[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) released from mechanosensitive bone cells plays a key role in the adaptation of bone structure to its mechanical usage. Despite its importance in bone, the mechanisms involved in NO mechanotransduction at the cellular level remain unknown. Using combined atomic force microscopy and fluorescence microscopy, we report both stimulation and real-time monitoring of NO responses in single osteoblasts induced by application of quantified periodic indenting forces to the osteoblast membrane. Peak forces ranging from 17 to 50 nN stimulated three distinct NO responses in the indented osteoblasts: (1) a rapid and sustained diffusion of NO from the perinuclear region, (2) diffusion of NO from localized pools throughout the osteoblast, and (3) an initial increase and subsequent drop in intracellular NO. Force-indentation characteristics showed considerable interosteoblast variation in elasticity. NO responses were associated with application of force to more rigid membrane sites, suggesting cytoskeletal involvement in mechanotransduction.
Journal of Orthopaedic Research 05/2008; 26(4):513-21. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been shown that a human salivary gland cell line (HSG) is capable of differentiation into gland-like structures, though little is known of how morphological features are formed or controlled. Here we investigated the changes in cell proliferation and apoptosis upon terminal differentiation of HSG cells in Matrigel, an extracellular matrix derivative. Changes in the expression of survivin, a prominent anti-apoptotic factor, and caspase-3, a key apoptotic factor were also measured. In order to better understand the involvement of key signal transduction pathways in this system we pharmacologically blocked the activity of tyrosine kinase, nuclear factor kappa B(NF kappa B), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and matrix metalloproteases (MMP). Results of these studies demonstrate that cytodifferentiation of HSG cells to an acinar phenotype is accompanied first by a decrease of cell proliferation and then by a massive programmed cell death, affected by multiple signal transduction pathways. Thus, Matrigel alone is insufficient for the full maturation and long term survival of the newly formed acini: the presence of other factors is necessary to complete the acinar differentiation of HSG cells.
Journal of Cellular Biochemistry 02/2008; 103(1):284-95. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Histatin-resistant derivatives of Candida albicans strain 132A, generated by successive exposure to increasing concentrations of histatin 3, were previously reported to be similar to the parent strain in their histatin binding, internalization, oxygen consumption, ATP efflux, and histatin degradation. Proteomic analysis of further histatin-resistant secondary derivatives of this series revealed that 59 proteins were differentially expressed compared to the parental strain. Of these 59 proteins, 3 were absent in histatin-resistant secondary derivatives and 11 were absent in the parent strain. Of the proteins absent in the histatin-resistant derivatives, the most notable was elongation factor 2, a target for the natural antifungal sordarin. Of the proteins absent in the parent strain but present in histatin-resistant derivatives, those identified included isocitrate lyase (Icl1p), fructose biphosphate aldolase (Fba1p), pyruvate decarboxylase (Pdc2p), and ketol-acid reductoisomerase (Ilv5p). The present secondary derivatives showed significantly decreased rates of oxygen consumption and histatin 3-mediated ATP release compared to the parent strain and also showed stability of the histatin-resistant phenotype. A significant (twofold) decrease in transcript levels of the potassium transporter encoded by TRK1, a critical mediator of histatin killing, was found in only one of the secondary histatin-resistant derivatives compared to the parent strain. The sequential exposure of C. albicans to histatin 3 described here resulted in the induction or selection of a phenotype with impaired metabolic function. The results support an important role for metabolic pathways in the histatin resistance mechanism and suggest that there may be several intracellular targets for histatin 3 in C. albicans.
Antimicrobial Agents and Chemotherapy 09/2007; 51(8):2793-800. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: A human salivary intercalated duct cell line (HSG) is capable of morphological change to acinar-type cells, and of salivary amylase (AMY1) expression, by culturing on basement membrane extracts (BME). The aim of this study was to determine the critical conditions for functional and morphological differentiation of HSG cells and to establish if the processes are related. Cells were grown on BMEs that had different protein concentrations and growth factor content, and then examined with respect to morphology and AMY1 expression. To investigate the role of intracellular calcium in amylase expression, a pcDNA3.1-TRPC1alpha construct was used to overexpress htrp1alpha, which mediates the store-operated calcium entry in HSG cells. Expression of the AMY1, TRPC1alpha and beta genes was quantified by means of real time RT-PCR. Growth factor-reduced BME (12.8 mg/ml) induced multicellular acinar structures with lumen formation but without stimulation of either AMY1 or TRPC1. HSG cells cultured on higher concentration BME (17.5 or 16.4 mg/ml) formed reticular networks. AMY1 expression increased both on growth factor-reduced BME (17.5 mg/ml: 3.0-fold, P < 0.001) and on regular BME (16.4 mg/ml: 3.7-fold, P < 0.001) accompanied by a slight increase in expression of TRPC1alpha and TRPC1beta. Overexpression of htrp1alpha did not cause any significant changes in AMY expression, though it attenuated the BME (17.5 mg/ml)-induced AMY1 upregulation. Overall, the higher protein concentration BME favors amylase expression in HSG cells, whereas the lower concentration causes marked morphological changes.
Journal of Cellular Physiology 08/2007; 212(2):416-23. · 4.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antifungal activity of histatin 3 against two Candida albicans clinical isolates was determined in assays containing rabbit submandibular gland saliva. Histatin 3 inhibited the cell growth and germination of both isolates dose-dependently (10-100 microg ml(-1)) with maximum inhibition occurring after 60 min incubation. Adding fresh histatin 3 after 60 min caused further reduction in the viable cell count. Higher histatin 3 concentrations (50-100 microg ml(-1)) and prolonged exposure to peptide were required to inhibit germination. Histatin 3 was rapidly degraded in rabbit submandibular gland saliva and this may explain why fresh addition of histatin 3 increases candidacidal activity.
[show abstract][hide abstract] ABSTRACT: Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event.
[show abstract][hide abstract] ABSTRACT: We examined the distribution and toxicity associated with a single salivary gland administration of a recombinant adenoviral vector, AdCMVH3, encoding human histatin 3.
Adult rats received different doses of AdCMVH3 (0, 106, 3 x 107, and 109 pfu; 50 microl) via the right submandibular gland and were followed for 15 days. Food consumption, weight gain, clinical appearance, and serum chemistry were monitored, and a necropsy was performed. Vector distribution was examined by polymerase chain reaction, and selected saliva samples were tested for replication-competent adenovirus (RCA).
All animals survived to sacrifice (days 2, 8, and 15), and appeared normal clinically. There were no differences in food consumption, weight gain, and serum chemistry. The only consistent necropsy findings were lymphoid infiltrates and necrosis in the target submandibular glands of high-dosage animals. AdCMVH3 detection was virus dose dependent, decreased with time, and at low dose preferentially observed in the targeted gland. No RCA was detected.
Salivary gland administration of 109 pfu AdCMVH3 elicits an initial focal pathologic response and wide tissue distribution. There is no associated systemic toxicity up to 15 days, and lower doses are primarily found in glands.
Journal of Oral Pathology and Medicine 09/2003; 32(7):414-21. · 2.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The antifungal mechanism of salivary histatin has been studied in Candida albicans and involves binding to a specific receptor, translocation across the membrane and targeting intracellularly. Cell death correlates with non-lytic release of ATP that may function as a cytotoxic mediator extracellularly. By sequential exposure to increasing concentrations of histatin 3, we generated histatin-resistant derivatives of C. albicans strain CA132A that show five-fold less killing at physiological concentrations of histatin 3. Protection against histatin killing in histatin-resistant derivatives is not due to alterations in binding, internalisation or degradation of histatin or efflux of ATP. These results indicate that protective mechanisms activated by exposure to histatin 3 may involve unidentified pathways downstream of binding and internalisation events.
[show abstract][hide abstract] ABSTRACT: Candida dubliniensis is a recently described Candida species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected patients and patients with AIDS. The majority of C. dubliniensis clinical isolates tested to date are susceptible to the commonly used antifungal drugs, including fluconazole, ketoconazole, itraconazole, and amphotericin B. However, the appearance of fluconazole-resistant C. dubliniensis strains in this patient group is increasing. Histatins are a family of basic histidine-rich proteins present in human saliva which have therapeutic potential in the treatment of oral candidiasis. The mechanism of action of histatin is distinct from that of commonly used azole and polyene drugs. Characterization of the antifungal activity of histatin has mainly been carried out using C. albicans but it is also effective in killing C. glabrata and C. krusei. Here we report that C. dubliniensis is also susceptible to killing by histatin 3. The concentration of histatin 3 giving 50% killing (the IC(50) value) ranged from 0.043 to 0.196 mg/ml among different strains of C. dubliniensis. The least-susceptible C. dubliniensis strain, P9224, was found to internalize histatin at a lower rate than the C. albicans reference strain CA132A. The dissociation constant (K(d)) for the least-susceptible strain (C. dubliniensis 9224) was ninefold higher than that for the C. albicans reference strain. These results suggest that histatin 3 may have potential as an effective antifungal agent, particularly in the treatment of oral candidiasis in HIV-infected patients and patients with AIDS in which resistance to the commonly used antifungal drug fluconazole has emerged.
Antimicrobial Agents and Chemotherapy 02/2003; 47(1):70-6. · 4.57 Impact Factor