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Joseph E Mazurkiewicz

Publications and source records attributed to Joseph E Mazurkiewicz.

12 recordsLinked to original sources
PubMed2006

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture.

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.

Bacterial Adhesion↗
PubMed2006

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture.

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.

Bacterial Proteins↗
PubMed2006

Serotonin 5-HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confocal fluorescence resonance energy transfer.

Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.

Bacterial Proteins↗
PubMed2005

Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein.

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.

Arginine↗
PubMed2005

Mitochondrial H2O2 regulates the angiogenic phenotype via PTEN oxidation.

Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2.

Animals↗
PubMed2005

Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells.

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.

Animals↗
PubMed2004

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells.

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.

Bacterial Proteins↗
PubMed2003

Differential expression of a cutaneous corticotropin-releasing hormone system.

We completed the mapping of a cutaneous CRH signaling system in two species with widely different determinants of skin functions, humans and mice. In human skin, the CRH receptor (CRH-R) 1 was expressed in all major cellular populations of epidermis, dermis, and subcutis with CRH-R1alpha being the most prevalent isoform. The CRH-R2 gene was expressed solely in hair follicle keratinocytes and papilla fibroblasts, whereas CRH-R2 antigen was localized predominantly in hair follicles, sebaceous and eccrine glands, muscle and blood vessels. In mouse skin, the CRH-R2 gene and protein were widely expressed in all cutaneous compartments and in cultured normal and malignant melanocytes. CRH-binding protein mRNA was present in dermal fibroblasts, melanoma cells, and sc fat of human skin and undetectable in mouse skin. The urocortin II gene was expressed equally in mouse and human skin. Taken together with our previous investigations, the present studies document the preferential expression of CRH-R1 in human skin, which mirrors CRH-R2 expression patterns in human and mouse skin. They are likely reflecting different functional activities of human and mouse skin. The adnexal location of CRH-R2 suggests a role for the receptor in hair growth. The differential interspecies CRH signaling expression pattern probably reflects adaptation to species-specific skin function determinants.

Alternative Splicing↗
PubMed2003

Endothelial expression of the alpha6beta4 integrin is negatively regulated during angiogenesis.

Development and homeostasis of the vascular system requires integrin-facilitated cellular adhesion, migration, proliferation and survival. A specific role for the alpha6beta4 integrin in the vasculature, however, has not been identified. Using immunohistochemistry, we observed alpha6beta4 expression on the dermal microvasculature of human foreskin. Analysis of individual cells isolated from trypsin-disrupted foreskin tissue indicated that alpha6beta4 was expressed by a subset of epithelial and endothelial cells, and not by smooth muscle cells. Expression of alpha6beta4 was also analyzed during new vessel growth using explants of human saphenous vein cultured in fibrinogen gels. The results indicate that alpha6beta4 is not expressed by outgrowing endothelial cells, and is downregulated by the original alpha6beta4-positive endothelial cells of the explant. To determine whether alpha6beta4 is expressed during angiogenesis in vivo, the expression of the beta4 subunit was analyzed during the development of the mouse mystacial (whisker) pad. Immunohistochemical staining of the whisker pad indicates that beta4 is expressed by the adult vasculature. To identify when and where beta4 is turned on in the vasculature, we examined the whisker pads from the developing embryo (E19.5 pc), and from postnatal days zero (P0), three (P3) and seven (P7) pups. The expression of alpha6beta4 was found to be turned on spatially and temporally from caudal to rostral regions and from the deep to superficial vasculature, correlating with the maturation of the whisker pad and its corresponding vasculature. Together, these findings suggest a potential role for alpha6beta4 as a negative component of the angiogenic switch, whereas expression of alpha6beta4 on the adult vasculature may indicate regions requiring additional adhesive mechanisms.

Animals↗
PubMed2003

Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury.

Endothelin-1 (ET-1) is a newly described pain mediator that is involved in the pathogenesis of pain states ranging from trauma to cancer. ET-1 is synthesized by keratinocytes in normal skin and is locally released after cutaneous injury. While it is able to trigger pain through its actions on endothelin-A (ET(A)) receptors of local nociceptors, it can coincidentally produce analgesia through endothelin-B (ET(B)) receptors. Here we map a new endogenous analgesic circuit, in which ET(B) receptor activation induces the release of beta-endorphin from keratinocytes and the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs, also named Kir-3) linked to opioid receptors on nociceptors. These results indicate the existence of an intrinsic feedback mechanism to control peripheral pain in skin, and establish keratinocytes as an ET(B) receptor-operated opioid pool.

Analgesia↗
PubMed2002

Similarities and differences in the innervation of mystacial vibrissal follicle-sinus complexes in the rat and cat: a confocal microscopic study.

Our confocal three-dimensional analyses revealed substantial differences in the innervation to vibrissal follicle-sinus complexes (FSCs) in the rat and cat. This is the first study using anti-protein gene product 9.5 (PGP9.5) immunolabeling and confocal microscopy on thick sections to examine systematically the terminal arborizations of the various FSC endings and to compare them between two species, the rat and the cat, that have similar-appearing FSCs but different exploratory behaviors, such as existence or absence of whisking. At least eight distinct endings were clearly discriminated three dimensionally in this study: 1) Merkel endings at the rete ridge collar, 2) circumferentially oriented lanceolate endings, 3) Merkel endings at the level of the ring sinus, 4) longitudinally oriented lanceolate endings, 5) club-like ringwulst endings, 6) reticular endings, 7) spiny endings, and 8) encapsulated endings. Of particular contrast, each nerve fiber that innervates Merkel cells at the level of the ring sinus in the rat usually terminates as a single, relatively small cluster of endings, whereas in the cat they terminate en passant as several large clusters of endings. Also, individual arbors of reticular endings in the rat ramify parallel to the vibrissae and distribute over wide, overlapping territories, whereas those in the cat ramify perpendicular and terminate in tightly circumscribed territories. Otherwise, the inner conical body of rat FSCs contains en passant, circumferentially oriented lanceolate endings that are lacking in the cat, whereas the cavernous sinus of the cat has en passant corpuscular endings that are lacking in the rat. Surprisingly, the one type of innervation that is the most similar in both species is a major set of simple, club-like endings, located at the attachment of the ringwulst, that had not previously been recognized as a morphologically unique type of innervation. Although the basic structure of the FSCs is similar in the rat and cat, the numerous differences in innervation suggest that these species would have different tactile capabilities and perceptions possibly related to their different vibrissa-related exploratory behaviors.

Animals↗