Epidermin is a large peptide antibiotic, which is synthesized in the ribosome via a precursor protein, followed by enzymatic modifications. It was isolated from the culture filtrate of Staphylococcus epidermidis TU 3298 by adsorption on Amberlite XAD-8. The basic heneicosapeptide amide was chromatographed on Sephadex LH-20 and purified to homogeneity via multiplicative counter-current distributions in one acidic and one neutral system. Tryptic digestion gave the soluble N-terminal fragment epidermin-(I -13)-peptide (Pl) and the insoluble Cterminal fragment 2-oxobutyryl-epidermin-(l5 -21)-peptide amide (P2), each possessing two sulfide ring systems. The heterodetic rings consisted of meso-lanthionine and (2S,3S,6R)-3-methyllanthionine (Pl), meso-lanthionine and C-terminally the new amino acid S-(2-aminovinyl)-~-cysteine (P2). The complex sequence was elucidated via a combination of desulfurization with Raney nickel, enzymatic and acidolytic degradations, gas-phase sequencing, fast-atom bombardment and field-desorption mass spectrometry and NMR spectroscopy.
We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.
A decosapeptide that is highly effective against acne has been isolated from a culture filtrate of Staphylococcus epidermidis Tü 3298. The molecule contains four disulfide bridges derived from meso‐lanthionine, β‐methyllanthionine, and the newly discovered amino acid S‐(2‐aminovinyl)‐D‐cysteine. The structure elucidation was achieved by enzymatic and hydrolytic fragmentation and desulfurization with Raney nickel followed by sequence analysis.
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