesis of the three encoded TDK polypeptides (TDHa, TDHb and TDHc, respectively) is not co-ordinately regulated and that TDHa is only synthesised as cells enter stationary phase, due to glucose starvation, or in heat-shocked cells. Furthermore, the synthesis of TDHb, but not TDHc, is strongly repressed by a heat shock. Hence, the TDHa enzyme may play a cellular role, distinct from glycolysis, that is required by stressed cells.     

Structures of glyceraldehyde 3‐phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co‐infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng‐Ct co‐infections. Development of a safe, effective, and inexpensive dual therapy for Ng‐Ct co‐infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X‐ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high‐throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.     

Variation in heat shock protein expression at the latitudinal range limits of a widely‐distributed species,the Glanville fritillary butterfly (Melitaea cinxia)

Studies of heat shock response show a correlation with local climate, although this is more often across altitudinal than latitudinal gradients. In the present study, differences in constitutive but not inducible components of heat shock response are detected among populations of the Glanville fritillary butterfly Melitaea cinxia L. that exist at the species' latitudinal range limits (Finland and Spain). The study demonstrates that macroclimatic differences between these sites should cause greater exposure of the Spanish population to higher temperatures. Thermal stress treatments are used to estimate differences in the expression of four genes potentially relevant for tolerating these temperatures. For the analysis, three heat‐shock proteins and glyceraldehyde‐3‐phosphate dehydrogenase (G3PDH), a glycolysis enzyme that also modulates cell growth based on metabolic state, are chosen. Two constitutive differences are found between the sites. First, insects from Spain have higher levels of Hsp 21.4 than those from Finland regardless of thermal stress treatment; this protein is not inducible. Second, insects from Finland have higher levels of G3PDH. The two remaining Hsps, Hsp20.4 and Hsp90, show dramatic up‐regulation at higher temperatures, although there are no significant differences between insects from the different populations in either constitutive levels or inducibility. In nature, differences between the study populations likely occur in the expression of all four genes that were studied, although these differences would be directly climate‐induced in Hsp20.4 and Hsp90 and constitutive in Hsp21.4 and G3PDH. Inducibility may mitigate the need for constitutive variation in traits that adapt insects to local climate.     

Operational intensification by direct product sequestration from cell disruptates: application of a pellicular adsorbent in a mechanically integrated disruption-fluidised bed adsorption process

A novel prototype adsorbent, designed for intensified fluidised bed adsorption processes, was assembled by the emulsification coating of 4% (w/v) porous agarose upon a zirconia-silica solid core. The adsorbent, designated ZSA (particle density 1.75 g/ml, maximum pellicle depth 40 microm), was subjected to physical and biochemical comparison with the performance of two commercial adsorbents (Streamline and Macrosorb K4AX). Bed expansion qualities and hydrodynamic characteristics (N, D(axl) and B(o)) of ZSA demonstrated a marked robustness in the face of elevated velocities (up to 550 cm/h) and biomass loading (up to 30% (ww/v)) disrupted yeast cells. Cibracron Blue derivatives of the pellicular prototype (ZSA-CB), evaluated in the batch and fluidised bed recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from unclarified yeast disruptates, exhibited superior capacities and adsorption/desorption performance to the commercial derivatives. These advanced physical and biochemical properties facilitated a demonstration of the direct, mechanical coupling of bead-milling and fluidised bed adsorption in a fully integrated process for the accelerated recovery of G3PDH from yeast. The generic application of such pellicular adsorbents and integrated processes to the recovery of labile, intracellular products is discussed.     

Centrosome function in normal and tumor cells

Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process.     

High‐resolution crystal structures of the photoreceptor glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) with three and four‐bound NAD molecules

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d ‐glyceraldehyde 3‐phosphate (G3P) into 1,3‐diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)₄, and bGAPDH(NAD)₃. The bGAPDH(NAD)₄ was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)₄ and bGAPDH(NAD)₃ models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54–56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.     

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

Background  

Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR.