Differential expression and regulation of TGF-beta1, TGF-beta2, TGF-beta3, TGF-betaRI, TGF-betaRII and TGF-betaRIII in cultured human corneal, limbal, and conjunctival fibroblasts.

PURPOSE. We have reported that three patterns of cytokine expression are potentially involved between epithelia and fibroblasts of the human ocular surface. The TGF-beta family is a prototypical fibrogenic cytokine responsible for fibroblast activation in wound healing. We investigated how the TGF-beta family is differentially expressed and regulated in cultured human corneal, limbal and conjunctival fibroblasts. METHODS. Human corneal (HCF), limbal (HLF) and conjunctival fibroblast (HJF) were cultured in DMEM-10% FBS until confluence and switched to serum-free DMEM-ITS for 48 h before adding 10 ng/ml of each of eight cytokines for 4 h in three separate experiments. Total RNA was isolated and subjected to Northern hybridization with GAPDH as a control. ELISA was used to determine TGF-beta1 and TGF-beta2 proteins in the media. RESULTS. All three isoforms of TGF-beta and three types of TGF-betaR were expressed by HCF, HLF and HJF. Expression of TGF-beta1 mRNA was strongest and upregulated by the three TGF-betas in all three types of fibroblast. PDGF-BB and TGF-alpha slightly increased TGF-beta1 mRNA. TGF-betas also upregulated TGF-beta3 mRNA in HJF. TGF-betaRI mRNA was the only receptor upregulated by TGF-betas. TGF-betaRII and TGF-betaRIII mRNA were not regulated by all cytokines tested. CONCLUSIONS. TGF-betas auto-induction is the major mechanism upregulating TGF-beta1 expression. Promotion of TGF-beta3 by the TGF-betas may have a special role in HJF. Differential expression and regulation of TGF-betas and TGF-betaRs suggest that each TGF-beta isoform may have specific functions in different ocular surface fibroblasts. No cytokine tested can downregulate TGF-beta1 and the TGF-betaRs.     

A male accessory gland protein that modulates female mosquito (Diptera: Culicidae) host-seeking behavior.

The male accessory gland product that modulates the host-seeking behavior of female Aedes aegypti (L.) mosquitoes was found to be a peptide of 7,600 mw. This peptide also prevented subsequent mating behavior and weakly stimulated oviposition. Neither whole glands nor gland fractions from Anopheles gambiae had any effect on Ae. aegypti females, but those from Aedes albopictus were active.     

The relationship of self-rated vision and hearing to functional status and well-being among seniors 70 years and older

PURPOSE: To describe the relationship between self-reported visual and hearing impairment and an index of global functional status among seniors age 70 years or older. METHODS: A total of 7,320 United States community-dwelling persons aged 70 years or older participating in the 1993 Assets and Health Dynamics of the Oldest Old Survey (AHEAD) completed detailed questionnaires about their demographic, socioeconomic, and health status. Multivariate analyses of functional status (using a global index of functional status based on self-reported limitations in 11 activities) were conducted, controlling for demographic and socioeconomic status and common medical conditions, as well as independently for hearing and vision. RESULTS: Of the respondents, 27% rated their vision as fair or poor, whereas 25% rated their hearing as fair or poor. Controlling for demographic factors, socioeconomic status, medical conditions, and general health status, limitations in both vision and hearing correlated independently with worsened functional status. Controlling for income, wealth, and education did not greatly reduce the strength of the association between visual and hearing impairment and function. CONCLUSIONS: Visual and hearing impairment appear to have a significant relationship to overall functioning in the oldest old, regardless of income or wealth. By confirming these findings across income and household wealth groups, adjusted for medical conditions and general health status, in a nationally representative population of Americans age 70 years or older, this study provides a powerful added impetus to efforts for improving vision and hearing for all other Americans, including the oldest old.     

Prevention of naphthalene-1,2-dihydrodiol-induced lens protein modifications by structurally diverse aldose reductase inhibitors.

The effects of aldose reductase inhibitors on lens protein modifications induced by naphthalene-1,2-dihydrodiol were investigated in vitro to confirm the role of aldose reductase on naphthalene cataract formation. HPLC analysis of naphthalene-1, 2-dihydrodiol incubated with aldose reductase and NAD+indicated the formation of a metabolite peak corresponding to 1,2-naphthoquinone. Soluble proteins from rat lenses prepared by gel filtration of crude lens extracts through Sephadex PD-10, incubated with naphthalene-1, 2-dihydrodiol in the presence of NAD+displayed an absorbance ca 450 nm and their spectra were essentially identical to those of 1, 2-naphthoquinone-protein adducts. Similar spectra were also obtained from proteins isolated from the intact rat lens after in vitro incubation in medium containing naphthalene-1,2-dihydrodiol. The spectra obtained from lens proteins incubated with 1, 2-dihydroxynaphthalene were distinct from those of either naphthalene-1,2-dihydrodiol or 1,2-naphthoquinone. Aldose reductase inhibitors possessing either hydantoin or carboxylic acid groups prevented protein modification induced by naphthalene-1, 2-dihydrodiol but not protein modification induced by 1, 2-dihydroxynaphthalene or 1,2-naphthoquinone. Therefore, the metabolite formed from naphthalene-1,2-dihydrodiol by aldose reductase is 1,2-naphthoquinone. Lens proteins modified by naphthalene-1,2-dihydrodiol appear essentially identical to protein adducts formed with 1,2-naphthoquinone and their formation can be prevented by both hydantoin and carboxylic acid containing aldose reductase inhibitors.     

Synthesized TGF-beta s in RPE regulates cellular proliferation

Retinal pigment epithelial (RPE) cells transdifferentiate in culture, a transition which is accompanied by a shift in biological activity. The present study investigates whether transforming growth factor (TGF)-beta has the same effects on morphologically transformed RPE cells that it has on primary RPE cells. It also evaluates the autocrine and paracrine activities of TGF-beta s synthesized by RPE cells as well as the anti-TGF-beta effect of mannose-6-phosphate (M-6-P). RPE cells were subcultured at the sixth passage to induce morphological change. The effect of second passaged RPE-conditioned medium (CM) on DNA synthesis was evaluated by the incorporation of 3H-thymidine in rabbit subconjunctival fibroblasts (SCFs) and primary RPE cells. The presence of TGF-beta in RPE-CM was determined using immunoblotting analysis. And the inhibitory effect of M-6-P on cell proliferation mediated by RPE-CM was also analyzed using 3H-thymidine incorporation into DNA. TGF-beta 1, TGF-beta 2, and TGF-beta 3 inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, but the spindle-shaped sixth passaged RPE cells were not inhibited by these growth factors. The medium conditioned by RPE cells stimulated the proliferation of SCFs and inhibited the proliferation of primary RPE cells, in a manner similar to TGF-beta. When this medium was precipitated with either anti-TGF-beta 1, anti-TGF-beta 2, or anti-TGF-beta 3 antibodies, all three TGF-beta s, with an apparent molecular size of 25 kDa, were detected. Mannose-6-phosphate significantly blocked the effect of RPE-CM on cell proliferation. These findings indicate that RPE cells produce biologically functional TGF-beta s and that M-6-P can block the inhibitory effect of RPE-CM on cell proliferation.     

YC-1 potentiates the antiplatelet effect of hydrogen peroxide via sensitization of soluble guanylate cyclase.

In the present study, we showed that 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a nitric oxide (NO)-independent activator of soluble guanylate cyclase, could potentiate H2O2-induced inhibition of platelet aggregation and increase of platelet cGMP levels. The synergistic effect of YC-1 and H2O2 on platelet aggregation and increases of cGMP were almost completely prevented by catalase and a selective soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or partially attenuated by the hydroxyl radical scavenger mannitol. In contrast, superoxide dismutase failed to influence H2O2/YC-1-induced inhibition of aggregation. Furthermore, YC-1 could enhance the activation of soluble guanylate cyclase caused by FeSO4/H2O2 and, this effect was prevented markedly by mannitol. These results suggest that YC-1 may enhance the antiaggregatory effect of H2O2 via the sensitization of platelet soluble guanylate cyclase. In addition, this phenomenon is, at least in part, dependent on H2O2-derived hydroxyl radical.     

The inhibitory effect of ambroxol on hypochlorous acid-induced tissue damage and respiratory burst of phagocytic cells

Ambroxol (100 microM and 1 mM) and the thiols (all 1 mM), glutathione, tiopronin and cysteine, significantly attenuated the myeloperoxidase, H(2)O(2) and Cl(-) system-caused destruction of alpha(1)-antiproteinase and the HOCl-induced destruction of collagen, whereas they did not affect the elastase-induced destruction of collagen. Glutathione, tiopronin and cysteine almost completely decomposed both HOCl and H(2)O(2), while ambroxol up to 1 mM did not show a scavenging action on H(2)O(2). Ambroxol (1 to 100 microM) and 1 mM thiol compounds markedly inhibited the HOCl-induced alteration of elastase activity. Thiol compounds significantly attenuated the HOCl production caused by degraded immunoglobulin G-activated neutrophils. Ambroxol depressed superoxide and H(2)O(2) production induced by degraded immunoglobulin G-activated neutrophils and by lipopolysaccharide-activated alveolar macrophages in a dose-dependent manner. The results show that ambroxol may interfere with oxidative tissue damage and decrease proteolytic tissue destruction by attenuation of oxidative stress-induced inactivation of alpha(1)-antiproteinase through both decomposition of HOCl and inhibition of the respiratory burst in phagocytic cells.     

Discovery and structure-activity relationships of imidazole-containing tetrahydrobenzodiazepine inhibitors of farnesyltransferase

2,3,4,5-Tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepines were found to be potent inhibitors of farnesyltransferase (FT). A hydrophobic substituent at the 4-position of the benzodiazepine, linked via a hydrogen bond acceptor, was important to enzyme inhibitory activity. An aryl ring at position 7 or a hydrophobic group linked to the 8-position through an amide, carbamate, or urea linkage was also important for potent inhibition. 2,3,4, 5-Tetrahydro-1-(1H-imidazol-4-ylmethyl)-7-(4-pyridinyl)-4-[2-(t rifluo romethoxy)benzoyl]-1H-1,4-benzodiazepine (36), with an FT IC(50) value of 24 nM, produced 85% phenotypic reversion of Ras transformed NIH 3T3 cells at 1.25 microM and had an EC(50) of 160 nM for inhibition of anchorage-independent growth in soft agar of H-Ras transformed Rat-1 cells. Selected analogues demonstrated ip antitumor activity against an ip Rat-1 tumor in mice.     

Second-generation peptidomimetic inhibitors of protein farnesyltransferase demonstrating improved cellular potency and significant in vivo efficacy.

The synthesis and evaluation of analogues of previously reported farnesyltransferase inhibitors, pyridyl benzyl ether 3 and pyridylbenzylamine 4, are described. Substitution of 3 at the 5-position of the core aryl ring resulted in inhibitors of equal or less potency against the enzyme and decreased efficacy in a cellular assay against Ras processing by the enzyme. Substitution of 4 at the benzyl nitrogen yielded 26, which showed improved efficacy and potency and yet presented a poor pharmacokinetic profile. Further modification afforded 30, which demonstrated a dramatically improved pharmacokinetic profile. Compounds 26 and 29 demonstrated significant in vivo efficacy in nude mice inoculated with MiaPaCa-2, a human pancreatic tumor-derived cell line.