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PubMed2005

In silico analysis of crustacean hyperglycemic hormone family.

Through multiple sequence alignment and phylogenetic analysis, the subgrouping of the crustacean hyperglycemic hormone (CHH) family was updated using the most complete, nonredundant sequence data set. All sequences from insects were clustered into a distinct subbranch with characters closer to CHH subfamily I. Several sequences that are controversial in their nomenclature and classification are discussed. The motif configuration of CHHs differs from that of molt-inhibiting hormone or gonad-inhibiting hormone in both N and C termini. These two motifs approach each other in tertiary structure models, and the motif preference reveals the critical roles of these regions in functional specificity. Two types of exon organizations of the CHH family genes were observed. Four-exon Chh genes were found in a wide range of pan-crustacean (crustacean and hexapod) taxa, except for the penaeid species, from which the 3-exon Chh genes were reported. Meanwhile, the 3-exon structure was found in the Mih gene and Moih genes from one brachyuran species. Combining gene scan skill and exon splicing rules found in this study, we define three more novel sequences from two insect genomes. The pattern of the exon-exon junction within the mature peptide segment is preserved in all CHH family members.

Amino Acid Motifs↗

PubMed1996

Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus.

Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent-human cell hybrids we localized the deoxyhypusine synthase gene to human chromosome 19.

Amino Acid Sequence↗

PubMed2006

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Animals↗

PubMed2004

Modeling of SEB-induced host gene expression to correlate in vitro to in vivo responses.

Detection of exposure to biological threat agents has relied on ever more sensitive methods for pathogen identification, but that usually requires pathogen proliferation to dangerous, near untreatable levels. Recent events have demonstrated that assessing exposure to a biological threat agent well in advance of onset of illness or at various stages post-exposure is invaluable among the diagnostic options. There is an urgent need for better diagnostic tools that will be sensitive, rapid, and unambiguous. Since human clinical cases of illness induced by biothreat agents are, fortunately, rare, use of animal models that closely mimic the human illness is the only in vivo option. Such studies can be very difficult and expensive; therefore, maximizing the information obtained from in vitro exposures to peripheral blood mononuclear cells (PBMCs) provide an opportunity to investigate dose/time variability in host responses. In our quest to study staphylococcal enterotoxin B (SEB) induced host gene expression patterns, we addressed two core issues using microarray analysis and predictive modeling. Our first objective was to determine gene expression patterns in human PBMCs exposed to SEB in vitro. Second, we compared the in vitro data with host responses gene expression patterns in vivo using PBMCs from an animal model of SEB intoxication that closely replicates the progression of illness in humans. We used cDNA microarrays to study global gene expression patterns in piglets intoxicated with SEB. We applied a supervised learning method for class prediction based on the k-nearest neighbor algorithm for the data obtained in piglets exposed to SEB in vivo against a training data set. This data set included gene expression profiles derived from in vitro exposures to eight different pathogens (Bacillus anthracis, Yersinia pestis, Brucella melitensis, SEB, cholera toxin, Clostridium botulinum toxin A, Venezuelan equine encephalitis, and Dengue-2) in PBMCs. We found that despite differences in gene expression profiles between in vitro and in vivo systems, there exists a subset of genes that show correlations between in vitro and in vivo exposures, which can be used as a predictor of exposure to SEB in vivo.

Algorithms↗

PubMed2005

The power of the pump: mechanisms of action of P-glycoprotein (ABCB1).

Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction.

A reverse transcription/polymerase chain reaction (RT/PCR) protocol for the rapid detection and identification of flaviviruses was developed using a set of universal oligonucleotide primers. These primers correspond to sequences in the 3' non-coding region and in the NS5 gene which are highly conserved among the mosquito-borne flaviviruses. The sequences of the resulting amplified products were analysed for dengue 1, dengue 2, dengue 3, dengue 4, Japanese encephalitis, West Nile, yellow fever and Zika viruses, and compared with the published sequences of other flaviviruses. The 291-297 nucleotides corresponding to the C-terminus of NS5 gene showed 56 to 76% similarity, whereas the 3' non-coding region (190 to 421 nucleotides) showed only 20 to 36% similarity. Genetic classification of the Zika virus supported its traditional serological grouping. Recombinant plasmids containing the flavivirus sequences were used in a nucleic acid hybridization test to identify the RT/PCR products derived from viral RNA extracted from experimentally infected mosquitoes. The plasmids were dotted on a strip of nitrocellulose membrane and incubated with the RT/PCR product labelled with digoxigenin during the PCR step. This is a valuable method for the rapid and specific identification of mosquito-borne flaviviruses in biological specimens and for subsequent sequence analysis.

Animals↗

PubMedDate not supplied

A biological consequence of variation in the site of D-JH gene rearrangement.

One mechanism which generates diversity in immunoglobulin variable (V) regions is flexibility in the site of recombination among the constituent genetic elements. Within a specific antibody family (that is, a particular VH-VL combination), variability in V-D-J rearrangement not only leads to sequence diversity at the boundary of the juxtaposed genes, but also enables the total length of the third complementarity-determining region (CDR-3) of the heavy chain to be conserved. We demonstrate here that the junctional diversity inherent in rearranged immunoglobulin genes can have consequences for the biology of the immune system. Sequence analysis of the expressed immunoglobulin genes of idiotypically variant as opposed to conventional B lymphocytes of a dominant antibody family showed that the variant B cells undergo a novel D-JH joining event such that an extra amino acid is inserted into the heavy chain CDR-3. The unique D-region conformation possessed by the variant B cells accounts for previous observations which showed that variant and conventional B cells could be differentially regulated in vivo by an autologous set of idiotope-specific B lymphocytes. Our findings indicate that D-region structure can determine the expression of regulatory idiotopes and suggest that the conservation of heavy-chain CDR-3 length within an antibody family may reflect regulatory as well as functional constraints.

Animals↗

PubMed2001

Abnormal hemoglobins: laboratory methods.

Laboratory methods allowing the detection and characterization of hemoglobin variants are reviewed. Protein chemistry techniques such as isoelectrofocusing, electrophoreses under various experimental conditions, cation exchange and reversed phase high performance liquid chromatography, are the most frequently used for the detection of variants. When associated with a few additional data they may lead to a presumptive diagnosis. DNA studies are also developed in many laboratories. Final identification of a variant may be achieved either by molecular biology techniques or by protein sequence analysis in which mass spectrometry now occupies a key position.

Chemistry Techniques, Analytical↗

PubMed2005

Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger.

Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.

Aspergillus niger↗

PubMed1991

Alpha 2-macroglobulin is not an acute-phase protein in the rat testis.

Earlier studies from this laboratory have shown that Sertoli cells actively synthesize and secrete a nonspecific protease inhibitor in vitro; N-terminal sequence analysis, subunit structural analysis, and other biological studies revealed that this protein is the homolog of serum alpha 2-macroglobulin. We have now quantified the relative distribution of alpha 2-macroglobulin in the reproductive compartments and their comparison with nonreproductive organs. In serum and all nonreproductive tissues examined, the concentration of alpha 2-macroglobulin progressively decreased with advancing age. However, in both the testis and epididymis, the levels of this protein increased with the age of the animals. Serum alpha 2-macroglobulin levels were consistently higher than those in any other tissues until 60 days when the concentrations of this protein were the highest in the epididymis. The distribution of alpha 2-macroglobulin in various nonreproductive tissues from female rats was similar to that observed for male rats in that its levels tended to decrease with age. However, uterine levels of alpha 2-macroglobulin increased progressively with advancing age, whereas ovarian levels of alpha 2-macroglobulin remained relatively stable with an increase in animal age. As serum alpha 2-macroglobulin is an acute-phase protein in the rat, the response of this protein in the testis to induced inflammation was examined. The concentration of alpha 2-macroglobulin in serum rose about 150-fold after injection of fermented yeast. By contrast, the levels of this protein in rete testis fluid, which is derived exclusively from seminiferous fluid, did not change in response to inflammation. These results suggest that there might be distinctive mechanisms that regulate this protein in the systemic circulation vs. the microenvironment behind the blood-testis barrier in the seminiferous epithelium.

Acute-Phase Proteins↗

PubMed2003

Beyond the human genome: examples of nuclear receptor analysis in model organisms and potential for drug discovery.

The nuclear receptor (NR) superfamily is a large group of related, pharmacologically important receptors, comprising the targets for over 10% of commonly prescribed drugs. Cross-genome analysis of NR sequence, structure, and biological function, provides an important source of information on the function of human NRs and thus plays a role in NR drug discovery. For example, research on the pregnane X receptor (PXR; NR1I2), constitutive androstane receptor (CAR; NR1I3), hepatocyte nuclear factor 4 (HNF4; NR2A1), and farnesoid X receptor (FXR) illustrate how the study of nonhuman orthologs has provided new insights into NR biology and has increased our understanding of human NRs and orphan NR function. Understanding differences between humans and pharmacological model species may provide useful tools for the development of new NR-binding drugs.

Amino Acid Sequence↗

PubMed2005

Studies of coltivirus in China.

OBJECTIVE: The purpose of this article is to review the developments of studies of Coltivirus in China. DATA SOURCES: The data used in this review was obtained mainly from the studies of Coltivirus reported from 1990 to 2003 in China. STUDY SELECTION: Relevant articles on studies of Coltivirus in domestic and foreign literature were selected. DATA EXTRACTION: Data were maily extracted from the articles which are listed in the reference section of this review. RESULTS: Many Coltiviruses have been isolated not only from blood samples of patients with unknown fever or from cerebrospinal fluid of patients with encephalitis in Xishuangbanna area in Yunnan province, but also from mosquitoes collected in many areas in China. In some patients diagnosed as Japanese encephalitis or unknown fever, an increase of Coltivirus IgG antibody of fourfold, or more, has been detected using ELISA. Similarly, Coltivirus IgM antibody was positive in some patients with Japanese encephalitis or viral encephalitis. From most Chinese patients, except the northeastern, the isolates of Coltiviruses belong to subgroup B2, according to RT-PCR amplification of the ninth and twelfth segments of the isolates and sequence analysis of their amplicons. Some biological properties of Chinese Coltiviruses isolates are different from that of North American Coltiviruses. CONCLUSIONS: The isolates of Coltiviruses from Chinese patients are one of the common agents causing viral encephalitis and unknown fever in summer-autumn season. It might be an important public health problem due to its high isolation rate and wide distribution in China. Mosquito is the main transmission vector of the virus.

Animals↗

PubMed2005

[Reduced intensity of BuCy conditioning regimen for transplantation in the treatment of malignant hematologic diseases].

OBJECTIVE: To evaluate the use of a new reduced intensity of BuCy conditioning regimen for the treatment of malignant hematologic diseases in aged or intolerable patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) from the siblings. METHODS: Twelve patients with acute lymphoblastic leukemia (ALL, n = 4), acute myelogenous leukemia (AML-M(2), n = 2), chronic myelogenous leukemia (CML, n = 4), and myelodysplastic syndromes-refractory anemia with excess blasts (MDS-RAEB, n = 2) were intolerant of conventional myeloablative therapy because of age (older than 50 years) or having severe concurrent diseases. The median age was 49 years (range 42-64 years). Seven were males and five females. Two of the 12 patients were HLA one antigen-mismatched and the rest HLA identical with their donors. The low dosage conditioning regimen consisted of busulfan (2 mg.kg(-1).d(-1) for 3 days), Ara-C (2 g.m(-2).d(-1) for 1 or 2 times), cyclophosphamide (1.0 g.m(-2).d(-1) for 2 days) and anti-T-lymphocyte globulin (ATG 2.5 mg.kg(-1).d(-1) for 4 days, -5 - -2 day). Granulocyte colony-stimulating factor mobilized bone marrow and peripheral blood stem cells (PBSC) were harvested (1 patient using PBSC alone). All patients received cyclosporin A, short-term MTX and mycophenolate mofetil (MMF) for prophylaxis of acute graft-versus-host disease (aGVHD). DNA short tandem repeat (STR) sequence analysis, cytogenetics and molecular-biologic technique were used to analyze chimerism. RESULTS: All the patients were well tolerated the regimen, with no severe regimen related toxicity. In all the 12 patients, absolute neutrophil count > or = 0.5 x10(9)/L was achieved in 11 to 17 (median 15) days and platelet count > 20 x 10(9)/L in 10 to 23 (median 15) days after transplantation. Complete chimerism was achieved in 11 patients and 1 patient was in mixed chimerism at one month after HSCT. With a median follow-up of 14.5 (4.0-24.0) months, 7 of the 12 patients (58.0%) were alive and 5 (42.0%) of the 7 were disease-free. The probabilities of OS and DFS at 12 months were 75.0% and 48.1%. Five patients (41.6%) had aGVHD and four had local chronic GVHD with a cumulative probability of chronic GVHD of 41.5%. CONCLUSION: This reduced intensity conditioning regimen is well tolerated and safe for HSCT in the older patients or patients with severe concurrent medical conditions and can achieve full chimerism and long-term disease-free survival.

Adult↗

PubMed2006

A universal algorithm for genome-wide in silicio identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis.

Short motifs of many cis-regulatory elements (CREs) can be found in the promoters of most Arabidopsis genes, and this raises the question of how their presence can confer specific regulation. We developed a universal algorithm to test the biological significance of CREs by first identifying every Arabidopsis gene with a CRE and then statistically correlating the presence or absence of the element with the gene expression profile on multiple DNA microarrays. This algorithm was successfully verified for previously characterized abscisic acid, ethylene, sucrose and drought responsive CREs in Arabidopsis, showing that the presence of these elements indeed correlates with treatment-specific gene induction. Later, we used standard motif sampling methods to identify 128 putative motifs induced by excess light, reactive oxygen species and sucrose. Our algorithm was able to filter 20 out of 128 novel CREs which significantly correlated with gene induction by either heat, reactive oxygen species and/or sucrose. The position, orientation and sequence specificity of CREs was tested in silicio by analyzing the expression of genes with naturally occurring sequence variations. In three novel CREs the forward orientation correlated with sucrose induction and the reverse orientation with sucrose suppression. The functionality of the predicted novel CREs was experimentally confirmed using Arabidopsis cell-suspension cultures transformed with short promoter fragments or artificial promoters fused with the GUS reporter gene. Our genome-wide analysis opens up new possibilities for in silicio verification of the biological significance of newly discovered CREs, and allows for subsequent selection of such CREs for experimental studies.

Abscisic Acid↗

PubMed2006

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average half-life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

Acetic Acid↗

PubMed2006

Application of functional genomics to primate endometrium: insights into biological processes.

Endometrium is a dynamic tissue that responds on a cyclic basis to circulating levels of the ovarian-derived steroid hormones, estradiol and progesterone. Functional genomics has enabled a global approach to understanding gene regulation in whole endometrial tissue in the setting of a changing hormonal milieu. The proliferative phase of the cycle, under the influence of estradiol, has a preponderance of genes involved in DNA synthesis and cell cycle regulation. Interestingly, genes encoding ion channels and cell adhesion, as well as angiogenic factors, are also highly regulated in this phase of the cycle. After the LH surge, different gene expression profiles are uniquely observed in the early secretory, mid-secretory (window of implantation), and late secretory phases. The early secretory phase is notable for up-regulation of multiple genes and gene families involved in cellular metabolism, steroid hormone metabolism, as well as some secreted glycoproteins. The mid-secretory phase is characterized by multiple biological processes, including up-regulation of genes encoding secreted glycoproteins, immune response genes with a focus on innate immunity, and genes involved in detoxification mechanisms. In the late secretory phase, as the tissue prepares for desquamation, there is a marked up-regulation of an inflammatory response, along with matrix degrading enzymes, and genes involved in hemostasis, among others. This monograph reviews hormonal regulation of gene expression in this tissue and the molecular events occurring therein throughout the cycle derived from functional genomics analysis. It also highlights challenges encountered in using human endometrial tissue in translational research in this context.

Animals↗

PubMed2007

Characteristics and value of directed algorithms in high content screening.

High content screening requires image processing algorithms that can accurately and robustly analyze large image numbers without requiring human intervention. Thus, a suite of algorithms that are directed by an understanding of the biology being studied was developed for the optimized automated acquisition and quantitation of cellular images. Two categories of directed algorithms were developed: Developer Tools for assay development and Specific Algorithms for turnkey screening of specific biological situations. The same basic sequence of analysis steps are used in these directed algorithms: 1. Primary object identification. 2. Measurement of primary object properties. 3. Identification and measurements of associated targets. 4. Analysis of raw measurements for specific biological problems. The detailed application of these steps is guided by the biology being studied and the expected phenotypic changes. Most cell biological problems to be analyzed using high content screening can be categorized by either the phenotype of the problem or labeling pattern, or by a standard biological response behavior of the cells. This enables application of directed algorithms optimized for these categories. Examples of the use of directed algorithms for specific categories are discussed, as well as the detailed analysis steps for a specific directed algorithm.

Algorithms↗

PubMed2003

The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community.

Arabidopsis thaliana is the most widely-studied plant today. The concerted efforts of over 11 000 researchers and 4000 organizations around the world are generating a rich diversity and quantity of information and materials. This information is made available through a comprehensive on-line resource called the Arabidopsis Information Resource (TAIR) (http://arabidopsis.org), which is accessible via commonly used web browsers and can be searched and downloaded in a number of ways. In the last two years, efforts have been focused on increasing data content and diversity, functionally annotating genes and gene products with controlled vocabularies, and improving data retrieval, analysis and visualization tools. New information include sequence polymorphisms including alleles, germplasms and phenotypes, Gene Ontology annotations, gene families, protein information, metabolic pathways, gene expression data from microarray experiments and seed and DNA stocks. New data visualization and analysis tools include SeqViewer, which interactively displays the genome from the whole chromosome down to 10 kb of nucleotide sequence and AraCyc, a metabolic pathway database and map tool that allows overlaying expression data onto the pathway diagrams. Finally, we have recently incorporated seed and DNA stock information from the Arabidopsis Biological Resource Center (ABRC) and implemented a shopping-cart style on-line ordering system.

Arabidopsis↗