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Biomedical subjects

Y F Chang

Publications and source records attributed to Y F Chang.

At least 73 records · Page 4Linked to original sources

Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi.

Twenty-two specific-pathogen-free beagles were vaccinated with recombinant OspA (ospA gene derived from Borrelia burgdorferi B31) alone or with adjuvant (QuilA, Montanide ISA25, or aluminum hydroxide) at 6 weeks of age. Thirteen dogs were used as nonvaccinated controls with or without adjuvant. Three dogs were kept as contact controls and received neither vaccine nor challenge. Six weeks or 6 months after the first vaccination, the vaccinated (20 of 22) and nonvaccinated dogs (13) were challenged by exposure to adult ticks (Ixodes scapularis) naturally that were infected with B. burgdorferi (tick infection rate, > or = 60%) and that were collected from Westchester County, N.Y. Protection from infection was evaluated by culture for B. burgdorferi from skin biopsies taken near the sites of tick bites. Skin biopsies were taken at monthly intervals for 3 months. B. burgdorferi was not isolated from any of the vaccinated dogs. In contrast, 12 of 13 control dogs challenged by exposure to the ticks were culture positive. The histopathology of the joint capsules 3 months after the challenge was used to evaluate protection from arthritis. Eight of 13 control dogs showed synovitis in single or multiple joints, while only 1 of the 22 vaccinated dogs had a single focus of mild inflammation in a single joint. At the time of the challenge, the vaccinated dogs had antibody to B. burgdorferi, which was demonstrable by kinetic enzyme-linked immunosorbent assay, Western blotting (immunoblotting), and a serum growth inhibition assay. The vaccinal antibody declined gradually after the challenge, especially in dogs vaccinated with OspA without adjuvants. Antibodies in the challenge control dogs were only detectable by 4 to 6 weeks after the challenge and remained at high levels until the termination of the study. Contact control dogs showed no antibody responses or histopathologic lesions and were culture negative. By Western blot analysis, antibodies to OspA first appeared in the sera of vaccinated dogs 3 weeks after the first vaccination. The absence of additional bands after the challenge suggests that infection in vaccinated dogs was blocked. Results from this study show that vaccination with recombinant OspA protected dogs against infection and disease after an experimental challenge with B. burgdorferi by exposure to ticks.

Animals↗

Sera from OspA-vaccinated dogs, but not those from tick-infected dogs, inhibit in vitro growth of Borrelia burgdorferi.

Dogs were challenged with Borrelia burgdorferi by exposure to ticks, with or without prior protection from infection by recombinant OspA (rOspA) vaccination. Sera from these dogs were tested for their capability to inhibit the growth of B. burgdorferi in vitro. Bacterial growth was detected by a color change in the culture medium, and the optical density was measured with a spectrophotometer in microtiter plates. By growth inhibition, which was complement dependent, the color change was lacking after 5 days of incubation. Over a 1-year study, nonvaccinated dogs infected by exposure to ticks showed high antibody titers to B. burgdorferi by kinetic enzyme-linked immunosorbent assay (KELA). The same sera did not inhibit spirochetal growth or did so only at a low dilution. These results corresponded to the lack of OspA and OspB antibodies seen in Western blots (immunoblots), and these dogs were not protected from infection or disease. In contrast, dogs immunized with rOspA prior to challenge with infected ticks produced high antibody titers, as determined by KELA, but their sera also had high growth-inhibiting antibody titers. Western blot analysis showed a strong band in the 32-kDa region when the sera of these dogs were tested. When adjuvant was administered with rOspA, antibody titers by both KELA and growth inhibition were higher and persisted longer in the immunized dogs. All dogs immunized with rOspA were protected from infection and disease.

Animals↗

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↗

Steroid hormone receptors in three human gastric cancer cell lines.

Steroid hormone receptors in three human gastric adenocarcinoma cell lines and their transplanted tumors (except nontumorigenic KATO-III) in nude mice were determined by dextran-coated charcoal assay. Progesterone receptors (PgR) were found in all cell lines, transplanted NUGC-3, and AZ 521 tumors. Estrogen receptors (ER) were found in KATO-III cells, transplanted NUGC-3, and AZ 521 tumors, whereas glucocorticoid receptors (GR) were found only in NUGC-3 tumor and no androgen receptor was found in any cell lines or transplanted tumors. Since NUGC-3 cells had ER, PgR, and GR, it was used for the study of the effects of steroid hormones on growth. The results showed the cell cycle phase distributions and growth rate of transplanted tumors were similar in hormone-treated and nontreated groups. The persistent expression of PgR in gastric cancer cell lines and tumors, and the slight increase of tumor volumes in the progesterone-treated group suggests that progesterone and its receptors may be important in the pathogenesis of gastric cancer, but their biological function remains to be elucidated.

Adenocarcinoma↗

Flowcytometric analysis of the effect of berberine on the expression of glucocorticoid receptors in human hepatoma HepG2 cells.

Berberine is an alkaloid found in many plants, including the Coptis chinensis and Arcangelisia flava. Berberine has been reported to have cytostatic effect on tumor growth. Previously, we have found that the level of glucocorticoid receptors (GR) was significantly higher in hepatoma than in adjacent liver tissues. Using human HepG2 hepatoma cells, we have found that GR were expressed not only in G0-G1 phases, but also in S and G2+M phases. The objective of the present study was to examine the effect of berberine on the expression of GR and its relation to cell cycle progression of HepG2 cells. Continuous exposure of HepG2 cells to various concentrations (1-50 microM) of berberine resulted in growth inhibition in a dose dependent manner. The viability of berberine-treated HepG2 cells was greater than 90% in all treatment groups. Flowcytometric analysis of berberine-treated HepG2 cells showed that the S phase fraction was significantly reduced. GR levels were higher in berberine-treated HepG2 cells than in vehicle (DMSO)-treated cells. In addition, the secretion of alpha-fetoprotein by HepG2 cells was inhibited by berberine. Finally, the berberine induced cell growth arrest was partially reversible in HepG2 cells.

Analysis of Variance↗

Hybridization of clinical Escherichia coli isolates from calves and piglets in New York State with gene probes for enterotoxins (STaP, STb, LT), Shiga-like toxins (SLT-1, SLT-II) and adhesion factors (K88, K99, F41, 987P).

Six hundred and sixty-six bovine and fifty-seven swine clinical isolates of E. coli from New York state were examined for the presence of enterotoxins (STaP, STb, LT, SLT-I, and SLT-II) and adhesins (K88, K99, F41, and 987P) using colony hybridization techniques. Three hundred and sixty-seven of the bovine isolates (45.2%) hybridized with at least one gene probe. Of these, two hundred and twenty-three (33.2%) hybridized with F41, one hundred twelve (16.7%) with K99, eighty-two (12.2%) with 987P, ninety-six (14.3%) with STaP, seven (1.1%) with STb, and none (0.0%) with LT and K88. A total of thirty-three (4.7%) of the isolates hybridized with SLT-I, and one (0.1%) with SLT-II. The major pathotypes among the 666 isolates from bovine were K99/F41/StaP (9.8%), K99/F41 (2.5%), p87P/F41 (2.1%) and 987P/K99/F41/StaP (1.4%). Of the swine clinical isolates, twenty-two hybridized with at least one gene probe. The major pathotypes among the isolates from piglets were K88/K99/F41/StaP (5.3%) and K88/F41 (5.3%).

Adhesins, Escherichia coli↗

L-pipecolic acid oxidation in rat: subcellular localization and developmental study.

By using a sensitive radioactive assay method, we present here evidence that L-pipecolic acid oxidase is localized in both mitochondria and peroxisomes of rat liver. Brain white matter contained a more than 2-fold higher activity of L-pipecolic acid oxidation than the brain cortex. Suborganellar fractionation studies indicate that while the enzyme is a matrix protein in mitochondria, it is membrane-associated in peroxisomes. Both rotenone and antimycin A completely inhibited the enzyme activity in mitochondria but not in peroxisomes. The enzyme was shown to be inducible in mitochondria and peroxisomes of rat liver and brain tissues by glucagon and di-(2-ethylhexyl)phthalate, respectively. We report here for the first time the developmental aspects of L-pipecolic acid oxidation activity in rat liver and brain tissues. L-Pipecolic acid oxidase activity was detectable in whole rat embryo at 10 days of gestation, suggesting active L-pipecolic acid metabolism early during development. In both liver and brain tissues L-pipecolic acid oxidation activity was highest at 15 days of gestation and decreased with age in prenatal and postnatal conditions.

Aging↗

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli.

Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi. This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD. The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains six histidyl residues in its N-terminus.

Antigens, Surface↗

Chronic L-lysine develops anti-pentylenetetrazol tolerance and reduces synaptic GABAergic sensitivity.

L-Lysine 10 mmol/kg given to mice for 1 to 10 days significantly increased clonic and tonic seizure latencies caused by 60 mg/kg pentylenetetrazol (PTZ). On day 1 the clonic and tonic seizure latencies were increased from 160.4 +/- 26.3 and 828.6 +/- 230.8 s to 286.1 +/- 103.3 and 982.3 +/- 98.6 s, respectively. Both clonic and tonic seizure latencies increased steadily with additional L-lysine treatment without significant change in survival rate. On day 10, the anticonvulsant effect reached its highest level with a block of tonic seizures and a survival rate of 100% without tolerance developing. Acute L-lysine treatment significantly increased the mean clonic latency from 85.8 +/- 5.24 to 128.2 +/- 9.0 s and the mean tonic seizure latency from 287.2 +/- 58.7 to 313.5 +/- 42.2 s with 80 mg/kg PTZ. On the day 10 of treatment, the anticonvulsant effect of L-lysine was highest, with a significant increase of 155 and 184% in clonic and tonic latencies over the control, respectively. After 15- and 20-day treatment, clonic and tonic seizures latencies and survival rate decreased, suggesting development of tolerance. Brain membranes from tolerant mice showed decreased enhancement by gamma-aminobutyric acid of specific [3H]flunitrazepam binding from 210 +/- 8 to 169 +/- 5% with EC50 values of 4.1 +/- 1.4 and 7.8 +/- 1.5 microM, respectively. Scatchard analysis of [3H] flunitrazepam binding showed no significant change of apparent binding affinity (KD) or binding density (Bmax) after chronic L-lysine exposure. L-Lysine enhanced the specific [35S]tert-butyl bicyclophosphorothionate (TBPS) binding in brain membranes dose dependently.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multiocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella cholerasuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.

Animals↗

Cross-reactivity between B. burgdorferi and other spirochetes affects specificity of serotests for detection of antibodies to the Lyme disease agent in dogs.

Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Crossreactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

Agglutination Tests↗

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↗

Experimental Lyme disease in dogs produces arthritis and persistent infection.

Lyme disease was reproduced in specific pathogen-free beagle dogs by exposure to Borrelia burgdorferi-infected ticks (Ixodes dammini). Seroconversion and disease frequency were higher after exposure to infected adult ticks than to infected nymphs. Young pups developed clinical disease more readily than older dogs. The incubation period lasted 2-5 months. Acute recurrent lameness with fibrinopurulent arthritis was the dominant clinical sign. Dogs recovered but developed persistent mild polyarthritis. B. burgdorferi persisted in recovered dogs for at least 1 year. Isolation of B. burgdorferi and detection by polymerase chain reaction was most successful from skin biopsies at the site of the tick bite. Antibody to B. burgdorferi antigens was first detected by ELISA and Western blots by 4-6 weeks after exposure. High serum levels persisted during 17 months of observation. In contrast to infection from ticks, inoculation of dogs with cultured B. burgdorferi resulted in seroconversion with a shorter duration of antibody persistence and no clinical disease.

Age Factors↗

Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes.

The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for Actinobacillus pleuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9, 10 and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxI operon will have the designations apxIC, apxIA, apxIB, and apxID for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII, ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular mass between 103 and 105 kDa. The genes of the apxII operon will have the designations apxIIC for the activator gene and apxIIA for the structural toxin gene. In the apxII operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxIB and apxID of the apxI operon. The designation ApxIII is proposed for the nonhaemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin III (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat).(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus pleuropneumoniae↗

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↗

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↗

Analysis of glucocorticoid receptors in human hepatocellular carcinoma and HepG2 cells.

Hepatocellular carcinoma is the leading cause of male cancer death in Taiwan. We have found that the level of glucocorticoid receptor in hepatocellular carcinoma is significantly higher than that in the peritumoral tissue. In this study, we used a rat liver glucocorticoid receptor complementary DNA probe to examine the expression of glucocorticoid receptor gene in 15 paired samples of hepatocellular carcinoma and their peritumoral tissues. No differences in genomic DNA patterns of the glucocorticoid receptor gene were found between the tumor and peritumoral tissues. The amount of glucocorticoid receptor was found to be significantly higher in hepatoma samples than in peritumoral liver samples. The levels of glucocorticoid receptor messenger RNAs were increased in most tumors compared with their peritumoral samples. To examine the function of glucocorticoid receptors in hepatoma, we examined the expression of glucocorticoid receptor and its relation to cell-cycle progression in human HepG2 cells. Using specific monoclonal antibodies and flow cytometric study, we found glucocorticoid receptor to be expressed constitutively in all cell-cycle phases. In addition, hydrocortisone treatment of HepG2 cells resulted in increased expression of glucocorticoid receptors and increased secretion of alpha-fetoprotein. RU-486, a glucocorticoid antagonist, blocked the hydrocortisone effect, indicating that glucocorticoid receptors are functional in HepG2 cells. Taken together, our data suggest that glucocorticoids and their receptors play an important role in the growth of hepatoma.

Adult↗