Biomedical subjects

L Brink

Publications and source records attributed to L Brink.

At least 19 recordsLinked to original sources

PubMed1997

Physiological responses of fathead minnow larvae to rice pesticides.

Newly hatched fathead minnow (Pimephales promelas) larvae were exposed for 4 days to two pesticides and ambient receiving waters to simulate conditions in the Sacramento River, California, during the striped bass spawning season which coincides with pesticide use in adjacent rice culture. Carbofuran and molinate were tested at two concentrations: a higher level approximating one-half the LC50 and a level much lower that is similar to that seen in the receiving waters of Colusa Basin Drain. Physiological measurements were made immediately after the exposures and again after a 10-day recovery period in noncontaminated waters. These included growth rate, swimming capacity, response to a mild electric shock, upper and lower lethal temperatures, and activity of acetylcholinesterase in whole-body homogenates. The higher concentrations of carbofuran and molinate caused reductions in swimming capacity, an increased sensitivity to the electric shock, and a reduction in upper lethal temperature. Acetylcholinesterase was reduced in those larvae exposed to the higher levels of carbofuran. In general, the lower levels of pesticide exposure caused no measureable effects nor did exposure to water from Colusa Basin Drain.

Acetylcholinesterase↗

PubMed1995

An active carbonyl formed during glycosylphosphatidylinositol addition to a protein is evidence of catalysis by a transamidase.

Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumable the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.

Acyltransferases↗

PubMed1994

Trials of Edmonston-Zagreb measles vaccine in Guinea-Bissau: serological responses following vaccination with Edmonston-Zagreb strain at 4-8 months versus vaccination with Schwarz strain at 9-12 months of age.

In two trials of measles vaccination in Guinea-Bissau, children were randomized to receive either the Edmonston-Zagreb (EZ) virus at age 4-8 months or, as a control group, a standard dose (5000 p.f.u.) of the Schwarz (SW) virus at 9-12 months. In the first trial a medium dose of EZ virus (40,000 p.f.u.) was used and in the later trial a high dose (150,000 p.f.u.). Pre- and postvaccination blood samples were analysed with a haemagglutination inhibition (HAI) test and an enzyme-linked immunosorbent assay (ELISA). For the ELISA the blood samples were collected on filter paper. Of 362 prevaccination specimens, 18 and 55%, respectively, were positive in the ELISA and HAI tests. At 18-20 months, the children who had received the EZ vaccine had a seropositivity of 91% by the ELISA method and one of 89% by the HAI method. The equivalent values for the SW group of children were 100 and 96% respectively. Antibody levels in the EZ group, as measured by either method, were significantly lower than the levels in the SW group. The serological results of the present study suggest that lowering the age at measles vaccination to below 9 months is feasible. However, further studies are needed to determine which virus strain, dosage and age at vaccination will prove to be optimal in countries where severe measles is common before the age of 9 months.

Antibodies, Viral↗

PubMed1994

Long-term survival in trial of medium-titre Edmonston-Zagreb measles vaccine in Guinea-Bissau: five-year follow-up.

A trial of protective efficacy which compared medium-titre Edmonston-Zagreb (EZ) measles vaccine (10(4.6) p.f.u.) from the age of 4 months with the standard Schwarz (SW) measles vaccine given from the age of 9 months was started in an urban community in Guinea-Bissau in 1985. Because trials of high-titre measles vaccine have found increased mortality among female recipients, we examined whether EZ medium-titre vaccine was associated with any long-term impact on mortality, suppression of T-cells, or growth. The mortality rate ratio over 5 years of follow-up was 1.12 for EZ children compared with children in the standard group (P = 0.63). Seventy-five percent of the children still residing in the area at 5 years of age took part in an immunological and anthropometric examination. There was no difference in T-cell subsets between the two groups. There was no difference in mid-upper-arm circumference, but EZ children were significantly shorter than the children in the standard group. In conclusion, medium-titre EZ was not associated with reduced survival or persistent immunosuppression.

Age Factors↗

PubMed1994

Use of pediatric physician extenders in pediatric and neonatal intensive care units.

OBJECTIVES: To determine present and future use of pediatric physician extenders in neonatal and pediatric intensive care units (ICUs). DESIGN: Descriptive, prospective, questionnaire survey. PARTICIPANTS: One hundred thirty hospitals represented by members of the Pediatric Section of the Society of Critical Care Medicine and 18 randomly selected hospitals identified as having no pediatric intensivist. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One hundred one (68.2%) of 148 responding institutions employed physician extenders and 69 (46.7%) employed pediatric physician extenders. Eighty percent of the hospitals using pediatric physician extenders employed pediatric nurse practitioners and 25% employed physician assistants. Of the 69 hospitals that employed pediatric physician extenders, 51 (73.9%) hospitals utilized them in neonatal ICUs and 12 (17.4%) hospitals used them in the pediatric ICUs. Institutions that did or did not employ pediatric physician extenders in pediatric ICUs were comparable in all factors studied, except for the perception of childcare physician staffing shortages. Duties competently performed by pediatric physician extenders did not differ between pediatric nurse practitioners and physician assistants and were similar to those duties of a second-year pediatric resident. More than 40% of institutions expected to increase the use of pediatric physician extenders in neonatal and pediatric ICUs and they expected to provide the majority of the specialty training required. CONCLUSIONS: Pediatric physician extenders are extensively employed in pediatric and neonatal ICUs. They are perceived to perform at the level of second-year pediatric residents and are strongly supported by staff physicians and residents. It appears that more pediatric physician extenders will be employed in pediatric and neonatal ICUs in the future.

Chi-Square Distribution↗

PubMed1993

Long-term survival after Edmonston-Zagreb measles vaccination in Guinea-Bissau: increased female mortality rate.

In an urban area of Guinea-Bissau, 384 children were enrolled in a randomized trial comparing morbidity and mortality rates after receiving high-titer Edmonston-Zagreb (EZ) measles vaccine administered from 4 months of age, with a control group receiving inactivated poliomyelitis vaccine at 4 months of age and the standard Schwarz vaccine from 9 months of age. Children were followed to the age of at least 3 years. The mortality ratio of the EZ vaccinees compared with control subjects was 1.79 (range, 1.06 to 3.02; p = 0.027) if children were excluded at the time of migration; if deaths after migration were included, the mortality ratio was 1.53 (range, 0.94 to 2.49; p = 0.087). Girls in the EZ group had significantly higher mortality rates than girls in the control group (mortality ratio = 1.95; range, 1.07 to 3.56; p = 0.027); there was no difference for the boys (mortality ratio = 0.98; range, 0.41 to 2.30). Adjustment for background factors in a Cox regression model did not modify these estimates. Furthermore, female recipients of EZ vaccine had more days with diarrhea (relative risk = 1.35; range, 1.17 to 1.56; p = 0.00003) and were more likely than control subjects to visit a health center in the month after vaccination (relative risk = 1.86; range, 1.05 to 3.31; p = 0.027); those who consulted were more likely to die subsequently (mortality ratio = 2.31; range, 0.99 to 5.41; p = 0.054). These observations were unplanned and require confirmation in larger studies.

Cause of Death↗

PubMed1993

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell-free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.

Alkaline Phosphatase↗

PubMed1992

Phosphatidylinositol-glycan (PI-G)-anchored membrane proteins: requirement of ATP and GTP for translation-independent COOH-terminal processing.

Placental alkaline phosphatase (PLAP) belongs to a class of proteins that are anchored to the plasma membrane by a COOH-terminal phosphatidylinositol-glycan (PI-G) moiety. Nascent forms of such proteins undergo NH2- and COOH-terminal processing to yield the mature PI-G-tailed proteins. We previously introduced a shortened engineered form of preproPLAP (preprominiPLAP) that permits monitoring in cell-free preparations its sequential processing to the pro form and then to the mature PI-G-tailed form. Previous studies were carried out by synthesizing the preproprotein cotranslationally in the presence of rough microsomal membranes (RM). Because of the complexity of the cotranslational system it was not possible to determine whether cofactors were required for processing. We have now prepared RM that are preloaded with prominiPLAP but contain little mature PI-G-tailed miniPLAP. Maximal processing requires supplementation with both ATP and GTP. Inhibitors of PI-G biosynthesis do not affect processing. Since cleavage and PI-G addition are presumably catalyzed by a transamidase, the nucleoside triphosphate requirements suggest that there are additional steps in prominiPLAP processing prior to transamidation with PI-G. These may involve translocation of the pro protein in a proper conformational state to the transamidase site.

Adenosine Triphosphate↗

PubMed1992

Biosynthesis of phosphatidylinositol-glycan (PI-G)-anchored membrane proteins in cell-free systems: PI-G is an obligatory cosubstrate for COOH-terminal processing of nascent proteins.

It is generally recognized that nascent proteins destined to be processed to a phosphatidylinositol-glycan (PI-G)-anchored membrane form contain a hydrophobic signal peptide at both their NH2 and COOH termini. In previous studies we showed that rough microsomal membranes (RM) prepared from CHO cells can carry out COOH-terminal processing. We have now investigated RM prepared from many additional cell types, including frog oocytes, B cells, and T cells, and found that all are competent with respect to COOH-terminal processing. Exceptions were certain mutant T cells that had been shown to be defective at various steps of PI-G anchor biosynthesis [Sugiyama, E., De Gasperi, R., Urakaze, M., Chang, H.-M., Thomas, L. J., Hyman, R., Warren, C. D. & Yeh, E. T. H. (1991) J. Biol. Chem. 266, 12119-12122]. In one such defective mutant, COOH-terminal processing activity of RM could be restored either by transfecting the intact cells with the gene for the deficient step in PI-G synthesis or by adding PI-G extracts to the RM in vitro. Cleavage of the COOH-terminal signal peptide in the RM is therefore dependent on the presence of intact PI-G incorporated into the mature protein.

Acyltransferases↗

PubMed1991

Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide.

Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G "transamidase" derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2- and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted beta carbon at the cleavage/attachment site that were found previously with wild type PLAP.

Acyltransferases↗

PubMed1991

Localization of gene for familial hypertrophic cardiomyopathy to chromosome 14q1 in a diverse US population.

BACKGROUND: Familial hypertrophic cardiomyopathy, an inherited primary cardiac abnormality characterized by ventricular hypertrophy, is the leading cause of sudden death in the young. Recent application of restriction fragment length polymorphism markers has provided provocative results, with localization to chromosome 18 (Japanese studies), 16 (Italian studies), 14 (US and French-Canadian studies), and two (National Institutes of Health studies) indicating genetic heterogeneity. Interpretation remains speculative until at least one of these loci is confirmed in unrelated pedigrees by independent investigators. METHODS AND RESULTS: We studied eight unrelated families of varied ethnic origins across the United States. DNA from each individual was digested with restriction enzymes TaqI or BamHI and analyzed by Southern blots followed by hybridization with probes T cell receptor alpha (TCRA), myosin heavy chain beta, D14S25, and D14S26. Multipoint linkage analysis showed a maximum lod score of 4.3, placing the locus 10 cM from D14S26 between D14S26 and TCRA, with an odds ratio of 20,000:1 and 90% confidence limits of 12 cM proximal to D14S25 to 4 cM distal to TCRA. The probability of linkage to 14q1 was more than 99%. CONCLUSIONS: These results indicate that the loci for familial hypertrophic cardiomyopathy in our families is primarily 14q1 but does not exclude other loci in a small proportion of the families. Thus, 14q1 appears to be the locus for familial hypertrophic cardiomyopathy in a significant proportion of the US population.

Cardiomyopathy, Hypertrophic↗

PubMed1989

The effect of astemizole on bronchial hyperresponsiveness and exercise-induced asthma in children.

The ability of the new generation H1-receptor antagonist, astemizole, to prevent histamine-induced airway obstruction and exercise-induced asthma (EIA) was studied in 20 children with asthma. The study was a randomised clinically controlled trial of oral astemizole versus placebo in a cross-over study. In each of the two treatment periods the children were tested at days 0, 6, 15 and 22 of therapy. The two treatment periods were separated by a washout period of 50 days, and at each visit a bronchial challenge with increasing concentrations of histamine followed by an exercise test was performed, and peak flow and asthmatic symptom score were recorded daily. The children tolerated significantly higher mean concentrations of histamine when treated with astemizole compared with placebo (P less than 0.001). Astemizole postponed the response to exercise, but no change in the maximal response was found. No differences between the treatment periods were found regarding frequency of asthmatic symptoms or the daily recording of peak flow.

Administration, Oral↗

PubMed1988

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein.

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.

Alkaline Phosphatase↗

PubMed1988

Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: application to placental alkaline phosphatase.

An immunological approach to the study of the structure and biogenesis of the phosphatidylinositol glycan (PI-G) membrane anchor at the carboxyl terminus of human placental alkaline phosphatase (PLAP) is described. Based on the protein sequence predicted from full length PLAP cDNA, two epitopes were chosen in the region of the carboxyl terminus for the production of site-directed antibodies. The exo site represents the last nine residues of preproPLAP, (res. 505-513), which is part of the sequence that is expected to be cleaved from the nascent protein during processing and addition of the PI-G tail. A second site, the endo sequence, was selected close to the expected carboxyl terminus in mature PI-G-tailed PLAP (res. 474-484 of proPLAP). The two peptides were synthesized, polyclonal antibodies to the conjugated peptides were prepared, and the antisera were characterized. Analytical methods for both synthetic peptides and proteins are presented. Preliminary applications to the isolation and characterization of the PI-G-linked carboxyl terminus of mature PLAP and to the characterization of nascent PLAP are described. The application of both carboxyl terminal-directed antibodies, and a third antibody directed to the amino terminus of mature PLAP, in studies employing mutant forms of PLAP and to the PI-G tailing process itself are discussed. The immunological approach used here for PLAP should be applicable generally to the study of other PI-G-tailed proteins.

Alkaline Phosphatase↗

PubMed1988

Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.

A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan.

Alkaline Phosphatase↗

PubMed1987

Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells.

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.

Antibodies, Neoplasm↗

PubMed1987

Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

A membrane-associated folate binding protein (FBP) and a soluble FBP, which is released into the culture medium, have been purified from human KB cells using affinity chromatography. By NaDodSO4/PAGE, both proteins have an apparent Mr of approximately 42,000. However, in the presence of Triton X-100, the soluble FBP eluted from a Sephadex G-150 column with an apparent Mr of approximately 40,000 (similar to NaDodSO4/PAGE) but the membrane-associated FBP eluted with an apparent Mr of approximately 160,000, indicating that this species contains a hydrophobic domain that interacts with the detergent micelles. The amino acid compositions of both forms of FBP were similar, especially with respect to the apolar amino acids. In addition, the 18 amino acids at the amino termini of both proteins were identical. The membrane FBP, following delipidation with chloroform/methanol, contained 7.1 mol of fatty acid per mol of protein, of which 4.7 mol was amide-linked and 2.4 mol was ester-linked. The soluble FBP contained only 0.05 mol of fatty acid per mol of protein. These studies indicate that the membrane FBP of KB cells contains covalently bound fatty acids that may serve to anchor the protein in the cell membrane.

Acylation↗

PubMed1986

Characterization of multiple forms of folate-binding protein from human leukemia cells.

Folate-binding proteins were isolated from the particulate fraction (44,000 X g pellet) and the soluble fraction (44,000 X g supernate) of the homogenate of a spleen obtained from a patient who had an acute leukemic (blast) transformation of chronic myelogenous leukemia. The folate-binding activity which was obtained from the particulate fraction by solubilization with 1% Triton X-100 could be resolved into two binding proteins (Mr 310,000 and 28,000) by gel filtration through Sephadex G-200 after incubation with excess [3H]pteroylglutamic acid (PteGlu). The folate-binding protein in the solubilized particulate fraction and the soluble folate-binding protein in the 44,000 X g supernatant cytoplasm were purified by affinity chromatography. Only a 32 kDa protein was identified by SDS-polyacrylamide gel electrophoresis in the final preparation of the purified folate-binding protein from the particulate, whereas two protein bands (Mr 42,000 and 32,000) were identified by SDS-polyacrylamide gel electrophoresis in the purified preparation of the soluble folate-binding protein. Both of these species were immunologically crossreacting. Both the purified folate-binding protein from the particulate fraction and the purified soluble form had higher affinity for oxidized folate than for the reduced folate cofactors, and both proteins had very low affinity for the antifolate compound, methotrexate. The amino-acid composition of the soluble folate-binding protein was similar with regard to the content of apolar amino acids to that reported for the membrane-derived folate-binding protein purified from milk and human placenta.

Amino Acids↗