Kidney-specific WNK1 isoform (KS-WNK1) is a potent activator of WNK4 and NCC.

Familial hyperkalemic hypertension (FHHt) can be mainly attributed to increased activity of the renal Na+:Cl- cotransporter (NCC), which is caused by altered expression and regulation of the with-no-lysine (K) 1 (WNK1) or WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/STE20-proline-alanine rich kinase (SPAK)/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK-interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylated. In contrast, this serine becomes phosphorylated when the intracellular chloride concentration ([Cl-]i) is reduced or when KS-WNK1 is coexpressed with WNK4. KS-WNK1-mediated activation of WNK4 is not due to a decrease of the [Cl-]i. Coimmunoprecipitation analysis revealed that KS-WNK1 and WNK4 interact with each other and that WNK4 becomes autophosphorylated at serine 335 when it is associated with KS-WNK1. Together, these observations suggest that WNK4 becomes active in the presence of KS-WNK1, despite a constant [Cl-]i.

The Effect of WNK4 on the Na+-Cl- Cotransporter Is Modulated by Intracellular Chloride.

It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.

The ß1-subunit of the MaxiK channel associates with the thromboxane A2 receptor and reduces thromboxane A2 functional effects.

The large conductance voltage- and Ca(2+)-activated K(+) channel (MaxiK, BK(Ca), BK) is composed of four pore-forming α-subunits and can be associated with regulatory ß-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A(2) prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK ß1-subunit in TP-MaxiK association. We found that the ß1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that ß1 and TP are closely associated at the cell periphery. The molecular mechanism of ß1-TP interaction involves predominantly the ß1 extracellular loop. As reported previously, TP activation by the thromboxane A(2) analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP(o)) as a function of voltage. However, the positive shift of the FP(o) versus voltage curve by U46619 relative to the control was less prominent when ß1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, ß1 gene ablation reduced the EC(50) of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the ß1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.

New tricks of an old pattern: structural versatility of scorpion toxins with common cysteine spacing.

Scorpion venoms are a rich source of K(+) channel-blocking peptides. For the most part, they are structurally related small disulfide-rich proteins containing a conserved pattern of six cysteines that is assumed to dictate their common three-dimensional folding. In the conventional pattern, two disulfide bridges connect an α-helical segment to the C-terminal strand of a double- or triple-stranded ß-sheet, conforming a cystine-stabilized α/ß scaffold (CSα/ß). Here we show that two K(+) channel-blocking peptides from Tityus scorpions conserve the cysteine spacing of common scorpion venom peptides but display an unconventional disulfide pattern, accompanied by a complete rearrangement of the secondary structure topology into a CS helix-loop-helix fold. Sequence and structural comparisons of the peptides adopting this novel fold suggest that it would be a new elaboration of the widespread CSα/ß scaffold, thus revealing an unexpected structural versatility of these small disulfide-rich proteins. Acknowledgment of such versatility is important to understand how venom structural complexity emerged on a limited number of molecular scaffolds.

Enhancing Free Cyanide Photocatalytic Oxidation by rGO/TiO2 P25 Composites.

Graphene-TiO2 composites have been investigated in various photocatalytic reactions showing successful synergy compared to pristine TiO2. In the present work, graphene oxide (GO) was synthesized by the Hummers method and then reduced graphene oxide-TiO2 composites (rGO/TiO2) were obtained by an in situ GO photoreduction route. X-ray diffraction, FTIR, Raman, UV-vis DRS, and photoluminescence were the main characterization techniques. The obtained composites containing 1 and 3 wt.% rGO were evaluated in the cyanide (50 mg/L) oxidation and Au-cyanide complex (300 mg/L) degradation under UV-A light. The composites showed higher photocatalytic activity than TiO2, mainly with the 1% rGO content. Cyanate and gold nanoparticles, deposited on the photocatalyst's surface, were the main byproducts during the photocatalyst assessment. The improved photocatalytic activity of the composites was attributed to a higher rate of electron transfer and a lower rate of charge recombination due to the chemical interaction of rGO with TiO2.

Bis-cations with two 2,3-diferrocenylcyclopropenium fragments stabilized with diamino-alkanes: Synthesis and cytotoxic activity.

Bis-cations with two 2,3-diferrocenylcyclopropenium fragments 3a-d, and the cis-2-(1,2-diferrocenylvinyl)-2-imidazolinium tetrafluoroborates 4a, d or the cis-2-(1,2-diferrocenylvinyl)-3,4,5,6-tetrahydropyrimidin-2-ium tetrafluoroborates 4b, c were obtained by interactions of 2,3-diferrocenyl-1-ethoxycyclopropenium tetrafluoroborate 1 with bis-1,4-N,N-(2a, d) or bis-1,5-N,N-(2b, c) nucleophiles. The reactions of 3a-d with sodium azide proceed with high regioselectivity, forming tetraferrocenyl-substituted compounds: N,N'-bis-(4',6'-diferrocenyl-1',2',3'-triazin-5'-yl)-piperazine 5a, N,N'-bis-(4',6'-diferrocenyl-1',2',3'-triazin-5'-yl)-N,N'-dialkyl-1,3- or 1,2-alkanediamines 5b-d. Sodium hydrogencyanamide reacts with 3a-d to form N,N'-bis-(1'-aza-1'-cyano-3',4'-diferrocenyl-1',3'-butadien-2'-yl)-piperazine 6a, N,N'-dialkyl-1,3- or 1,2-alkanediamines 6b-d and N-(1'-cyano-3',4'-diferrocenyl-1'-aza-1',3'-butadien-2'-yl)-N,N'-dialkyl-alkanediamines 7a-d. The characterization of new compounds was done by IR, 1H and 13C NMR spectroscopy, mass-spectrometry, elemental analysis, and X-ray diffraction analysis only for the compounds 4b, 4d, and 7a. The biological activity of compounds 5a, 6a, 6b, 6c was assessed regarding anticancer activity against U-251, K-562, SKLU-1, HCT-15, and MCF-7 cell lines. All tested compounds showed good activity but compounds 6a and 6b had the best anticancer activity against U-251 (human glioblastoma) and SKLU-1 (human lung adenocarcinoma) cultures.

Mapping the receptor site for ergtoxin, a specific blocker of ERG channels.

We show here that ergtoxin (ErgTx) is a bona fide, specific blocker of the human ether-a-go-go-related gene (HERG) channels. It does not affect the function of either M-eag or M-elk channels. A chimeric construction containing a segment of the P-region of M-eag channel inserted into the HERG channel drastically diminished or completely abolished the inhibitory effect of ErgTx, whereas chimeras of the P-region of HERG channel into M-eag channels recovered the inhibitory effect. From the P-region point mutants of HERG channel assays, only the mutant N598Q shows about 25% decrement of the ErgTx inhibitory effect. ErgTx recognizes the P-region of HERG channels, blocking the channel function with a K(d) in the order of 12 nM.

Biochemical, genetic and physiological characterization of venom components from two species of scorpions: Centruroides exilicauda Wood and Centruroides sculpturatus Ewing.

Current literature concerning the taxonomic names of two possibly distinct species of scorpions from the genus Centruroides (sculpturatus and/or exilicauda) is controversial. This communication reports the results of biochemical, genetic and electrophysiological experiments conducted with C. exilicauda Wood of Baja California (Mexico) and C. sculpturatus Ewing of Arizona (USA). The chromatographic profile fractionation of the soluble venom from both species of scorpions is different. The N-terminal amino acid sequence for nine toxins of C. exilicauda was determined and compared with those from C. sculpturatus. Lethality tests conducted in mice support the idea that C. exilicauda venom should be expected to be medically less important than C. sculpturatus. Thirteen genes from the venomous glands of the scorpion C. exilicauda were obtained and compared with previously published sequences from genes of the species C. sculpturatus. Genes coding for cytochrome oxidase I and II of both species were also sequenced. A phylogenetic tree was generated with this information showing important differences between them. Additionally, the results of electrophysiological assays conducted with the venom from both species on the Ca(2+)-dependent K(+)-channels, showed significant differences. These results strongly support the conclusion that C. exilicauda and C. sculpturatus are in fact two distinct species of scorpions.

Gel de hidróxido de aluminio: análisis comparativo de métodos de separación sólido-líquido que se utilizan en su producción / Aluminum hydroxide: a comparative analysis of the solid-liquid separation methods used in its production

Se analizó de forma comparativa diferentes variantes de separación sólido-líquido para la obtención del gel de hidróxido de aluminio como sedimentación, filtración al vacío (por lotes y continua), filtración a presión y centrifugación. Se presentan las ventajas y desventajas de cada variante incluyendo un análisis técnico-económico de éstas. Se concluye que el uso de un filtro rotatorio al vacío satisface los requerimientos establecidos

Mejoramiento del proceso de obtención de citrato de Piperacina / Improvement of the piperazine citrate obtention process

Se describió un procedimiento industrial mediante el cual se obtuvo citrato de piperacina de calidad farmacéutica, al hacer reaccionar a 60ºC una solución de piperacina anhidra con otra solución de ácido cítrico monohidratado. Se realizó a la solución copulada tratamiento de carbón, filtración en caliente y posterior cristalización y centrifugación. Se determinó la concentración adecuada para la cristalización, se introdujo el control de la concentración mediante lectura refractométrica como control de proceso, se modificó la carga utilizada en la preparación del lote cuando en él se emplearon los líquidos madres colectados en el lote anterior. Se analizaron los resultados del rendimiento acumulado en la medida que aumentaba el número de reproducciones. Se incluyeron los datos obtenidos en los ensayos al nivel de producción experimental. Se concluye que el procedimiento por su factibilidad técnica y económica es adecuado para la obtención del citrato de piperacina

Procedimiento para la obtención de Cimetidina Polimorfo "A" a partir de Cimetidina Polimorfo "B" impuro / Method for obtaining \"A\" polymorph cimetidine from impure \"B\" polymorph cimetidine

Se describen procedimientos que permiten mediante cristalización en mezclas alcohol etílico-agua (solventes de producción nacional), obtener cimetidina polimorfo a que cumple los requerimientos de la Farmacopea de los EE.UU. a partir de polimorfos A y b impuros. Se estableció que si el contenido de impureza era igual o menor al doble del permitido, podía utilizarse alcohol etilico absoluto con un rendimiento en peso superior al 70 por ciento, pero si el contenido de impurezas estaba entre 2 y 3 veces, era necesario utilizar alcohol etílico con un contenido de agua del 6 por ciento y un rendimiento en peso del 60 al 65 por ciento. Se presentan además los resultados obtenidos durante su comprobación a escala industrial

Acidos biliares VIII: Los ácidos biliares mayoritarios presentes en bilis de cerdos cubanos / Major bile acids present in bile of Cuban pigs

Para establecer la composición de los ácidos biliares mayoritarios presentes en la bilis de cerdos cubanos, se emplearon métodos cromatográficos en capa delgada, cromatográficos por columna, así como procedimientos colorimétricos. Se estudiaron bilis de cerdos criados en empresas estatales y cerdos criados por pequeños agricultores. Se concluye que la bilis de cerdo doméstico presenta como componente fundamental el ácido hyodesoxicólico, mostrando en la mezcla de ácidos biliares crudos una distribución de ácidos mayoritarios similar a la de cerdos convencionales. En cambio, la bilis de cerdo estatal muestra como componente mayoritario el ácido quenodesoxicólico, coincidiendo la composición del crudo con la de cerdos genobióticos (animales libres de gérmenes). Se discuten las posibles causas de dichas diferencias y se evalúa de forma preliminar la posibilidad de explotación de las bilis

Separación sólido-líquido de la harina de henéquen obtenida por hidrolisis ácida del jugo del Agave fourcroides / Solid-liquid separation of henequen meal obtained by acid hydrolysis of the juice from Agave fourcroides

El proceso de obtención de coffe-ground se basa en la hidrólisis ácida del jugo de henéquen, transformándose las saponinas presentes en sapogeninas insolubles, con posterior separación del sólido por filtración, lavado y secado. El proceso inicialmente utilizado para la separación sólido-líquido era la filtración al vacío, el cual, bajo las condiciones de operación empleada, requería de 10 a 14 días para filtrar y lavar 1 000 L de iodo utilizado. Se describe el estudio del comportamiento de filtración del iodo, y se determinan las principales caracterísiticas de éste y su comportamiento a nivel de banco al emplear diferentes métodos de separación como : filtración al vacío, filtración a presión y centrifugación. A partir de los datos obtenidos se procedió a escalar dichos resultados los cuales se comprobaron a escala semiindustrial. Se presentan las condiciones óptimas para la explotación del equipo existente en la instalación y se discuten variables de separación más adecuadas para el iodo a procesar

La placenta humana: estudio bibliográfico de su composición química y sus extractos / Human placenta: bibliographic study of its chemical composition and extracts

Se presentan los resultados de la revisión bibliográfica realizada hasta el año 1987 sobre la composición química de la placenta humana, así como de los diferentes extractos reportados que tienen aplicación en la terapéutica o en la elaboración de cosméticos. Una gran parte de la literatura consultada contempla solamente publicaciones referativas como el Chemical Abstracts. Debido a lo amplio del tema a tratar, sólo se profundiza en algunos tópicos que se han considerado los más importantes, mientras que en otros se han mencionado los trabajos sin detallar los mismos. La placenta es un órgano que contiene gran variedad de sustancias que forman un conjunto biológico bastante complejo. En este trabajo se exponen aquellas de mayor importancia de las tantas reportadas en la literatura