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PubMed1991

Software and hardware integration of a microprogrammable state machine for NMR imaging.

We have integrated a commercially available microprogrammable state machine (Tecmag PULSkit) for use as a magnetic resonance pulse programmer. Providing the capability for active research environment imaging protocols, it features timing resolution of 100 nsec, ten 16-bit loop counters, and individually addressable look-up tables. This integration involved hardware and software integration with a VAX 11/750 at several levels. Hardware: Each of the three gradient channels employs three digital-to-analog converters (DACs). An 8-bit, 4-quadrant, multiplying DAC generates the gradient waveform shape. A 12-bit DAC generates the multiplying DAC scaling voltage, controlling gradient amplitude and sign. A third 12-bit DAC produces a gradient offset (shim) voltage. An eddy current compensation network is present for each gradient channel. Software: The software design philosophy was to create a flexible interface (interactive window environment), while not constraining complex manipulation of the hardware (direct use of the pulse-sequence compiler primitives and microprogramming). The software levels include (a) pulse-sequence microprogramming, (b) pulse-sequence compiler, (c) interactive parameter specification, and (d) canned pulse-sequence microcode library.

Computer Systems↗
PubMed1993

Expression and characterization of cDNAs for cecropin B, an antibacterial protein of the silkworm, Bombyx mori.

To analyze the induction mechanism of antibacterial protein gene expression, cDNAs coding for cecropin B have been cloned from the B. mori fat body cDNA library. Nucleotide sequences of two positive clones were determined and their amino acid sequences deduced. They revealed that these clones coded for the same cecropin, which is identical to purified cecropin B. However, the cDNAs contained different nucleotides at the third codon position and 5' or 3' non-coding regions. Results obtained by Northern blot analysis showed that the gene expression of B. mori cecropin B was rapidly induced by Escherichia coli and reached maximum levels 8 h after immunization. The expression of cecropin B gene occurred specifically in tissues, mainly in the fat body and hemocytes.

Amino Acid Sequence↗
PubMed2003

Elevated levels of beta defensin-1 mRNA in diabetic kidneys of GK rats.

We previously described the isolation of CDK4, representing the 3(')end of an unknown mRNA, using differential display [Biophys. Res. Commun. 232 (1997) 49]. To determine the identity of the CDK4 gene, full-length CDK4 cDNA clones were isolated from a diabetic kidney cDNA library. DNA sequencing revealed that CDK4 represents the rat beta defensin-1 gene (rBD-1). rBD-1 mRNA was detected in rat kidney, heart, lung, and skeletal muscle using RT-PCR. Using Northern blot analysis, elevated levels of the 0.5kb mRNA transcript were detected in the kidneys of 6-, 16-, 26-, and 40-week-old GK rats when compared with age-matched Wistar controls. A novel 2.0kb mRNA transcript was also detected in all kidneys examined from GK rats in none of the age-matched control kidneys. Biglycan and TGF-beta1 mRNAs were significantly up-regulated in kidneys of GK rats at 26 weeks compared to 16 and 6 weeks, showing that the kidneys of GK rats mimic the gene expression pattern described for human and experimental DN. These data suggest that overexpression of beta-defensin-1 mRNA may play a role in diabetic nephropathy.

3' Untranslated Regions↗
PubMed2006

Electrochemical and ligand binding studies of a de novo heme protein.

Heme proteins can perform a variety of electrochemical functions. While natural heme proteins carry out particular functions selected by biological evolution, artificial heme proteins, in principle, can be tailored to suit specified technological applications. Here we describe initial characterization of the electrochemical properties of a de novo heme protein, S824C. Protein S824C is a four-helix bundle derived from a library of sequences that was designed by binary patterning of polar and nonpolar amino acids. Protein S824C was immobilized on a gold electrode and the formal potential of heme-protein complex was studied as a function of pH and ionic strength. The binding of exogenous N-donor ligands to heme/S824C was monitored by measuring shifts in the potential that occurred upon addition of various concentrations of imidazole or pyridine derivatives. The response of heme/S824C to these ligands was then compared to the response of isolated heme (without protein) to the same ligands. The observed shifts in potential depended on both the concentration and the structure of the added ligand. Small changes in structure of the ligand (e.g. pyridine versus 2-amino pyridine) produced significant shifts in the potential of the heme-protein. The observed shifts correlate to the differential binding of the N-donor molecules to the oxidized and reduced states of the heme. Further, it was observed that the electrochemical response of the buried heme in heme/S824C differed significantly from that of isolated heme. These studies demonstrate that the structure of the de novo protein modulates the binding of N-donor ligands to heme.

Binding Sites↗
PubMed2003

cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai.

An alginate lyase, termed HdAly in the present paper, was isolated from the hepatopancreas of abalone, Haliotis discus hannai, by ammonium sulfate fractionation, followed by TOYOPEARL CM-650M column chromatography. Enzymatic properties of HdAly were similar to those of previously reported Haliotis and Turbo poly(M) lyases, e.g., it preferentially degraded a poly(beta-D-mannuronate)-rich substrate with an optimal pH and temperature at pH 8.0 and 45 degrees C, respectively. In order to determine the primary structure of abalone lyase that is still poorly understood, cDNAs for HdAly were cloned by PCR from the abalone hepatopancreas cDNA library and sequenced. From the nucleotide sequences of the cDNAs, the sequence of 909 bp in total was determined, and the amino acid sequence of 273 residues was deduced from the translational region of 822 bp locating at nucleotide positions 27-848. The N-terminal region of 16 residues, except for the initiation Met in the deduced sequence, was regarded as the signal peptide since it was absent in the HdAly protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretary proteins. This suggests that HdAly is initially produced as a precursor possessing the signal peptide in hepatopancreatic cells and then secreted into digestive tract as the mature form. Thus, the mature HdAly was regarded to consist of 256 residues with the calculated molecular mass of 28895.5 Da. The amino acid sequence of HdAly showed 85 and 28% identity to those of Turbo cornutus alginate lyase SP2 and the C-terminal region of Chlorella virus lyase-like protein CL2, respectively, while it showed no significant identity to those of any bacterial alginate lyases. In order to provide the basis for the structure-function studies and various applications of the abalone lyase, a bacterial expression system was constructed by means of the HdAly-cDNA and pET-3a expression plasmid. Although the active recombinant HdAly was hardly produced at a cultivation temperature 37 degrees C in Escherichia coli BL21 (DE3), a small amount of soluble and active enzyme could be produced when the temperature was lowered to 19 degrees C.

Alginates↗
PubMed2005

Molecular characterization and expression of the gene encoding aspartate aminotransferase from the Pacific oyster Crassostrea gigas exposed to environmental stressors.

A partial cDNA encoding cytosolic aspartate aminotransferase (AST) (EC 2.6.1.1) was isolated from a Crassostrea gigas digestive gland library. This sequence was used to design specific primers to amplify the AST genomic sequence. We obtained a complete gene, 5054 bp in length, encoding cytosolic AST and containing a 404 amino acid open reading frame. Phylogenetic analysis showed that C. gigas AST sequence constitutes a branch distinct from homologous sequences from other invertebrate groups. We also investigated AST mRNA expression in different tissues of oysters exposed to hydrocarbons, pesticides, hypoxia and hypo-salinity stress. The results showed that AST expression responds to hydrocarbon exposure, hypoxia and salinity stress, but not to pesticide exposure in an organ and time-specific manner. Use of AST as a potential molecular biomarker for monitoring of disturbed ecosystems is discussed.

Amino Acid Sequence↗
PubMed2006

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion.

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289-296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.

Amino Acid Sequence↗
PubMed2006

Cloning and characterization of a cDNA encoding walleye (Sander vitreum) beta-2 microglobulin.

Beta(2)-microglobulin (beta(2)m) is an essential component of the MHC class I antigen presentation pathway, necessary for heavy chain folding and peptide binding. In this study a beta(2)m cDNA isolated from a walleye head kidney library was sequenced and found to be similar to other known beta(2)m sequences. The clone encodes a predicted protein sequence of 116 residues, with a mature peptide 100 amino acids in length. This represents an inconsistency with other predicted beta(2)m protein sequences as walleye contains an additional 3 residues at the N terminus of the mature protein. Southern blot analysis showed that beta(2)m is present in one copy in the walleye genome and northern blot analysis showed expression of a 1.2Kb transcript, with high expression in kidney, spleen, gills and intestine. Characterization of this gene will enable further studies of immune function in walleye, which comprises both an important fishery and an emerging aquaculture species in Canada.

Amino Acid Sequence↗
PubMed2005

Characterization and functional significance of myotrophin: a gene with multiple transcripts.

The underlying mechanism for the development of cardiac hypertrophy that advances to heart failure is not known. Many factors have been implied to play a role in this process. Among others, we have isolated and identified myotrophin, a factor that stimulates myocytes growth, from spontaneously hypertensive rat (SHR) heart and patients with dilated cardiomyopathy. The gene encoding myotrophin has been cloned and expressed in E. coli. Recently, myotrophin gene has been mapped and shown to be a novel gene localized in human chromosome 7q-33. To define the characteristics of each transcript and its pathophysiological significance, we examined transcripts of myotrophin in SHR heart during progression of hypertrophy. Northern blot analysis of myotrophin mRNA showed multiple transcripts. We isolated and characterized various myotrophin cDNA clones corresponding to the multiple transcripts by 5' "stretch plus" rat heart cDNA library screening. Sequence analysis of these cDNA clones indicates that each clone has a unique 5' UTR and multiple 3' UTR with varying lengths, repeated ATTTA motifs and many polyadenylation signals. In vitro transcripts generated from all these myotrophin-specific cDNA clones translate in vitro to a 12-kD protein. Among pathophysiological significance, we determined mRNA expression in 9 days old, 3 weeks old and 31 weeks old and observed a linear increased during the progression of hypertrophy. In WKY, this mRNA level remained the same throughout the growth and development of hypertrophy. Our data strongly suggest that myotrophin appears to be a candidate gene for cardiac hypertrophy and heart failure.

Amino Acid Sequence↗
PubMedDate not supplied

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes.

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.

Animals↗
PubMed2006

Vibrio fischeri and its host: it takes two to tango.

The association of Vibrio fischeri and Euprymna scolopes provides insights into traits essential for symbiosis, and the signals and pathways of bacteria-induced host development. Recent studies have identified important bacterial colonization factors, including those involved in motility, bioluminescence and biofilm formation. Surprising links between symbiosis and pathogenesis have been revealed through discoveries that nitric oxide is a component of the host defense, and that V. fischeri uses a cytotoxin-like molecule to induce host development. Technological advances in this system include the genome sequence of V. fischeri, an expressed sequence tagged library for E. scolopes and the availability of dual-fluorescence markers and confocal microscopy to probe symbiotic structures and the dynamics of colonization.

Animals↗
PubMed2004

The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization.

Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, Src kinases are involved in regulating the cell architecture. In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening. Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase. Its predicted size of 71 kDa was confirmed by Western blot analysis. Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development. Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female. Its enzymatic activity was confirmed by functional analyses. In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein. Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms. These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.

Amino Acid Sequence↗
PubMed2004

Application of laser capture microdissection to phage display peptide library screening.

OBJECTIVE: When identifying important regulatory genes using methods such as phage display peptide library screening it is critical to select such peptides from cells and tissues in their native state. Here, we report a novel approach to screen tumors using phage display and laser capture microdissection (LCM). STUDY DESIGN: A phage peptide library was screened directly on fresh oral tumor tissue, such that specifically bound peptides were selected from fresh tumor cells in the native tissue state. Tissue processing conditions were modified to ensure the survival of the bacteriophage. RESULTS: Our results demonstrate that live phage-peptide conjugates can be recovered from laser capture microdissected cells in a form suitable for additional cycles of amplification. CONCLUSION: Thus, LCM will be a valuable adjunct to phage display studies.

Animals↗
PubMed2005

The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: characterization of its most prominent polypeptide (IAM38).

A consequence of the acrosome reaction is to expose the inner acrosomal membrane (IAM), which is a requirement for the sperm's ability to secondarily bind to and then penetrate the zona pellucida (ZP) of the mammalian oocyte. However, the proteins on the IAM responsible for binding and presumably penetrating the zona have not been identified. This issue can be resolved if direct information is made available on the composition of the IAM. For this purpose, we devised a methodology in order to obtain a sperm head fraction consisting solely of the IAM bound to the detergent-resistant perinuclear theca. On the exposed IAM surface of this fraction, we defined an electron dense protein layer that we termed the IAM extracellular coat (IAMC), which was visible on sonicated and acrosome-reacted sperm of several mammalian species. High salt extraction removed the IAMC coincident with the removal of a prominent 38 kDa polypeptide, which we termed IAM38. Antibodies raised against this polypeptide confirmed its presence in the IAMC of intact, sonicated and acrosome-reacted sperm. By immunoscreening of a bovine testicular cDNA library and sequencing the resulting clones, we identified IAM38 as the equivalent of porcine Sp38 [Mori, E., Kashiwabara, S., Baba, T., Inagaki, Y., Mori, T., 1995. Amino acid sequences of porcine Sp38 and proacrosin required for binding to the zona pellucida. Dev. Biol., 168, 575-583], an intra-acrosomal protein with ZP-binding ability, whose precise localization in sperm was unknown. The blockage of IVF at the level of the zona with anti-IAM38 antibodies and the retention of IAM38 after sperm passage through the zona support its involvement in secondary sperm-zona binding. This study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM for identifying other candidates for sperm-zona interactions.

Acrosin↗
PubMed2005

Aptamers with fluorescence-signaling properties.

Aptamers are single-stranded DNA or RNA molecules with ligand-binding capabilities. Signaling aptamers refer to aptamers or modified aptamers with recordable signal generation ability. Fluorescence-signaling aptamers, in particular, are valuable molecular tools that can be used to establish important techniques or assays for the interrogation of identities and concentrations of proteins and metabolites. Since standard DNA and RNA aptamers themselves are not inherently fluorescent, modification methods are required for rationally converting non-fluorescent aptamers into fluorescent reporters or for selecting fluorescent aptamers directly from random-sequence DNA libraries by in vitro selection. This article will provide a brief review of various signaling aptamer design strategies as well as a detailed description of methods that can be used to generate, by both rational design and in vitro selection, a special class of signaling aptamers dubbed "structure-switching signaling aptamers." This class of signaling aptamers are designed to function by switching structures from a pre-formed, lowly fluorescent duplex assembly to a ligand-aptamer complex having a higher level of fluorescence.

Adenosine Triphosphate↗
PubMed2003

Folding of a highly conserved diverging turn motif from the SH3 domain.

Recent NMR structural characterization studies showed that a seven-residue segment (FKKGERL) from the src SH3 domain adopts the nativelike diverging type II beta-turn in aqueous solution in support of the prediction based on the I-sites library of sequence structural motifs. We study the conformational variability and folding/unfolding thermodynamics of this peptide in explicit solvent using replica-exchange molecular dynamics simulations, which greatly enhances the sampling of the conformational space. This peptide samples three main free energy basins (nativelike, intermediate, and unfolded) separated by small barriers. The nativelike basin is fractionally populated (DeltaG(300K) = 0.4 kcal/mol) with structures that satisfy a subset of the NMR-derived constraints. The intrinsic stability of the diverging turn is examined in relationship to the nature of three specific contacts: a turn-hydrogen bond, a mainchain-to-sidechain hydrogen bond, and an end-to-end hydrophobic contact. We have carried out simulations of mutants at the highly conserved GE positions in the sequence. The mutation E5D destabilizes the isolated diverging turn motif, contrary to the observation that this mutation stabilizes the fyn SH3 domain. The G4T mutation also destabilizes the isolated diverging turn; however, the extent of destabilization is smaller than that of the reverse mutation in the drk SH3.

Amino Acid Motifs↗
PubMed1999

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation.

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow-derived DC (BM-DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM-DC and its expression was upregulated following stimulation with LPS or TNF-alpha. Transfection experiments using COS-7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one-dimensional NMR data strongly support that the protein is structurally folded.

Amino Acid Sequence↗