Effects of manganese on Streptococcus mutans planktonic and biofilm growth.

Streptococcus mutans, an agent of dental caries, was tested for growth in the presence or absence of manganese (Mn), since studies have linked Mn levels with cariogenic potential. Seven S. mutans serotype c strains were grown in chemically defined medium under different atmospheric conditions: 5% CO2, O2-enriched 5% CO2 (shaking) and anaerobic. There was significant strain variability with respect to Mn requirements under the various conditions tested. Both sucrose-dependent and sucrose-independent biofilm growth by strain UA159 were affected by the absence of Mn. S. mutans strains show highly variable responses to both high and low Mn concentrations.

Pharmacological properties of the Cys23Ser single nucleotide polymorphism in human 5-HT2C receptor isoforms.

The human serotonin 2C (5-HT2C) receptor undergoes extensive RNA editing, generating multiple isoforms; the most prominent isoform in the human brain is the extensively edited VSV isoform. In addition, a naturally occurring single nucleotide polymorphism (SNP) is found in the coding region of the 5-HT2C receptor gene, which converts cysteine to serine at the 23rd amino acid (C23S). To elucidate the functional consequences, pharmacological properties were evaluated in cells expressing C23 or S23 in the nonedited, INI, or edited, VSV, isoform. Confocal imaging of HEK293 cells expressing the C23 and S23 variants revealed no apparent difference in cellular localization, which was confirmed in NIH-3T3 fibroblasts by surface biotinylation. Competition binding experiments revealed comparable high-affinity agonist binding for the C23 and S23 receptors and no difference in ligand affinities in either the INI or VSV backbones. The dose-response functions for 5-HT and (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI) to elicit phosphoinositide hydrolysis did not differ in either HEK293 or NIH-3T3 fibroblasts expressing the receptor variants. Constitutive activity, evaluated in COS-7 and HEK293 cells, also was not different. Lastly, fluorescence resonance energy transfer demonstrated homodimerization of C23 receptors, which was reproduced in cells expressing the S23 variant. We conclude that the C23S SNP in the 5-HT2C receptor has no functional consequences, even when evaluated in the most common, edited receptor backbone. Therefore, positive associations between this polymorphism and disease states may be a consequence of linkage disequilibrium with another SNP that is involved in the disease.

The Meissner corpuscle revised: a multiafferented mechanoreceptor with nociceptor immunochemical properties.

Meissner corpuscles (MCs) in the glabrous skin of monkey digits have at least three types of innervation as revealed by immunofluorescence. The previously well known Aalphabeta-fiber terminals are closely intertwined with endings from peptidergic C-fibers. These intertwined endings are segregated into zones that alternate with zones containing a third type of ending supplied by nonpeptidergic C-fibers. Although MCs are widely regarded as low-threshold mechanoreceptors, all three types of innervation express immunochemical properties associated with nociception. The peptidergic C-fiber endings have readily detectable levels of immunoreactivity (IR) for calcitonin gene-related peptide (CGRP) and substance P (SP). The Aalphabeta endings have relatively lower levels of IR for CGRP and SP as well as the SP neurokinin 1 receptor and vanilloid-like receptor 1. Both the Aalphabeta and peptidergic C-fiber endings were also labeled with antibodies for different combinations of adrenergic, opioid, and purinergic receptors. The nonpeptidergic C-fiber endings express IR for vanilloid receptor 1, which has also been implicated in nociception. Thus, MCs are multiafferented receptor organs that may have nociceptive capabilities in addition to being low-threshold mechanoreceptors.

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors.

Studies in mammalian skin have shown expression of the genes for corticotropin-releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH-Rs). These CRH-Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)-mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypothalamic-pituitary-adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/POMC system, the CRH-Rs may be a central element.

An in vitro model for studying the contributions of the Streptococcus mutans glucan-binding protein A to biofilm structure.

The method described here for analyzing biofilms was sensitive enough to allow the detection of differences formed by pure cultures of S. mutans or a GbpA knockout strain. Other strains have also been tested, and the differences in biofilm structure were sometimes even more extensive (data not shown). The advantages of this method are that it is quick, inexpensive, and adaptable to almost any laboratory setting. The constant rotation of the cultures, which was employed to simulate salivary flow, appears to be a critical element for establishing biofilm differences. An analysis of protein profiles confirmed that the biofilm bacteria were metabolically distinct from the planktonic phase bacteria. For the strains tested, the variations in biofilm architecture could be visualized with or without magnification. Staining of the bacteria was not required, though we typically stained the biofilms with either crystal violet or Schiff's reagent. Altogether, this in vitro method for generating biofilms allowed the evaluation of visual, quantitative (confocal microscopy), and functional (antimicrobial susceptibility) differences. We have employed these methods in a reductionist approach to understanding the contribution of individual proteins to dental plaque development. These methods may also be useful in the screening of mutants that would be of greatest for testing in multispecies biofilms, animal models, or more complex biofilm models.

Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H(2)O(2).

Manganese superoxide dismutase (MnSOD) overexpression has been shown to reverse the malignant phenotype in a variety of tumor cell lines. The inhibition of proliferation and reversal of the malignant phenotype has been attributed to an increase in H(2)O(2) production as a result of the dismutation reaction. However, direct evidence in support of this hypothesis has not been forthcoming. To evaluate the contribution of H(2)O(2) in the regulation of cell growth in response to MnSOD overexpression, control and MnSOD-overexpressing HT-1080 fibrosarcoma cells were transfected with constructs that direct catalase to either the mitochondrial or cytosolic compartments. Overexpression of catalase in either compartment reversed the proliferative and clonogenic inhibition associated with MnSOD overexpression, blocked the increase in the steady state levels of H(2)O(2) as measured by flow cytometric analysis of 2', 7'-dichlorofluorescein diacetate, and increased protection from the cytotoxicity of H(2)O(2). In addition, mitochondrial or cytosolic catalase enhances respiration through complex I and II in both control and MnSOD overexpressing cell lines and reverses a MnSOD-dependent decrease in net ATP production. Thus, catalase reverses the proliferative inhibition associated with MnSOD overexpression and may also play an important role in metabolic regulation.

Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells.

The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.

Spatiotemporal expression, distribution, and processing of POMC and POMC-derived peptides in murine skin.

In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides beta-endorphin, ACTH, beta-MSH, and alpha-MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, beta-endorphin, beta-MSH, and alpha-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin.

Nitric oxide enhances the manganese superoxide dismutase-dependent suppression of proliferation in HT-1080 fibrosarcoma cells.

The overexpression of manganese superoxide dismutase (MnSOD), an enzyme that catalyzes the removal of superoxide (O2*-) from the mitochondria, has been shown to be closely associated with tumor regression in vivo and loss of the malignant phenotype in vitro. To investigate the mechanism by which MnSOD overexpression mediates this reversal, we have established 29 independent, clonal MnSOD-overexpressing HT-1080 fibrosarcoma cells. MnSOD activity is inversely correlated with cell proliferation in our cell lines. Incubating cells in 3% oxygen can prevent the inhibition of cellular proliferation mediated by MnSOD, suggesting that oxygen is a prerequisite component of the MnSOD-dependent proliferative inhibition. Confocal laser microscopy was used in combination with the oxidant-sensitive fluorescent dyes dihydrorhodamine-123, dihydroethidium, and 2',7'-dichlorodihydrofluorescein diacetate to determine the oxidizing capacity of the MnSOD-overexpressing cells. When compared with parental or control cell lines, there was a significant decrease in the rate of oxidation of the fluorophores in the MnSOD-overexpressing cell lines. Thus, an increase in the oxidizing capacity of the cells does not appear to mediate the inhibition of proliferation associated with MnSOD overexpression. Superoxide dismutase has also been shown to enhance the cytotoxic activity of NO* toward tumor cells. In this study, we have shown that MnSOD overexpression enhances the cytostatic action of the NO* donors, sodium nitroprusside, 3-morpholinosydnonomine, and (Z)-1-[2-aminethyl)-N-(2-ammonioethyl)amino]diazen-1-+ ++ium-1,2-diolate in a dose-dependent manner. In addition, the NO* toxicity is blocked by oxyhemoglobin, a NO* scavenger. Our findings suggest that NO* may play a role in the reversal of tumorigenicity associated with MnSOD overexpression.

Inactivation of the gbpA gene of Streptococcus mutans alters structural and functional aspects of plaque biofilm which are compensated by recombination of the gtfB and gtfC genes.

Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180-2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.

Expression of proopiomelanocortin (POMC)-derived melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) peptides in skin of basal cell carcinoma patients.

We proposed that local expression and production of proopiomelanocortin (POMC) peptides may play a role in human skin physiology and pathology, including the development and progression of skin cancers. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting hybridization techniques were used to study gene expression. Reversed-phase (RP) high-pressure liquid chromatography (HPLC) separation with subsequent radioimmunoassays were used to identify alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) peptides. Immunocytochemistry (IHC) was used to localize ACTH, alpha-MSH, and beta-MSH antigens in skin. RT-PCR, RP-HPLC, and IHC analyses documented the expression of POMC mRNA and production of ACTH and alpha-MSH peptides in lesional and perilesional skin of basal cell carcinoma (BCC) patients and in cultured keratinocytes, which was accompanied by the expression of the MC1-R gene encoding the receptor activated by MSH and ACTH. Thirty specimens were analyzed by IHC. Immunoreactive alpha-MSH, beta-MSH, and ACTH were detected, in 21 of 21, in 11 of 20, and in 6 of 8 of lesional skin, and in 6 of 6, in 5 of 7, and in 6 of 8 perilesional skin specimens analyzed, respectively. Antigen distribution was heterogenous and present in BCC, epidermis, hair follicles, dermal mononuclear cells, and extracellular matrix. We conclude that messenger RNA for POMC, MC1-R, and the peptides MSH and ACTH are produced in skin of BCC patients. Because keratinocytes are a target for MSH and ACTH bioregulation, the production of these peptides is stimulated by UVB, and the peptides can act as immunosupressors, we suggest that MSH and ACTH may facilitate development of BCC.

Characterization of corticotropin-releasing hormone (CRH) in human skin.

We have confirmed the expression of CRH and CRH receptor type 1 genes in human skin, cultured HaCaT keratinocytes, squamous cell carcinoma, and melanoma cells. The size of CRH messenger ribonucleic acid (mRNA), estimated by Northern blot hybridization, was 1.5 kilobases. CRH peptide was identified by reverse phase high pressure liquid chromatography separation in both whole skin and cultured cells. Forskolin and dexamethasone at concentrations of 10 micromol/L stimulated and inhibited, respectively, CRH peptide production in squamous cell carcinoma and melanoma cells, but had no significant effect on the CRH mRNA level. In melanoma cells, stimulation of melanogenesis down-regulated CRH receptor type 1 mRNA expression, but was without effect on CRH mRNA production. We suggest that in human skin the CRH signaling system is both operative and under regulatory control.

16S mitochondrial ribosomal RNA degradation is associated with apoptosis.

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.

Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin.

Evidence is provided that human skin, the largest body organ exposed to multiple stressors, expresses proopiomelanocortin (POMC), corticotropin releasing hormone (CRH) and CRH-receptor (CRHR) genes in vivo. In vitro studies show that POMC and CRHR mRNAs are transcribed in melanocytes, cells derived from the neural crest, and in keratinocytes, cells derived from the ectoderm. CRH mRNA is transcribed in cultured melanocytes but not in keratinocytes. It is proposed that an equivalent of the 'hypothalamus-pituitary axis' composed of the CRH-CRHR-POMC loop is conserved in mammalian skin.

Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin.

We investigated the presence of proopiomelanocortin (POMC) products in sections of skin from normal subjects and patients with neoplastic and non-neoplastic cutaneous disorders. Antibodies specific against adrenocorticotropin, beta-melanotropin, and beta-endorphin were used for detection and characterization of cell types bearing POMC peptides. POMC products were not observed in sections of normal skin from the corporal (non-scalp) areas (six cases), whereas the hair follicles of scalp skin exhibited positive immunostains that were readily apparent (four cases). POMC products were frequently detected in corporal skin affected by diseases (13 of 26 cases), for example, psoriatic keratinocytes, the inflammatory infiltrate in scarring alopecia, nevocytes, the epithelial cell nests of basal cell carcinoma, and melanoma cells. Further tests were performed in keloids, a primary reactive skin disorder, to evaluate whether POMC accumulation represented a disease-related phenomenon or an expression of normal cutaneous reactivity. POMC products were consistently detected (10 of 11 cases) in the keratinocytes and mononuclear cells at keloid lesions. Thus these observations indicate that POMC products may accumulate locally in lesional skin representing, presumably, a novel cutaneous response to injury. The broad spectrum of POMC products detected suggests that these arise from production in situ (expression of the POMC gene itself) by human skin.

Cytoplasmic inclusions in spinal neurons of the motor neuron degeneration (Mnd) mouse. I. Light microscopic analysis.

The motor neuron degeneration (Mnd) mutation in the mouse is a late onset, autosomal dominant, neurodegenerative disease in which ventral horn neurons have been shown to contain numerous, large cytoplasmic inclusions. Histochemical and immunocytochemical studies performed on spinal cord from Mnd/Mnd mice in late stages of the disease showed the inclusions to contain protein, lipid and carbohydrate moieties. Spinal neurons, especially those in spinal lamina IX, contained increased beta-glucuronidase activity in the form of large cytoplasmic inclusions. Such inclusions also contained increased acid phosphatase and trimetaphosphatase activity. When immunostained with antiubiquitin antibodies, intracellular ubiquitin deposits were present as accumulations of varying size; some were amorphous while others contained small granules. Extraneuronal ubiquitin deposits were detected in the neuropil. Immunostaining with monoclonal antibody ML30, used here to assay for the presence of a mitochondrial epitope in the inclusions, was widespread and punctate in white and grey matter from Mnd/Mnd and age-matched control spinal cords. The overall pattern of staining was similar for both tissue sources and did not correspond to any of the other probes which reacted with the inclusions in Mnd neurons. The presence of increased levels of lysosomal hydrolases and ubiquitinated molecules suggests that the two general systems for intracellular digestion are activated in Mnd/Mnd spinal neurons.

Transfection of murine P19S18 embryonal carcinoma cells with the oncogene neu induces an epithelioid phenotype.

An embryonal carcinoma cell line, P19S18, was transfected with the rat oncogene neu to investigate the function of its protein product, p185*, in a multipotential cellular environment. Levels of message for p185* were determined by in situ hybridization analysis and two highly expressing clones, PnnA and PnnB, were isolated. As demonstrated by indirect immunofluorescence and immunoprecipitation, these neu-transfected cells synthesized a full length rat p185*. The transfectants do not resemble typical embryonal carcinoma cells either before or after differentiation is induced by retinoic acid treatment. They are much larger, flatter, "epithelioid" cells. These cells have lost the expression of stage specific embryonic antigen-1 (SSEA-1), but do synthesize and assemble the basement membrane components laminin and fibronectin. These results suggest that expression of the neu oncogene in a multipotential cell line may induce the synthesis of proteins indicative of an epithelioid phenotype due to the presence of p185*.

Neurofilament distribution is altered in the Mnd (motor neuron degeneration) mouse.

Motor neuron degeneration (Mnd) is a genetic neurodegenerative disease of the mouse that is characterized by a progressive increase in motor dysfunction, moving from hind to fore limbs, leading to paralysis. An immunocytochemical analysis of the neurofilament distribution in spinal motor neurons in Mnd mice from all stages of the disease, including the presymptomatic, was performed using antibodies to different neurofilament subunits with different degrees of phosphorylation. Perikarya that stained with antibodies to phosphorylated neurofilaments were present in Mnd and control spinal cords, but the number of stained perikarya in Mnd was not significantly different from controls. There was a marked redistribution of neurofilaments within the cytoplasm of some motor neurons in Mnd cords. In Mnd but not controls, the immunoreaction product appeared marginated, leaving areas in the cytoplasm absent of immunostaining. These areas were observed in all stages of the disease, but less predictably in presymptomatics. Both the size of the areas and the number of motoneurons containing these areas appeared to increase with the severity of the disease. The number of anterior horn neurons in the hind limb region of lamina IX in spinal segment L4 of Mnd was lower than in controls, suggesting there is a loss of neurons in Mnd.

Ubiquitin deposits are present in spinal motor neurons in all stages of the disease in the motor neuron degeneration (Mnd) mutant of the mouse.

Monoclonal antibodies to ubiquitin were used in an immunocytochemical analysis of spinal cord from the Mnd (motor neuron degeneration) mouse, an animal model for motor neuron disease. Tissue from mice with mild, moderate and severe disease, from presymptomatic mice, and age-matched controls were analyzed. Ubiquitin deposits were observed in spinal neurons from presymptomatic animals and all stages of the disease. No immunoreactive deposits were observed in control mice at the concentration of the antibodies used. The presence of ubiquitin immunopositivity in presymptomatic spinal motor neurons suggests that ubiquitination might play a primary role in the pathogenesis of motor neuron disease in the Mnd mouse, and perhaps of motor neuron disease in general.