Collagen Synthesis in Cultured Human Skin Fibroblasts: Effect of Ascorbic Acid and Its Analogs

In confluent human skin fibroblasts maintained in 0.5% serum-supplemented medium, L-ascorbate specifically stimulated the rate of incorporation of labeled proline into total collagenase-sensitive protein, without changing the specific activity of the intracellular free proline. This influence of ascorbate reached a maximum at 30 μM and continued for at least 4 days, resulting in a 4-fold increase. The ascorbate effect occurred in cells at both confluent and subconfluent densities and was evident at all serum concentrations from 0.5–20%. The effect was independent of duration of the radioactive pulse between 2–6 h. D-Ascorbate, D-isoascorbate, and L-dehydroascorbate also stimulated collagen synthesis but at considerably higher concentrations, i.e., 250–300 μM. The stimulation of collagen synthesis by ascorbate and its analogs was accompanied by a decline in prolyl hydroxylase activity and a rise in lysyl hydroxylase activity; again L-ascorbate was found to be most effective. Dimethyltetrahydropterine and L-lactate failed to produce these effects

Characterization of a Partial cDNA for Lysyl Hydroxylase from Human Skin Fibroblasts; Lysyl Hydroxylase mRNAs Are Regulated Differently by Minoxidil Derivatives and Hydralazine

Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that catalyzes the formation of hydroxylysine required for intermolecular crosslinking of collagen. We have isolated a partial (2.2-kb) cDNA for LH from human skin fibroblasts using PCR. DNA sequencing revealed 72% homology of the human coding sequence with the chick LH sequence at the nucleotide level and 76% homology predicted at the amino acid level. The LH cDNA hybridized strongly with two mRNA species of 2.4 and 3.4kb on Northern blots of normal fibroblast RNA. Administration of minoxidil decreased both mRNA species without affecting levels of the mRNAs for the β subunit of prolyl 4-hydroxylase (PH) or α1(I) collagen. Two derivatives of minoxidil (3' hydroxyminoxidil and 4' hydroxyminoxidil) produced similar decreases in LH mRNAs. In contrast hydralazine increased the mRNAs for LH in parallel with its previously reported effect on the mRNA for the β subunit of PH. This effect is accompanied by virtual elimination of the α1(I) collagen mRNAs. These results on the action of minoxidil and hydralazine at the pretranslational level correlate well with their previously reported effect on enzyme activity and collagen biosynthesis and indicate that changes in steady-state mRNA levels can account directly for changes at the protein level. Moreover, the unique action of minoxidil in specifically decreasing LH mRNAs contrasts with the less specific stimulatory effects of hydralazine and suggests that these pharmaceuticals arc regulating expression of LH at a pretranslational level by different mechanisms

Calcofluor White Combination Antifungal Treatments for <em>Trichophyton rubrum</em> and <em>Candida albicans</em>

<div><p>Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte <em>Trichophyton rubrum</em> and <em>Candida albicans</em> to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both <em>T. rubrum</em> and <em>C. albicans</em> to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV_{365 nm} and bind specifically to fungal cell walls, we tested whether CFW combined with UV_{365 nm} irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV_{365 nm} irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in <em>C. albicans</em>. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.</p> </div

Combination treatments with CFW and UV_{365 nm} irradiation.

a<p>80% growth inhibition for UV treatment alone was 56.2 J/cm² for <i>T. rubrum</i> and 149.6 J/cm² for <i>C. albicans</i>. 99% and 95% fungicidal UV treatment alone was 74.8 J/cm² for <i>T. rubrum</i> and 149.6 J/cm² for <i>C. albicans</i>, respectively.</p

Tabele 3. <i>T. rubrum</i> ATCC MYA-4438 and <i>C. albicans</i> SC5314 combination drug treatments with CFW.

a<p>Some experiment-to-experiment differences in CFW MIC₈₀s for <i>C. albicans</i> were observed, such as for experiments where the MIC₈₀s were read after 24 h instead of 48 h.</p>b<p>Not applicable. When the MIC₈₀ for any drug was higher than the highest concentration tested, the FIC could not be determined.</p

Disc diffusion assays showing enhanced <i>C. albicans</i> inhibition when treated with CFW and ergosterol biosynthesis inhibitors.

<p>Starting at the bottom right disc and moving clockwise, the amounts of each drug added per disc included 14.11, 3.52, 1.41 and 0 ng of itraconazole, 8.32, 2.08, 0.83, and 0 ng of miconazole, and 6.07, 1.52, 0.61, and 0 ng of fenpropimorph. Drugs were diluted in DMSO, and DMSO comprised the no-drug control.</p