Myocardial torsion and cardiac fulcrum.

OBJECTIVE: The development of the myocardial band shows that it starts and ends at the origin of the great vessels and that the myocardium joins to these rings but does not inserted into them. We always considered that there should be a fixed end of the muscle band that would allow it a helical rotation to fulfill its fundamental movements of shortening-torsion (systole) and elongation-distortion (suction). MATERIAL AND METHODS: Seven young-bovine hearts (800-1000g) and seven human hearts (one embryo, 4g; one 10 years, 250g and five adult, 300g/average) were used for a detailed macrocoscopic and microscopic study. RESULTS: We have found in all the bovine and human hearts studied a nucleus underlying the right trigone, whose osseus, chondroid or tendinous histological structure depends on the specimen analyzed. The microscopic analysis revealed in the hearts a trabecular osteochondral matrix (fulcrum) with segmental lines in bovines and in the ten-year-old human. In the fetus, it was found pre-chondroid areas in a myxoid stroma. In the adult human hearts, the histological analysis revealed a matrix similar to that of a tendon. All the hearts studied presented myocardial attachment to the rigid structure of the fulcrum. Myocardiocytes were not found neither at the left or rigth trigonous nor at the base of the valves. CONCLUSIONS: The finding of the fulcrum gives support to the spiral myocardial band being the point of fixation that allows the helicoidal torsion.

DNA paternity tests in Spain without the mother's consent: the legal responsibility of the laboratories.

It is technically feasible to perform paternity diagnosis testing solely involving an alleged father and his descendent. However, there are serious legal and ethical problems for forensic genetics laboratories when it comes to paternity testing cases for investigating the alleged father-child relationship if the biological mother has not given consent to access her genetic information. Based on the Spanish Constitution, the new Code of Ethics of the Spanish Medical Association includes several articles on studies about genetic information and their acceptance by all the individuals involved. This problem is greater when the child is a minor, mentally incapacitated or psychologically incapable, because current Spanish law requires informed consent from legal representatives, but the law does not typify what happens when one parent gives consent (the putative father) and the other parent (the mother) does not agree. The aim of this study is to put forward legal solutions to avoid potential legal problems.

A small molecule cell-impermeant Hsp90 antagonist inhibits tumor cell motility and invasion.

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains function of numerous intracellular signaling nodes utilized by cancer cells for proliferation and survival. Hsp90 is also detected on the plasma membrane of tumor cells and its expression has been suggested to correlate with metastatic potential. Given the abundance and diverse functions of the intracellular pool of this protein, the precise contribution of cell surface Hsp90 to cell motility and tumor metastasis remains to be determined. In this study we utilized the small molecule DMAG-N-oxide, a novel cell-impermeable Hsp90 inhibitor, to specifically examine the role of cell surface Hsp90 in cell motility. We observed that, while not affecting intracellular Hsp90 function, DMAG-N-oxide significantly retarded tumor cell migration and integrin/extracellular matrix-dependent cytoskeletal reorganization. Concomitant with these findings, targeting cell surface Hsp90 significantly inhibited tumor cell motility and invasion in vitro, and had a dramatic impact on melanoma cell lung colonization in vivo. These data indicate that cell surface Hsp90 plays an important role in modulating cancer cell migration that is independent of the function of the intracellular Hsp90 pool, and that small molecule inhibitors of surface Hsp90 may provide a new approach to targeting the metastatic phenotype.

Aproximación diagnóstica a las inmunodeficiencias combinadas primarias / Diagnostic approach of primary immunodeficiencies

Las inmunodeficiencias primarias son un grupo deenfermedades amplio, poco frecuente y de expresiónclínica muy variable que hacen difícil su diagnóstico.Por su gravedad, esta revisión se ha centradoen las Inmunodeficiencias Combinadas y orientadoal pediatra general, principal responsable de sudetección, que fundamentalmente se basa en lasospecha clínica que se analiza con detenimiento.También se repasan los principios básicos de la respuestainmune innata y adquirida normal, lo que nosayudará a reconocer las consecuencias esperablescuando fallen cualquiera de sus elementos, y orientarnuestro diagnóstico.Basados en la sospecha clínica inicial, se recomiendaun estudio inmunitario básico fácilmente accesible(hemograma, cuantificación de inmunoglobulinas,subpoblaciones linfocitarias y serologíaVIH) con el que se pueden detectar el 80 % de loscasos y, sobre todo, excluir las formas más severascon abolición del sistema inmune que suponen unaverdadera urgencia pediátrica. Posteriormente seránnecesarias otras exploraciones propias de centrosespecializados en inmunología infantil para confirmary concretar el diagnóstico preciso.Por último se presentan tres casos clínicos ilustrativosde IDC con la aplicación práctica de lo descrito

Primary immunodeficiencies are a large, infrequentgroup of diseases whose variable clinical expressionmakes them difficult to diagnose. Giventheir seriousness, this revision has been focusedon combined immunodeficiencies (CID) and directedat the general pediatrician, who is the main responsiblefor their detection, usually based on a carefullyanalyzed clinical suspicion. The basic principlesof the innate and normally acquired immunologicresponses are also reviewed; they will help usto recognize the consequences to be expectedwhen any of their elements fail, and will guide ourdiagnosis.Based on initial suspicion, we recommend an easilyaccessible basic immunological study (completeblood count, quantification of immunoglobulins,lymphocyte subsets y and HIV serology), which willallow to identify 80 % of the cases and, especially, toexclude more severe forms of immunologic systemabolition which are a real pediatric urgency. On a laterstage, further examinations will be required, to becarried out in centers specialized in pediatric immunology,in order to confirm and obtain a more precisediagnosis.Lastly, we present three illustrative clinical casesof IDC with practical application of the method described

Genetically engineered analogs of ascomycin for nerve regeneration.

The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/520-calcineurin ternary complex. They also have calcineurin-independent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13- and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13- and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. A representative compound was also shown to accelerate nerve regeneration and functional recovery in the rat sciatic nerve crush model.

Precursor-directed production of erythromycin analogs by Saccharopolyspora erythraea.

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 degree) Saccharopolyspora erythraea strains to produce 13-substituted erythromycin analogs. This direct feeding process potentially represents a simplified production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concentration up to a precursor-specific saturation level. However, the rate of product formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Streptomyces coelicolor. Several strategies were pursued to address the issue of these high diketide consumption rates: (1) elucidation of the locale of diketide degradation, (2) addition of beta-oxidation inhibitors to the cultures, and (3) addition of a sacrificial diketide enantiomer to occupy putative degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythromycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs to a large-scale production system for these compounds.

An FKBP12 binding assay based upon biotinylated FKBP12.

A binding assay was developed for measuring the affinity of FKBP12 ligands. A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase. The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with [3H]FK506 for FKBP12 sites on the plate. The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values. The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules.

Manipulation of polyketide biosynthesis for new drug discovery.

Modular polyketide synthases (PKS) are large multifunctional proteins which direct the condensation of activated short chain carboxylic acids into products of defined length and functionality using a dedicated set of active sites, or module, for each step in the polymerization. The structure of the product is directly related to the number, content and sequence of modules in a PKS. Technology is described which allows the rational manipulation of the biosynthesis of these compounds and enables the generation of specific novel polyketide structures. Examples of polyketide drugs whose structures may be manipulated using this technology are given.

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogs in Streptomyces coelicolor: understanding precursor effects.

A fermentation process employing precursor-directed biosynthesis is being developed for the manufacture of 6-deoxyerythronolide B (6-dEB) analogues. Through a plasmid-based system in Streptomyces coelicolor, 6-dEB synthesis is catalyzed by 6-dEB synthase (DEBS). 6-dEB synthesis is abolished by inactivation of the ketosynthase (KS) 1 domain of DEBS but can be restored by providing synthetic activated diketides. Because of its inherent catalytic flexibility, the KS1-deficient DEBS is capable of utilizing unnatural diketides to form various 13-substituted 6-dEBs. Here we characterize process variables associated with diketide feeding in shake-flask experiments. 13-R-6-dEB production was found to depend strongly on diketide feed concentrations, on the growth phase of cultures at feeding time, and on the R-group present in the diketide moiety. In all cases a major portion of the fed diketides was degraded by the cells.

Development and screening of a polyketide virtual library for drug leads against a motilide pharmacophore.

A virtual library of macrocyclic polyketide molecules was generated and screened to identify novel, conformationally constrained potential motilin receptor agonists ("motilides"). A motilide pharmacophore model was generated from the potent 6,9-enol ether erythromycin and known derivatives from the literature. The pharmacophore for each molecular conformation was a point in a distance-volume space based on presentation of the putative binding moieties. Two methods, one fragment based method and the other reaction based, were explored for constructing the polyketide virtual library. First, a virtual library was assembled from monomeric fragments using the CHORTLES language. Second, the virtual library was assembled by the in silico application of all possible polyketide synthase enzyme reactions to generate the product library. Each library was converted to low-energy 3D conformations by distance geometry and standard minimization methods. The distance-volume metric was calculated for low-energy conformations of the members of the virtual polyketide library and screened against the enol ether pharmacophore. The goal was to identify novel macrocycles that satisfy the pharmacophore. We identified three conformationally constrained, novel polyketide series that have low-energy conformations satisfying the distance-volume constraints of the motilide pharmacophore.

Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase.

A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.

Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase.

The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.

NO2/NO3 and cytokine plasma profiles under different postoperative parenteral nutrition regimens.

Both nitric oxide and cytokines are considered mediators of the acute-phase response in humans, and their early postoperative period plasma levels have been found to be of prognostic value. On the other hand, it has been suggested that the fatty emulsions used in total parenteral nutrition (TPN) may induce changes in macrophage function. In the present study we investigated the postoperative evolution of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), and nitrate/nitrite plasma levels under three different TPN regimens. Twenty-one patients diagnosed with upper digestive tract neoplasm, without preoperative TPN, and having undergone radical surgery, were randomly assigned to three groups: Group I, all nonprotein calories supplied by hypertonic glucose solution: Group II, 55% of the nonprotein calories supplied by glucose and 45% by 20% long-chain triacylglycerides emulsion (LCT) (Intralipid 20%, Kabi-Pharmacia); Group III, same as Group II, but a 20% emulsion of a mixture of medium-chain and long-chain triacylglycerides (MCT/LCT) (Lipofundina MCT/LCT 20%, B. Braun) was used instead of LCT. Blood samples were obtained on postoperative Days 1-5 and 10, 3 h after ending the lipid infusion. In all the three groups IL-1, IL-6, and TNF-alpha levels rose after surgery, peaking at Day 2, whereas NO2/NO3 levels had their peak at Day 3. Day-to-day comparison of plasma levels of cytokines and NO2/NO3 between the investigated groups did not show any statistical significance. Differences between group means were not found when the areas under the curve over the first 5 postoperative days were compared (1.72 +/- 0.25, Group I; 1.88 +/- 0.34, Group II; and 2.52 +/- 0.50, Group III, for TNF-alpha; 1.79 +/- 0.12, Group I; 1.92 +/- 0.18, Group II; and 1.50 +/- 0.12, Group III, for NO2/NO3). We conclude that the different parenteral nutrition regimens studied do not evoke alterations in cytokine and NO2 + NO3 levels in the patient groups investigated in this study.

The catalytic mechanism and structure of thymidylate synthase.

Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.

[Major abdominal surgery and the clearance of lipid emulsions]. / Cirugía abdominal mayor y aclaramiento de emulsiones lipídicas.

The use of lipid emulsions for TPN remains controversial. Although experimental studies show that medium chain triglycerides (MCT) are beneficial over long chain triglycerides (LCT) clinical studies are contradictory. With the aim to study the clearance of two lipid emulsions a prospective study was designed. 21 patients, submitted to resective surgery because of upper gastrointestinal tract malignancy were randomized in three groups. In group I (without lipids): all caloric intake was supplied by hypertonic glucose solution. In group II (LCT group) 55% of caloric intake was supplied by glucose and 45% by a LCT 20% emulsion (Intralip[id 20%, Kabi-Pharmacia). In group III (MCT group) 55% of caloric intake was supplied by hypertonic glucose solution and 45% by a physical mixture of LCT and MCT at 20% concentration (Lipofundina MCT, B. Braun). Our results show that in postoperative period of major abdominal surgery both lipid emulsions are cleared of in a similar way. When these emulsions are administered during 12 hours per day plasmatic triglycerides are completely cleared before to start the next-day infusion of lipid emulsion. Differences in total cholesterol were not found between groups. Nevertheless LDL-cholesterol rose significatively between first and tenth postoperative day in LCT group.

[Lymphocyte subpopulations and surgery. The role of postoperative parenteral nutrition]. / Subpoblaciones linfocitarias y cirugía. Papel de la nutrición parenteral postoperatoria.

The effects of LCT-based lipid emulsions used in TPN on immune system remains controversial. In this prospective study we research the effects of three types of TPN on T-lymphocyte subsets and NK cells. 21 patients diagnosed because of upper gastrointestinal carcinoma (UGIC), and amenable of curative surgery were included in the study. TPN support was maintained 10 postoperative days at least. All patients received 35 non-proteic Kcal/KG BW/day. Group I (without lipid): received 100% of caloric intake (CI) by glucose. Group II (LCT): received 55% of CI by glucose and 45% by LCT at 20% emulsion. Group III (MCT/LCT): received 55% of CI by glucose and 45% by MCT/LCT at 20% mixture. T-lymphocyte subsets were determined by flow cytometry preoperatively and in first and tenth postoperative days. Our results suggest that patients diagnosed of UGIC present alterations of cellular immunity. These alterations are increased by the age and by surgical act. The changes found are independent of the type of TPN. LCT-based emulsions have similar effects on T-lymphocyte subsets that MCT/LCT-based emulsions.