Biosynthesis of Largimycins in Streptomyces argillaceus Involves Transient ß-Alkylation and Cryptic Halogenation Steps Unprecedented in the Leinamycin Family.

Largimycins A1 and A2 are key members of a recently identified family of hybrid nonribosomal peptide polyketides belonging to the scarcely represented group of antitumor leinamycins. They are encoded by the gene cluster lrg of Streptomyces argillaceus. This cluster contains a halogenase gene and two sets of genes for the biosynthesis and incorporation of ß branches at C3 and C9. Noticeably, largimycins A1 and A2 are nonhalogenated compounds and only contain a ß branch at C3. By generating mutants in those genes and characterizing chemically their accumulated compounds, we could confirm the existence of a chlorination step at C19, the introduction of an acetyl-derived olefinic exomethylene group at C9, and a propionyl-derived ß branch at C3 in the biosynthesis pathway. Since the olefinic exomethylene group and the chlorine atom are absent in the final products, those biosynthetic steps can be considered cryptic in the overall pathway but essential to generating keto and epoxide functionalities at C9 and C18/C19, respectively. We propose that chlorination at C19 is utilized as an activation strategy that creates the precursor halohydrin to finally yield the epoxy functionality at C18/C19. This represents a novel strategy to create such functionalities and extends the small number of natural product biosynthetic pathways that include a cryptic chlorination step.

Targeted photodynamic therapy using reconstituted high-density lipoproteins as rhodamine transporters.

Reconstituted high-density lipoprotein (rHDL) nanoparticles are excellent transporters of molecules and very useful for targeted therapy as they specifically recognize the scavenger receptor, class B1 (SR-B1) that is present on the surface of a wide range of tumor cells. However, they have rarely been employed to transport photosensitizers (PS) for photodynamic therapy (PDT). Rhodamine (R) compounds have been dismissed as useful PSs for PDT due to their low 1O2 production, excitation wavelengths with little tissue penetration, and poor selectivity for tumor cells. It was recently demonstrated that when irradiating at 532 nm or with Cerenkov radiation (CR) from a ß-emitting radionuclide, R123, R6G, and RB undergo electron transfer reactions (type I reaction) with folic acid. R6G also produces type I reactions with O2. In this work, the photodynamic effects of the rHDL-R system were evaluated in vitro. rHDL nanoparticles loaded with R123, R6G, and RB were synthesized, and the PS was internalized into T47D tumor cells. When cells were irradiated with a 532-nm laser in the presence of an rHDL-R systems, a cytotoxic photodynamic effect was obtained in the order R6G > R123 > RB. In the presence of CR from a 177Lu source, cytotoxicity showed the order R6G > RB > R123. The higher cytotoxicity induced by R6G in both cases corresponds to higher cellular internalization and larger production of type I and II reactions. Thus, in this work, it is proposed that rHDL-R/177Lu system can be applied in theragnostics as a multimodal radiotherapy-PDT-imaging system (imaging by SPECT or Cerenkov) and in hypoxic solid tumors in which external radiation is not effective and 177Lu-CR acts as light source.

Discovery of Cryptic Largimycins in Streptomyces Reveals Novel Biosynthetic Avenues Enriching the Structural Diversity of the Leinamycin Family.

Largimycins are hybrid nonribosomal peptide-polyketides that constitute a new group of metabolites in the leinamycin family of natural products displaying unique structural features such as containing an oxazole instead of a thiazole ring or being oxime ester macrocycles, unprecedented in nature, rather than macrolactams. Their discovery in Streptomyces argillaceus and Streptomyces canus has relied on the activation of two homologous silent gene clusters by overexpressing a transcriptional activator and cultivating in specific media. The proposed biosynthesis of largimycins includes the key action of the oxidoreductase LrgO, responsible for the formation of the oxime group involved in macrocyclization, and two putative cryptic biosynthetic steps consisting of chlorination of l-Thr by the NRPS loading module and incorporation of an olefinic exomethylene group by LrgJ PKS. The discovery of largimycins uncovers novel biosynthetic avenues employed in nature to enrich the structural diversity of leinamycins and provides tools for combinatorial biosynthesis.

The role of the trithorax group TnaA isoforms in Hox gene expression, and in Drosophila late development.

Regulation of developmental gene expression in eukaryotes involves several levels. One of them is the maintenance of gene expression along the life of the animal once it is started by different triggers early in development. One of the questions in the field is when in developmental time, the animal start to use the different maintenance mechanisms. The trithorax group (TrxG) of genes was first characterized as essential for maintaining homeotic gene expression. The TrxG gene tonalli interacts genetically and physically with genes and subunits of the BRAHMA BAP chromatin remodeling complex and encodes TnaA proteins with putative E3 SUMO-ligase activity. In contrast to the phenocritic lethal phase of animals with mutations in other TrxG genes, tna mutant individuals die late in development. In this study we determined the requirements of TnaA for survival at pupal and adult stages, in different tna mutant genotypes where we corroborate the lack of TnaA proteins, and the presence of adult homeotic loss-of-function phenotypes. We also investigated whether the absence of TnaA in haltere and leg larval imaginal discs affects the presence of the homeotic proteins Ultrabithorax and Sex combs reduced respectively by using some of the characterized genotypes and more finely by generating TnaA defective clones induced at different stages of development. We found that, tna is not required for growth or survival of imaginal disc cells and that it is a fine modulator of homeotic gene expression.

Uncovering production of specialized metabolites by Streptomyces argillaceus: Activation of cryptic biosynthesis gene clusters using nutritional and genetic approaches.

Sequencing of Streptomyces genomes has revealed they harbor a high number of biosynthesis gene cluster (BGC), which uncovered their enormous potentiality to encode specialized metabolites. However, these metabolites are not usually produced under standard laboratory conditions. In this manuscript we report the activation of BGCs for antimycins, carotenoids, germicidins and desferrioxamine compounds in Streptomyces argillaceus, and the identification of the encoded compounds. This was achieved by following different strategies, including changing the growth conditions, heterologous expression of the cluster and inactivating the adpAa or overexpressing the abrC3 global regulatory genes. In addition, three new carotenoid compounds have been identified.

Participation of NMDA-glutamatergic receptors in hippocampal neuronal damage caused by adult-onset hypothyroidism.

We analyzed the participation of N-methyl-d-aspartate (NMDA) receptors in the neuronal damage caused by adult-onset hypothyroidism. Wistar rats were randomly assigned into four groups. The euthyroid group received tap water. The hypothyroid group received methimazole (60 mg/kg) in their drinking water to induce hypothyroidism. Two more groups of rats received the antithyroid treatment and were injected daily with the NMDA antagonist ketamine (15 mg/kg, sc) or MK-801 (0.5mg/kg, ip). Treatments were administered during 4 weeks. At the end of the respective treatments rats were deeply anaesthetized and perfused intracardially with 0.9% NaCl followed by 4% paraformaldehyde. The brains were removed from the skull, and coronal brain sections (7microm thick) were obtained. Neurons were counted in the CA1, CA2, CA3, and CA4 hippocampal regions differentiating between normal and atrophic cells by an experimenter blind to the treatment. The percentage of neuronal damage found in the MMI group was significantly greater in the hippocampal regions compared to the euthyroid group. In contrast, both NMDA antagonists were able to prevent the neuronal damage secondary to hypothyroidism in all hippocampal regions. Our results suggest that the neuronal damage caused in the hippocampus of adult-onset hypothyroid rats requires activation of NMDA channels.

Differential aromatase (CYP19) expression in human arteries from normal and neoplasic uterus: an immunohistochemical and in situ hybridization study.

Aromatase CYP19 catalyzes the synthesis of estrogen from androgens in a tissue-specific manner. This enzyme is present in several tissues, including gonads, brain and fatty tissue. More recently, its presence has been described in vessels. Here, we describe the expression of aromatase in human uterine artery and compare its expression with that found in arteries of estrogen-dependent uterine leiomyomata from women. To do this, we employed immunohystochemical and in situ hybridization techniques. We used, a polyclonal antibody raised against the carboxyl terminus of aromatase (ARO) and RNAm probes, of the exon 1 of ARO. We found an increased immunoreactivity of ARO in uterine arteries of patients with leiomyoma as compared with control group. Probe showing positive signal in skin fibroblasts (1b), showed positive hybridization signal in normal artery, while probes with positive signal in placenta (1a), ovary (1c) and testis (1d) were over-expressed in arteries of leiomyomas.

Influence of high-calorie (cafeteria) diets on the population of Paneth cells in the small intestine of the rat.

A high-calorie (cafeteria) diet is known to cause changes in the intestinal morphology and functioning that seem to be related to calorie overfeeding. Among the cell lineages found in the small intestine epithelium, the Paneth cell (PC) population is known to be influenced by factors related mainly to the intestinal microbiota. The role of PCs in the intestinal cell concert remains unclear, because experimental evidence suggests PC involvement in local processes other than protection against pathogens. Participation of PC in digestive mechanisms has been proposed on this basis. We have analyzed the effect of high-carbohydrate (HC) and high-fat (HF) cafeteria diets on the PC population in the small intestine of the adult rat. For 8 weeks, both HC and HF diets caused a gain in body weight, but whereas the HC-fed rats showed reduced counts of intestinal crypts per 5-mum section, the HF-fed group showed the opposite. In control rats, the number of crypts per section showed a slight tendency to decrease along the duodenum - ileum axis, whereas the number of PCs per crypt was increased towards the ileum. As a result, the number of PCs per section (calculated from these data) remained constant along the three segments of the intestine. The hypercaloric diets did not modify the general tendencies seen in the crypt and PC counts, but reduced the number of PCs per section in the duodenum by 50%. HC-fed, but not HF-fed, rats showed a similar reduction in jejunum also. These changes do not correlate particularly with any of the predictable effects of diet composition, so that a multifactorial control of PC density is proposed.

Prueba de equilibrio peritoneal, análisis de 80 casos, en el Servicio de Nefrolología del Hospital Juárez de México de la Secretaría de Salud y Asistencia / Peritoneal equilibration test. Analysis of 80 cases in the nefrology service of the Hospital Juarez de Mexico, SSA

La prueba de equilibrio peritoneal (PET) fue descrita en 1987 por Z. Twardovsky, como una prueba para conocer la difusión de solutos y capacidad de ultrafiltración (UFR) en la membrana peritoneal (MP). La PET ha permitido clasificar el tipo de transporte peritoneal (TP), en los pacientes que se encuentran en diálisis peritoneal (DP) en: promedio alto, promedio bajo, alto y bajo. El presente trabajo tiene como objetivo, presentar el análisis prospectivo de 80 casos de PET, realizadas en el Servicio de Nefrología del hospital Juárez de México.Se incluyeron a 80 pacientes en DP, de ambos sexos, adultos, sin peritonitis, ni inestabilidad cardiovascular, que tuvieran por lo menos 6 meses en el programa de diálisis peritoneal continua ambulatoria (DPCA). Se les hizo PET en la unidad ambulatoria de diálisis del hospital. Después de hacer la PET. los resultados fueron: 35 pacientes como promedio alto, 15 promedio bajo, 25 alto y 5 transporte bajo. 25 pacientes con falla tipo I de ultrafiltración y 55 con UFR adecuada.La PET, es una prueba muy útil para clasificar el tipo de transporte peritoneal y conocer la tasa de UFR. Permite adecuar la DP y decidir en qué tipo de modalidad de DP puede estar el paciente.