Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.Macrolide antibiotics consist of a polyketide macrolactone ring to which one or more deoxysugars are attached. They inhibit protein synthesis by binding to the large subunit of the ribosome and can also, in some cases, interfere with the assembly of the large subunit (reviewed in reference19). As with many other natural products, glycosylation of macrolides is essential for their biological activity. The determination of the three-dimensional structure of the 50S ribosomal unit in complex with various macrolides showed that the sugar moieties are involved in specific interactions with the 23S rRNA (11, 38). All macrolides block the ribosomal peptide exit tunnel, thus preventing peptide chain elongation. In addition to their role in binding, the sugars are also important for steric hindrance in the tunnel. For instance, it was shown that the C-5 disaccharide moieties of some 16-membered macrolides, such as spiramycin or tylosin, extend in the tunnel toward the peptidyl transferase center, and a correlation could be established between the length of the macrolide saccharide side chain and the number of amino acids which could be incorporated before peptide chain elongation was blocked (11).One of the most common resistance mechanisms to macrolide antibiotics encountered in pathogenic bacteria involves target modification. These modifications could include mutation or m...

Background. Mesenchymal stem cells (MSCs), isolated from bone marrow, adipose tissue, and umbilical cord tissue, have been known to differentiate into hepatocyte-like cells. MSCs can also be easily obtained from umbilical cord lining membrane (CLMSCs). CLMSCs are more primitive MSCs than those isolated from other tissue sources.Objectives. The aim of this study was to investigate thein vitrodifferentiation of CLMSCs into hepatocyte lineage.Materials and Methods. In this study, CLMSCs were isolated through a tissue attachment method. Cells were characterized for expression of MSC-specific markers and differentiation potency. CLMSCs were induced to differentiate into hepatocytes by a simple two-step protocol. Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions.Results. CLMSCs expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. RT-PCR, real-time qRT-PCR, Western blot, and immunocytochemistry analyses demonstrated that differentiated CLMSCs, having hepatocyte-like morphology, expressed several liver-specific markers, such as ALB, AFP, CK18, and CK19, at both mRNA and protein levels following hepatocyte differentiation. Furthermore, periodic acid-Schiff staining and low-density lipoprotein (LDL) uptake assay showed that differentiated cells could store glycogen and uptake LDL.Conclusion. This study demonstrated that CLMSCs can differentiate into functional hepatocyte-like cells. CLMSCs can serve as a favorable cell source for tissue engineering in the treatment of liver disease.

Streptomyces ambofaciens synthesizes the macrolide antibiotic spiramycin. The biosynthetic gene cluster for spiramycin has been characterized for S. ambofaciens. In addition to the regulatory gene srmR (srm22), previously identified (M. Geistlich et al., Mol. Microbiol. 6:2019-2029, 1992), three putative regulatory genes had been identified by sequence analysis. Gene expression analysis and gene inactivation experiments showed that only one of these three genes, srm40, plays a major role in the regulation of spiramycin biosynthesis. The disruption of srm22 or srm40 eliminated spiramycin production while their overexpression increased spiramycin production. Expression analysis was performed by reverse transcription-PCR (RT-PCR) for all the genes of the cluster in the wild-type strain and in the srm22 (srmR) and srm40 deletion mutants. The results from the expression analysis, together with the ones from the complementation experiments, indicated that Srm22 is required for srm40 expression, Srm40 being a pathway-specific activator that controls most, if not all, of the spiramycin biosynthetic genes.Streptomyces species are Gram-positive, soil-inhabiting, filamentous bacteria that undergo a complex process of morphological differentiation and produce a great variety of secondary metabolites, including antibiotics with important applications in human medicine and in agriculture. These secondary metabolites are synthesized by complex pathways that utilize primary metabolites as building blocks. The genes required for