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Biomedical subjects

D P Ma

Publications and source records attributed to D P Ma.

At least 19 recordsLinked to original sources
PubMed2001

Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone.

OBJECTIVE: To develop a method to experimentally induce Borrelia burgdorferi infection in young adult dogs. ANIMALS: 22 healthy Beagles. PROCEDURE: All dogs were verified to be free of borreliosis. Twenty 6-month-old dogs were exposed to Borrelia burgdorferi-infected adult ticks and treated with dexamethasone for 5 consecutive days. Two dogs not exposed to ticks were treated with dexamethasone and served as negative-control dogs. Clinical signs, results of microbial culture and polymerase chain reaction (PCR) testing, immunologic responses, and gross and histologic lesions were evaluated 9 months after tick exposure. RESULTS: Predominant clinical signs were episodic pyrexia and lameness in 12 of 20 dogs. Infection with B burgdorferi was detected in microbial cultures of skin biopsy specimens and various tissues obtained during necropsy in 19 of 20 dogs and in all 20 dogs by use of a PCR assay. All 20 exposed dogs seroconverted and developed chronic nonsuppurative arthritis. Three dogs also developed mild focal meningitis, 1 dog developed mild focal encephalitis, and 18 dogs developed perineuritis or rare neuritis. Control dogs were seronegative, had negative results for microbial culture and PCR testing, and did not develop lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Use of this technique successfully induced borreliosis in young dogs. Dogs with experimentally induced borreliosis may be useful in evaluating vaccines, chemotherapeutic agents, and the pathogenesis of borreliosis-induced arthritis.

Animals↗
PubMed2001

Cloning and expressionanalysis of two cotton (Gossypium hirsutum L.) genes encodingcell wall proline-rich proteins.

Two cotton (Gossypium hirsutum L.) genes, ghprp1 and ghprp2, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized. The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP. The GhPRP1 has an 80% identity in aa sequence with that of GhPRP2. Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units. Northern blot analyses showed that the ghprpl gene is predominantly expressed in the fiber during the elongation stage of fiber development. Reverse transcription (RT)-PCR analysis showed that ghprpl is expressed in both fiber and root tissues, whereas ghprp2 is in roots only. The ghprpl gene was shown to be present in the A1, A2, D1 and D5 genomes of Gossypium by PCR amplification, whereas the ghprp2 gene is only present in the A1 and A2 genomes. The ghprpl gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1). Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers. However, the 50 kDa protein was absent in other cotton tissues.

Amino Acid Sequence↗
PubMed2000

Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3(1).

A cotton Ltp3 gene and its 5' and 3' flanking regions have been cloned with a PCR-based genomic DNA walking method. The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3). The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3. The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli beta-glucuronidase gene (GUS) as a reporter. The results of histochemical and fluorogenic GUS assays indicate that the 5' flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter.

Amino Acid Sequence↗
PubMed1999

Molecular phylogenetic studies of Brassica, rorippa, arabidopsis and allied genera based on the internal transcribed spacer region of 18S-25S rDNA.

The phylogenetic relationships of nine genera in four tribes of the family Brassicaceae were estimated from the sequences of the internal transcribed spacer region (ITS) of the 18S-25S nuclear ribosomal DNA. The entire ITS region of 16 accessions belonging to 10 species of seven genera was sequenced. Eight published sequences of Brassicaceae were also used. A total of 27 sequences were included in this study; four of them were found to be pseudogenes. Both the neighbor-joining and the parsimony trees suggest that the nine genera can be divided into three groups: (1) Arabidopsis, Cardaminopsis, Capsella, and Lepidium; (2) Rorippa and Cardamine; and (3) Brassica, Sinapis, and Raphanus. In contradiction to the proposal that Cardaminopsis and Arabidopsis be put into an expanded tribe Arabideae, our data show that these two genera are more closely related to Capsella and Lepidium (tribe Lepidieae) than to Rorippa and Cardamine (tribe Arabideae). Further, our data show that within the tribe Brassiceae, Raphanus is more closely related to B. nigra than to the B. oleracea/B. rapa clade. This result is in agreement with the nuclear data obtained in several studies, but is in conflict with the RFLP data of mitochondrial and chloroplast DNA. As pointed out by previous authors, it is possible that Raphanus is a hybrid between the B. nigra and B. oleracea/B. rapa lineages with the latter as the maternal parent.

Arabidopsis↗
PubMed1998

Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris.

A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions.

Aldehyde Reductase↗
PubMed1997

Cloning and characterization of a cotton lipid transfer protein gene specifically expressed in fiber cells.

A cotton genomic library was screened using a fiber-specific cDNA (GH3) encoding a lipid transfer protein (LTP). One genomic clone (1.7 kb DNA insert) containing the Ltp gene (Ltp6) was sequenced and characterized. The Ltp6 contains an open reading frame of 360 bp, which is interrupted by a single intron (136 bp) located in the region corresponding to the C-terminal of the protein. The derived amino-acid sequence of LTP6 is 64% homologous to that of GH3. Like the GH3 gene, the Ltp6 is specifically expressed in fiber cells in a temporal manner. However, its expression level is lower than that of GH3.

Amino Acid Sequence↗
PubMed1995

Differential expression of a lipid transfer protein gene in cotton fiber.

A full-length cDNA clone, GH3, has been isolated from a cotton fiber cDNA library using a differential screening method. The nucleotide and derived amino acid sequence data show that GH3 encodes a lipid transfer protein (LTP) of 120 amino acids. The presence of a transmembrane signal peptide at the N-terminal of the protein would suggest its possible outer cellular location in fiber cells. Northern analysis indicates that the GH3 gene is developmentally regulated.

Amino Acid Sequence↗
PubMed1995

Sequence analysis of leukotoxin secretion determinants from a Pasteurella haemolytica-like organism.

The pHLBD genes encoding the secretion functions for the 105 kDa RTX leukotoxin of Pasteurella haemolytica-like (PHL) organism has been cloned and sequenced. Like analogous genes from other RTX determinants, the pHLBD genes lie immediately downstream from the leukotoxin structural gene, pHLA. Although isolated from a diverse group of gram-negative organisms, the pHLBD genes and the characterized RTX BD genes from other organisms exhibit a high degree of homology at both the DNA and predicted amino acid sequence levels. We have previously reported the cloning of the leukotoxin gene (pHLCA) (Chang et al., Infect. Immun. 61:2089-2095), which encodes a 105-kda polypeptide with cytotoxic activity. DNA sequence analysis of the pHLBD genes shows 83.93% and 86.05% homologous to that of P. haemolytica IktBD genes, respectively.

ATP-Binding Cassette Transporters↗
PubMed1993

Evolution of a noncoding region of the chloroplast genome.

The relative rate of occurrence of nucleotide substitutions versus indel (insertion/deletion) events is investigated by comparing complete DNA sequence data from the noncoding portion of the chloroplast genome that maps between the genes rbcL and atp beta. The sequence data are obtained from nine species that represent three tribes of the grass family. Indels could be categorized by those that are deletions or duplications of adjacent or proximal sequences and those that do not appear to be permutations of adjacent sequences. The first category represents 82% of the recorded indels. These indels may also be characterized by being direct duplications of one to several bases usually within runs of As or Ts or by being duplications or deletions of more complex sequences. When viewed from within groups of closely related taxa, indel events appear to occur at an equal or slightly faster rate than do nucleotide substitution events. However, the apparent rate of accumulation of indels in more distantly related species is significantly slower than that of nucleotide substitutions. This difference in apparent accumulation rates between indel events and nucleotide substitutions suggests that the proportion of superimposed changes has been higher among all indel events than among all nucleotide substitution events. Indeed the indels involving more complex sequences were found to be confined across taxa to a number of highly labile sites. Independent, though similar, indel events occur at identical sites in unrelated taxa, yet may not be shared among related taxa, resulting in a type of molecular parallelism. As a result, the phylogenetic tree based on indel events represents an evolutionary hypothesis which is inconsistent with the accepted phylogeny of these grasses. The phylogenetic tree based on nucleotide substitutions is consistent with accepted phylogeny.

Base Sequence↗
PubMed1993

Structure and evolution of opossum, guinea pig, and porcupine cytochrome b genes.

We have sequenced the mitochondrial cytochrome b gene from the guinea pig, the African porcupine, and a South American opossum. A phylogenetic analysis, which includes 22 eutherian and four other vertebrate cytochrome b sequences, indicates that the guinea pig and the porcupine constitute a natural clade (Hystricomorpha) that is not a sister group to the clade of mice and rats (Myomorpha). Therefore, the hypothesis that the Rodentia is paraphyletic receives additional support. The artiodactyls, the perissodactyls, and the cetaceans form a group that is separated from the primates and the rodents. The 26 sequences are used to study the structure/function relationships in cytochrome b, whose function is electron transport. Most of the amino acid residues involved in the two reaction centers are well conserved in evolution. The four histidines that are believed to ligate the two hemes are invariant among the 26 sequences, but their nearby residues are not well conserved in evolution. The eight transmembrane domains represent some of the most divergent regions in the cytochrome b sequence. The rate of nonsynonymous substitution is considerably faster in the human and elephant lineages than in other eutherian lineages; the faster rate might be due to coevolution between cytochrome b and cytochrome c.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of the Actinobacillus pleuropneumoniae RTX toxin-III gene cluster.

Actinobacillus pleuropneumonia strains that secrete three different exotoxins (ApxI, ApxII, and ApxIII) have been implicated in the etiology of porcine pleuropneumonia. To understand the role of these toxins in the pathogenesis of this disease, we have previously reported the cloning of the hemolysin gene (apxII) (Chang et al., 1989a), which encodes a 110-kD polypeptide with hemolytic and cytotoxic activity. To clone the third toxin gene (apxIII), a new genomic library using A. pleuropneumoniae serotype 2 chromosomal DNA was constructed. A series of five overlapping recombinant phage clones carrying the gene (apxIII) for this 120-kD antigen were identified using a DNA probe containing sequences from the Pasteurella haemolytica lktBD genes. Sequence analysis of a region of the cloned DNA reveals four open reading frames encoding proteins with predicted masses of 20.4, 112.5, 80.3, and 54.7 kD. These genes, designated apxIIC, apxIIIA, apxIIIB, and apxIIID, respectively, are similar in sequence to the RTX (repeat of toxin) toxin family. The toxin produced by the cloned gene kills BL-3 cells and is not hemolytic in vitro.

Actinobacillus pleuropneumoniae↗
PubMed1993

Molecular characterization of a leukotoxin gene from a Pasteurella haemolytica-like organism, encoding a new member of the RTX toxin family.

A Pasteurella haemolytica-like organism, a new species of bacterium isolated from piglets with diarrhea, secretes a leukotoxin into the culture media. Western blot (immunoblot) analysis indicated that this leukotoxin cross-reacted with antileukotoxin antibody derived from cattle immunized with P. haemolytica. Five overlapping recombinant bacteriophages carrying the gene for this 105-kDa polypeptide were identified with a DNA probe containing sequences from the P. haemolytica lktCA genes from a P. haemolytica-like organism strain 5943 genomic library. Sequence analysis of a region of the cloned DNA revealed two open reading frames encoding proteins with predicted masses of 19.4 and 101.6 kDa. These genes, which we designate pllktC (P. haemolytica-like organism leukotoxin C gene) and pllktA (A gene), respectively, are similar in sequence to the RTX (repeat of toxin) toxin family. The structure of the 101.6-kDa protein derived from the DNA sequence shows three transmembrane domains in the N-terminal part of the protein, 13 glycine-rich repeat domains in the second half of the protein, and a hydrophobic C-terminal part. pllktC and pllktA are strongly homologous to P. haemolytica lktC and lktA genes. However, this leukotoxin kills both BL-3 and pig leukocytes and is not hemolytic.

Amino Acid Sequence↗
PubMed1993

Cloning, sequencing and expression of a Pasteurella haemolytica A1 gene encoding a PurK-like protein.

A membrane protein antigen of Pasteurella haemolytica A1 encoded on the recombinant plasmid pYFC13 is isolated and characterized. Nucleotide sequence analysis of the insert DNA in pYFC13 identified the gene mpa1, which codes a protein of approximately 45 kDa without signal sequence. The deduced amino acids from the DNA sequence are homologous to Bacillus subtilis PurK by 29.4%; to Schizosaccharomyces pombe Pur6 by 29.34%, to Saccharomyces cerevisiae Pur6 by 25.867%; and to E. coli PurK by 25.223% identity, respectively. The purK and pur6 from these organisms are responsible for the activity of 5'-phosphoribosyl- 5-amino-4-imidazole carboxylase which is involved in de novo purine biosynthesis. The protein was over-expressed in E. coli by its own promoter. The antigen we designated as Mpa1, could be localized to the cytoplasmic membrane of both P. haemolytica A1 and E. coli TB1 harbored pYFC13. The Mpa1 was antigenic in rabbit and in cattle since both animals produced antibody against this protein.

Amino Acid Sequence↗
PubMed1992

Amplification and characterization of an inverted repeat from the Chlamydomonas reinhardtii mitochondrial genome.

Based on the nucleotide (nt) sequences of cob and L2a, two oligodeoxyribonucleotides (oligos) were synthesized and used in the polymerase chain reaction (PCR) to amplify the termini of the Chlamydomonas reinhardtii mitochondrial (mt) genome. A 0.8-kb PCR product was detected by agarose-gel electrophoresis when using unligated mt DNA as the template for PCR. This may have indicated the presence of a naturally occurring circular mt DNA molecule that acted as the PCR template. The 0.8-kb DNA could also be amplified from the linear mt DNA via an intramolecular jump during PCR. The sequence data from the 0.8-kb PCR product, and the right 0.6-kb and left 1-kb terminal fragments of the linear mt DNA, along with Southern hybridization analysis, indicated that a 0.49-kb inverted repeat (IR) sequence is present at the right and left termini of the linear mt DNA. The IR contains A+T-rich clusters, as well as numerous short direct repeats (DR) and IR, and might be involved in the recombination, replication and expression of the C. reinhardtii mt genome.

Animals↗
PubMed1992

The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease.

The mitochondrial DNA molecules of two interfertile algal species, Chlamydomonas smithii and C. reinhardtii, are co-linear except for a 1075 bp intron (the alpha-insert) that is present in the cob gene of C. smithii. The alpha-insert, a group I intron (Cs cob.1) containing an open reading frame (ORF) which encodes a basic, hydrophilic protein of 237 amino acids, is unidirectionally transmitted to all diploid progeny during interspecific crosses. In this report, we show that the Cs cob.1-encoded protein is a site-specific endonuclease (I-Csm I) which could mediate the intron transfer via the gene conversion mechanism. The Cs cob.1 ORF was cloned into the vector pMALcr1 and over-expressed as a hybrid protein fused to maltose-binding protein (MBP). This fusion protein exhibited an in vivo endonuclease activity which specifically cleaved the intron homing site within the intronless cob gene.

Algal Proteins↗
PubMed1992

Characterization of plasmids with antimicrobial resistant genes in Pasteurella haemolytica A1.

Two R plasmids, pYFC1 and pYFC2, from Pasteurella haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFC1 consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, RSF1010, pGS05, pFM739, pHD148 and pGS03B. pYFC1 encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which is homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range IncQ plasmid RSF1010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 is similar to the plasmid from P. haemolytica LNPB51 isolated in France by partial restriction enzyme mapping. pYFC1 and pYFC2 have the same size of 4.2 kbp.

Amino Acid Sequence↗
PubMed1991

Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region.

IRBP is a photoreceptor-specific glycoprotein that has been suggested as a retinoid carrier in the visual process. Previous research has shown that 1.3 kb of 5'-flanking sequence from the human IRBP gene is sufficient to promote photoreceptor-specific expression of reporter genes in transgenic mice. To define more narrowly the sequences that promote tissue-specific expression, chimeric constructs with shorter promoters were used to generate transgenic mice. The bacterial CAT gene was fused to fragments of 706 bp or 212 bp from the 5' end of the human IRBP gene. Analysis of the three transgenic families bearing the 706 bp IRBP promoter revealed that CAT expression was confined to the neuro-retina and the pineal gland. Analysis of the four transgenic families bearing the 212 bp IRBP promoter revealed the same tissue-specific CAT expression in three families. These results establish that tissue-specific expression of IRBP can be regulated by a short 212 bp promoter which has been conserved between humans and mice.

Amino Acid Sequence↗