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L T Hu

Publications and source records attributed to L T Hu.

30 records · Page 2Linked to original sources
PubMed1993

Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli.

The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided.

Amino Acids↗
PubMed1992

Can test statistics in covariance structure analysis be trusted?

Covariance structure analysis uses chi 2 goodness-of-fit test statistics whose adequacy is not known. Scientific conclusions based on models may be distorted when researchers violate sample size, variate independence, and distributional assumptions. The behavior of 6 test statistics is evaluated with a Monte Carlo confirmatory factor analysis study. The tests performed dramatically differently under 7 distributional conditions at 6 sample sizes. Two normal-theory tests worked well under some conditions but completely broke down under other conditions. A test that permits homogeneous nonzero kurtoses performed variably. A test that permits heterogeneous marginal kurtoses performed better. A distribution-free test performed spectacularly badly in all conditions at all but the largest sample sizes. The Satorra-Bentler scaled test statistic performed best overall.

Female↗
PubMed1992

Relationship of ethnicity to psychiatric diagnosis.

The purpose of this study was to determine the relationship of ethnic identity to psychiatric diagnosis in white, black, Latino, and Asian clients of the Los Angeles County mental health system. The sample (N = 26,400) consisted of adult inpatient and outpatient clients seen in county mental health facilities between January 1983 and August 1988. Logistic regression analysis was used to determine the relationship of ethnicity to diagnosis in both outpatient and inpatient samples. The covariates included in the analysis were age, gender, socioeconomic status, and primary language. Ethnicity had a significant and consistent relationship to diagnosis in both outpatient and inpatient samples, with black and Asian clients having a greater proportion of psychotic diagnoses than whites, and Latinos a lesser proportion than whites. None of the covariates included in the analysis had a consistent relationship to diagnosis. Whites and Asians received more diagnoses of major affective disorders than blacks or Latinos; blacks and Asians received more diagnoses of schizophrenia and other psychoses than whites, and Latinos received fewer of these diagnoses than whites. Substance abuse was lower for Asians than for the other three groups. Based on the findings, it was concluded that there continues to be a difference in psychiatric diagnosis that is related to ethnicity. Clinical practice issues and recommendations for further research are considered in relationship to these findings.

Adult↗
PubMed1992

Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB.

Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.

Apoenzymes↗
PubMed1992

Use of polymerase chain reaction-amplified Helicobacter pylori urease structural genes for differentiation of isolates.

Helicobacter pylori has been demonstrated as an etiologic agent of human gastritis and peptic ulcer formation. However, there is no straightforward basis to distinguish different isolates. We used the polymerase chain reaction (PCR) to amplify the urease structural subunit genes, ureA and ureB, which, when digested with appropriate restriction endonucleases, allow the differentiation of patterns on agarose gels. PCR amplification was possible with DNA rapidly extracted from H. pylori by alkaline lysis and phenol-chloroform. The 2.4-kb PCR products amplified from 22 clinical isolates and subjected to HaeII restriction endonuclease digestion produced 10 distinct patterns on agarose gels, with two patterns being shared between five and six strains. PCR amplification of the urease genes may enable the differentiation of closely related H. pylori strains by restriction digest analysis of PCR-amplified ureA and ureB genes.

Bacterial Typing Techniques↗
PubMed1992

Racial/ethnic identity and amount and type of psychiatric treatment.

OBJECTIVE: The purpose of this study was to examine the relationship of racial/ethnic identity to the amount and type of psychiatric treatment received by white, black, Latino, and Asian patients in the Los Angeles County mental health system. METHOD: The patients studied (N = 19,400) consisted of all adult inpatients and outpatients seen in all county mental health facilities between January 1983 and August 1988. Multiple regression analysis was used to test the relationship between race/ethnicity and four measures of treatment received: number of treatment sessions, treatment modality, treatment setting, and therapist's discipline. The covariates included in the analyses were age, sex, socioeconomic status, primary language, diagnosis, and measures of treatment when these were logical predictors and were not acting as dependent variables. RESULTS: Race/ethnicity did not have a consistent significant relationship to the treatment variables studied. However, diagnosis had a consistent and highly significant relationship to all four measures of treatment. A psychotic diagnosis was related to receiving more treatment sessions, greater use of medication, greater use of inpatient treatment, and less treatment by a professional therapist. Socioeconomic status and primary language also had consistent and significant relationships to three of the treatment variables. CONCLUSIONS: In considering modifications to the service delivery system, clinicians must evaluate whether the type of treatment provided to psychotic patients is the treatment of choice in terms of effectiveness and efficiency or whether it involves bias in service delivery. Similarly, the issue of bias in treatment of lower socioeconomic patients must be addressed.

Adolescent↗
PubMed1991

Community mental health services for ethnic minority groups: a test of the cultural responsiveness hypothesis.

This study investigated services received, length of treatment, and outcomes of thousands of Asian-American, African-American, Mexican-American, and White clients using outpatient services in the Los Angeles County mental health system. It tested the hypothesis that therapist-client matches in ethnicity and language are beneficial to clients. Results indicate that Asian Americans and Mexican Americans underutilized, whereas African Americans overutilized, services. African Americans also exhibited less positive treatment outcomes. Furthermore, ethnic match was related to length of treatment for all groups. It was associated with treatment outcomes for Mexican Americans. Among clients who did not speak English as a primary language, ethnic and language match was a predictor of length and outcome of treatment. Thus, the cultural responsiveness hypothesis was partially supported.

Adult↗
PubMed1991

Helicobacter pylori urease: properties and role in pathogenesis.

Urease (urea amidohydrolase, EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbon dioxide. Research on this enzyme has gained momentum since the discovery of Helicobacter pylori as a causative agent of human gastritis. The remarkably high urease activity of each organism has served as the basis of diagnostic tests for the presence of the organism in the urease biopsy test and urea breath test. Urease undoubtedly plays a central role in H. pylori pathogenesis. Hydrolysis of urea with generation of ammonia may enable survival of this acid-sensitive organism in the gastric mucosa. Ammonia generated by urea hydrolysis may also produce severe cytotoxic effects within gastric epithelium. The enzyme also elicits a strong immune response during acute infection, suggesting that this abundant antigen is readily available to the immune system. An increase in serum IgG titer is predictive of ongoing infection. Much progress has been made with regard to the molecular biology of urease. The high molecular weight protein (estimated by several investigators to be 300-520 kDa) has been purified, revealing two distinct subunits of 29.5 kDa and 66 kDa, a unique subunit structure as compared with other microbial ureases. However, amino acid sequences are nevertheless well conserved when compared with other bacterial ureases and that of the jack bean, Canavalia ensiformis. Furthermore, genes encoding urease of H. pylori have been cloned, sequenced, and amplified by the polymerase chain reaction.

Animals↗
PubMed1990

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor.

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.

Acid Phosphatase↗
PubMed1990

Purification and N-terminal analysis of urease from Helicobacter pylori.

Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureases, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera raised against the 66-kDa subunit of H. pylori urease, demonstrating that at least some antigenic determinants were conserved among ureases from different species.

Amino Acid Sequence↗
PubMed1990

Morganella morganii urease: purification, characterization, and isolation of gene sequences.

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.

Amino Acid Sequence↗