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Biomedical subjects

P Mertz

Publications and source records attributed to P Mertz.

14 recordsLinked to original sources
PubMed2000

Calcineurin: form and function.

Calcineurin is a eukaryotic Ca(2+)- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and a tightly associated, myristoylated, Ca(2+)-binding subunit, calcineurin B. The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved from yeast to mammals. As a serine/threonine protein phosphatase, calcineurin participates in a number of cellular processes and Ca(2+)-dependent signal transduction pathways. Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein. Many studies have used these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organisms such as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function. Recent advances regarding calcineurin structure include the determination of its three-dimensional structure. In addition, biochemical and spectroscopic studies are beginning to unravel aspects of the mechanism of phosphate ester hydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studies which may lead to new calcineurin inhibitors. This review provides a comprehensive examination of the biological roles of calcineurin and reviews aspects related to its structure and catalytic mechanism.

Amino Acid Motifs↗
PubMed1999

Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)]2 cluster.

The interaction of bacteriophage lambda protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage lambda protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases.

Bacteriophage lambda↗
PubMed1997

Kinetic and spectroscopic analyses of mutants of a conserved histidine in the metallophosphatases calcineurin and lambda protein phosphatase.

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage lambda protein phosphatase is also considered to be a member of this family based on sequence comparisons (Lohse, D. L., Denu, J. M., and Dixon, J. E. (1995) Structure 3, 987-990). Using EPR spectroscopy, we demonstrate that lambda protein phosphatase accommodates a dinuclear metal center. Calcineurin and lambda protein phosphatase likewise contain a conserved histidine that is not a metal ligand but is within 5 A of either metal in calcineurin. In this study the conserved histidine in calcineurin was mutated to glutamine and the mutant protein analyzed by EPR spectroscopy and kinetic methods. Parallel studies with an analogous lambda protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in kcat of 460- and 590-fold for the calcineurin and lambda protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease in kcat of 1,300-fold for calcineurin. With the analogous lambda protein phosphatase mutant, kcat decreased 530-fold compared with wild type lambda protein phosphatase using phenyl phosphate as a substrate. EPR studies of the iron-reconstituted enzymes indicated that although both mutant enzymes can accommodate a dinuclear metal center, spectroscopic differences compared with wild type proteins suggest a perturbation of the ligand environment, possibly by disruption of a hydrogen bond between the histidine and a metal-coordinated solvent molecule.

Animals↗
PubMed1995

Calcineurin subunit interactions: mapping the calcineurin B binding domain on calcineurin A.

Recombinant forms of the A and B subunits of the protein phosphatase calcineurin were produced in Escherichia coli, reconstituted into a heterodimer and purified to homogeneity. The reconstituted heterodimer exhibited properties like that of bovine brain calcineurin. This included calmodulin-stimulated activity and a subunit stoichiometry and Stokes radius consistent with native-like structure. In order to map the region on the A subunit where calcineurin B binds, a series of overlapping 20-residue peptides corresponding to this putative domain were synthesized. Using isolated calcineurin A and B subunits, an assay that relied upon peptide inhibition of calcineurin B stimulation of calcineurin A activity was developed. All five peptides, but not a control peptide, inhibited calcineurin B-dependent stimulation of calcineurin A although with different potencies. The three most effective inhibitory peptides spanned calcineurin A residues 338-377. These three peptides also altered the electrophoretic mobility of the isolated calcineurin B subunit during native polyacrylamide gel electrophoresis indicating a direct interaction between these peptides and calcineurin B. The peptide corresponding to residues 348-367 was also able to block binding of calcineurin B to the catalytic subunit.

Amino Acid Sequence↗
PubMed1993

Human neoplastic submandibular intercalated duct cells express an acinar phenotype when cultured on a basement membrane matrix.

Culture of the human neoplastic submandibular gland intercalated duct cell line, HSG, on the basement membrane extract Matrigel induces dramatic morphologic changes and cytodifferentiation. Transmission electron microscopy demonstrated an acinar cell phenotype with polarized cells containing a well-developed Golgi apparatus, multiple microvilli-like projections from the apical surfaces into a lumenal-like area, and numerous granule-like organelles. Amylase, an acinar cell marker, was detected by both immunocytochemical and Northern blot analyses. A 50% reduction in [3H]thymidine incorporation by cells cultured on Matrigel, as compared to cells cultured on tissue culture plates, confirmed the differentiated phenotype of the cells. Multiple components of Matrigel appear to contribute to the morphologic differentiation of the HSG cells since antibodies to both laminin and collagen IV, as well as the laminin-derived bioactive peptide containing SIKVAV, have potent inhibitory effects on HSG cell organization on Matrigel. Collectively, these data indicate that culture of HSG cells on Matrigel is a useful model to study salivary gland acinar development.

Amylases↗
PubMed1992

The effects of occlusive dressings on the recruitment of mononuclear cells by endothelial binding into acute wounds.

Mononuclear cell infiltration is a characteristic feature of wounds and may play an important role in the healing process. We have previously shown that the recruitment of peripheral blood mononuclear cells into wounded skin is an active, dynamic, and regulated process mediated at least in part by specific interactions between mononuclear leukocytes and specialized dermal microvascular endothelial cells in the wounded skin. The purpose of this study was to investigate the capacity of dermal microvascular endothelial cells in wounds exposed to air or covered with occlusive dressings to promote the adhesion of monocytes and lymphocytes. The results showed that dermal microvascular endothelial cells in both air-exposed and occluded wounds were capable of supporting peripheral blood mononuclear cell and monocyte adherence. However, in comparison with air exposed wounds, the level of peripheral blood mononuclear cell binding was significantly higher in occluded biopsies obtained at 0 to 9, 12, 15, and 21 days after wounding. In addition, monocyte and peripheral blood mononuclear cells binding to occluded wounds peaked earlier than peripheral blood mononuclear cell binding to air exposed wounds. These studies provide evidence that differences in the kinetics and magnitude of mononuclear cell adherence may account at least in part for the beneficial effects of occlusive dressing on wound healing.

Animals↗
PubMed1992

Ciliated respiratory-like epithelium forming cystic conjunctival lesions in a patient with Stevens-Johnson syndrome.

A woman with severe cicatricial conjunctival changes secondary to allopurinol-induced Stevens-Johnson syndrome developed bilateral palpebral conjunctival cysts. Pathologic examination of these lesions revealed respiratory-like pseudostratified ciliated epithelium in the walls of several cysts of one eye. To our knowledge, this is the first report of this phenomenon.

Aged↗
PubMed1992

c-myc mRNA expression in minor salivary glands of patients with Sjögren's syndrome.

c-myc protooncogene is implicated in the pathogenesis of B cell lymphoid malignancies and high levels of c-myc mRNA expression are observed in activated blood mononuclear cells. Sjögren's syndrome (SS) is characterized by lymphocytic infiltrates of exocrine glands, remarkable B cell hyperreactivity and a strong predisposition to B cell neoplasia. In this study, c-myc protooncogene mRNA expression in 29 labial minor salivary gland biopsies from patients with primary SS and 15 controls was examined using in situ hybridization histochemistry. Two 40mer oligonucleotides from the 1st and the 2nd exon of the c-myc gene, labeled with 35S, were used as probes. To detect the origin of the cell hybridized with a c-myc probe, a combined immunochemistry in situ hybridization histochemistry technique was used. High c-myc mRNA expression was detected on acinar epithelial cells. c-myc did not correlate with c-fos and c-jun protein expression. Stronger c-myc mRNA expression was detected in labial salivary glands of patients with longer disease duration (p less than or equal to 0.002) and more intense T lymphocyte infiltrates (p less than 0.05) although these patients revealed no hypergammaglobulinemia. No correlation was observed between c-myc mRNA and B lymphocyte monoclonicity or lymphoma. In conclusion, strong c-myc mRNA expression was observed on epithelial cells of labial salivary glands from patients with primary SS. Our findings may indicate the presence of a reactivated virus hosted in these cells.

Adult↗
PubMed1990

Topical erythromycin and zinc therapy for acne.

A double-blind, 12-week study was undertaken to determine the safety and efficacy of a formulation of 4% erythromycin plus 1.2% zinc acetate compared with its vehicle. The study was continued for 40 weeks after the 12-week double-blind phase by switching vehicle-treated patients to active treatment and continuing to give patients treated with active drug the same treatment. Seventy-three female patients started the study; 39 completed 1 full year of study. In the first 12 weeks statistically significant differences were noted in the efficacy of the erythromycin-zinc compared with vehicle for acne severity grades (global assessment) and for papule, pustule, and comedo counts. After crossover, the vehicle-treated group receiving active therapy duplicated the improvement of the group initially treated with erythromycin-zinc. No clinical problems with superinfection or secondary infection occurred during 1 year of treatment in 39 patients.

Acne Vulgaris↗
PubMedDate not supplied

Collagen fiber orientation as quantified by small angle light scattering in wounds treated with transforming growth factor-beta2 and its neutalizing antibody.

The purpose of this study was determine quantitative differences in collagen fiber orientation in a wound healing model in the presence of transforming growth factor-beta2 and anti-transforming growth factor-beta2,3 antibody. Full-thickness wounds were made in the paravertebral area of two young pigs. Wounds were treated once, topically, with either transforming growth factor-beta2 or anti-transforming growth factor-beta2 antibody, or with methylcellulose gel. Control wounds were left untreated. Tissue biopsies were obtained from each wound on days 7, 14 and 46 post wounding. Tissue sections were stained with hematoxylin and eosin, and collagen fiber preferred orientation was quantified using small angle light scattering. Our results indicated that wounds treated with transforming growth factor-beta2 and anti-transforming growth factor-beta2,3 antibody had a significantly higher degree of orientation of collagen fibers than normal unwounded skin on days 7, 14 and 46 (p < 0.001). Transforming growth factor-beta2- treated wounds had a higher degree of orientation of collagen fibers than control wounds on days 7 and 14 (p < 0.001), and control wounds displayed a higher degree of orientation than wounds treated with anti-transforming growth factor-beta2,3 and normal unwounded skin at all time points (p < 0.001). These results suggest that differences in the dermal collagen degree of orientation correlate with scarring, and show that small angle light scattering can be used quantitatively to assess differences in the collagen fiber architecture of dermal wounds.

Administration, Topical↗
PubMedDate not supplied

The pig as a model for human wound healing.

The medical literature describes numerous in vitro and in vivo wound-healing models. The selection of an animal model depends on a number of factors including availability, cost, ease of handling, investigator familiarity, and anatomical/functional similarity to humans. Small mammals are frequently used for wound healing studies, however, these mammals differ from humans in a number of anatomical and physiological ways. Anatomically and physiologically, pig skin is more similar to human skin. The many similarities between man and pig would lead one to believe that the pig should make an excellent animal model for human wound healing. The purpose of this paper is to review the existing literature for evidence of this supposition and determine how well the various models correlate to human wound healing. Studies of wound dressings, topical antimicrobials, and growth factors are examined. Over 180 articles were utilized for this comparative review. Our conclusion is that the porcine model is an excellent tool for the evaluation of therapeutic agents destined for use in human wounds.

Animals↗