Enfermedad de Hailey-Hailey, adecuada respuesta a doxiciclina / Good Response to Doxycycline in Hailey-Hailey Disease

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Trombocitopenia en probable relación con acitretina / Thrombocytopenia Probably Induced by Acitretin

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Blakeslea trispora genes for carotene biosynthesis.

We cloned the carB and carRA genes involved in beta-carotene biosynthesis from overproducing and wild-type strains of Blakeslea trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase. The estimated transcript sizes for carB and carRA were 1.8 and 1.9 kb, respectively. CarB from the beta-carotene-overproducing strain B. trispora F-744 had an S528R mutation and a TAG instead of a TAA stop codon. The overproducing strain also had a P143S mutation in CarRA. Both B. trispora genes could complement mutations in orthologous genes in Mucor circinelloides and could be used to construct transformed strains of M. circinelloides that produced higher levels of beta-carotene than did the nontransformed parent. The results show that these genes are conserved across the zygomycetes and that the B. trispora carB and carRA genes are functional and potentially useable to increase carotenoid production.

Biotechnological lycopene production by mated fermentation of Blakeslea trispora.

A semi-industrial process (800-l fermentor) for lycopene production by mated fermentation of Blakeslea trispora plus (+) and minus (-) strains has been developed. The culture medium was designed at the flask scale, using a program based on a genetic algorithm; and a fermentation process by means of this medium was developed. Fermentation involves separate vegetative phases for (+) and (-) strains and inoculation of the production medium with a mix of both together. Feeding with imidazole or pyridine, molecules known to inhibit lycopene cyclase enzymatic activity, enhanced lycopene accumulation. Different raw materials and physical parameters, including dissolved oxygen, stirring speed, air flow rate, temperature, and pH, were checked in the fermentor to get maximum lycopene production. Typical data for the fermentation process are presented and discussed. This technology can be easily scaled-up to an industrial application for the production of this carotenoid nowadays widely in demand.

Evaluación de regiones organizadoras nucleolares argirófilas (AgNORs) en neoplasias foliculares tiroideas / Evaluation of argirophilic nucleolar organizer regions (AgNORs) in thyroid follicular neoplasias

Introducción: Las Regiones Organizadoras Nucleolares (NOR) son segmentos de DNA que codifican el RNA ribosómico, el cual está directamente relacionado con la síntesis proteica y la proliferación celular. Mediante técnicas de impregnación argéntica se pueden detectar las proteinas asociadas a los NORs (AgNORs), siendo su cantidad proporcional a la actividad proliferativa. Esto parece tener importancia para distinguir entre tumores benignos y malignos. El objetivo de este trabajo es determinar la utilidad de la expresión de AgNORs en el diagnóstico diferencial de neoplasias foliculares tiroideas. Material y métodos: La técnica se realizó sobre piezas quirúrgicas correspondientes a 10 adenomas y 7 carcinomas foliculares tiroideos. La técnica de plata fue la original de Ploton (1986) con viraje con cloruro de oro, fijación con tiosulfato sódico y contraste con rojo nuclear extra. Se evaluaron mediante microscopía óptica en 100 células tumorales por caso: número de puntos AgNOR por núcleo, localización, distribución y número de núcleos con 5 o más puntos AgNOR. Resultados: El número de puntos AgNOR por núcleo resultó significativamente mayor en los carcinomas que en los adenomas foliculares, así como el número de núcleos con 5 o más puntos AgNOR por cada 100 células. El número de núcleos con puntos AgNOR agrupados (versus dispersos) fue mayor en los carcinomas que en los adenomas, mostrando los carcinomas mayor cantidad de puntos localizados en el centro del núcleo y menor proporción de casos con un punto AgNOR único. La antigüedad de los casos de archivo no afectó negativamente los resultados de la técnica. Conclusiones: En las neoplasias foliculares tiroideas el número medio de puntos AgNOR por núcleo, el número de núcleos con 5 o más puntos AgNOR y la disposición agrupada de los mismos pueden ser útiles en la distinción entre adenomas y carcinomas tiroideos (AU)

Background: The Nucleolar Organizer Regions (NOR) are segments of DNA that encode ribosomal RNA which is directly related to protein synthesis and cellular proliferation. Silver-staining methods allow detection of AgNOR proteins with the amount of silver-stained proteins proportional to cell proliferative activity. This seems to be important in distinguishing between benign and malignant tumors. The aim of this study is to determine the utility of the expresion of AgNORs in the differential diagnosis of follicular thyroid neoplasms. Material and Methods: 17 surgical specimens of follicular thyroid neoplasms (10 adenomas and 7 carcinomas) were studied. Ploton’s original silver technique (1986) with gold toning, fixation with sodiumthiosulfate and counterstaining with nuclear red extra was carried out. One hundred cells per case were evaluated, taking into account the mean number of Agnor dots per nucleus, their location, distribution and the number of cells with five or more dots. Results: The total mean number of AgNOR dots per nucleus was significantly higher in carcinomas than in adenomas as was the number of nuclei with 5 or more AgNOR dots. Carcinomas were frequently found to present AgNOR dots grouped in a central cluster. Adenomas showed small, dispersed and peripheral dots which were often unique. The technique applied did not appear to have been adversely affected by the time elapsed in the older cases. Conclusions: The mean total number of AgNOR dots per nucleus, the number of nuclei with 5 or more AgNOR dots and the grouped distribution of them may be useful in the differential diagnosis of follicular adenoma and carcinoma of thyroid (AU)

Direct enzymatic synthesis of natural penicillins using phenylacetyl-CoA: 6-APA phenylacetyl transferase of Penicillium chrysogenum: minimal and maximal side chain length requirements.

In vitro synthesis of different natural penicillins (hexanoyl, heptanoyl and octanoyl-penicillin) have been carried out by direct acylation of 6-aminopenicillanic acid (6-APA) with several fatty acid-CoA derivatives (hexanoyl-CoA, heptanoyl-CoA and octanoyl-CoA). The reactions were catalyzed by the enzyme Acyl-CoA: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum AS-P-78. This enzyme only accepts as substrate, aliphatic side chain precursors whose carbon length is between 6 and 8 atoms. Although the enzymatic synthesis of octanoylpenicillin has been previously reported the in vitro synthesis of hexanoyl and heptanoyl penicillins is described here for the first time.

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum.

The content of alpha-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into alpha-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of alpha-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing alpha-aminoadipic acid accumulation. The repression of alpha-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of alpha-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts alpha-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.

Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay.

The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341. The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20 degrees C. The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 microM, respectively. When octanoyl-CoA was employed as substrate similar results were obtained. In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with beta-lactamase. Reactions performed in the presence of Escherichia coli penicillin acylase did not generated active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA. Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours; thereafter the level of the enzyme slowly decreased.

Cyclization of phenylacetyl-L-cysteinyl-D-valine to benzylpenicillin using cell-free extracts of Streptomyces clavuligerus.

Benzylpenicillin, a typical antibiotic produced by some species of fungi, was obtained by direct cyclization of the heteropeptide phenylacetyl-L-cysteinyl-D-valine using cell-free extracts of Streptomyces clavuligerus. This is the first description of evidence of the synthesis of benzylpenicillin from a non natural molecule using a bacterial enzyme.

Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N.

The tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, an intermediate in the penicillin biosynthetic pathway, is converted to isopenicillin N by isopenicillin N synthetase (cyclase) of Penicillium chrysogenum. The cyclization required dithiothreitol and was stimulated by ferrous ions and ascorbate. Co2+ and Mn2+ completely inhibited enzyme activity. Optimal temperature and pH were 25 degrees C and 7.8, respectively. The reaction required O2 and was stimulated by increasing the dissolved oxygen concentration of the reaction mixture. Purification of the enzyme to a single major band in polyacrylamide gel electrophoresis was achieved by protamine sulfate precipitation, ammonium sulfate fractionation (50 to 80% of saturation), DEAE-Sephacel chromatography, and gel filtration on Sephacryl S-200. The estimated molecular weight was 39,000 +/- 1,000. The apparent Km of isopenicillin N synthetase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.13 mM. The enzyme activity was strongly inhibited by glutathione, which acts as a competitive inhibitor. A good correlation was observed between the isopenicillin N synthetase activity in extracts of four different strains of P. chrysogenum (with widely different penicillin-producing capability) and the amount of penicillin production by these strains.

Carbon catabolite repression of penicillin biosynthesis by Penicillium chrysogenum.

The addition of glucose to batch cultures of Penicillium chrysogenum AS-P-78 reduced the biosynthesis of penicillin. This regulatory effect was also observed in penicillin biosynthesis by nitrogen-limited resting cells when cultures were previously grown in high concentrations of glucose. The effect of glucose was concentration-dependent in the range of 28-140 mM. Incorporation of L-[U-14C]valine into penicillin in nitrogen-limited resting cultures was reduced by 70% when cells were grown on 140 mM glucose, as compared with that grown on lactose. It was not affected when the sugar was added to the resting cell system, in which penicillin biosynthesis took place without growth. Fructose, galactose and sucrose exerted the regulatory effect to the same extent as glucose (64 to 70%). Lactose did not exert suppression of penicillin biosynthesis. Penicillin-synthesizing activity in control cultures with lactose reached a peak at 24 hours of incubation and decreased slowly thereafter, as studied with resting cell cultures in which further protein synthesis was blocked with cycloheximide. Glucose repressed the formation of penicillin-synthesizing enzymes, but had no effect on the activity of these enzymes. These results suggest that glucose represses but does not inhibit penicillin biosynthesis.

A study of the biosynthesis of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in a beta-lactam-negative mutant of Cephalosporium acremonium.

A cell-free extract of Cephalosporium acremonium (Takeda N-2) was obtained that synthesized the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and also the dipeptide delta-(L-alpha-aminoadipyl)-L-cysteine from the corresponding L-amino acids.