Search

The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.

FAPESP (2012/08338-8; 14/50315-0). Universidade de S?o Paulo. Faculdade de Medicina. Departamento de Patologia. S?o Paulo, SP, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade de S?o Paulo. Faculdade de Medicina. Departamento de Patologia. S?o Paulo, SP, Brazil. Universidade de S?o Paulo. Faculdade de Medicina. Departamento de Patologia. S?o Paulo, SP, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade de S?o Paulo. Instituto de Bioci?ncias. Departamento de Fisiologia. S?o Paulo, SP, Brazil. Universidade de S?o Paulo. Instituto de Bioci?ncias. Departamento de Fisiologia. S?o Paulo, SP, Brazil. Universidade de S?o Paulo. Faculdade de Medicina. Departamento de Patologia. S?o Paulo, SP, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade de S?o Paulo. Faculdade de Medicina. Departamento de Patologia. S?o Paulo, SP, Brazil. Universidade de S?o Paulo. Instituto de Bioci?ncias. Departamento de Fisiologia. S?o Paulo, SP, Brazil. Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered., Author summary Protozoan parasites of the genus Leishmania are able to undergo genetic exchange during their growth. The previous description of hybrids in Peru and the recent discovery of unexpected genetically complex strains having characteristics of both hybrid and mito-nuclear discordance in its neighbouring country (Ecuador) with a similar eco-epidemiological situation led us to consider that the genetic structure of Leishmania strains in Peru is more complicated than previously thought. In an effort to revise the data on Leishmania strain dispersion in Peru and to search for evidence of genetic recombination, the present study was conducted. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis targeting the mannose phosphate isomerase (mpi) gene sequence was performed to identify the infecting parasite at the species level in 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Most results (121/134) including 4 hybrids between L. (V.) braziliensis and L. (V.) peruviana showed agreement between PCR-RFLP of the mpi gene and cyt b gene sequence analysis; however, 13 of 134 samples revealed the distribution of strains with hybrids and mito-nuclear discordance. The results demonstrate that genetically complex Leishmania strains are present in Peru. These findings indicate that Leishmania strain dispersion in Peru is genetically more complex than previously considered. Further prospective studies including larger samples and the isolation of parasite strains are required to update the available data.

The mannose phosphate isomerase-congenital disorder of glycosylation (MPI-CDG) is caused by phosphomannose isomerase deficiency. Clinical features include hyperinsulinaemic hypoglycaemia, protein losing enteropathy, hepatomegaly and hepatic fibrosis, digestive symptoms and coagulation abnormalities. The condition is treated with mannose supplementation. Long-term outcomes in adults are not well described. We present a case of an adult female patient who discontinued mannose therapy in her adolescence. In adulthood she developed gastrointestinal problems, chronic anaemia and osteophytes in her knees.

Mannose phosphate isomerase-congenital disorder of glycosylation (MPI-CDG) deficiency is a rare subtype of congenital disorders of protein N-glycosylation. It is characterised by deficiency of MPI caused by pathogenic variants in MPI gene. The manifestation of MPI-CDG is different from other CDGs as the patients suffer dominantly from gastrointestinal and hepatic involvement whereas they usually do not present intellectual disability or neurological impairment. It is also one of the few treatable subtypes of CDGs with proven effect of oral mannose. This article covers a complex review of the literature and recommendations for the management of MPI-CDG with an emphasis on the clinical aspect of the disease. A team of international experts elaborated summaries and recommendations for diagnostics, differential diagnosis, management, and treatment of each system/organ involvement based on evidence-based data and experts' opinions. Those guidelines also reveal more questions about MPI-CDG which need to be further studied.

Aims: Nondiabetic patients were studied to determine whether modest elevations in plasma mannose may be associated with a greater incidence of coronary artery disease (CAD). Materials and Methods: Plasma insulin, mannose, glucose, hexokinase 1-2, GLUT1-GLUT4 levels, and serum mannose phosphate isomerase enzyme levels were evaluated with respect to subsequent CAD using records from 120 nondiabetic CAD patients and 120 healthy volunteers. CAD was identified from myocardial infarction and new diagnoses of angina. Results: Of 120 nondiabetic CAD patients studied, their plasma GLUT4 and HK1 levels were significantly lower than those of the control group. In addition, a significant increase in plasma mannose levels was found in the patient group compared to the control group. Conclusion: Our findings showed that elevated baseline mannose levels in plasma are associated with an increased risk of CAD over time. © Copyright 2020, Mary Ann Liebert, Inc., publishers 2020.

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru., Author summary Leishmaniasis, a neglected tropical disease (NTD) caused by the intracellular protozoa of the genus Leishmania, affects at least 12 million people in 96 countries. Peru is one of the most highly endemic countries for cutaneous leishmaniasis (CL), and our previous study identified Leishmania (Viannia) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis in the tropical rainforest, in the Andean highlands, and in the northern and central rainforest areas, respectively, as the main CL-causative agents. In addition, distribution of L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis has been identified. Of these, one case each of L. (V.) shawi infection was reported from the Departments of Junin and Madre de Dios, while clinical cases due to the hybrid of L. (V.) braziliensis and L. (V.) peruviana were recorded only in the Department of Huanuco. To further elucidate the current geographic distribution of causative Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where little information on causative parasites is available, was performed based on the cytochrome b (cyt b) gene sequence and PCR-RFLP analysis of the mannose phosphate isomerase (mpi) gene by using FTA (Flinders Technology Associates) card-spotted samples and smear slides as DNA sources. In addition to current epidemiological observations, the current study revealed that 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana was identified, for the first time, outside the Department of Huanuco, 2) L. (V.) lainsoni, an uncommon CL-causative species in Peru, was predominantly found in the Department of Puno, where causative Leishmania species are not well-studied, and 3) New endemic foci of L. (V.) shawi were identified in central to southern rainforest areas of Peru.

Background Leishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing. Results Partial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples. Conclusions A HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated. Electronic supplementary material The online version of this article (10.1186/s13071-018-3143-7) contains supplementary material, which is available to authorized users.

In an attempt to investigate the genetic and biochemical causes underlying resistance to anthelmintics, the trichostrongylid nematode Haemonchus contortus was examined by an allozymic analysis of nine candidate geneenzyme systems. Two strains resistant to benzimidazoles, two multidrug-resistant strains, and three strains susceptible to anthelmintics isolated from three continents were examined by isoelectric focusing. Two enzymes (mannose phosphate isomerase and peptidase-D) had polymorphic patterns. The peptidase-D zymograms indicated an association with the selection for resistance. Susceptible isolates gave more complex patterns due to the formation of a heterodimer controlled by the two active loci, whereas in resistant isolates, only a single locus with one electromorph was responsible for the profiles. A possible link between dipeptidases and the acquisition of resistance through the turnover of proteinrich collagen, and changes in membrane-associated genes that influence drug efflux, are discussed.

Usin seven allozyme loci as gene markers, we studied the genetic structure of ten natural popula� tions of the sea urchin Strongylocentrotus intermedius in the northwestern Sea of Japan that differ in the pro� portion of individuals with different spawning seasonality (early spawning at the end of May-June and late spawning in September-early October). This study revealed the spatial genetic heterogeneity of the sea� urchin populations (Fst = 0.051); its degree was independent of the geographic distance between populations. Out of the seven loci that were surveyed, four loci (peptidase Pep�1, Pep�2, mannose phosphate isomerase Mpi, and inorganic pyrophosphatase Ipp) accounted for the differences between pairs of samples; different loci contributed to the variation in each sample pair. The genetic similarity between pairs of populations ranged from 0.905 to 0.988. The genetic heterogeneity of the seaurchin populations seems to be a result of both genetic drift and natural selection. At the same time, no significant genetic differences were found between specimens of S. intermedius with different spawning times (the genetic similarity was 0.988-0.991). The shift in spawning season from autumn to early summer in the populations of S. intermedius inhabiting the areas of Peter the Great Bay adjacent to Vladivostok city can be explained by a phenotypic response of this species to environmental changes caused by chronic anthropogenic pollution of the bay.

We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.

Fibroblasts from a pig with a spontaneous reciprocal translocation involving chromosome 7, were used to prepare a set of pig-mouse somatic cell hybrids. The isozyme analysis strongly indicated that in pigs, the NP (nucleoside phosphorylase) gene is located on the distal part of the long arm of chromosome 7, (q21----qter), while the MPI (mannose phosphate isomerase) gene is in the region q21----pter. This confirmed previously reported chromosomal assignment of these genes in pigs and that this synteny has been evolutionarily conserved in several different animal species.

A PCR-Restriction Fragment Length Polymorphism (RFLP) targeting the mannose phosphate isomerase gene was established to differentiate Leishmania species distributed near the Department of Huanuco, Peru. The technique was applied to 267 DNA samples extracted from Giemsa-stained smears of cutaneous lesions taken from patients suspected for cutaneous leishmaniasis in the area, and the present status of causative Leishmania species was identified. Of 114 PCR-amplified samples, 22, 19, 24 and 49 samples were identified to be infected by Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana, respectively, and the validity of PCR-RFLP was confirmed by sequence analysis. Since PCR-RFLP is simple and rapid, the technique will be a useful tool for the epidemiological study of leishmaniasis.

Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263 kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres.

The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area., Author Summary Leishmaniasis is a protozoan disease caused by members of the genus Leishmania, which are distributed worldwide, especially in tropical and subtropical areas. More than 20 species of Leishmania are described as causative agents of human leishmaniases and clinical features are largely associated with the infective species. The parasites are transmitted by bites of female sand flies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Approximately 800 sand fly species have been recorded; of these, fewer than 10% have been confirmed as vectors of Leishmania and only a restricted number of species transmit specific Leishmania species. Therefore, sand fly surveillance is important for predicting risk and spread of leishmaniasis in endemic and surrounding areas. Cutaneous leishmaniasis caused by a hybrid of L. (Viannia) braziliensis/L. (V.) peruviana have been reported in the Department of Huanuco of Peru; however, the vector species has not been identified. In the present study, approximately 3,000 sand flies were examined, and the sand fly infected by the hybrid Leishmania strain was successfully identified.

A reciprocal mussel seed transfer experiment was conducted involving two farm sites (Charles Arm and Thwart Island) whose seedstocks differed significantly in their respective compositions of Mytilus edulis L., M. trossulus Gould and hybrids. Electrophoretic variation at the mannose phosphate isomerase (Mpi) enzyme locus was used to classify the two species and their hybrids. Survival and growth in shell length, wet weight, shell weight and flesh weight were monitored over a 14-mo period. Both stock groups originating from Thwart Island seed (TI reared on its native site and the transferred TI-X stock) and the transferred stock group originating from Charles Arm seed (CA-X) showed no evidence of genotype-dependent variability in survival whereas, in the Charles Arm seed reared on its native site (CA), the proportion of M. edulis declined in relation to M. trossulus. There was no evidence of either a survival advantage or disadvantage in hybrids. After 14 mo, both transferred stocks (CA-X and TI-X...

Background. Tegumentary leishmaniasis in Latin America is caused mainly by Leishmania viannia braziliensis complex parasites. L. braziliensis and Leishmania viannia peruviana are the 2 predominant Leishmania species in Peru. L. braziliensis is more virulent, because it can cause mucocutaneous leishmaniasis, known as espundia, that results in severe facial destruction. Early identification of the species that causes the initial cutaneous infection would greatly help to prevent mucocutaneous leishmaniasis, because it would allow more aggressive treatment and follow-up. However, because of the close genetic similarity of L. braziliensis and L. peruviana, there currently exists no simple assay to distinguish between these species. Methods. We cloned the mannose phosphate isomerase gene from both L. braziliensis and L. peruviana. It is the only known isoenzyme capable of differentiating between L. braziliensis and L. peruviana in multilocus enzyme electrophoresis. Interestingly, only a single nucleotide polymorphism was found between the mannose phosphate isomerase genes from L. braziliensis and L. peruviana, resulting in an amino acid change from threonine to arginine at amino acid 361. A polymerase chain reaction assay was developed to distinguish the single nucleotide polymorphism of the mannose phosphate isomerase gene to allow for the specific identification of L. braziliensis or L. peruviana. Results. This assay was validated with 31 reference strains that were previously typed by multilocus enzyme electrophoresis, successfully applied to patient biopsy samples, and adapted to a real-time polymerase chain reaction assay. Conclusions. This innovative approach combines new genetic knowledge with traditional biochemical fundamentals of multilocus enzyme electrophoresis to better manage leishmaniasis in Latin America.

The primary objective of this research was to determine if the genotype of the enzymes glucose phosphate isomerase (Gpi), phosphoglucomutase (Pgm), or mannose phosphate isomerase (Mpi) are factors affecting survival during acute endosulfan, fluoranthene, and chromium(VI) exposures. Palaemonetes pugio were exposed in the laboratory to 6.3 μg/L endosulfan, 100 mg/L chromium(VI), or 0.6 mg/L fluoranthene. Dead shrimp were removed at approximately 15–30 min intervals and the individual's genotypes for the Gpi, Mpi, and Pgm enzymes were determined. These data were used to establish whether allozyme genotype was related to tolerance as analyzed with non-parametric methods (i.e. development of survival curves using the Kaplan–Meier method of estimation) and parametric methods (i.e. proportional hazards models). Since the sex and/or size of the organism was expected to affect tolerance, these variables were accounted for in the proportional hazards models. Results indicated individuals that were heterozygous (MF) for the Gpi allozyme survived longer and had less overall mortality than the homozygous MM genotype when exposed to chromium(VI) and to fluoranthene. No allozyme genotypes were related to tolerance during the endosulfan exposure when sex was included as a covariate in the analysis. These results support the hypothesis that there is a genetic basis for tolerance in P. pugio during acute exposures to chromium(VI) and to fluoranthene. Although a relationship between Gpi genotype and contaminant tolerance has been identified in previous studies, no such relationship has been documented in P. pugio or with chromium(VI) as the contaminant.

We sampled and analyzed European flounder (Platichthys flesus) from two highly contaminated estuaries (Seine and Loire, France) and one moderately contaminated estuary (reference site: Ster, France). Significant and convergent modifications of the allelic frequencies for the loci phosphoglucomutase (PGM), glucose phosphate isomerase 2 (GPI-2), mannose phosphate isomerase (MPI), and aspartate aminotransferase (AAT-2) were evident for fish in the contaminated sites versus fish from the reference site. Back-calculation from otoliths showed that the average growth rate of fish between the first and the second winter was greater at the reference site (approximately 150 mm/year) than at the contaminated sites (approximately 100 mm/year). Flounder from the reference site also had a higher condition factor (somatic wt/(fish length)3) compared to fish from the two contaminated sites. However, the observed pattern of growth rate and condition factor might be biased by particular environmental conditions other than contaminants and must be confirmed by more extensive study. Flow cytometry analysis of fish blood revealed a significant difference in the frequency of abnormal profiles for fish from the Seine (20%) versus from the Ster (3%). We interpret this result as a marked genotoxic effect of contaminants on fish in the Seine system. Some genotypes, such as PGM 85/85, appeared to be linked to the measured components of fitness, particularly to DNA integrity. Thus, these genotypes might be considered to be more tolerant to pollutants. The frequency of the PGM 85 allele was clearly elevated in flounder from the more contaminated sites, compared to flounder from the reference site.

In Atlantic Canada, both Mytilus edulis and M. trossulus occur naturally in sympatric populations and readily hybridize forming genotypically complex assemblages that vary widely in relative species composition throughout the region. We monitored growth in single year-class cohorts of rope-cultured Mytilus spp. during a 14-month period from sleeving of seed to harvest at three commercial mussel farms in Notre Dame Bay, Newfoundland, Canada. Electrophoretic variation at the mannose phosphate isomerase (Mpi) enzyme locus was used to categorize the two species (M. edulis and M. trossulus) and their hybrids. Seed populations varied significantly among sites in relative proportions of each species and hybrids. Growth in shell length, wet weight and shell weight varied significantly with Mpi genotype at all three farm sites. Growth in flesh weight varied significantly with Mpi genotype at two of the three sites. Growth rates for all weight variables were consistently significantly greater in M. edulis than in sympatric M. trossulus. Growth in shell length was significantly different between sympatric M. edulis and M. trossulus at only one farm site. Growth rates of hybrids were typically intermediate between sympatric M. edulis and M. trossulus, but were often statistically similar to one or other of M. edulis or M. trossulus. We conclude that the production of rope-cultured blue mussels in Atlantic Canada is significantly influenced by genotype-dependent growth variation in naturally occurring mixed-species stocks. Our results also suggest that farm site production may be increased and intrasite variability reduced at farm sites currently using indigenous mixed-species stocks by substitution and growout of transplanted seed from unispecific M. edulis stocks.

To detect cell cross-contamination and verify the origin of the cells of an artificial organ, the sensitive isoenzyme assay was chosen to monitor the quality test of cell-based devices. Authoritative cell evaluation of artificial skin products has been established in this study. Human and porcine cell suspensions with total cell counts of between I x 10 5 and 4 x 10 6 were individually tested to determine the activity of isoenzymes. Human fibroblast, mixed with 1% to 100% of porcine fibroblast, could be significantly distinguished in the isoenzyme assay. Based on the glucose-6-phosphophate-dehydrogenase analysis, the human fibroblast tested in this study belonged to the B type human cells. Lactate dehydrogenase (LD), malate dehydrogenase (MD) and mannose phosphate isomerase isoenzyme (MPI) activities obviously revealed that a different pattern corresponds to the percentage of human and porcine cell mixtures. The discriminatory limit of MPI, LD and MD activity can reach up to 1% of sensitivity of the isoenzyme analysis. This sensitive isoenzyme analysis method allows us to routinely test cellular biomaterials whether interspecies cell line cross-contamination has occurred in the development of artificial organs.

Summary Electrophoretic studies of isoenzymes in three chromosomally distinct forms (A, B and C) of the mosquito Anopheles sundaicus Theobald (Diptera: Culiciae) were undertaken on wild samples collected from six geographically isolated populations in Indonesia. Analyses of 12 enzyme systems comprising 15 loci revealed significant allelic variations, genetic polymorphism, within and among the populations of the An. sundaicus complex. Phylogenetic dendrograms produced by analysis using the Biosys-1 program based on upgma methods show that all the populations of form A fall into one cluster, which is closely related to the form C cluster, whereas the populations of form B belong to a more distinct cluster. Allelic frequency and Wright’s F statistics of Mpi (mannose phosphate isomerase) are sufficient to identify individuals of each cytological form. This isozyme data correlates with our previous cytological evidence for the existence of three isomorphic species within the taxon An. sundaicus in Indonesia. These three species of the An. sundaicus complex were found together sympatrically at one locality, Asahan in North Sumatra.

Mytilus edulis L. and M. galloprovincialis Lmk. are two forms of mussels that live and are cultivated on the Northeastern Atlantic coasts and where they coexist they hybridise in varying proportions. Up to date, the form or forms of Mytilus that inhabit the Atlantic coast of northern Africa have not been reliably determined. Four partially diagnostic allozyme loci for M. edulis and M. galloprovincialis (esterase-D, leucine aminopeptidase-1, mannose phosphate isomerase and octopine dehydrogenase) were investigated in mussel samples from five northern African sites in the Atlantic Ocean and the Alboran Sea. Pure European M. galloprovincialis and M. edulis samples were also studied for comparison. Evidence from diagnostic enzyme loci indicates that M. galloprovincialis occurs on the Atlantic and SW Mediterranean coasts of northern Africa, and no evidence for the occurrence of M. edulis, or hybrids between M. edulis and M. galloprovincialis, has been found in this area. The results are discussed in relation to the geographical distribution of both forms of Mytilus on Northeastern Atlantic coasts.

A cellulose acetate electrophoresis system was used to study the isozyme polymorphism of the Anopheles gambiae complex in a rural village and a city in southwestern Burkina Faso. In both areas A. gambiae Giles was the dominant species (95%) whereas A. arabiensis Patton represented only 5%. Both species were separated readily by octanol dehydrogenase Odh and mannose phosphate isomerase (Mpi) even if they shared some alleles at these two loci. Polymorphism analysis (13 loci) at the intraspecific level of A. gambiae showed a significant difference between the specimens collected in the city from those collected in the village in their allelic and genotypic frequencies of isocitrate dehydrogenase-1 and malate dehydrogenase-1 and in their allelic frequencies for Mpi. No genetic difference was observed between the human biting A. gambiae collected inside or outside the houses in either the village or the city. The Plasmodium falciparum-infected A. gambiae differed from the noninfected ones in their allelic and genotypic frequencies at Mpi and acid phosphatase (Acp). A two-fold difference in infection rate was found for the genotype Mpi130/130 and Acp110/100 compared with other genotypes. However, infected anophelines were found in all genotypes that belonged to these two enzyme systems. Consequently, no refractory mechanism occurs in these natural populations.

Four populations of Leishmania (Viannia) braziliensis from Central America, Colombia, Peru and Brazil were analyzed and compared for up to 20 enzyme loci. Each of the 180 isolates could be identified as L. braziliensis using combined data from glucose phosphate isomerase and mannose phosphate isomerase. When the most common enzyme band was present at a frequency of < or = 0.95, the populations were polymorphic (more than a single allomorph for an enzyme) for more than 50% of the loci. Included were diagrammatic representations of the enzyme polymorphisms. Comparisons of levels of enzyme polymorphism and of genetic similarity among other Leishmania populations, L. tropica, L. major, L. mexicana, and L. donovani sensu lato, were discussed. The mean +/- SD level of genetic similarity among the four populations was 0.924 +/- 0.036 (range 0.878-0.966), which indicates that L. braziliensis is probably one reproductive population from Mexico in the north to Brazil and Peru in the south.

An electrophoretic study of erythrocyte allozymes and serum proteins representing 32 genetic loci in 32 hamadryas baboons ( Papio h.hamadryas ) from Cologne and Frankfurt zoos revealed biallelic polymorphism in phosphoglucomutase (PGM), mannose phosphate isomerase (MPI) and transferrin (Tf). Polymorphism amounted to P = 0.097 over 31 loci and observed heterozygosity to H o = 0.011, indicating significant reduction from the respective gene diversity parameters encountered in free-living hamadryas baboons. Polygynous mating reduced numbers of heterozygotes at the Tf and the PGM-II loci significantly below HardyWeinberg expectations. This results support the importance of active genetic management in programmes for captive breeding and species conservation.

Electrophoresis of 28 protein-coding loci in 51 roe deer (Capreolus capreolus) from two isolated Dutch populations in urbanized areas revealed percentage allozyme polymorphism of P = 0.093, and mean Hardy-Weinberg expectation of heterozygosity of He = 0.019. Adenylate kinase and mannose phosphate isomerase displayed biallelic polymoiphism. Results are discussed against the background of population management in fragmented habitats.

Leishmania parasites isolated from two patients with cutaneous leishmaniasis from geographically different localities in Paraguay have been characterized by enzyme electrophoresis (zymodeme) and digestion profiles of kinetoplast DNA with restriction enzymes (schizodeme). Both Paraguayan isolates showed identical zymodeme profiles to each other using 14 enzymes (glutamic pyruvate transaminase, glutamic oxaloacetic transaminase, enolase, fumarate hydratase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, malic enzyme, mannose phosphate isomerase, nucleoside phosphorylase, peptidase-D, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and pyruvate kinase). Although two Paraguayan isolates showed different zymodeme profiles from those of six Leishmania reference strains of Old and New World Leishmania species, they showed identical zymodeme profiles to those of an L. major-like parasite from Ecuador. These observations were confirmed by schizodeme analysis using three restriction endonucleases (Msp I, Hae III, and Taq I). These results indicate that Leishmania parasites isolated in Paraguay are identified as an L. major-like parasite, and it is necessary to consider the existence of L. major-like parasites when classifying Leishmania isolates from the New World.

Electrophoretic data at 10 allozyme loci from the offspring of six single pair matings of Mytilus edulis were analysed for linkage. Significant deviations from expected octopine dehydrogenase (Odh)/phosphoglycerate kinase (Pgk) and leucine aminopeptidase (Lap)/esterase D (EsD) di-locus genotype frequencies were evident in two families. In the former case, the recombination frequency was 0.463 suggesting epistasis but in the latter case a recombination frequency of 0.022 indicated almost complete linkage between the Lap and EsD loci. Other loci showing significant linkage were Odh/Lap and Odh/glucose phosphate isomerase (Gpi). Based on these data and other known linked loci in Mytilus a linkage group comprising Lap, EsD, Odh, mannose phosphate isomerase (Mpi) and possibly also Gpi is proposed. It is noted with interest that the closely related taxa M. edulis, M. galloprovincialis and M. trossulus are distinguished by significant allele frequency variations almost exclusively at loci in this linkage group.

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.

Different allozyme genotypes at the mannose phosphate isomerase (Mpi) locus in the northern acorn barnacle (Semibalanus balanoides) show a strong association with distinct intertidal microhabitats. In estuaries along the Maine Coast, the FF homozygote has higher fitness in exposed, high-tide level microhabitats while the SS homozygote has higher fitness under algal cover or at low-tide microhabitats. These patterns are consistent with a Levene (1953) model of balancing selection. In these same samples, polymorphisms at the glucose phosphate isomerase locus (Gpi) and mitochondrial DNA (mtDNA) show no fitness differences among microhabitats, providing intra-genomic controls supporting selection at or near Mpi. Here we report a similar analysis of genotype-by-microhabitat associations at sites in Narragansett Bay, Rhode Island, close to the southern range limit of S. balanoides. Genotype zonation at Mpi between high- and low-tide microhabitats is significantly different between Maine and Narragansett Bay due to opposite zonation patterns for the SF and FF genotypes. Enzyme activity data are consistent with this "reverse" zonation. At Gpi, there is significant microhabitat zonation in Narragansett Bay, while this locus behaves as a neutral marker in Maine. Mt DNA shows no significant microhabitat zonation in either Rhode Island or Maine. The Mpi data suggest that Levene-type selection for alternative genotypes in alternative habitats may operate at scales of both 10's of meters and 100's of kilometers. The Gpi data show how an apparently neutral locus can exhibit non-neutral variation under different environmental conditions. We argue that both Mpi and Gpi provide important genetic variation for adaptation to environmental heterogeneity that is recruited under distinct conditions of stress and carbohydrate substrate availability.

A rapid and highly significant drop in frequency of the azinphosmethyl resistant phenotype of Epiphyas postvittana (Walker) was detected along the coastline away from the center of a small group of apple orchards. This decline was detected by three assessment methods: (1) topical application of males attracted by sex pheromone, (2) bioassays of the offspring from tethered susceptible females mated to wild males, and (3) bioassays of the offspring from mated, field-collected females. Frequencies of susceptible-susceptible crosses were not significantly different in (2) and (3) assuming Hardy-Weinberg equilibrium. The estimated size of the selection coefficient favoring the susceptible phenotype was 0.004; the slope of the cline was estimated to be 0.7 x 103 units of susceptible phenotype frequency per m. We used electrophoresis to study allelic frequencies from four sites in the Nelson district and one in Canterbury. Five polymorphic loci were detected for phosphoglucomutase (PGM), phosphoglucose isomerase (GPI), isocitrate dehydrogenase (IDH A and B), and mannose phosphate isomerase (MPI). Samples from four sites in the Nelson district (three at Mariri) showed no difference in allelic frequencies with PGM, GPI, and IDH, although a difference between sites 20 km apart was detected for MPI. The average heterozygosity per locus fit the expected values for an interbreeding population, indicating that Hardy-Weinberg criteria were met at scrub and orchard sites at Mariri (where the localized insecticide-resistant population is present).

SummaryIn Guatemalan villages people commonly rear pigs, and both hosts may be infected withAscaris. This study was designed to ask whether both humans and pigs are potential hosts in a single parasite transmission cycle in such villages, or alternatively, if there are two separate transmission cycles, one involving pigs and one involving human hosts. Parasites were collected from both host species from locations in the north and south of Guatemala. Allelic variation in the nuclear genome ofAscariswas measured using enzyme electrophoresis, while mitochondrial DNA (mtDNA) sequence variation was quantified using restriction mapping. Low levels of enzyme polymorphism were found inAscaris, but allele frequencies at two loci, mannose phosphate isomerase and esterase, suggest that there is little gene exchange between parasite populations from humans and pigs. MtDNA haplotypes fall into two distinct clusters which differ in sequence by 3–4%; the two clusters broadly correspond to worms collected from humans and those collected from pigs. However, some parasites collected from humans have mtDNA characteristic of the ‘pigAscaris’ haplotype cluster, while some parasites collected from pigs have mtDNA characteristic of the ’ haplotype cluster. These shared haplotypes are unlikely to represent contemporary cross-infection events. Patterns of phylogenetic similarity and geographical distribution of these haplotypes suggest, instead, that they are the result of two historical introgressions of mtDNA between the two host-associatedAscarispopulations. The results clearly demonstrate thatAscarisfrom humans and pigs are involved in separate transmission cycles in Guatemala.