Histochemical method for the demonstration of myosin adenosine triphosphatase in muscle tissues

Zusammenfassung In der vorliegenden Arbeit wird die Wirkung der Antiandrogene Cyproteron und Cyproteronacetat auf einige Enzyme in den Leydigzellkomplexen (LZK) des Hodens infantiler und ausgewachsener Ratten untersucht. Neben einer Vergrößerung der LZK — Ausdruck einer vermehrten Gonadotropin-Ausschüttung als Folge eines Kastrations-effektes — ergab sich eine Aktivitätssteigerung der Enzyme des Steroidstoffwechsels (-Hydroxybuttersäuredehydrogenase, Steroid-3-ol-Dehydrogenase), des Energiestoffwech-sels (Laktatdehydrogenase, Succinodehydrogenase) und der Atmungskette (NADH-Cyto-chrom-c-Reduktase). Die Glukose-6-Phosphatdehydrogenase wies dagegen keine Aktivitätsveränderung auf. Unbeeinflußt blieben auch die unspezifischen Esterasen und die sauren Phosphatasen. Der erwartete Unterschied zwischen Cyproteron und Cyproteronacetat blieb aus. Die Gründe hierfür werden diskutiert.

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The effect of the antiandrogens cyproterone and its acetate on the interstitial cells of juvenile and adult rat testes was studied histochemically

Summary Under both steroids, the interstitial cell complex enlarged which is considered evidence of increased gonadotropin secretion mediated by an antiandrogen blockade of hypothalamic androgen receptors. There was also an increase in the activity of enzymes involved in steroid metabolism (-hydroxy-butyrate and 3-hydroxy-steroid-dehydrogenase) and energy metabolism (lactic and succinic dehydrogenases) as well as an increase in NADH-cytochrome-c-reductase activity. On the other hand, no changes in the activity of glucose-6-phosphate dehydrogenase, non-specific esterases and acid phosphatases were found. Unexpectedly, there was no difference between the effects of cyproterone and cyproterone acetate. The reasons for this are discussed.

     

The Contribution of Mere Recognition to the P300 Effect in a Concealed Information Test

In two experiments, we investigated the role of mere recognition in a P300 based CIT. Mere recognition was isolated by having participants respond based on an irrelevant dimension of the stimuli. In Experiment 1 stimuli consisted of familiar and unfamiliar faces, with a dot placed on the left or the right cheeck. Participants responded according to dot location. In the second experiment, participants were presented with autobiographical information, alternated with irrelevant stimuli, while instructed to respond based on the case of the stimuli. Results showed that with both familiar faces, and autobiographical information, mere recognition was sufficient to elicit a P300.

Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake

Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.     

Gastric cancers of Western European and African patients show different patterns of genomic instability

Background

Infection with H. pylori is important in the etiology of gastric cancer. Gastric cancer is infrequent in Africa, despite high frequencies of H. pylori infection, referred to as the African enigma. Variation in environmental and host factors influencing gastric cancer risk between different populations have been reported but little is known about the biological differences between gastric cancers from different geographic locations. We aim to study genomic instability patterns of gastric cancers obtained from patients from United Kingdom (UK) and South Africa (SA), in an attempt to support the African enigma hypothesis at the biological level.

Methods

DNA was isolated from 67 gastric adenocarcinomas, 33 UK patients, 9 Caucasian SA patients and 25 native SA patients. Microsatellite instability and chromosomal instability were analyzed by PCR and microarray comparative genomic hybridization, respectively. Data was analyzed by supervised univariate and multivariate analyses as well as unsupervised hierarchical cluster analysis.

Results

Tumors from Caucasian and native SA patients showed significantly more microsatellite instable tumors (p < 0.05). For the microsatellite stable tumors, geographical origin of the patients correlated with cluster membership, derived from unsupervised hierarchical cluster analysis (p = 0.001). Several chromosomal alterations showed significantly different frequencies in tumors from UK patients and native SA patients, but not between UK and Caucasian SA patients and between native and Caucasian SA patients.

Conclusions

Gastric cancers from SA and UK patients show differences in genetic instability patterns, indicating possible different biological mechanisms in patients from different geographical origin. This is of future clinical relevance for stratification of gastric cancer therapy.

     

Diazonium inactivation in simultaneous-coupling and product inhibition in post-coupling azo-techniques for demonstrating activity of acid hydrolases

Summary In this communication results are presented of an investigation in which the activity of the hydrolytic enzymes acid phosphatase, -glucuronidase, non specific arylesterase, microsomal arylsulphatase, -galactosidase, -N-acetylglucosaminidase, acid -glucosidase and aminopeptidase M are demonstrated in tissue sections with simultaneous- and post-coupling azo-techniques. Semipermeable membrane techniques are used to hamper enzyme diffusion during the incubation period. From the histochemical and biochemical findings it appeared that an advantage of the post-coupling techniques over the simultaneous-coupling techniques is that inactivation of the enzymes by the coupling reagents is avoided. On the other hand post-coupling techniques are subject to product inhibition. With kinetic inhibition studies it is found that for microsomal arylsulphatase and non-specific arylesterase this product inhibition is non-competitive. This product inhibition may be a problem for histochemical quantitative post-coupling techniques for the determination of acid hydrolase activity.     

Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)