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Biomedical subjects

Eduardo Rodriguez

Publications and source records attributed to Eduardo Rodriguez.

11 recordsLinked to original sources
PubMed2005

Arterialized venous flow-through flap for simultaneous reconstruction of a radial artery defect and palmar forearm soft-tissue loss from sarcoma resection.

A 50-year-old right-handed male presented with a previously incompletely excised low-grade fibrous histiocytoma on his distal radial palmar forearm. Reoperative wide resection resulted in a segmental defect of the radial artery and a large soft-tissue defect with exposed tendons denuded of paratenon. An arterialized venous fasciocutaneous flow-through flap, measuring 8 x 3 cm, was harvested from the dorsal ipsilateral hand and used to reconstruct both the soft-tissue and the segmental radial-artery defects. A full-thickness skin graft was harvested from the ipsilateral groin to cover the dorsal hand wound. Moderate venous congestion was noted in the flap on postoperative day 1 and treated with four days of leeches. Approximately 10 percent of the flap was lost due to the venous congestion. The flap healed well without further complications. At 33 months the flap remained well-perfused, with excellent flow through the reconstructed radial artery evidenced by both clinical examination and by color vascular Doppler exam. The patient had full range of motion in his right hand despite initial postoperative stiffness and reported no cold intolerance in the right hand. There has been no local recurrence of the sarcoma. The authors believe that this is the largest arterialized venous flow-through flap currently reported to have survived, as well as the first reported case of its use for the simultaneous reconstruction of a radial artery defect and an associated soft-tissue loss.

Combined Modality Therapy↗
PubMed2004

Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae.

Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors--malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA--used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG-K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.

Acyl Carrier Protein↗
PubMed2004

Kienböck's disease in an elderly patient treated with proximal row carpectomy.

Kienböck's disease most often occurs in the dominant hand of young men with a history of manual labor. We report an atypical presentation of stage III Kienböck's disease in the nondominant hand of a 70-year-old woman. The patient was managed successfully by proximal row carpectomy. The risk factors and cause of Kienböck's disease in the elderly may differ from those in the typical younger population. The detection of Kienböck's disease in the elderly and the use of proximal row carpectomy in advanced cases is emphasized.

Aged↗
PubMed2003

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains.

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms- Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively-to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site ( attB) for phiC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.

Anti-Bacterial Agents↗
PubMed2003

A model of structure and catalysis for ketoreductase domains in modular polyketide synthases.

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea.

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301-105B, compared to its parent. In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.

Bacterial Proteins↗