Development of a chemically defined medium for the production of the antibiotic platensimycin by Streptomyces platensis.

The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semi-defined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7.

Neuropeptide (calcitonin gene-related peptide) induction of nitric oxide in human keratinocytes in vitro.

Nitric oxide (NO) is an important signaling molecule in both the central nervous system and the periphery, where it is involved in neurotransmission, vascular and bronchial tone, inflammation, and cutaneous immune function. More recently, NO has been implicated in intracellular signaling and may have a role in cellular differentiation, cytokine expression, and apoptosis. The experiments described herein examined the effect of calcitonin gene-related protein (CGRP), a cutaneous nerve neuropeptide, on NO production in human keratinocytes in vitro. CGRP stimulated two distinct increases in NO production: one within 30 minutes and a second at 24 hours. CGRP stimulated a modest increase in inducible nitric oxide synthase (iNOS) at 3-6 hours. Experimental evidence suggested that CGRP stimulated both constitutive NOS activity and generation of NO via nitrosothiol degradation within the first hour. Production of NO was paralleled by a decrease in nitrosothiol levels for 2 hour, suggesting that immediate NO release may originate from pre-existing stores. Nitrosothiols are ubiquitous molecules that comprise an important NO pool and have intracellular regulatory roles, particularly linked to oxidative stress. The present data indicate that, in addition to its known cAMP signaling pathway, CGRP may act to regulate keratinocyte biology through intracellular NO by modulation of S-nitrosothiol stores and stimulation of NOS activity.