Search

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.

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.

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., (Copyright 2010 S. Karger AG, Basel.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

SDS-PAGE and iso-enzyme analysis of 11 human isolates of Blastocystis hominis revealed at least two variants with different polypeptide patterns and two zymodemes, respectively. This is the first iso-enzyme and the second protein analysis to indicate strain differences in B. hominis.

Three recent reports have suggested that rat liver microsomal glucose-6 phosphatase (Glc6Pase) should be a 62-64 kDa polypeptide. In this work, we examine the possibility that the 62-64 kDa could represent a functional dimeric form of the 36.5 kDa glucose-6 phosphate (Glc6P)-phosphohydrolase, previously identified [Countaway et al. (1988) J. Biol. Chem. 263, 2672-2678]. From 32P-labeling experiments with 32P-Glc6P and analysis of 32P-labeled protein by SDS-PAGE and autoradiography, we show that three different rat liver microsomal polypeptides, the apparent molecular masses of which are 62, 54, and 37 kDa, may be specifically labeled with 32P-Glc6P. We demonstrate that the 62 kDa polypeptide is a microsome-bound form of cytosolic phosphoglucomutase, by combining labeling competition experiments and enzymatic assay. It should likely not account for a putative dimeric form of Glc6P-phosphohydrolase. The 37 kDa polypeptide fulfills the criteria of Glc6P-phosphohydrolase. We have not obtained any definitive evidence for its assembly as a dimer under functioning conditions. The 32P-Glc6P-labeling characteristics of the 54 kDa polypeptide are those expected for a protein displaying affinity in the millimolar range of concentration and a high binding capacity for Glc6P. They are consistent with those of a 54 kDa microsomal polypeptide, previously suggested to be involved in Glc6Pase activity.

Natural selection is one of the most fundamental processes in biology. However, there is still a controversy over the importance of selection in microevolution of molecular traits. Despite the general lack of data most authors hold the view that selection on molecular characters may be important, but at lower rates than selection on most phenotypic traits. Here we present evidence that natural selection may contribute substantially to molecular variation on a scale of meters only. In populations of the marine snail Littorina saxatilis living on exposed rocky shores, steep microclines in allele frequencies between splash and surf zone groups are present in the enzyme aspartate aminotransferase (allozyme locus Aat; EC. 2.6.1.1). We followed one population over 7 years, including a period of strong natural perturbation. The surf zone part of the population dominated by the allele Aat100 was suddenly eliminated by a bloom of a toxin-producing microflagellate. Downshore migration of splash zone snails with predominantly Aat120 alleles resulted in a drastic increase in surf zone frequency of Aat120, from 0.4 to 0.8 over 2 years. Over the next four to six generations, however, the frequency of Aat120 returned to the original value. We estimated the coefficient of selection of Aat120 in the surf zone to be about 0.4. Earlier studies show similar or even sharper Aat clines in other countries. Thus, we conclude that microclinal selection is an important evolutionary force in this system.

A total of 77 mentally retarded male inpatients residing in a psychiatric institution in northern Italy were screened for the presence of stool parasites, Entamoeba histolytica particularly. Parasitological stool examination showed Entamoeba spp. (E. histolytica and/or E. dispar) in 26 cases (33.7%). In vitro culture on Robinson's medium was positive in 16 cases (61.1%); in 11 cases we could stabilize and clone the isolates and proceed to electrophoretic assays. In all cases, patterns of pathogenic zymodemes were found (zymodeme II, 3 isolates; zymodeme XII, 4 isolates; zymodeme XIV, 4 isolates). All isolates were therefore identified as E. histolytica.

The enzymatic characterization of sarcoplasmic reticulum membrane fragments from rabbit skeletal muscle presented in this paper shows that glycogen phosphorylase, as well as other enzymes (e.g., creatine kinase, myokinase, phosphorylase kinase, glycosidase, AMP-deaminase, phosphoglucomutase) are associated with these membrane preparations. Amongst these enzymes, the highest activity associated with sarcoplasmic reticulum membranes is that of glycogen phosphorylase, which is mostly (at least 95%) in its b form (dephosphorylated form), since its activity in sarcoplasmic reticulum membranes is largely dependent upon AMP. A protocol is presented to quantify the amount of phosphorylase bound to sarcoplasmic reticulum membranes from fluorimetric measurements of the content of its coenzyme, pyridoxal 5'-phosphate. The content of phosphorylase ranged from 0.03 to 0.37 mg phosphorylase per mg of membrane protein, in sarcoplasmic reticulum membrane preparations made following several of the protocols most commonly used and also depending upon the length of the starvation period of the animal before killing. We also show that dilution of sarcoplasmic reticulum membranes to 0.1-0.2 mg protein per ml in a buffer containing 50 mM Tes-KOH (pH 7.4), 0.1 M KCl and 0.25 M sucrose removes at least 95% of glycogen phosphorylase from these membrane fragments, as well as other enzymes like myokinase and glycosidase. On these grounds, we suggest to introduce a final dilution step as indicated above in protocols of sarcoplasmic reticulum membrane preparations.

We report the results of enzymatic patterns of isolates of Leishmania cultured from patients referred to Department of Dermatology of the American University of Beirut Medical Center in Lebanon. The results reveal that these Lebanese isolates are all very similar despite variable clinical presentations of the patients and differences in geographic origin. Leishmania donovani sensu lato is the dominant species present in the skin lesions observed; thus, clinical manifestations and/or geographic distribution cannot be used as reliable criteria for identifying Leishmania parasites from this geographic area. Enzyme data should be combined with these parameters before definitive identification can be made.

The electrophoretic analysis of five natural populations of Aedes detritus has shown that the two sibling species A and B of the complex, described in 1977 in an other geographical area by Pasteur et al., cohabit on Atlantic coastline in France.

Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co-infections with Trypanosoma cruzi, demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of T. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.

Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a closely spaced doublet in SDS-gels. The proteins (60/63 kDa) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and are immunologically closely related, if not identical. No N-terminal sequence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanogen bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified polyclonal antibodies and three different monoclonal antibodies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomutase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 kDa proteins. Peptide maps of the 60/63 kDa proteins and phosphoglucomutase 1 are markedly different, and the purified proteins have no detectable phosphoglucomutase activity. Staining of cultured smooth muscle cells and fibroblasts with antibodies to 60/63 kDa proteins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. About 60% of the cellular 60/63 kDa proteins were found in the detergent-insoluble fraction, suggesting a physical association with the cytoskeleton. The highest levels of protein immunoreactivity were found in muscles. The antigen is concentrated in muscle adherens junctions, including smooth muscle dense plaques, cardiomyocyte intercalated disks, and striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocytes where immunofluorescent staining was localized in cell-to-cell adherens junctions. Expression of the 60/63 kDa proteins in vascular smooth muscle cells is developmentally regulated and correlates with the differentiated contractile phenotype of these cells.

Paired human teeth and fresh blood samples were analysed for the genetic markers ABO, PGM1, GLO-I, EsD, EAP, AK, ADA, 6-PGD, G-6-PD and CA-II. ABO blood groups were successfully determined from dentine, pulp and cement samples whereas only dental pulp could be typed for different isozymes. Studies on the persistence of these genetic markers in teeth stored at room temperature showed that ABO blood groups were the most stable, followed by PGM1 and ADA isozymes, whereas CA-II isozymes were the least stable.

Ninety-one Chilean, 15 Bolivian, and 9 Argentinian Trypanosoma cruzi stocks, isolated from various hosts and vectors, were characterized by schizodeme analysis with EcoRI and MspI endonucleases. The three major similar pattern groups that emerged from this sample correlated with results of isoenzyme analysis. This result confirms previous work and supports the hypothesis of the clonal structure of natural populations of T. cruzi, fully defined at the level of isoenzyme analysis, quantitative kinetoplast DNA restriction fragment length polymorphism, and kinetoplast DNA hybridization analysis. In Chile, sylvatic and domestic cycles of T. cruzi transmission appear to be mainly independent: genetically different families of natural clones are specific to these cycles. Nevertheless, the possibility of overlap remains unclear. Results described here indicate that natural clones inhabiting Chilean regions appear genetically related to the natural clones identified in neighboring countries. In Chile the more frequently sampled parasite types are natural clone 39 and a genetically closely related clone NP13. In this work an evaluation of T. cruzi natural clone mixtures in T. cruzi stocks from Chile was performed for the first time by schizodeme analysis before and after serial transfer in mouse maintenance. The results indicate that six of nine stocks are composed of two or more natural clones. This observation raises the relevant question of whether specific T. cruzi natural clones generate different clinical features of Chagas' disease.

No differences were observed in the isozyme patterns of 4 enzymes examined between fresh samples stored at -80 C and samples stored at room temperature for 10 days after lyophilization, which supports the validity of comparing lyophilized samples to fresh frozen tissue. Mature proglottids as well as plerocercoids of Spirometra erinacei from Japan and Australia were indistinguishable by comparison of isozyme patterns after isoelectric focusing. The isozyme patterns of acid phosphatase, glucosephosphate isomerase (GPI), and mannosephosphate isomerase from plerocercoids of Spirometra mansonoides were distinctly different from those of plerocercoids of S. erinacei. The adenylate kinase isozyme patterns of the mature proglottids of S. mansonoides were also distinctly different from those of the mature proglottids and the plerocercoids of S. erinacei. The GPI isozyme pattern of the mature proglottids of S. mansonoides was also distinguishable from the GPI patterns of those of S. erinacei. These electrophoretic data suggest that the S. erinacei from Japan and Australia are closely related, if not identical, but that S. mansonoides is genetically distinct from S. erinacei.

PGM-1 and its isoenzymes had been studied in the human cornea, in cornea cell cultures, in erythrocytes and lymphocytes. Qualitative and quantitative comparisons between cornea of healthy persons and cornea cell cultures of patients with cornea dystrophies showed differences between certain phenotypes and their signals of activity.

Isoenzyme analysis using isoelectrofocusing in polyacrylamide gels was used to distinguish Hammondia hammondi and Toxoplasma gondii sporozoites. Five enzyme systems were studied: aconitase (EC 4.2.1.3), aspartate aminotransferase (EC 2.6.1.1), glucose phosphate isomerase (EC 5.3.1.9), lactate dehydrogenase (EC 1.1.1.27), and phosphoglucomutase (EC 2.7.5.1). Three stocks of T. gondii belonging to 3 zymodemes were compared to 1 stock of H. hammondi. Hammondia hammondi differed from T. gondii at all 5 loci analyzed. This was observed for all 3 zymodemes of T. gondii. These results indicated clear genetic differences between the 2 species.

We previously reported that a 64-kDa protein (p64) in human polymorphonuclear leukocytes (PMN) was phosphorylated with [gamma-32P]ATP under a micromolar concentration of glucose in a cell-free system. The present paper presents the results of analysis of phosphorylation reaction and the identification of phosphoprotein. The findings that p64 was also phosphorylated with glucose-6-[32P]phosphate and that phosphorylation was inhibited with mannoheptulose suggested that the reaction was mediated by hexokinase. In fact, it was found that [32P]phosphate in glucose-6-[32P]phosphate was incorporated into either p64 or rabbit muscle phosphoglucomutase and that glucose-6-phosphate formation from glucose and ATP was detected in over 100-kDa fraction of PMN cytosol. These results showed that p64 was phosphoglucomutase in PMN and that phosphate incorporation into p64 was a conversion of a phosphate group in glucose-6-phosphate produced by hexokinase. It was further demonstrated by analysis of two-dimensional electrophoresis that p64 phosphorylated with glucose induction was different from another 64-kDa protein phosphorylated by stimulation with formyl-methionyl-leucyl-phenylalanine in vivo.

Five clones of axenic E. histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. Isoenzymes of these 5 clones of E. histolytica (HMI) were investigated in starch gel electrophoresis. There were no differences in the electromobility of maleate NADP oxidoreductase and glucosephosphoisomerase amongst the five clones and uncloned cultures of axenic E. histolytica. The relative electromobility (rf) of a single phosphoglucomutase (PGM) band of uncloned Mexican E. histolytica (HMI) and Indian axenic E. histolytica (KCG: 0986: 11) cultures and cloned E. histolytica HMI-C121, HMI-C145 was 0.087 while a single PGM band of uncloned E. histolytica (NIH: 200) and cloned E. histolytica HMI-C131, HMI-C143 and HMI-C144 cultures had rf of 0.075. Isoenzyme characterization of four cloned HMI-C121, HMI-C131, HMI-C143, HMI-C144 cultures of axenic E. histolytica (HMI) revealed existence of three bands of hexokinase (HK). The additional third band of HK was located close to the place of application of lysate and had rf ranging from 0.11-0.14. The data indicated that parent axenic E. histolytica (HMI) consisted of several populations and each population expressed different isoenzyme pattern without an association of amoebic cultures with any bacterial species.

This report concerns the rape of a woman by a stranger. The complainant stated that, during the course of the rape, penetration took place without ejaculation: these details were later confirmed by a suspect. This suspect, who was identified by the complainant, confessed and was convicted. Laboratory findings, however, indicated that the suspect could not have been the source of the semen found on the complainant's underpants. Further, a vaginal swab taken from the complainant contained intact sperm cells, an indication of a recent sexual contact. In spite of the presence of semen on the underwear which could not have come from the suspect, and the nonoccurrence of an ejaculation during the rape, the complainant maintained that she had not had a sexual contact for a month prior to the rape. This apparent contradiction and its implications are discussed.

1. The zymodeme and virulence of 31 Entamoeba histolytica isolates obtained from asymptomatic human subjects in Calcutta, India are described. 2. Virulence was measured by the extent of lesion diameter (mm) induced by each isolate in the liver of golden hamsters and resistance of isolates to non-immune hamster sera in vitro. 3. Two nonpathogenic zymodemes, III (N = 17) and IV (N = 14), were detected among 31 isolates by isoenzyme electrophoresis. Most of the zymodeme III isolates were moderately to highly virulent while a quarter of the zymodeme IV were invasive although with low virulence. 4. The virulence of the isolates was found to have a significant positive correlation (r = 0.96, P < 0.001) with their greater resistance to complement-mediated lysis. 5. The data suggest that the virulence of E. histolytica is probably not related to its zymodeme.

Electrophoretic techniques have been used to survey five different field-collected isolates (i.e., Anhui, Hubei, Guangxi, Sichuan and Yunnan) of S. japonicum from the mainland of China for genetic variation at 7 isozymes markers representing 9 loci. Polymorphic loci were observed for LDH-1, LDH-2, MDH and PGM, respectively, the proportion of polymorphic loci being 44.4%. Average heterozygosity varied between 0.223 and 0.425, with a mean of 0.332. Nei's genetic distance (D) among the populations of 5 isolates gave values between 0.001 and 0.039 with an average of 0.023, indicating that these isolates are very closely related.

Isoenzymes of hexokinase, phosphoglucomutase and glucosephosphate isomerase were examined, using agarose gel isoelectrofocusing, in populations of Phlebotomus ariasi (France), P. neglectus (Greece), P. perfilewi (Italy & Greece) and P. tobbi (Greece). Comparison between enzymatic profiles by numerical analysis separated Larroussius from Phlebotomus and Paraphlebotomus subgenera and brought together P. ariasi and P. neglectus. Nei genetic coefficients indicated similarities between populations of P. perfiliewi from Corfu and central Italy and between Corfuan and Cretan P. neglectus samples.

An isoenzymatic analysis using thin-layer agarose gel isoelectrofocusing on laboratory strains of Echinostoma trivolvis and Echinostoma caproni adults showed characteristic monomorphic phenotypes for phosphoglucomutase and glucose phosphate isomerase. The fixed allelic variation observed between these 2 taxa is consistent with their current classification as distinct species.

The first day after birth, 22 conventional piglets were experimentally infected with the oocysts of the coccidia of I. suis (infection dose 200,000 oocysts). The activity of 5-nucleotidase (5-ribonucleotide phosphohydrolase, EC.3.1.3.5) and phosphoglucomutase (alpha-D-glucoso-1-phosphate phosphotransferase, EC.5.4.2.2) was densitometrically assessed in the mucosa of the small intestines of these piglets. Enzyme activities were studied in the infected piglets during the 2nd to 10th day after infection. The same histochemical examination was simultaneously performed in the intestinal mucosa of five control conventional piglets at an age of 2-14 days. 5-nucleotidase and phosphoglucomutase were found to have a high density in the mucosa of the small intestine of the control piglets: the high-density locations of these enzymes include, first of all, the supranuclear area of the absorption cells, the microvillous zone of enterocytes and the smooth muscle elements of lamina muscularis mucosae. The experimentally infected piglets showed a marked decline of the density of both enzymes during the infection. The deficit affected, for a transient period, the microvillous zone and the supranuclear region of enterocytes; the musculature of the mucous layer was affected permanently. The inactivity was more protracted in the case phosphoglutamase (especially 5 to 9 days after infection). The density of 5-nucleotidase showed a partial return to the normal already the 7th day after infection, with an interruption of resumption of activity on the 10th day. Resumption of enzyme activity in the lamina muscularis mucosae was not recorded during the infection. In the three locations under study, the density of none of the enzymes did reach parameters comparable with the controls at the end of the trial (10 days after infection).

Electrophoretic mobility patterns of six enzymes, viz. alkaline phosphatase E.C. 3.1.3.1., acid phosphatase E.C. 3.1.3.2., malic enzyme E.C. 1.1.1.40., phosphoglucomutase E.C. 2.7.5.1., isocitrate dehydrogenase E.C. 1.1.1.42., glucose-6-phosphate dehydrogenase E.C. 1.1.1.49 of two axenically cultured human Giardia lamblia isolated from India (PD-1 and PD-2) and one strain from Portland, Oregon, USA (P-1) were compared using polyacrylamide gel electrophoresis (PAGE). Based on the difference in the mobility patterns of the enzymes phosphoglucomutase, isocitrate dehydrogenase and malic enzyme, the PD-1 and PD-2 isolates appeared to be quite different from P-1. In the present study, the isocitrate dehydrogenase and alkaline phosphatase enzymes were used for the first time for differentiation of Giardia isolates. In the case of PD-1, two alkaline phosphatase bands could be seen whereas only one band was observed in PD-2 and P-1. Thus, the three strains could be grouped into three different zymodemes. These findings reveal the significant heterogeneity in G. lamblia isolates both from widely separated areas and within a single region. Heterogeneity among G. lamblia strains may explain the variable clinical manifestations, host response and treatment efficacy characteristic of human giardiasis.

Using agarose gel electrophoresis, the electrophoretic patterns of four enzymes: malate NADP oxidoreductase (malic enzyme) (ME), glucosephosphate isomerase (GPI), phosphoglucomutase (PGM) and hexokinase (HK), were compared and analysed for five strains of Entamoeba histolytica, which had been obtained from patients with acute amebic dysentery or asymptomatic cyst carrier in Beijing, Tianjin and Fujian Province. The results showed that the electrophoretic patterns of all the five strains of E. histolytica belong to pathogenic zymodeme, zymodeme XIV, which is suggested to be the main pathogenic zymodeme in our country.

Entamoeba histolytica isolated from human can be associated with either symptomatic disease or with asymptomatic carriers. Pathogenic and nonpathogenic strains can be distinguished on the basis of differences in the electrophoretic patterns of four isoenzymes (zymodeme). With glucose phosphate isomerase and phosphoglucomutase, we observed variation of the expression of their gamma bands as a function of starch volume in culture. We cultured E. histolytica strains from different zymodemes in Robinson medium using both a low (2-4 mg/bottle) and a high (12-15 mg/bottle) content of rice starch as supplement. These cultures were monitored by electrophoresis of glucose phosphate isomerase and phosphoglucomutase. Strains having gamma or delta bands exhibited those bands when a high content of starch was used in culture, but did not do so with a low content. Contrarily, strains that never exhibited those bands did not express them when the amount of starch in the culture was increased. However, alpha and beta bands of the same isoenzymes were always present and never showed any variation. The results suggest that expression of gamma and delta bands of glucose phosphate isomerase and phosphoglucomutase are subject to culture conditions and that genes coding for those isoenzymes may be different from those coding for the alpha and beta bands.

Stool samples containing Entamoeba histolytica were obtained from two sources: an urban hospital and an urban slum. Using the enzyme patterns of three enzymes (E.C.2.7.1.1 hexokinase, E.C.5.3.1.9 glucose phosphate isomerase and E.C.2.7.5.1 phosphoglucomutase) stained after cellulose acetate electrophoresis, four zymodemes (I, II, XIV and XVI) were identified in 71 isolates. Zymodemes considered to be pathogenic (II and XIV) were identified from 31 of 34 isolates from the urban hospital, and these zymodemes were strongly associated with dysentery and trophozoites containing ingested red blood corpuscles. Blood visible to the naked eye was more commonly seen in stools from which zymodeme XIV was isolated than in those containing zymodeme II (P less than 0.001). Zymodemes considered to be non-pathogenic (I and XVI) were identified in 34 of 37 isolates from slum dwellers and were not associated with blood in the stools.

Enzyme electrophoresis indicated that all Schistosoma mattheei eggs passed in the urine of humans derive from S. mattheei females in copula with S. haematobium males. It appears that S. mattheei males do not reach sexual maturity in man; however, S. haematobiumxS. mattheei males possibly do.

Trials were conducted to determine the accuracy of separating the sympatric sand fly species Phlebotomus papatasi (Scopoli) and P. langeroni Nitzulescu by means of cellulose acetate enzyme electrophoresis. Malic enzyme, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and fumarate hydratase were each evaluated in laboratory-reared and field-collected populations of the two species. Each of the four enzyme-based identifications was highly sensitive (greater than 97%) and specific (greater than 93%). Identifications based upon fumarate hydratase were in perfect agreement with morphological identifications, and evidence was obtained which indicates that this enzyme may be the most stable of the four enzymes tested. The application of enzyme-based vector identification is discussed in relation to classical and novel survey procedures for Leishmania promastigote detection in sand flies.

A non-cloned and a cloned isolate of non-pathogenic (zymodeme I) Entamoeba histolytica derived from homosexual males were incubated in Diamonds' TP-S-1 medium in the presence of Crithidia for 52 d. A single addition of Escherichia coli irradiated at 1000 Gy rendered the culture axenic. Analysis of zymodeme patterns established that both isolates had transformed to zymodeme II. The re-establishment of cultures in the presence of microflora from the original cultures resulted in a return to zymodeme I, whereas incubation with microflora from an amoebic isolate exhibiting a zymodeme II pattern maintained the pathogenic pattern.