Structural studies on a non-toxic homologue of type II RIPs from bitter gourd: Molecular basis of non-toxicity, conformational selection and glycan structure

The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPs, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPs of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The N-glycosylation of the lectin involves a plant-specific glycan while that in toxic type II RIPs of known structure involves a glycan which is animal as well as plant specific.

Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology

Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdh(Q111)/Hdh(Q111) cells, a cell model of HD and compared with STHdh(Q7)/Hdh(Q7) cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. Biological significance: In this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock -in CAG repeats in their two alleles. The 111Q containing cell line (STHdh(Q111)/Hdh(Q111)) mimics diseased condition, whereas the 7Q containing ones (STHdh(Q7)/Hdh(Q7)), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, Hdh(Q) knock -in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential expressions of proteins in these two cell lines. In this study, we annotated 76 proteins from STHdh(Q7)/Hdh(Q7) and STHdh(Q111)/Hdh(Q111) cells using 2D-gel/mass spectrometry. Next, by performing 2D-DIGE, we observed differential expressions of 31 proteins (16 upregulated and 15 downregulated) between these two cell lines. We also performed customized qRT-PCR array focused to HD pathway and found differential expressions of 47 genes (8 gene exptessions increased and 39 genes were decreased significantly). A total of 77 genes/proteins (Htt downregulated in both the studies) were found to be significantly altered from both the experimental paradigms. We validated the differential expressions of Vim, Hypk, Ran, Dstn, Hspa5 and Sod2 either by qRT-PCR or Western blot analysis or both. Out of these 77, similar trends in alteration of 19 out of 31 and 38 out of 47 proteins/genes were reported in earlier studies. Thus our study confirmed earlier observations on differential gene/protein expressions in HD and are really useful. Additionally, we observed differential expression of some novel genes/proteins. One of this was Hypk, a Htt-interacting chaperone protein with the ability to solubilize mHtt aggregated structures in cell lines. We propose that downregulation of Hypk in STHdh-Qm (Q111)/Hdh(Q111) has a causal effect towards HD pathogenesis. Thus the novel findings from our study need further research and might be helpful to understand the molecular mechanism behind HD pathogenesis. (C) 2015 Elsevier B.V. All rights reserved.

Differential dynamics of the serotonin(1A) receptor in membrane bilayers of varying cholesterol content revealed by all atom molecular dynamics simulation

The serotonin(1A) receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target in neuropsychiatric disorders. The receptor has been shown to require membrane cholesterol for its organization, dynamics and function. Although recent work suggests a close interaction of cholesterol with the receptor, the structural integrity of the serotonin(1A) receptor in the presence of cholesterol has not been explored. In this work, we have carried out all atom molecular dynamics simulations, totaling to 3s, to analyze the effect of cholesterol on the structure and dynamics of the serotonin(1A) receptor. Our results show that the presence of physiologically relevant concentration of membrane cholesterol alters conformational dynamics of the serotonin(1A) receptor and, on an average lowers conformational fluctuations. Our results show that, in general, transmembrane helix VII is most affected by the absence of membrane cholesterol. These results are in overall agreement with experimental data showing enhancement of GPCR stability in the presence of membrane cholesterol. Our results constitute a molecular level understanding of GPCR-cholesterol interaction, and represent an important step in our overall understanding of GPCR function in health and disease.

Molecular Mechanism Underlying ATP-Induced Conformational Changes in the Nucleoprotein Filament of Mycobacterium smegmatis RecA

RecA plays a central role in bacterial DNA repair, homologous recombination, and restoration of stalled replication forks by virtue of its active extended nucleoprotein filament. Binding of ATP and its subsequent recognition by the carboxamide group of a highly conserved glutamine (GIn196 in MsRecA) have been implicated in the formation of active RecA nucleoprotein filaments. Although the mechanism of ATP-dependent structural transitions in RecA has been proposed on the basis of low-resolution electron microscopic reconstructions, the precise sequence of events that constitute these transitions is poorly understood. On the basis of biochemical and crystallographic analyses of MsRecA variants carrying mutations in highly conserved Gln196 and Arg198 residues, we propose that the disposition of the interprotomer interface is the structural basis of allosteric activation of RecA. Furthermore, this study accounts, for the contributions of several conserved amino acids to ATP hydrolysis and to the transition from collapsed to extended filament forms in Mycobacterium smegmatis RecA (MsRecA). In addition to their role in the inactive compressed state, the study reveals a role for GIn196 and Arg198 along with Phe219 in ATP hydrolysis in the active extended nucleoprotein filament. Finally, our data suggest that the primary, but not secondary, nucleotide binding site in MsRecA isomerizes into the ATP binding site present in the extended nucleoprotein filament.

Modelling within-host spatiotemporal dynamics of invasive bacterial disease

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics. © 2008 Grant et al.

Clemaps: Multiple Alignment Of Protein Structures Based On Conformational Letters

CLEMAPS is a tool for multiple alignment of protein structures. It distinguishes itself from other existing algorithms for multiple structure alignment by the use of conformational letters, which are discretized states of 3D segmental structural states. A letter corresponds to a cluster of combinations of three angles formed by C-alpha pseudobonds of four contiguous residues. A substitution matrix called CLESUM is available to measure the similarity between any two such letters. The input 3D structures are first converted to sequences of conformational letters. Each string of a fixed length is then taken as the center seed to search other sequences for neighbors of the seed, which are strings similar to the seed. A seed and its neighbors form a center-star, which corresponds to a fragment set of local structural similarity shared by many proteins. The detection of center-stars using CLESUM is extremely efficient. Local similarity is a necessary, but insufficient, condition for structural alignment. Once center-stars are found, the spatial consistency between any two stars are examined to find consistent star duads using atomic coordinates. Consistent duads are later joined to create a core for multiple alignment, which is further polished to produce the final alignment. The utility of CLEMAPS is tested on various protein structure ensembles.

Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model : mechanism for deficient glutamatergic transmission?

Astrocytes are fundamental for brain homeostasis and the progression and outcome of many neuropathologies including Alzheimer's disease (AD). In the triple transgenic mouse model of AD (3xTg-AD) generalised hippocampal astroglia atrophy precedes a restricted and specific beta-amyloid (A beta) plaque-related astrogliosis. Astrocytes are critical for CNS glutamatergic transmission being the principal elements of glutamate homeostasis through maintaining its synthesis, uptake and turnover via glutamate-glutamine shuttle. Glutamine synthetase (GS), which is specifically expressed in astrocytes, forms glutamine by an ATP-dependent amination of glutamate. Here, we report changes in GS astrocytic expression in two major cognitive areas of the hippocampus (the dentate gyrus, DG and the CA1) in 3xTg-AD animals aged between 9 and 18 months. We found a significant reduction in Nv (number of cell/mm(3)) of GS immunoreactive (GS-IR) astrocytes starting from 12 months (28.59%) of age in the DG, and sustained at 18 months (31.65%). CA1 decrease of GS-positive astrocytes Nv (33.26%) occurs at 18 months. This Nv reduction of GSIR astrocytes is paralleled by a decrease in overall GS expression (determined by its optical density) that becomes significant at 18 months (21.61% and 19.68% in DG and CA1, respectively). GS-IR Nv changes are directly associated with the presence of A beta deposits showing a decrease of 47.92% as opposed to 23.47% in areas free of A beta. These changes in GS containing astrocytes and GS-immunoreactivity indicate AD-related impairments of glutamate homeostatic system, at the advanced and late stages of the disease, which may affect the efficacy of glutamatergic transmission in the diseased brain that may contribute to the cognitive deficiency.

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

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Coral Disease and Health Workshop: Coral Histopathology II

The health and continued existence of coral reef ecosystems are threatened by an increasing array of environmental and anthropogenic impacts. Coral disease is one of the prominent causes of increased mortality among reefs globally, particularly in the Caribbean. Although over 40 different coral diseases and syndromes have been reported worldwide, only a few etiological agents have been confirmed; most pathogens remain unknown and the dynamics of disease transmission, pathogenicity and mortality are not understood. Causal relationships have been documented for only a few of the coral diseases, while new syndromes continue to emerge. Extensive field observations by coral biologists have provided substantial documentation of a plethora of new pathologies, but our understanding, however, has been limited to descriptions of gross lesions with names reflecting these observations (e.g., black band, white band, dark spot). To determine etiology, we must equip coral diseases scientists with basic biomedical knowledge and specialized training in areas such as histology, cell biology and pathology. Only through combining descriptive science with mechanistic science and employing the synthesis epizootiology provides will we be able to gain insight into causation and become equipped to handle the pending crisis. One of the critical challenges faced by coral disease researchers is to establish a framework to systematically study coral pathologies drawing from the field of diagnostic medicine and pathology and using generally accepted nomenclature. This process began in April 2004, with a workshop titled Coral Disease and Health Workshop: Developing Diagnostic Criteria co-convened by the Coral Disease and Health Consortium (CDHC), a working group organized under the auspices of the U.S. Coral Reef Task Force, and the International Registry for Coral Pathology (IRCP). The workshop was hosted by the U.S. Geological Survey, National Wildlife Health Center (NWHC) in Madison, Wisconsin and was focused on gross morphology and disease signs observed in the field. A resounding recommendation from the histopathologists participating in the workshop was the urgent need to develop diagnostic criteria that are suitable to move from gross observations to morphological diagnoses based on evaluation of microscopic anatomy. (PDF contains 92 pages)

Preprocess and data analysis techniques for affymetrix DNA microarrays using bioconductor: a case study in Alzheimer disease

DNA microarray, or DNA chip, is a technology that allows us to obtain the expression level of many genes in a single experiment. The fact that numerical expression values can be easily obtained gives us the possibility to use multiple statistical techniques of data analysis. In this project microarray data is obtained from Gene Expression Omnibus, the repository of National Center for Biotechnology Information (NCBI). Then, the noise is removed and data is normalized, also we use hypothesis tests to find the most relevant genes that may be involved in a disease and use machine learning methods like KNN, Random Forest or Kmeans. For performing the analysis we use Bioconductor, packages in R for the analysis of biological data, and we conduct a case study in Alzheimer disease. The complete code can be found in https://github.com/alberto-poncelas/ bioc-alzheimer

Control of Disease in Aquaculture: Proceedings of the Nineteenth U.S.-Japan Meeting on Aquaculture, Ise, Mie Prefecture, Japan, 29-30 October 1990, Satellite Symposium: 2 November

The United States and Japanese counterpart panels on aquaculture were formed in 1969 under the United States-Japan Cooperative Program in Natural Resources (UJNR). The panels currently include specialists drawn from the federal departments most concerned with aquaculture. Charged with exploring and developing bilateral cooperation, the panels have focused their efforts on exchanging information related to aquaculture which could be of benefit to both countries. The UJNR was begun during the Third Cabinet-Level Meeting of the Joint United States-Japan Committee on Trade and Economic Affairs in January 1964, In addition to aquaculture, current subjects in the program include desalination of seawater, toxic microorganisms, air pollution, energy, forage crops, national park management, mycoplasmosis, wind and seismic effects, protein resources, forestry, and several joint panels and committees in marine resources research, development, and utilization. Accomplishments include increased communication and cooperation among technical specialists; exchanges of information, data, and research findings; annual meetings of the panels, a policy-coordinative body; administrative staff meetings; exchanges of equipment, materials, and samples; several major technical conferences; and beneficial effects on international relations. (PDF file contains 150 pages.)

Intranasal Delivery of Plasma and Platelet Growth Factors Using PRGF-Endoret System Enhances Neurogenesis in a Mouse Model of Alzheimer’s Disease

[EN] Neurodegeneration together with a reduction in neurogenesis are cardinal features of Alzheimer’s disease (AD) induced by a combination of toxic amyloid-β peptide (Aβ) and a loss of trophic factor support. Amelioration of these was assessed with diverse neurotrophins in experimental therapeutic approaches. The aim of this study was to investigate whether intranasal delivery of plasma rich in growth factors (PRGF-Endoret), an autologous pool of morphogens and proteins, could enhance hippocampal neurogenesis and reduce neurodegeneration in an amyloid precursor protein/presenilin-1 (APP/PS1) mouse model. Neurotrophic and neuroprotective actions were firstly evident in primary neuronal cultures, where cell proliferation and survival were augmented by Endoret treatment. Translation of these effects in vivo was assessed in wild type and APP/PS1 mice, where neurogenesis was evaluated using 5-bromodeoxyuridine (BdrU), doublecortin (DCX), and NeuN immunostaining 5 weeks after Endoret administration. The number of BrdU, DCX, and NeuN positive cell was increased after chronic treatment. The number of degenerating neurons, detected with fluoro Jade-B staining was reduced in Endoret-treated APP/PS1 mice at 5 week after intranasal administration. In conclusion, Endoret was able to activate neuronal progenitor cells, enhancing hippocampal neurogenesis, and to reduce Aβ-induced neurodegeneration in a mouse model of AD.

Control of bacterial disease in small scale fresh-water aquaculture: Project R0754. Project completion report December 1998 - May 1999

The work presented here represents an 18-month study to examine the relationship between environmental conditions, bacterial load in the water and bacteria levels in tissue macrophages of a range of clinically healthy freshwater fish species, farmed in a range of culture systems in Thailand and Vietnam. Preliminary assessment was made of the clinical significance of the macrophage bacterial load. The aim of this work was to improve production in fresh-water aquaculture through the control of clinical bacterial disease and subclinical infection, and to identify management practices most effective in promoting fish health. [PDF contains 37 pages]