Biomedical subjects

M A Clark

Publications and source records attributed to M A Clark.

At least 91 records · Page 5Linked to original sources

PubMed1996

Ribosomal protein S1 (RpsA) of Buchnera aphidicola, the endosymbiont of aphids: characterization of the gene and detection of the product.

Buchnera aphidicola is a prokaryotic endosymbiont found in specialized cells of the aphid Schizaphis graminum. Many of the previously cloned B. aphidicola genes are preceded by a poor ribosome-binding site. Ribosomal protein S1 (RpsA) allows the translation of messenger RNAs that lack or have a poor ribosome binding site. We have cloned and sequenced a 4.5-kilobase (kb) B. aphidicola DNA fragment containing four open reading frames corresponding to aroA-rpsA-himD-tpiA. The deduced amino acid sequence of B. aphidicola RpsA was 75% identical to that of the Escherichia coli protein. The major difference was in the number of basic amino acids, which were present in higher numbers in B. aphidicola RpsA. Antiserum to E. coli RpsA was prepared and used to detect B. aphidicola RpsA in cell-free extracts of aphids. During the first 12 days of aphid growth there is a slight decrease in the amount of RpsA per unit of aphid weight. The three additional genes found on the 4.5-kb DNA fragment encoded for proteins involved in aromatic amino acid biosynthesis (aroA), DNA bending (himD), and carbohydrate metabolism (tpiA). The presence of these genes in B. aphidicola is additional evidence of its similarity to free-living bacteria.

Amino Acid Sequence↗

PubMed1996

Suggestions for crops grown in controlled ecological life-support systems, based on attractive vegetarian diets.

Assuming that crops grown in controlled ecological life-support systems (CELSS) should provide a basis for meals that are both nutritious and attractive (to taste and vision), and that CELSS diets on the moon or Mars or in space-craft during long voyages will have to be mostly vegetarian, a workshop was convened at the Johnson Space Center, Houston, Texas, U.S.A. on 19 to 21 January, 1994. Participants consisted of trained nutritionists and others; many of the approximately 18 presenters who discussed possible diets were practicing vegetarians, some for more than two decades. Considering all the presentations, seven conclusions (or points for discussion) could be formulated: nutritious vegetarian diets are relatively easily to formulate, vegetarian diets are healthy, variety is essential in vegetarian diets, some experiences (e.g., Bios-3 and Biosphere 2) are relevant to planning of CELSS diets, physical constraints will limit the choice of crops, a preliminary list of recommended crops can be formulated, and this line of research has some potential practical spinoffs. The list of crops and the reasons for including specific crops might be of interest to professionals in the field of health and nutrition as well as to those who are designing closed ecological systems.

Crops, Agricultural↗

PubMed1996

Invasion of murine intestinal M cells by Salmonella typhimurium inv mutants severely deficient for invasion of cultured cells.

We have examined the role of the Salmonella typhimurium inv locus in invasion of the murine intestine. Previous studies have demonstrated that M cells within the lymphoid-follicle-associated epithelia are the primary site of intestinal invasion by S. typhimurium. In this study, we show that mutants possessing defects in one of two inv genes, invA or invG, which render them severely deficient for invasion of polarized epithelial MDCK cells, retain their ability to actively invade mouse Peyer's patch M cells. The interaction of these mutants with M cells was associated with apical membrane remodelling resembling that induced by wild-type strains. These data demonstrate that Salmonella invasion in vivo can proceed via mechanisms other than those previously defined in cultured cells.

Animals↗

PubMed1996

Cholera toxin induces synthesis of phospholipase A2-activating protein.

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.

Amino Acid Sequence↗

PubMed1996

Biological properties of renal oncocytoma cells in culture.

This paper sought to determine biological properties of oncocytoma cells, both in vivo and in vitro, which may serve as useful diagnostic indicators. Cell lines were grown in short-term culture from two renal oncocytomas following enzymatic dissociation, characterised by immunohistochemistry and electron microscopy, and further studied for abnormal p53 or retinoblastoma genes. Cells in culture were shown to maintain the morphological attributes of the primary lesion, including the overproduction of mitochondria. Despite the absence of vimentin staining in the primary tumour, cells in culture were shown to express this intermediate filament. Immunohistochemistry for P53 protein demonstrated an overexpression in one of the tumours, suggesting that mutation had occurred. Restriction fragment length polymorphisms of the retinoblastoma and the p53 genes were not demonstrated. Mutation of the p53 gene does occur in oncocytomas. The maintenance of the phenotype of oncocytoma cells in culture suggests that in vitro studies may be useful in the identification of unique properties of the tumour.

Adenoma, Oxyphilic↗

PubMed1996

Choosing plants to be grown in a Controlled Environment Life Support System (CELSS) based upon attractive vegetarian diets.

Space explorers on the Moon, Mars, or even in a space craft might grow plants in a CELSS to remove CO2 and provide O2 and food. Selection of crops to be studied has been rather arbitrary but should be based on plants that can provide a balanced and attractive, mostly vegetarian diet. Additional selection criteria include ease of growth in artificial environments and sufficient variety provided over long intervals. This article is based on a workshop convened to study vegetarian diets for use in a CELSS. Participants included nutritional scientists, practicing vegetarians, and interested employees of the Johnson Space Center. It was concluded that diets meeting the criteria could be formulated, and a list of suitable crops was compiled.

Crops, Agricultural↗

PubMed1996

Targeting to intestinal M cells.

The specialised, antigen-transporting, epithelial M cells in the follicle-associated epithelium (FAE) overlying gut-associated lymphoid tissues constitute the primary target for oral delivery of vaccines. Our studies have shown that polystyrene microspheres selectively bind to, and are efficiently transcytosed by, rabbit Peyer's patch M cells in closed intestinal loops. Binding of biodegradable poly(DL-lactide-co-glycolide) microspheres to rabbit Peyer's patch FAE is an order of magnitude lower than that of polystyrene microspheres. Although poly(DL-lactide-co-glycolide) microspheres are not selectively targeted to M cells, a high proportion of those which bind to M cells are transcytosed, supporting the potential of such microspheres as vehicles for oral vaccine delivery. Comparison of the binding of polystyrene microspheres by murine FAE revealed this to be markedly less extensive than by rabbit FAE. These data demonstrate that microsphere binding by M cells depends on the surface properties of both cells and microspheres and suggest that surface modification may enhance the efficacy of microsphere delivery vehicles. One such approach is the incorporation of molecules with inherent binding specificity for M cells. Lectin-binding studies have revealed that M cells exhibit pronounced regional and species variation in glycoconjugate expression. In murine intestine, certain lectins bind selectively to M cells either in Peyer's patches or caecum, or at both sites. Selective targeting to, and transcytosis of, lectin-conjugates by M cells in ligated segments of murine intestine have also been demonstrated. While several lectins display strong selectivity for rabbit caecal M cells, none to date have been identified with specificity for rabbit or rat Peyer's patch M cells. Knowledge of human M cells is limited and no lectin has yet been identified with specificity for these cells. However, at least one lectin exhibits binding specificity for FAE in the human ileum. In the future, knowledge of the regional patterns of M cell carbohydrate expression within a species may allow lectins to be utilised to target selectively antigenic material to the mucosal immune system at specific locations.

Administration, Oral↗

PubMed1995

Presence in human eosinophils of a lysophospholipase similar to that found in the pancreas.

The supernatant fraction from lysed human eosinophils, when separated by gel-filtration chromatography, contains a protein with lysophospholipase activity of approximate molecular mass 74 kDa. This mass differs substantially from the 17 kDa of a previously cloned eosinophil lysophospholipase (Charcot-Leyden crystal protein), but is similar to that reported for a pancreatic enzyme. We have therefore further characterized this pancreatic-like lysophospholipase in human eosinophils. A rabbit polyclonal antibody was produced against a synthetic peptide consisting of amino acids 325-349 from the 74 kDa rat pancreatic lysophospholipase. Western-blot analysis of eosinophil extracts indicate that this antibody recognizes a single 74 kDa band in these preparations. Incubation of the supernatant fraction from sonified eosinophils with this antibody, followed by precipitation of antibody-antigen complexes with Protein A, removes the majority of the lysophospholipase activity. Indirect immunofluorescence examination with this antibody indicates this protein to be localized to granules of eosinophils and not in other leucocytes. Moreover, reverse transcriptase PCR of polyadenylated RNA from eosinophils and from rat pancreatic tissue with primers to rat pancreatic lysophospholipase resulted in readily detectable 1 kb DNA products in both samples. Sequencing revealed this DNA fragment to be identical with the human pancreatic lysophospholipase cDNA sequence. Taken together, these data indicate that eosinophils contain a lysophospholipase that is similar to the human pancreatic enzyme.

Amino Acid Sequence↗

PubMed1995

A novel pyrrolidine analog of histamine as a potent, highly selective histamine H3 receptor agonist.

Employing classical conformational analysis on a known H3 agonist, (R)-alpha-methylhistamine (1), a series of conformationally constrained H3 agonists were proposed and synthesized. Pyrrolidine (+/-)-4a, a compound proposed to mimic the anti-conformation of (R)-alpha-methylhistamine (1), was found to be a potent and selective H3 agonist. The pyrrolidine (+/-)-4a was resolved, and its (+) enantiomer, immepyr [(+)-4a], showed a greater separation of H3 and H1 activities in vivo (H3/H1 ratio >> 550) than (R)-alpha-methylhistamine (1) (H3/H1 ratio = 17), the standard H3 agonist. In fact, no evidence of H1 activity was detected at doses of immepyr [(+)-4a] as high as 100 mg/kg i.v. This pyrrolidine, immepyr [(2R,3S)-(+)-4a], represents, to our knowledge, the first reported cyclic, conformationally restricted analog of histamine to possess selective in vivo H3 agonist activity.

Histamine↗

PubMed1995

Hutchinson-Gilford progeria types defined by differential binding of lectin DSA.

Hutchinson-Gilford Progeria Syndrome (progeria) is an extremely rare childhood disorder characterized by precocious senility which presents features similar to those seen in human aging. We have previously described a consistent increase of the glycoprotein gp200 in progeria skin fibroblasts in vitro. Here we extend these glycosylation studies and present evidence for the existence of two types of progeria skin fibroblasts. These two forms, called D- and D+, are distinguished by their response to the lectin DSA. In the D- group, DSA bound glycoproteins from progeria fibroblast strains AG03513B and AG10750 with markedly lower affinities compared with glycoproteins from three control fibroblast strains. In the D+ group, DSA binding to glycoproteins from four other progeria strains AG01972A, AG06297A, AG06917 and AG03198, was comparable to controls. Discrimination by DSA is the most distinctive feature of the D- and D+ groups, in contrast to binding of lectins Con A, GNA, PHA-L, RCA120, AAA and PNA which show no such selectivity. The data are consistent with a model of altered glycosylation in the D- type of progeria fibroblasts.

Adolescent↗

PubMed1995

Phospholipase A2-activating protein induces the synthesis of IL-1 and TNF in human monocytes.

Phospholipase A2-activating protein (PLAP) is an important mediator of eicosanoid generation. PLAP can also be found in high concentrations in synovial fluid from patients with rheumatoid arthritis, and injection of PLAP into animal joints results in an inflammatory, rheumatoid-like lesion. We have demonstrated previously that TNF-alpha and IL-1 beta stimulate formation of PLAP before phospholipase A2 (PLA2) enzyme activation and production of eicosanoids. To further explore the mechanisms found in the inflammatory response, we examined the ability of PLAP to stimulate release of TNF and IL-1 from human peripheral blood monocytes. TNF and IL-1 protein levels were measured by ELISA, and IL-1 and TNF mRNA were determined by Northern blotting. PLAP, PLAP peptide, and melittin, a bee venom PLA2 activator with homology with PLAP, all increased IL-1 and TNF production in a time- and dose-dependent manner. Heat-denatured PLAP and actin (an irrelevant protein) failed to exert this effect. PLAP stimulation of TNF and IL-1 could be enhanced with co-treatment of cells with free fatty acids, such as arachidonic or linoleic acid, but it was not blocked completely by PLA2 inhibitors. These results demonstrate not only that synthesis of PLAP can be stimulated by cytokines, but also that PLAP may regulate cytokine synthesis and thus perpetuate an immune or inflammatory response.

Amino Acid Sequence↗

PubMed1995

Purification of a phosphatidic-acid-hydrolysing phospholipase A2 from rat brain.

A phosphatidic-acid-hydrolysing phospholipase A2 was purified from rat brain and characterized. This phospholipase A2 was purified by sequential cation, hydrophobic, heparin and gel-filtration chromatography. The purified protein had a mass of approximately 58 kDa as assayed by SDS/PAGE, had a pH optimum of 6.0, and was Ca(2+)-independent. This enzyme was apparently phosphatidic-acid-selective and had little measurable catalytic activity when phosphatidylcholine, phosphatidylethanolamine or diacylglycerol was used as substrate. On the basis of its physical and catalytic properties, we conclude that this phospholipase A2 is unique from those previously purified, and we speculate that it may be important for the production of the bioactive lipid lysophosphatidic acid.

Animals↗

PubMed1995

Differential effect of cytokines on the phenobarbital or 3-methylcholanthrene induction of P450 mediated monooxygenase activity in cultured rat hepatocytes.

Cultured rat hepatocytes have been used to compare the relative activities of cytokines to inhibit the phenobarbital (PB) or 3-methylcholanthrene (MC) induction of cytochrome P4502B1 and 2B2 (P4502B1/2) or P4501A1 and 1A2 (P4501A1/2), respectively. Recombinant cytokines tested were human interleukin-6 (IL-6), interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta, respectively), and rat gamma-interferon (INF gamma). Hepatocytes were cultured in the presence of 2 mM PB or 1 microgram MC/mL culture medium for 24 hr with or without the cytokines. Benzyloxyresorufin and ethoxyresorufin O-dealkylase (BROD and EROD, respectively) activities were determined as indices of P4502B1/2 and P4501A1/2, respectively. All cytokines produced a concentration-dependent inhibition of the PB induction of BROD activity. IL-1 beta and IL-6 were approximately equipotent with IC50 values of 1-2 U/mL, causing greater than 90% inhibition of PB induction of BROD activity at a concentration of 50 U/mL culture medium. IL-1 alpha tended to be less active. PB induction of BROD activity was also inhibited by INF gamma, but higher concentrations (62.5 to 500 U/mL culture medium) were required. All cytokines were less effective in inhibiting the MC induction of EROD activity than the PB induction of BROD activity. IL-1 beta and IL-6, at 50 U/mL culture medium, inhibited EROD induction by only 35% compared with the greater than 90% inhibitory effect on the PB induction of BROD activity. INF gamma was ineffective in inhibiting EROD activity at the concentrations studied. Western immunoblot analysis indicated that the cytokines prevented the ability of the inducers to increase the expression of P4502B1/2 and P4501A1/2 immunoreactive proteins, and this effect correlated with their inhibitory effect on induction of enzyme activity. The results suggest that inducible isoforms of cytochrome P450 differ in their susceptibility to regulation by the cytokines, and that cytokines possess differential activity to inhibit the induction of P450 isoforms, with IL-1 beta and IL-6 being the most effective.

Animals↗

PubMed1995

Attitudes and practices for smoking cessation counseling by provider type and patient age.

Attitudes and self-reported practices for smoking cessation counseling among 145 providers at a health maintenance organization were compared among two provider groups, physicians/nurse practitioners and registered/licensed practical nurses, and across three patient age groups, < 50, 50-64, and > or = 65. Smoking cessation attitudes did not differ by provider type but they did differ by patient age, especially among the registered/licensed practical nurses, whose attitudes were least favorable for the oldest smokers (> or = 65). While smoking cessation practices did not differ by patient age, they did differ by provider type. Self-reported performance of the 4 As of smoking cessation practice (Ask, Advise, Assist, Arrange) was more frequent among the physicians/nurse practitioners than among the registered/licensed practical nurses. However, among both groups, asking and advising practices were reported more often than were assisting and arranging. In all cases, different attitudes were correlated with different practice behaviors for the two provider groups. Also, there were more significant correlations between age-specific attitudes and practices among the registered/licensed practical nurses than among the physicians/nurse practitioners. This was true especially regarding the oldest patients. The findings suggest a need for provider education, especially among registered/licensed practical nurses, about the benefits of smoking cessation for patients of all ages and the potential effectiveness of provider-based intervention strategies that are targeted toward specific age groups. The findings also suggest that assisting and arranging practices in particular need improvement among all types of providers.

Adult↗

PubMed1995

Age and smoking cessation behaviors.

BACKGROUND: Predictors of smoking cessation behaviors during a 3-month period were examined among smokers in three age groups: 18 to 29, 30 to 49, and 50 years or older. METHODS: Using random-digit dialing, telephone interviews were completed with 1,644 smokers in the Chicago metropolitan area at baseline and at a 3-month follow-up. Multiple logistic regression was used to identify factors that predicted trying to quit smoking and quitting smoking within each age group and by stage of readiness to quit smoking. RESULTS: There were no significant differences by age in trying to quit smoking or in quitting smoking among smokers in the contemplation or preparation stages. However, there were important age differences in the factors that predicted those behaviors. In general, different factors predicted smoking cessation behaviors in each age group at each of three stages of the smoking cessation process (contemplation, preparation, and action). Also, within each age group, different factors predicted the behavior at each stage. CONCLUSIONS: The findings suggest that smokers of all ages are equally appropriate targets for smoking cessation interventions. Moreover, there are important age-related differences among smokers over the course of their smoking careers that should be considered when planning and implementing smoking cessation interventions.

Adolescent↗

PubMed1995

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

Actins↗

PubMed1995

Lectin binding defines and differentiates M-cells in mouse small intestine and caecum.

M-cell surface glycoconjugate expression was investigated by applying a panel of lectins to whole fixed mouse Peyer's and caecal patches. While the majority of lectins failed to identify mouse M-cells, the lectin Euonymus europaeus differentially stained the surface of M-cells in both mouse Peyer's and caecal patches, and the lectins Ulex europaeus II and Bandeiraea simplicifolia I isolectin B4 identified M-cells in the Peyer's and caecal patch follicle associated epithelium, respectively. These three mouse M-cell markers failed to identify rat and rabbit Peyer's patch M-cells, although both Euonymus europaeus and Ulex europaeus II differentially stained M-cells in the periphery of rabbit caecal patch domes. These site and species related variations in M-cell surface glycoconjugate expression may reflect the local microorganism populations and will have important implications if orally delivered vaccines and drugs are to be targeted to M-cells via their surface glycoconjugates.

Animals↗

PubMed1995

Joint coordination deficits in limb apraxia.

Competing models of the basis of limb apraxia were tested through analysis of joint coordination deficits in three apraxic subjects with lesions that included the left parietal lobe. Three-dimensional shoulder, elbow, wrist and hand trajectories were recorded for repetitive 'slicing' gestures made in a series of conditions in which contextual cues were introduced in a graded fashion. The apraxic subjects showed marked deficits in joint coordination across context conditions. Even when actually manipulating a tool and object, the apraxic subjects failed to show proper joint synchronization, failed to apportion their relative joint amplitudes properly, and failed to produce the correct phase relationships among pairs of arm angles. Thus, apraxic subjects not only have deficits in the spatial plan for the movement, but they also have deficits in translating those plans into the details of the angular motions at the joints, even when actually manipulating a tool and object. These data support a model of apraxia in which apraxia can result from either the destruction of visuo-kinaesthetic motor representations of learned movement, stored in posterior association cortex, or from a separation of these representations from premotor or motor areas.

Aged↗