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R C Johnson

Publications and source records attributed to R C Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1998

P-CIP1, a novel protein that interacts with the cytosolic domain of peptidylglycine alpha-amidating monooxygenase, is associated with endosomes.

The cytosolic domain of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) contains signals that direct its trafficking in the secretory and endosomal pathways. Using the yeast two-hybrid system, Alam et al. (Alam, M. R., Caldwell, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636) identified three proteins that interact with a fragment of the PAM cytosolic domain containing these targeting signals. We cloned the rat and human cDNAs encoding PAM COOH-terminal interactor protein-1 (P-CIP1). Both cDNAs contain an open reading frame that encodes a novel protein of 435 amino acids. The P-CIP1 protein is highly conserved from rat to human (85% identity) but does not display significant homology to proteins in the GenBank data base. In vitro, P-CIP1 interacts with the cytosolic domain of wild type PAM-1, but does not interact with mutant PAM-1 proteins that fail to target correctly when expressed in endocrine cells. P-CIP1 contains multiple consensus serine/threonine phosphorylation sites and a region predicted to form a coiled-coil at the COOH terminus. When expressed in endocrine cells or fibroblasts, P-CIP1 is distributed in a punctate pattern in the perinuclear area but does not significantly overlap the distribution of transfected wild type PAM-1. The distribution of P-CIP1 displays significant overlap with the distribution of the secretory carrier membrane proteins, internalized Texas Red-conjugated transferrin, and Rab11. The data suggest that P-CIP1 associates with vesicles in the recycling endosomal pathway, and may play a role in regulating the trafficking of integral membrane PAM.

Amino Acid Sequence↗
PubMed1998

Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion.

The Hin DNA invertase becomes catalytically activated when assembled in an invertasome complex containing two Fis dimers bound to an enhancer segment. The region of Fis responsible for transactivation of Hin contains a mobile beta-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating beta-arm are sufficient to form catalytically competent invertasomes. Analysis of homodimer and heterodimer mixes of different Hin mutants suggests that Fis must activate each subunit of the two Hin dimers that participate in catalysis. These experiments also indicate that all four Hin subunits must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is independent of the catalytic steps of recombination. We propose a molecular model for the invertasome structure that is consistent with current information on protein-DNA structures and the topology of the DNA strands within the recombination complex. In this model, a single Fis activation arm could contact amino acids from both Hin subunits at the dimer interface to induce a conformational change that coordinately positions the active sites close to the scissile phosphodiester bonds.

Carrier Proteins↗
PubMed1998

Hydrological studies of schistosomiasis transport in Sichuan Province, China.

Schistosomiasis is a water-bourne parasitic disease endemic to Sichuan Province of China. Long-term studies of infection and disease ecology in catchments in Sichuan have been supplemented by detailed hydrometric measurements to produce a model of water velocity and flow in an irrigation system. The model provides a means of estimating travel times of two infectious stages of the parasite from source sites to water contact exposure sites for individuals of both the human population and the intermediate vector snail populations. The hydrological transport model will be part of an overall model of schistosomiasis transmission in the catchments. A GIS system is used to manage spatial data of the drainage network, land use, infection sources and population centres. The development of the Three Gorges Dam in China will increase marshlands and irrigation in areas currently free of schistosomiasis. The potential for the spread of schistosomiasis into these new areas is a major concern. Hydrological models can be of particular importance in assessing future environmental risk.

Animals↗
PubMed1998

Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents.

Immunoisolation of allogeneic cells within a membrane-bound device is a unique approach for gene therapy. We employed an immunoisolation device that protects allograft, but not xenograft, cells from destruction, to implant a human fibroblast line (MSU 1.2) in athymic rodents. Cells, transduced with the MFG-human factor IX retroviral vector, and expressing 0.9 microg/10(6) cells/day in vitro, were implanted in rats (four 40-microl devices, each containing 2 x 10(7) cells, two subcutaneously, two in epididymal fat) and in mice (two 20-microl devices, each containing 2 x 10(6) cells, subcutaneously). Plasma factor IX levels increased for 50 days, reaching maxima of 203 ng/ml (rat) and 597 ng/ml (mouse), and both continued at greater than 100 ng/ml for more than 140 days. A clone derived from the transduced cells, making 5 microg of factor IX/10(6) cells/day, was implanted within a device (one 20-microl device containing 2.5 x 10(6) cells), or without a device (1 x 10(7) cells implanted freely), either subcutaneously or in epididymal fat. The freely implanted cells expressed transiently, reaching more than 100 ng/ml in each site by day 4, but dropped to zero by day 20 (subcutaneous) or day 90 (epididymal fat). In devices, levels gradually increased to 100 ng/ml (subcutaneous) or 300 ng/ml (epididymal fat), remaining high for more than 100 days. These results show long-term, high-level expression of a human protein: (1) when cells are implanted within a cell transplantation device, but not when the cells are freely implanted, and (2) from a transgene driven by a viral promoter. An alloprotective device will enable the use of cloned cell lines that can be subjected to stringent quality control assessment that is impossible to achieve with autologous approaches.

Animals↗
PubMed1998

Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and liver histology in tumor xenograft models.

Type 5 adenoviral (Ad) vectors have been the "vector-of-choice" for preclinical studies on p53 tumor suppressor gene therapy of cancer. Previous studies have examined the in vivo efficacy of p53 Ad when given intratumorally. However published information does little to guide clinicians in the design of intraperitoneal (i.p.) dosing trials for i.p. tumors, e.g., ovarian, or clinical trials using regional organ perfusion, e.g., for lung tumors. Therefore, we examined several parameters with special significance for these routes of administration. Lung metastases from p53mut MDA-MB-231 mammary xenografts were treated with therapeutic levels of intravenous buffer, beta-galactosidase (beta-Gal) Ad, or p53 Ad. Treatment with intravenous p53 Ad significantly reduced the number of metastases per lung and there was a dramatic reduction in the surface area occupied by these tumors as compared to control groups. Two types of i.p. tumor xenografts were used for preclinical modeling of i.p. gene therapy, the p53null SK-OV-3 ovarian and the p53mut DU-145 prostate human cancers. In a study examining the effect of different vehicle volumes on the efficacy of a constant drug dose, all mice treated with p53 Ad had reduced tumor burden compared to controls. Dosing volumes between 0.2 and 1 ml were equally effective and all were more effective than a dosing volume of 0.1 ml. However, reduced efficacy was observed when a volume of 1.5 ml was used. When the effect of dosing frequency on antitumor efficacy was examined, fractionated doses of p53 Ad had somewhat greater efficacy than fewer, bolus injections. One of the significant elements in the emerging toxicology associated with recombinant adenoviruses is the hepatocyte pathology caused by high systemic concentrations of adenovirus. For recombinant Ad used in this study, there was a pronounced dose-dependence for the liver response, with very high, repeated doses causing significant hepatocellular insult. Expression of cytoplasmic beta-Gal protein coincided with areas of greatest damage in mice treated with high doses of beta-Gal Ad. Ultrastructural examination of hepatocyte intranuclear inclusions revealed moderately electron-dense, tightly packed granular material interspersed with more electron-dense nuclear material. Human tumor xenografts, but not mouse tissues, expressed viral hexon protein. In summary, hepatic toxicity caused by high concentrations of recombinant adenovirus was observed in murine cancer models. However, therapeutic levels of p53 Ad could be achieved which had dramatic efficacy without significant pathology.

Adenoviridae↗
PubMed1998

Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine.

The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Deltanhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain. Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Deltanhp6a/b mutants in vivo.

Amino Acid Sequence↗
PubMed1998

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala.

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.

Amino Acid Sequence↗
PubMed1998

Prediction of major depression and dysthymia from CES-D scores among ethnic minority adolescents.

OBJECTIVE: The Native Hawaiian Mental Health Research Development Program is an epidemiological longitudinal study of adolescents residing in Hawaii. This article examines the utility of the Center for Epidemiologic Studies-Depression Scale (CES-D) for predicting DSM-III-R diagnoses of major depression (MD) and dysthymic disorder (DD) and investigates whether prediction differs by gender and ethnicity. METHOD: Diagnostic Interview Schedule for Children interviews were conducted with 556 adolescents randomly selected from among more than 7,000 students who had completed the CES-D. RESULTS: Six-month prevalence rates were as follows: MD = 8.5%, DD = 4.7%, either (MDDD) = 9.9%. Prevalence rates were significantly higher among females, but after CES-D scores were accounted for, gender no longer predicted depression in most analyses. When a cutoff score of 16 was used, classification accuracy was lower for Native Hawaiians than non-Hawaiians. However, after group differences in gender and grade level were accounted for, the predictive validity of the CES-D did not differ by ethnicity. CES-D factor 1 scores identified MD, DD, and MDDD about as well as the total score or all three factors together. CONCLUSIONS: These results support the validity of the CES-D for screening for depression among adolescents of Native Hawaiian and other minority backgrounds.

Adolescent↗
PubMed1998

Immunodiagnosis of human granulocytic ehrlichiosis by using culture-derived human isolates.

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused by Ehrlichia chaffeensis, Rickettsia rickettsii, and Coxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples from R. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests for Borrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n = 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure to Ehrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.

Antibodies, Bacterial↗
PubMed1998

Selective alteration of personality and social behavior by serotonergic intervention.

OBJECTIVE: The authors sought to test the causal hypothesis that serotonergic function modulates aspects of the normal spectrum of individual differences in affective experience and social behavior in humans. METHOD: A selective serotonin reuptake inhibitor (SSRI), paroxetine, 20 mg/day (N = 26), or placebo (N = 25) was administered to normal volunteers in a double-blind manner for 4 weeks, and personality variables and social behavior were assessed at baseline and at weeks 1 and 4 of treatment. RESULTS: Relative to placebo, SSRI administration reduced focal indices of hostility through a more general decrease in negative affect, yet did not alter indices of positive affect. In addition, SSRI administration increased a behavioral index of social affiliation. Changes in both negative affect and affiliative behavior were significantly related to volunteers' plasma SSRI levels at the end of the experiment. CONCLUSIONS: Central serotonergic function may modulate a dimension of normal personality characterized by reduced negative affective experience and increased affiliative behavior. SSRI administration has significant and detectable effects on these measures even in the absence of baseline clinical depression or other psychopathology.

Adult↗
PubMed1998

Depressive symptoms among Filipino American adolescents.

Minority ethnic status has been found to be related to higher levels of depressive symptoms among adolescents and adults. The present study examined the rates of depressive symptoms (as measured by the Center for Epidemiologic Studies Depression Scale: CES-D Scale) of 270 Filipino American adolescents residing in rural and small-town areas of Hawaii. CES-D scores were compared with scores of a White group, and no ethnic differences were found. Compared with Filipino males, Filipino females were found to have higher CES-D scores, with higher mean scores on the majority of the CES-D items. The few Filipino students who reported attempting suicide had moderately high to very high levels of reported depressive symptoms. Lack of ethnic differences may be due to Hawaii's unique cultural mix, where there is no single "majority group" and a high rate of cultural interaction.

Adolescent↗
PubMed1998

Double heterozygosity for mutations in the BRCA1 and BRCA2 genes in a breast cancer patient.

BACKGROUND: Germline mutations in the tumor suppressor genes BRCA1 and BRCA2 confer substantial increased lifetime risk for breast cancer, and in the case of BRCA1, for ovarian carcinoma as well. These two genes alone account for the vast majority of hereditary breast cancer families. Numerous mutations have been described in each gene, the majority of which are small insertions or deletions resulting in expression of a truncated protein. MATERIALS AND METHODS: Several common mutations can be detected using a polymerase chain reaction-mediated, site-directed mutagenesis assay, which transforms the amplicon derived from either the wild-type or mutant allele by adding or removing a restriction endonuclease site. We screened 49 putative sporadic breast tumors using this methodology, targeting four BRCA1 mutations (185delAG, 5382insC, R1443X, and E1250X) and a single BRCA2 mutation (6174delT). RESULTS: Using the polymerase chain reaction-mediated, site-directed mutagenesis assay, we identified two mutations, namely, a 185delAG mutation (BRCA1) and a 6174delT mutation (BRCA2). Interestingly, these two mutations were found in the same sample. None of the remaining 48 breast tumors showed evidence of these mutations. Allele-specific oligonucleotide probes were then employed in conjunction with the Universal GeneComb Test Kit, which confirmed the presence of mutations. CONCLUSIONS: Our data suggest that the common germline BRCA1 and BRCA2 mutations are infrequently encountered in sporadic breast cancers. The one case with dual BRCA1 and BRCA2 mutations suggests that this tumor may be hereditary in origin, despite the lack of a positive family history. Double heterozygosity for mutations in BRCA1 and BRCA2 may have increasingly significant implications with regard to predisposition to breast cancer.

BRCA2 Protein↗
PubMed1998

Religious affiliation, religious involvement, and alcohol use in Korea.

This article is the first study of religious commitment and involvement of homeland Koreans as related to alcohol use. Religious affiliation and involvement are predictive of abstemiousness, but chiefly for women, who are at low risk for alcohol abuse and dependency, and not for men, who are at high risk. The frequent abstemiousness of Buddhist women results chiefly from data having to do with mothers and probably is a consequence of the women's traditional lifestyles, being both Buddhist and abstemious. Among the probably more Westernized Christians, religious involvement is most clearly associated with abstemiousness among daughters, a group at a relatively low but increasing risk for alcohol use.

Adult↗
PubMed1997

The transactivation region of the fis protein that controls site-specific DNA inversion contains extended mobile beta-hairpin arms.

The Fis protein regulates site-specific DNA inversion catalyzed by a family of DNA invertases when bound to a cis-acting recombinational enhancer. As is often found for transactivation domains, previous crystal structures have failed to resolve the conformation of the N-terminal inversion activation region within the Fis dimer. A new crystal form of a mutant Fis protein now reveals that the activation region contains two beta-hairpin arms that protrude over 20 A from the protein core. Saturation mutagenesis identified the regulatory and structurally important amino acids. The most critical activating residues are located near the tips of the beta-arms. Disulfide cross-linking between the beta-arms demonstrated that they are highly flexible in solution and that efficient inversion activation can occur when the beta-arms are covalently linked together. The emerging picture for this regulatory motif is that contacts with the recombinase at the tip of the mobile beta-arms activate the DNA invertase in the context of an invertasome complex.

Amino Acid Sequence↗
PubMed1997

Activation of RpoS-dependent proP P2 transcription by the Fis protein in vitro.

The proP gene, encoding a transporter of the osmoprotecting compounds proline and glycine betaine, is expressed from two promoters. Transcription of the P2 promoter occurs at a transient period in late exponential phase and is dependent upon Fis and the RpoS (sigma38) sigma factor. Here we characterize Fis-mediated activation of the P2 promoter in vitro. We find that this promoter displays unusually high specificity for sigma38. Fis strongly activates P2 when bound to site I centered at -41 within the promoter region. There is a complex relationship involving DNA supercoiling and potassium glutamate concentration on Fis activation, but most efficient transcription occurs under high salt conditions when the superhelical density is above -0.03. The major stimulatory effect of DNA supercoiling occurs between superhelical densities of 0 to -0.02 suggesting that, while supercoiling is mechanistically important, it may not be a physiologically relevant controlling factor. However, the stimulation of transcription by high potassium glutamate concentrations may contribute to the osmotic inducibility of the P2 promoter. We show that Fis and E sigma38 bind cooperatively on supercoiled DNA to form a stable complex at P2 that involves promoter melting. Fis also binds to a second site within the proP regulatory region. While binding to this site appears to play no role in Fis activation of the P2 promoter, it functions as a repressor of transcription initiating from the P1 promoter by either sigma70 or sigma38.

Bacterial Proteins↗
PubMed1997

Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme.

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways.

Amino Acid Sequence↗