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Biomedical subjects

D J Schnell

Publications and source records attributed to D J Schnell.

At least 19 recordsLinked to original sources
PubMed1996

Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

Animals↗
PubMed1995

A regression method for analysing ordinal data from intervention trials.

We consider the case of a community intervention trial evaluated with a series of cross-sectional surveys and having outcomes measured on an ordinal scale. We propose a modelling procedure that combines ridit analysis and linear regression methods. We use the multinomial distribution as the basis for variance estimation of the mean ridits and then use simple regression models to estimate differences (for example, between intervention and comparison areas) among the ridits. We illustrate this procedure with data from a community intervention trial promoting condom use, with the adoption of consistent condom use measured on a 5-point ordinal scale.

Clinical Trials as Topic↗
PubMed1994

Isolation of components of the chloroplast protein import machinery.

Components of the protein import machinery of the chloroplast were isolated by a procedure in which the import machinery was engaged in vitro with a tagged import substrate under conditions that yielded largely chloroplast envelope-bound import intermediates. Subsequent detergent solubilization of envelope membranes showed that six envelope polypeptides copurified specifically and, apparently, stoichiometrically with the import intermediates. Four of these polypeptides are components of the outer membrane import machinery and are associated with early import intermediates. Two of these polypeptides have been characterized. One is a homolog of the heat shock protein hsp70; the other one is a channel-protein candidate.

Adenosine Triphosphate↗
PubMed1994

Identification of two GTP-binding proteins in the chloroplast protein import machinery.

Two of four proteins that associated with translocation intermediates during protein import across the outer chloroplast envelope membrane were identified as guanosine triphosphate (GTP)-binding proteins. Both proteins are integral membrane proteins of the outer chloroplast membrane, and both are partially exposed on the chloroplast surface where they were accessible to thermolysin digestion. Engagement of the outer membrane's import machinery by an import substrate was inhibited by slowly hydrolyzable or non-hydrolyzable GTP analogs. Thus, these GTP-binding proteins may function in protein import into chloroplasts.

Adenosine Triphosphate↗
PubMed1994

Biosynthesis of DB58 lectin in cell suspension cultures of Dolichos biflorus.

The stem and leaf lectin of the legume, Dolichos biflorus, was found to be expressed in cell suspension cultures derived from calli from this plant. The lectin is present at levels equivalent to the amount of lectin in the plant and its expression is correlated with the exponential growth phase of the cells. In vitro translation of mRNA isolated from these cultures, followed by immunoprecipitation with antibodies to the lectin, yields a single polypeptide precursor for this lectin. In vivo pulse chase labeling of the DB58 lectin yields a single glycosylated precursor that ultimately gives rise to the mature alpha and beta subunits of this heterodimer. Chemical deglycosylation of the labeled precursors and products shows that the alpha and beta subunits do not differ simply by their extent of glycosylation. Antibodies generated against a synthetic peptide representing the deduced COOH-terminus of the nascent protein react only with the alpha subunit. These data support a mechanism of lectin subunit generation involving differential carboxyl terminal modification of a single polypeptide precursor.

Cells, Cultured↗
PubMed1993

An evaluation of sexual behaviour change using statistical and cognitive models. The AIDS Community Demonstration Projects.

We evaluate sexual behaviour change among homosexual men enrolled in the cohorts in four AIDS Community Demonstration Projects. Behaviour change is classified following the stages of behaviour change model and described using a Markov model. Predictors of behaviour change are identified and evaluated using logit models for correlated data. Sexual behaviour change within the cohort could be modelled as a first-order Markov process. In addition, predictors suggested by models of health behaviour were correlated with particular patterns of sexual behaviour change. Our evaluation revealed a variety of patterns of sexual behaviour change in the cohorts and suggests multi-faceted interventions for promotion of behaviour change.

Adult↗
PubMed1993

Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites.

We have used a hybrid precursor protein to study the pathway of protein import into chloroplasts. This hybrid (pS/protA) consists of the precursor to the small subunit of Rubisco (pS) fused to the IgG binding domains of staphylococcal protein A. The pS/protA is efficiently imported into isolated chloroplasts and is processed to its mature form (S/protA). In addition to the mature stromal form, two intermediates in the pathway of pS/protA import were identified at early time points in the import reaction. The first intermediate represents unprocessed pS/protA bound to the outer surface of the chloroplast envelope and is analogous to a previously characterized form of pS that is specifically bound to the chloroplast surface and can be subsequently translocated in the stroma (Cline, K., M. Werner-Washburne, T. H. Lubben, and K. Keegstra. 1985. J. Biol. Chem. 260:3691-3696.) The second intermediate represents a partially translocated form of the precursor that remains associated with the envelope membrane. This form is processed to mature S/protA, but remains susceptible to exogenously added protease in intact chloroplasts. We conclude that the envelope associated S/protA is spanning both the outer and inner chloroplast membranes en route to the stroma. Biochemical and immunochemical localization of the two translocation intermediates indicates that both forms are exposed at the surface of the outer membrane at sites where the outer and inner membrane are closely apposed. These contact zones appear to be organized in a reticular network on the outer envelope. We propose a model for protein import into chloroplasts that has as its central features two distinct protein conducting channels in the outer and inner envelope membranes, each gated open by a distinct subdomain of the pS signal sequence.

Biological Transport↗
PubMed1992

Men's disclosure of HIV test results to male primary sex partners.

We evaluated disclosure of human immunodeficiency virus (HIV) antibody status to a main sex partner and the impact on the relationship in men who have sex with men and who are enrolled in the Acquired Immunodeficiency Syndrome (AIDS) Community Demonstration Projects cohorts. Eighty-nine percent of both seronegative and seropositive men disclosed the results to their main sex partner. Seventy percent of the seronegative men and 82% of the seropositive men who did so reported that the relationship remained "as strong as ever" after 6 months. Most men who did not disclose their test results to their main partner reported being "single" after 6 months.

Adult↗
PubMed1991

Evidence for the effects of HIV antibody counseling and testing on risk behaviors.

OBJECTIVE: To review published abstracts, journal articles, and presentations for evidence of the effects of human immunodeficiency virus (HIV) antibody counseling and testing on risk behaviors. Studies reviewed focused on homosexual men, intravenous drug users in treatment programs, pregnant women, and other heterosexuals. DATA SOURCES: Peer-reviewed journals (January 1986 through July 1990) and published abstracts and oral presentations from the second (1986) through the sixth (1990) International Conferences on AIDS. STUDY SELECTION: We identified 66 studies that included data on the behavioral effects of HIV antibody counseling and testing. By consensus of the authors, 16 of these were excluded because of small sample size or inadequate study design. DATA EXTRACTION: Studies were assessed by the authors according to methodological strength (sample selection, inclusion of appropriate comparison groups, and inclusion of statistical tests of significance). DATA SYNTHESIS: All longitudinal studies of homosexual men reported reductions in risky behavior among both tested and untested men, and a few reported greater decreases among seropositive men than among seronegative men and those untested or unaware of their serostatus. For intravenous drug users in treatment, we found reductions in intravenous drug use and sexual risk behaviors regardless of counseling and testing experience. We found little evidence for the impact of counseling and testing on pregnancy and/or pregnancy termination rates for either seropositive or seronegative high-risk women. We noted substantial risk reduction among heterosexual couples with one infected partner. Findings among other heterosexuals at increased risk were scanty and mixed. CONCLUSIONS: Further studies should specifically address the behavioral consequences of counseling and testing in various settings.

Counseling↗
PubMed1991

Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope.

We have used synthetic peptides representing segments of the signal sequences of preferredoxin (pFd) and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) to study interactions with the signal sequence recognition system at the chloroplast surface. Peptides representing the COOH-terminal 30 amino acids of the pFd and pS signal peptides were able to completely and reversibly inhibit the import of their homologous precursors into isolated chloroplasts at a 2.5 microM concentration. Import was blocked at the level of precursor binding to the chloroplast. This inhibition of precursor binding and import was not due to disruption of chloroplast integrity as incubation of isolated chloroplasts with the peptides did not cause measurable perturbation of the envelope membranes. The peptides also were able to block the import of the heterologous precursor protein, suggesting that pS and pFd share a common signal sequence recognition system. Visualization of the bound peptides at the chloroplast surface by indirect immunofluorescence microscopy using antipeptide antibodies gave a marked punctate staining pattern. This pattern is consistent with the localization of chloroplast import receptor(s) at contact zones between the inner and outer envelope membranes.

Amino Acid Sequence↗
PubMed1990

Two lectin genes differentially expressed in Dolichos biflorus differ primarily by a 116-base pair sequence in their 5' flanking regions.

Previous studies in our laboratory have shown that the Dolichos biflorus plant contains two similar lectins, a seed lectin and a stem and leaf lectin called DB58, that are present at different stages in the plant's life cycle. We have now established that each of these lectins is encoded by a separate gene by isolating these lectin genes from a library of D. biflorus nuclear DNA. Restriction mapping and nucleotide sequencing analyses show that the seed lectin and DB58 genes are located in the same transcriptional orientation within 3-kilobase pairs of one another. The lectin genes contain no introns and show greater than 90% nucleotide sequence identity in their protein coding and untranslated regions. This sequence similarity extends to both the 5' and 3' flanking regions of the genes; the major exception is that a 116-base pair segment located at position -215 to -100 from the transcription start site of the seed lectin gene is missing in the 5' flanking region of the DB58 gene. The possible significance of this segment with respect to the differential expression of these genes is discussed.

Base Composition↗
PubMed1990

The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites.

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5-bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flügge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flügge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296-304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of receptors for protein import into chloroplasts and mitochondria.

An anti-idiotypic antibody approach was used to identify chloroplast and mitochondrial protein component(s) which interact with the corresponding signal sequence. The proteins thus identified can be operationally defined as receptor(s) for import of proteins into chloroplasts and mitochondria. The import receptor(s) was found in "contact sites" between the outer and inner membrane of chloroplast envelope or of mitochondria.

Antibodies, Anti-Idiotypic↗
PubMed1989

Time series analysis of syphilis surveillance data.

To manage a public health programme effectively it is necessary to set objectives and priorities according to the resources available. To utilize available resources optimally in a disease control programme we should both estimate the present and predict the future magnitude of the health problem. The CDC has an effective programme to control syphilis, a sexually transmitted disease. To keep track of control activities, CDC receives the number of syphilis cases from all states every three months. Primary and secondary syphilis cases declined from an all time high of 106,539 cases in 1947 to 6399 cases in 1956. Since then, syphilis cases increased to 27,921 in 1986. Congenital syphilis cases declined from 17,600 in 1941 to 239 in 1983, but increased to 408 in 1986. We built time series models for primary and secondary syphilis cases in men and women and congenital syphilis cases in children under one year of age. These models were used to forecast syphilis cases in all three categories. This analysis suggests that no change in the trend of male syphilis cases has occurred, but syphilis cases in women and congenital syphilis cases in children under one year of age during 1987 have increased.

Data Interpretation, Statistical↗
PubMed1988

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus.

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.

Amino Acid Sequence↗
PubMed1987

cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin.

The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.

Amino Acid Sequence↗