Nitric oxide and C-type atrial natriuretic peptide stimulate primary aortic smooth muscle cell migration via a cGMP-dependent mechanism: relationship to microfilament dissociation and altered cell morphology. (1/359)

Migration of aortic smooth muscle cells is thought to be of essential importance in vascular restenosis, remodeling, and angiogenesis. Recent studies have shown that NO donors inhibit the migration of subcultured aortic smooth muscle cells. However, there is evidence that NO elicits opposite effects on cell proliferation in primary versus subcultured cells, indicating fundamental differences among different models of aortic smooth muscle cell cultures. The purpose of the current study was to investigate the effect of NO donors on migration of primary cultures of rat aortic smooth muscle cells and to compare and contrast their response with those in subcultured cells. A second purpose was to investigate some of the underlying mechanisms associated with NO-induced effects on cell migration. We report that 2 NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 2, 2-(hydroxynitrosohydrazino)bis-ethanamine, stimulated the migration of primary cells in a wounded-culture model as well as in a transwell migration model. The effect of NO donors was mimicked by 2 cGMP analogues and C-type natriuretic peptide and blocked by a specific inhibitor of guanyl cyclase, 1H-(1,2,4)oxadiazolo[4,3, -a]quinoxalin-1-one, indicating the involvement of cGMP as second messenger. Moreover, neither NO donors nor cGMP analogues altered migration of primary cultures stimulated by either FBS or angiotensin II. In contrast to its effect in primary cultures, SNAP did not alter basal or stimulated migration of subcultured cells, except at a relatively high concentration of 1 mmol/L, at which migration was inhibited. The migration-stimulatory effect of NO donors and cGMP was associated with altered cell morphology and dissociation of actin filaments, consistent with recent studies indicating that cell morphology and cytoskeletal organization influence cell migration. The results suggest the possible involvement of NO-induced cell migration in vascular injury or remodeling, representing conditions in which vascular NO levels would be expected to be elevated.  (+info)

Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture. (2/359)

BACKGROUND: Adherence of crystals to the surface of renal tubule epithelial cells is considered an important step in the development of nephrolithiasis. Previously, we demonstrated that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection against the adherence of calcium oxalate monohydrate crystals. We now examined whether this property is cell type specific. The susceptibility of the cells to crystal binding was further studied under different culture conditions. METHODS: Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohydrate crystal binding to LLC-PK1 cells and to two MDCK strains cultured on either permeable or impermeable supports. These cell lines are representative for the renal proximal tubule (LLC-PK1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and present, respectively. RESULTS: Whereas relatively large amounts of crystals adhered to subconfluent MDCK cultures, the level of crystal binding to confluent monolayers was reduced for both MDCK strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired a higher degree of protection than on impermeable surfaces. Crystals avidly adhered to LLC-PK1 cells, irrespective of their developmental stage or growth substrate used. CONCLUSIONS: These results show that the prevention of crystal binding is cell type specific and expressed only by differentiated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of its in situ equivalent, the renal distal tubule/collecting ducts.  (+info)

Active and higher intracellular uptake of 5-aminolevulinic acid in tumors may be inhibited by glycine. (3/359)

Topical 5-aminolevulinic acid is used for the fluorescence-based diagnosis and photodynamic treatment of superficial precancerous and cancerous lesions of the skin. Thus, we investigated the kinetics of 5-aminolevulinic acid-induced fluorescence and the mechanisms responsible for the selective formation of porphyrins in tumors in vivo. Using amelanotic melanomas (A-Mel-3) grown in dorsal skinfold chambers of Syrian golden hamsters fluorescence kinetics were measured up to 24 h after topical application of 5-aminolevulinic acid (1%, 3%, or 10%) for 1 h, 4 h, or 8 h by intravital microscopy (n = 54). Maximal fluorescence intensity in tumors after 1 h application (3% 5-aminolevulinic acid) occurred 150 min and after 4 h application (3% 5-aminolevulinic acid) directly thereafter. Increasing either concentration of 5-aminolevulinic acid or application time did not yield a higher fluorescence intensity. The selectivity of the fluorescence in tumors decreased with increasing application time. Fluorescence spectra indicated the formation of protoporphyrin IX (3% 5-aminolevulinic acid, 4 h; n = 3). The simultaneous application of 5-aminolevulinic acid (3%, 4 h) and glycine (20 microM or 200 microM; n = 10) reduced fluorescence in tumor and surrounding host tissue significantly. In contrast, neither decreasing iron concentration by desferrioxamine (1% and 3%; n = 10) nor inducing tetrapyrrole accumulation using 1, 10-phenanthroline (7.5 mM; n = 5) increased fluorescence in tumors. The saturation and faster increase of fluorescence in the tumor together with a reduction of fluorescence by the application of glycine suggests an active and higher intracellular uptake of 5-aminolevulinic acid in tumor as compared with the surrounding tissue. Shorter application (1 h) yields a better contrast between tumor and surrounding tissue for fluorescence diagnosis. The additional topical application of modifiers of the heme biosynthesis, desferrioxamine or 1,10-phenanthroline, however, is unlikely to enhance the efficacy of topical 5-aminolevulinic acid-photodynamic therapy at least in our model.  (+info)

A partial catalog of proteins secreted by epidermal keratinocytes in culture. (4/359)

Proteins secreted by epidermal keratinocytes are known to engage in functions other than those directly associated with barrier formation. We have used a previously published culture model to collect proteins secreted by adult human epidermal keratinocytes. Electrophoresis and microsequencing allowed us to identify 20 proteins. The list of proteins includes those known to be produced by keratinocytes (beta-2 microglobulin, betaIG-H3, calgranulin A, cathepsin B and D, E-cadherin, gelatinase B, gelsolin, interstitial collagenase, laminin B2t, plasminogen activator inhibitor-1, protein 14-3-3epsilon, SCC antigen, stratifin, and translationally controlled tumor protein) as well as those not previously known to be secreted by keratinocytes (epididymis secretory protein, maspin, and anti-neoplastic urinary protein). In addition, two proteins were identified that are not known to be secreted (glutathione-S-transferase and heat shock protein 27/28 kDa). The varied nature of the proteins identified suggests that epidermal keratinocytes have physiologic functions that have yet to be identified.  (+info)

Efficacy of beta-lactam and inhibitor combinations in a diffusion chamber model in rabbits. (5/359)

Using a diffusion chamber in rabbits, we evaluated therapy with the combination of ceftriaxone plus the beta-lactamase inhibitor tazobactam in comparison with ceftriaxone alone. One sensitive and one resistant strain of Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae were inoculated into one of the six diffusion chambers, implanted in the same animal. In order to simulate pharmacokinetics in humans, both substances were administered in decreasing doses. Ceftriaxone was given 0, 2, 4 and 6 h after infection in dosages of 45, 35, 25 and 15 mg/kg of body weight, while tazobactam was administered either in one dose at 0 h, or divided into two doses at 0 and 1 h or 0 and 4 h, or divided into three doses at 0, 1 and 4 h after infection. The ratio of ceftriaxone:tazobactam was fixed at 8:1. Ceftriaxone, in combination with tazobactam, given in one dose immediately after infection showed a significant reduction in bacterial count. All other combinations of ceftriaxone and tazobactam did not differ from ceftriaxone in monotherapy. Co-administration of the beta-lactamase inhibitor tazobactam significantly enhanced the activity of ceftriaxone against all three tested species.  (+info)

Growth, cellular differentiation and virulence factor expression by Proteus mirabilis in vitro and in vivo. (6/359)

A uropathogenic strain of Proteus mirabilis was grown in vitro in human and mouse urine and brain-heart infusion broth (BHIB) and in vivo in subcutaneous open chambers (SOC) in mice, intraperitoneal diffusion chambers (IPC) in rats and by ascending urinary tract infection in mice in order to compare growth pattern, cellular differentiation and expression of virulence factors. Although the growth rate was slower in vivo than in vitro, the extent of growth was similar after 24 h. PR mirabilis differentiated into filamentous swarmer cells in all in-vitro culture conditions, but no filamentous cells were observed in either of the in-vivo chamber models. Transurethrally infected mice showed a rapid release or loss of filamentous cells and these could not be seen in kidney or bladder homogenates 7 days after infection. Bacteria showed increasing haemagglutination titres for fresh and tanned red blood cells after subculturing in BHIB, but bacteria grown in vivo did not show haemagglutination. An increasing resistance to normal serum was found when bacteria were grown in vivo. Significant haemolytic activity was detected with bacteria grown in BHIB and IPC, but almost no activity was found when bacteria had grown in urine. These findings improve the understanding of the role of P. mirabilis uropathogenic virulence factors in vivo.  (+info)

Penetration of anticancer drugs through solid tissue: a factor that limits the effectiveness of chemotherapy for solid tumors. (7/359)

Penetration of anticancer agents to cells distant from the vascular system is required for efficacy of cancer chemotherapy against solid tumors. Many solid tumors have a poorly formed blood vascular system with variable rates of blood flow and much larger intercapillary distances than those found in normal tissues. The requirement for drugs to penetrate several layers of tissue might pose a barrier to the effective treatment of solid tumors. Multicellular layers (approximately 200 microm thick) were grown in vitro on Teflon membranes from EMT6 murine and MCF7 human tumors and have been used to quantitate the penetration of four widely used anticancer drugs through solid tissue. The penetration of doxorubicin and mitoxantrone was limited and very slow (<10% of the rate of penetration through the Teflon membrane alone). The penetration of methotrexate and 5-FU was more rapid (approximately 30-50% of the rate of penetration through the Teflon membrane alone), but remains a substantial barrier to the effectiveness of these drugs. Strategies to improve the penetration of anticancer drugs through poorly vascularized tumor tissue have considerable potential to improve the outcome of chemotherapy for solid tumors.  (+info)

HIV type 1 Nef protein is a viral factor for leukocyte recruitment into the central nervous system. (8/359)

Recombinant HIV-1 Nef protein, but not Tat, gp120, and gp160, provoked leukocyte recruitment into the CNS in a rat model. The strong reduction of bioactivity by heat treatment of Nef, and the blocking effect of the mAb 2H12, which recognizes the carboxy-terminal amino acid (aa) residues 171-190 (but not of mAb 3E6, an anti-Nef Ab of the same isotype, which maps the aa sequence 168-175, as well as a mixture of mAbs to CD4) provided evidence for the specificity of the observed Nef effects. Using a modified Boyden chamber technique, Nef exhibited chemotactic activity on mononuclear cells in vitro. Coadministration of the anti-Nef mAb 2H12, as well as treatment of Nef by heat inhibited Nef-induced chemotaxis. Besides soluble Nef, chemotaxis was also induced by a Nef-expressing human astrocytoma cell line, but not by control cells. These data suggest a direct chemotactic activity of soluble Nef. The detection of elevated levels of IL-6, TNF-alpha, and IFN-gamma in rat cerebrospinal fluid 6 h after intracisternal Nef injection hint at the additional involvement of indirect mechanisms in Nef-induced leukocyte migration into rat CNS. These data propose a mechanism by which HIV-1 Nef protein may be essential for AIDS neuropathogenesis, as a mediator of the recruitment of leukocytes that may serve as vehicles of the virus and perpetrators for disease through their production of neurotoxins.  (+info)