Potential effects of long-term abuse of anabolic androgen steroids on human skeletal muscle.

BACKGROUND: We have previously evaluated muscle functions and morphology in power athletes of long term (5 to15 years) abuse of anabolic androgen steroids (AAS; Doped) and in clean power athletes (Clean), and observed significant improvements in both muscle morphology and muscle functions in Doped. To our knowledge, the effects of long term AAS abuse on human muscle protein profile have never been studied. METHODS: The study examined further the muscle biopsies using a two-dimensional difference gel electrophoresis (2D DIGE) for proteomic screening and protein expression. Cellular localization/distribution of specific proteins identified by proteomic analysis was examined using immunohistochemistry (IHC). RESULTS: Different protein profiles were observed between Doped and Clean, and a valid orthogonal projection of latent structure discriminant analysis model was built (N.=16, x=5, R2=0.88/Q2=0.84, P=0.0005), which separated Doped from Clean. Liquid chromatography followed by tandem spectrometry identified 14 protein spots (representing nine different proteins) of significant difference in relative quantity (P<0.05), of which nine spots were down-regulated in Doped compared with Clean. IHC revealed no significant alteration in cellular localization in phosphoglucomutase-1 and heat shock protein beta-1, but indeed in two reference proteins desmin and F-actin in Doped. CONCLUSIONS: Long term abuse of AAS in combination with training is potentially associated with alterations in skeletal muscle protein profile and protein expression, and structural proteins rather than non-structural proteins are preferentially affected in cellular localization/distribution.

Development of assays using hexokinase and phosphoglucomutase gene sequences that distinguish strains of Leishmania tropica from different zymodemes and microsatellite clusters and their application to Palestinian foci of cutaneous leishmaniasis.

BACKGROUND/OBJECTIVES: Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307. METHODOLOGY/PRINCIPLE FINDINGS: Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system 'correctly' identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter. CONCLUSIONS/SIGNIFICANCE: Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.

Dermoscopy and in vivo reflectance confocal microscopy of a congenital nevus of the nipple.

We report a 26-year-old male with a 4 mm diameter, asymmetric, irregularly pigmented and bordered, brown maculopapular lesion on the right nipple present since childhood with enlargement of the lesion within the last 3 months. Dermoscopy revealed a global globular pattern with the presence of focally light brown globules and irregular black globules in its centre. In vivo reflectance confocal microscopy (RCM) revealed dense junctional and dermal melanocytic nests of different sizes and shapes that appeared as sharply demarcated round to oval reflective structures; cellular outlines of single melanocytes were not always detected. In the centre of the lesion within the upper dermis, irregularly shaped, homogeneously reflecting structures were observed. As a clear differentiation between clusters of melanophages and melanocytic nests could not be made with certainty, an excisional biopsy was performed to establish the diagnosis of compound nevus with features of congenital nevus. Therefore, to prove that dermoscopic globules correlated with melanophages, the correlation between dermoscopic RCM and histopathology was necessary.

Characterization of macrophage subpopulations and microvessel density in carcinomas of the gastrointestinal tract.

BACKGROUND: The role of tumor associated macrophages (TAMs) in tumor angiogenesis and inflammation and the interactions between TAMs and tumor cells as well as lymphocytes appear to be critical factors in the development and progression of cancer. PATIENTS AND METHODS: Carcinomas of the gastrointestinal tract have been analysed by tissue microarrays. TAMs and vessels were characterized by immunohistochemistry using the antibodies PG-M1, KP1, MRP8, MRP14, MRP8/14 and CD31, CD34, respectively. RESULTS: The number of all macrophages was significantly higher and lymphocyte densities were lower in tumor tissues than in tumor-free tissues. The MRP-antibodies identified a minority population of macrophages and a low numbers of these macrophages tended to occur in more advanced cancers. There was a positive correlation between the number of macrophages and the number of microvessels in all tumors, but no correlation between macrophages and vessel counts in tumor-free tissues. CONCLUSION: The results indicated a suppressed immune response towards the tumors. The observed common characteristics regarding macrophage attraction, lymphocyte suppression and microvessel density suggested that these mechanisms are regulated similarly in all carcinomas of the GI-tract.

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus.

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.

Genetic responses to metal contamination in two clams: Ruditapes decussatus and Ruditapes philippinarum.

Coastal ecosystems are subjected to a wide variety of disturbances, including those due to xenobiotics of agricultural and industrial origin. These pollutants as heavy metals can modify the genetic diversity of populations by favouring or counter-selecting certain alleles or genotypes by differential mortality. In the present study, two genetic markers (phosphoglucomutase and glucosephosphate isomerase) and a protein marker (metallothionein) were monitored in order to determine the impact of heavy metals in different clam populations. Analysis of the genetic structure of the clam populations examined reveals that those inhabiting environments contaminated by heavy metals exhibit a higher allelic diversity and possess alleles at PGM loci that could be selected by the presence of heavy metals. The evaluation of metallothionein levels using a specific polyclonal antibody developed in the Pacific oyster (Crassostrea gigas) demonstrated the existence of a relationship between metallothionein concentrations and the level of metal pollution for clam populations sampled from different sites. An inter-specific difference was also detected between Ruditapes decussatus and Ruditapes philippinarum living in sympatry at the same site, suggesting a differential response of these two species upon exposure to an identical heavy metal concentration.

Allozyme markers for the identification of Lecithochirium rufoviride and Lecithochirium furcolabiatum (Trematoda: Hemiuridae), parasites of Conger conger and Anguilla anguilla from Atlantic Spanish waters.

The allozyme variation between 2 sympatric populations of Lecithochirium furcolabiatum and L. rufoviride was examined with the aim to investigate if the 2, morphologically similar, helminths are reproductively distinct. Three enzyme loci, Gpd, Pgm-1, and Idh were found to be diagnostic for the 2 species. These results support the conclusion that the worms are different biological species. Only the Gpi and Gdh loci showed no differences. Marked genetic subdivision was detected at the Idh locus in the L. rufoviride population. Given that the genotypic distribution at Pgm-I showed no significant deviations from Hardy-Weinberg expectations, it does not appear that the absence of heterozygotes for Idh was caused by the reproductive structure of the population. Although there are several other possible reasons for the deficiency of heterozygotes in natural populations, the existence of a new noninterbreeding group within the population studied constitutes a plausible explanation.

Unusual electrophoretic patterns for phosphoglucomutase and fumarase in a population of Lecithochirium rufoviride (Trematoda: Hemiuridae), a parasite of Conger conger.

Electrophoretic analyses of phosphoglucomutase (PGM) and fumarase (FH) in a population of Lecithochirium rufoviride parasitizing Conger conger, revealed 2 independent activity zones for each enzyme on starch gel electrophoresis. However, some individuals exhibited only 1 activity zone for 1 or both enzymes. The banding patterns observed strongly suggest that (1) PGM is coded by 2 polymorphic loci, Pgm-1 (expressed in all individuals) with allelic frequencies not significantly different from those expected under Hardy-Weinberg equilibrium, and Pgm-2 (expressed in a subset of individuals); and (2) FH is coded by 2 loci, Fh-2 (monomorphic and expressed in all individuals) and Fh-1 (expressed in a subset of individuals). A high degree of concordance (88.75%) was observed between the expression and nonexpression of Pgm-2 and Fh-1. The most likely explanations for these findings are either variation in enzyme expression with developmental stage or the presence of null alleles at high frequencies in the population.

Biological and biochemical characterization of isolates of Trypanosoma evansi from Pantanal of Matogrosso--Brazil.

Ten isolates of Trypanosoma evansi from the Pantanal region of Brazil, recently derived from coati (Nasua nasua, carnivora, Procyonidae), horses and dogs, were characterized on the basis of biological (experimental infections in Wistar rats) and biochemical (multilocus enzyme eletrophoresis) data. Biological data were analyzed by Nested analysis of variance and Kruskal-Wallis. Marked heterogeneity in virulence was observed in the isolates. Some of the isolates showed an undulating parasitaemia, typical for African trypanosomes. This biological heterogeneity did not correspond with the biochemical homogeneity observed in the T. evansi isolates. T. evansi has one of the widest distributions and greatest range of mammalian hosts and is widely recognized to have evolved from Trypanosoma brucei. Adaptability of T. evansi was not reflected in the variability of biochemical and molecular parameters studied to date. The variability in virulence was very significant, but not correlated with the host from which it was derived. These data suggested that, in the region studied, T. evansi is transmitted among both domestic and sylvatic animals in one single transmission cycle.

Contribution to the knowledge of Hysterothylacium aduncum through electrophoresis of the enzymes glucose phosphate isomerase and phosphoglucomutase.

A total of 163 Hysterothylacium aduncum specimens, obtained from two gadoids and one percid, were studied by electrophoresis of the enzymes glucose phosphate isomerase and phosphoglucomutase. The two loci deviated significantly from the Hardy-Weinberg equilibrium, both when considering all specimens and when distinguishing the hosts. This could suggest that there is no single species in either case.

Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella.

Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3-10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellanii, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM. Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.

Isoenzymes of Eimeria from the domestic fowl: electrophoretic variants among species, strains and clones.

Electrophoretic variation of enzymes in five Eimeria spp. of the domestic fowl, including nine strains, ten single-sporocyst clones and two single-sporozoite clones of E. acervulina, three strains each of E. maxima and E. tenella, two strains of E. praecox and one strain of E. necatrix, were assayed using cellulose acetate electrophoresis. Ten enzymes [aldehyde oxidase (AO), alkaline phosphatase (ALP), amylase (AMY), fumarate hydratase (FUM), glucose-6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase (GPI), glutamate-oxaloacetate transferase (GOT), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) and phosphoglucomutase (PGM)] were analyzed for their ability to distinguish between these species and strains. Enzymatic activity of G6PDH, GPI, IDH, MDH and PGM was detected in all the Eimeria spp. examined. Strains within each species were characterized by the same electrophoretic variant of G6PDH. Electrophoretic variants of GPI and PGM were the most valuable in the identification of inter- and intra-specific variation, particularly in the field strains of E. acervulina and E. tenella. These two enzymes were used to examine single-sporocyst and single-sporozoite clones derived from two strains of E. acervulina. The enzymes in E. maxima appeared to be conserved, showing no variation among strains with the five enzymes detected. Relative mobilities, calculated as described in this paper, were found to be consistent between different electrophoresis runs and may serve as a reference when this medium is used.

First isolation and characterization in humans of Entamoeba histolytica (laboratory-made) zymodeme XX.

Isoenzyme analysis by starch-gel electrophoresis has proved to be a useful method for the biochemical differentiation of pathogenic Entamoeba histolytica and non-pathogenic E. dispar isolates. Of the known 24 zymodemes, 3 are laboratory-made and have not previously been identified in humans. Parasitology screening was carried out in a psychiatric institution. Two amebic stocks were isolated and characterized that had never previously been found in humans and that have protein patterns identical to that of the laboratory-made zymodeme XX.

Cutaneous leishmaniasis: iso-enzyme characterisation of Leishmania tropica.

In order to identify and characterise the organisms responsible for Cutaneous Leishmaniasis, parasites were isolated from active lesions, grown in-vitro cultures and identified by iso-enzyme characterisation. Thirteen isolates from different patients were typed as L. tropica. Seven of these isolates were from Afghan refugees encamped in the suburbs of Islamabad, 3 were from patients in Multan, 1 was from a patient from Azad Jammu and Kashmir and 1 was from Besham (Swat, NWFP). The study confirms the presence of anthroponotic Cutaneous Leishamaniasis caused by L. Tropica in Pakistan.

Giardia duodenalis: exposure to metronidazole inhibits competitive interactions between isolates of the parasite in vitro.

The competitive interactions of genetically distinct isolates of Giardia duodenalis with different growth rates were studied in vitro. Electrophoretic analysis of mixed cultures showed that competition between 2 cloned isolates occurs under normal in vitro culture conditions, with faster-growing isolates outcompeting those with slower growth rates. The addition of sublethal concentrations of metronidazole to clonal mixtures in vitro prevented the competitive exclusion, which was seen in normal culture. This apparently occurred because the drug reduced the growth rate of the faster-growing but not the slower-growing clone.

Isozymes of a rattan species and their genetic interpretation.

A combination of a modified Feret' (Silvae Genet. 1971, 20, 46-50) extraction buffer and two types of electrophoresis with acrylamide and starch gels were used to characterize allozymes in mature vegetative tissue of a commercially high value species of rattans (Calamus subinermis). From the analysis of allelic segregation from single maternal rattans and their offspring, genetic control of the 16 observed banding zones, which were consistently scorable, was assumed. Seventeen gene loci were identified. The percentage of polymorphic loci within Calamus subinermis was much higher (70.5%) than expected levels of genetic diversity for tropical woody and non-woody species. It is thought that the protocol described may be applied to the analysis of the genetic diversity of all the endangered Calamus species.

Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi.

Thirty six stocks of Trypanosoma cruzi isolated from sylvatic mammals (32 Didelphis marsupialis and one Philander opossum) and triatomine bugs (Rhodnius robustus and one unidentified bug) in the Amazonian forest by Carajas, Brazil were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis as belonging to principal zymodeme '1 (Z1). Two different homozygous phenotypes and the corresponding heterozygous phenotype were found for phosphoglucomutase with an observed frequency almost identical with that predicted by the theoretical Hardy-Weinberg distribution. Parental and hybrid profiles were also suggested by RAPD analysis, which allowed exclusion of mixed parental strains from the hybrids: isoenzyme and RAPD profiles of biological clones were also indistinguishable from those of uncloned stocks. Trypanosoma cruzi stocks from widely separated geographic origins in Central and South America gave similar RAPD profiles that allowed them to be recognized as being Z1. These results indicate that genetic exchange could contribute to the generation of genetic diversity during the sylvatic cycle of T. cruzi, and this may have epidemiologic and taxonomic implications.

Comparative electrophoretic investigation of Rana temporaria, R. graeca and R. dalmatina.

The results of comparative electrophoretic analysis on muscle esterases, superoxide dismutases, lactate dehydrogenases, malate dehydrogenases-NAD-dependent, phosphoglucomutases and myogens in the brown frogs Rana temporaria, R. graeca and R. dalmatina are described. The coefficient of similarity and indices of divergence among the species investigated were estimated. It was established that in relation to the protein systems analysed R. graeca differed more significantly from R. dalmatina than from R. temporaria. It is suggested that the higher electrophoretic similarity of R. temporaria and R. dalmatina is a result of a greater divergence of the latter in comparison with R. graeca.

Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndromes are genetic multisystemic disorders characterized by defective N-glycosylation of serum and cellular proteins. The activity of phosphomannomutase was markedly deficient (< or = 10% of the control activity) in fibroblasts, liver and/or leucocytes of 6 patients with CDG syndrome type I. Other enzymes involved in the conversion of glucose to mannose 1-phosphate, as well as phosphoglucomutase, had normal activities. Phosphomannomutase activity was normal in fibroblasts of 2 patients with CDG syndrome type II. Since this enzyme provides the mannose 1-phosphate required for the initial steps of protein glycosylation, it is concluded that phosphomannomutase deficiency, which is first reported here for higher organisms, is a cause, and most likely the major one, of CDG syndrome type I.