Relation between coronary artery disease, aortic stiffness, and left ventricular structure in a population sample

With the use of the degenerated nucleotides that contain the conserved sequence of G protein-coupled receptor, we have identified a 648-bp clone (HDGRC02) from human genomic DNA with significant sequence homology to human neurotransmitter receptors. HDGRC02 was then used as a probe for the screening of full length gene. From human Lambda DASH II genomic library, a 1.6 Kb clone encoded a full length gene was isolated and named putative neurotransmitter receptor (PNR). PNR has a single open reading frame which predicts a 38.3 KD protein of 338 amino acids with seven transmembrane domain topography. The amino acid sequence of PNR exhibits considerable homology to the rat 5-HR1D receptor with 35% amino acid identity and 56% amino acid similarity. PNR also shows significant sequence homology to the 5-HT1D receptor from Japanese puffer fish fugu, to the 5-HT4L receptor from mouse, to the alpha-2 adrenergic receptor and to the D2 dopamine receptor. Northern blot analysis indicates that PNR is expressed in skeletal muscle and selected areas of the brain. A chromosome mapping study located the PNR gene with human chromosome band of 6q23. The findings in the present study demonstrate that PNR is a putative neurotransmitter receptor.     

Functional magnetic resonance imaging of median nerve stimulation in rats at 2.0 T

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54,633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin 1 of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Strategy for identifying the gene encoding the DNA polymerase of molluscum contagiosum virus type 1

Molluscum contagiosum virus (MCV) is a member of the family Poxviridae and pathogenic to humans. MCV causes benign epidermal tumors mainly in children and young adults and is a common pathogen in immunecompromised individuals. The viral DNA polymerase is the essential enzyme involved in the replication of the genome of DNA viruses. The identification and characterization of the gene encoding the DNA polymerase of molluscum contagiosum virus type 1 (MCV-1) was carried out by PCR technology and nucleotide sequence analysis. Computer-aided analysis of known amino acid sequences of DNA polymerases from two members of the poxvirus family revealed a high amino acid sequence homology of about 49.7% as detected between the DNA polymerases of vaccinia virus (genus Orthopoxvirus) and fowlpoxvirus (genus Avipoxvirus). Specific oligonucleotide primers were designed and synthesized according to the distinct conserved regions of amino acid sequences of the DNA polymerases in which the codon usage of the MCV-1 genome was considered. Using this technology a 228 bp DNA fragment was amplified and used as hybridization probe for identifying the corresponding gene of the MCV-1 genome. It was found that the PCR product was able to hybridize to the BamHI MCV-1 DNA fragment G (9.2 kbp, 0.284 to 0.332 map units). The nucleotide sequence of this particular region of the MCV-1 genome (7267 bp) between map coordinates 0.284 and 0.315 was determined. The analysis of the DNA sequences revealed the presence of 22 open reading frames (ORFs-1 to -22). ORF-13 (3012 bp; nucleotide positions 6624 to 3612) codes for a putative protein of a predicted size of 115 kDa (1004 aa) which shows 40.1% identity and 35% similarity to the amino acid sequences of the DNA polymerases of vaccinia, variola, and fowlpoxvirus. In addition significant homologies (30% to 55%) were found between the amino acid sequences of the ORFs 3, -5, -9, and -14 and the amino acid sequences of the E6R, E8R, E10R, and a 7.3 kDa protein of vaccinia and variola virus, respectively. Comparative analysis of the genomic positions of the loci of the detected viral genes including the DNA polymerases of MCV-1, vaccinia, and variola virus revealed a similar gene organization and arrangement.     

Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract

Two genes for Ca2+-dependent protein kinases, PCaPK-alpha and PCaPK-beta, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK-alpha encoded 481 amino acids and that of PCaPK-beta encoded 493 amino acids, predicting molecular masses of 55603 Da and 57131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK-alpha and PCaPK-beta were closely related to those of the Ca2+-dependent protein kinases (CDPKs) from Plasmodium, Eimeria, and several plants, and the catalytic region of the Ca2+/calmodulin-dependent protein kinase family (35-48% identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C-terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30-34% identity). Two Ca2+-dependent protein kinases previously characterized from Paramecium (52 kDa CaPK-1, and 50 kDa CaPK-2) are activated by Ca2+ in the micromolar concentration range and they directly bind Ca2+ in a 45Ca2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca2+-binding motifs encoded in PCaPK-alpha and PCaPK-beta, and the immunological cross-reaction of expressed cloned fragments of these genes with CaPK-2, suggest that they encode proteins of the same family.     

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.     

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.     

Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases

We have identified a Drosophila gene, Dror, which encodes a putative receptor tyrosine kinase (RTK) and maps to cytological location 31B/C on the second chromosome. In embryos, this gene is expressed specifically in the developing nervous system. The Dror protein appears to be a homolog of two human RTKs, Ror1 and Ror2. Dror and Ror1 proteins share 36% amino acid identity in their extracellular domains and 61% identity in their catalytic tyrosine kinase (TK) domains. Ror1 and Ror2 were originally identified on the basis of the similarity of their TK domains to the TK domains of members of the Trk family of neurotrophin receptors. The Dror protein shows even greater similarity to the Trk proteins within this region than do the human Ror proteins. In light of its similarity to trk and its neural-specific expression pattern, we suggest that Dror may encode a neurotrophic receptor that functions during early stages of neural development in Drosophila.     

Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco

A cDNA and a corresponding genomic clone encoding a protein with partial identity to type II proteinase inhibitors from potato, tomato and Nicotiana alata, were isolated from tobacco libraries. The protein of 197 amino acids contains a putative signal peptide of 24 residues and three homologous domains, each with a different reactive site. The tobacco PI-II gene is not expressed in leaves of healthy plants, but is locally induced in leaves subjected to different types of stress (TMV infection, wounding, UV irradiation) and upon ethephon treatment. As opposed to the analogous PI-II genes of potato and tomato, the tobacco gene is not systemically induced by wounding or pathogenic infection. A far-upstream region in the PI-II promoter, containing various direct and indirect repeats, shares considerable sequence similarity to a similar region in the stress-inducible Cu/Zn-superoxide dismutase gene of N. plumbaginifolia.     

Molecular cloning of capillary permeability-increasing enzyme-2 from Agkistrodon caliginosus (Korean viper)

The gene of capillary permeability-increasing enzyme-2 (CPI enzyme-2) was cloned from the cDNA library of Agkistrodon caliginosus and its nucleotide sequence was determined. Its sequence indicates that CPI enzyme-2 is synthesized as a pre-zymogen of 258 amino acid residues, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acids. The amino terminal sequence deduced from the cDNA sequence was exactly consistent with that of CPI enzyme-2 except for the substitution of an amino acid (Gly27-->Ser). The open reading frame is very similar to those of plasminogen activator and thrombin-like proteases cloned from other snakes. The clone encoding CPI enzyme-2 belongs to the serine protease family. The active site of the enzyme is highly conserved at His41, Asp86 and Ser180. Its possible glycosylation sites, Asn-X-Thr/Ser, are located at amino acid residues 20-22, 55-57, 79-81 and 97-99.     

Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.     

Functional analysis of vaccinia virus B5R protein: role of the cytoplasmic tail

Vaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread both in vitro and in vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation.     

Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.