Isolation, identification and expression of a Fasciola hepatica cDNA encoding a 2.9-kDa recombinant protein for the diagnosis of ovine fasciolosis.

A 400-bp Fasciola hepatica cDNA clone was isolated from an expression library by immunological screening using rat sera taken 2 weeks after experimental infection. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 78 bp which encoded a 25 amino acid polypeptide with a predicted molecular weight of 2.9 kDa. This polypeptide was expressed in bacteria as a GST-fusion protein and used for the production of specific antigen. The 2.9 kDa recombinant protein (APS) was evaluated against sera from experimentally infected sheep using an indirect ELISA, and the results were compared to those obtained using F. hepatica excretory/secretory products (ESP). The pattern of IgG was very similar both against the recombinant and the native proteins, increasing early following the infection. After treatment with triclabendazole, the IgG response against the APS seroreverted to negative values, whereas it remained elevated against the ESP. We conclude that this recombinant protein could be used in diagnostic assays for the identification of recently infected sheep.

Clinical challenges and controversies in the management of HIV/HCV-coinfected individuals

The natural history of HIV infection has been dramatically changed by the highly active antiretroviral therapies, reducing complications, morbidity and mortality of the disease. Approximately 25% of persons infected with HIV are co-infected with hepatitis C, and some high risk populations have a prevalence of HCV of more than 75%. Liver disease has become one of the principal causes of morbidity and mortality in this population. Co-infection increases viremia of hepatitis C, with increase in fibrosis progression, cirrhosis and death related to hepatitis C. The permanent state of chronic immune activation related to the persistent hepatitis C virus favors transcription of HIV in infected cells and causes a more rapid destruction of T4 and absolute lymphocytes. In addition, the immunologic response after the start of highly active antiretroviral therapy for HIV is less than in mono-infected patients. The role of liver biopsy in the management of co-infected patients is controversial. Many of these patients, even with normal transaminases, show fibrosis in liver biopsy. Predictive factors for advanced fibrosis include male sex, alcohol consumption in excess of 50 grams per day, age over 35, and HIV infection of more than 15 years with CD4 lymphocytes less than 400/ mm3. The treatment of hepatitis C is limited and sustained viral response is less than 30% for genotypes 1 and 4. This response is even less in the more advanced stages of HIV and hepatitis C. The determination of when to start treatment and the increased toxicity when combining pegylated interferon plus ribavirin and antiretroviral medications makes the management of these patients more difficult. The development of more potent, safe and tolerated medications is required. Management strategies for patients unresponsive to conventional therapy are geared towards improving liver histology and delaying progression to cirrhosis, hepatocellular cancer and liver transplantation.

Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.

A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex.

Increased chitin synthesis in response to type II myosin deficiency in Saccharomyces cerevisiae.

We reported previously that the chitin content in cell walls of type II myosin-deficient Saccharomyces cerevisiae strains is increased relative to wild-type cells suggesting that increased chitin synthesis is induced in these strains. In the present study, we have performed enzyme activity assays for chitin synthases 1, 2, and 3 to determine the enzyme isoform(s) involved. To determine if transcriptional regulation is involved, we conducted quantitative mRNA assays of the corresponding chitin synthase genes. We show that the enzyme activities of all three chitin synthases increase substantially over the wild-type strain while eight- and twofold increases in the mRNA levels for chitin synthases 1 and 3 were detected. Increases in enzyme activities and mRNA levels were not proportional. We conclude that the enzyme activities for all three chitin synthases are elevated in this strain and that this increase is mediated mainly by a posttranslational mechanism(s). The heightened sensitivity to osmotic stress and the corresponding increase in cell wall chitin content reported in these strains are consistent with a compensatory "stress response" mechanism induced by abnormal cell wall assembly.

Molecular analysis of the Saccharomyces cerevisiae YHR076w gene.

Our results demonstrate that open reading frame (ORF) YHR076w on chromosome VIII of Saccharomyces cerevisiae that was previously described as a hypothetical gene is expressed. This ORF is transcribed as an mRNA of approximately 1,100 nucleotides. A 41.2-kDa polypeptide and three others predicted to be modified forms of Yhr076wp are detected by Western blot with a Yhr076wp-specific antibody. Promoter activity assays indicate that YHR076w transcription is regulated by carbon source and primarily by ethanol. Consistent with this observation, we have identified two potential ADR1 regulatory elements in the YHR076w upstream DNA region. Potential YAP1 and HSP elements are also identified in this region, suggesting other forms of regulation. YHR076w knockout strains do not exhibit any measurable growth or morphological phenotype under any conditions tested. However, increased YHR076w gene dosage confers a growth advantage to both wild-type and YHR076w knockout strains on 2% ethanol or 2% galactose medium in a low O2 growth environment. The fluorescence emitted by a Yhr076wp protein fusion to A. aquorin GFP colocalizes with the mitochondria in vivo.

Elevated expression of chitinase 1 and chitin synthesis in myosin II-deficient Saccharomyces cerevisiae.

To determine if the attached cells formed in Myosin II-deficient Saccharomyces cerevisiae result from deficient chitinase 1 (CTS1) expression, the activity of chitinase 1 was assayed. Secretion of this enzyme was not prevented by a MYO1 gene deficiency, and soluble and cell wall-associated Cts1p activity were increased approximately 5-fold and 20-fold, respectively, in these cells. The increase in soluble activity was correlated with an increase in enzyme levels. Likewise, intracellular chitinase activity was increased approximately 22-fold, and the chitin content of cell walls was elevated 2-fold. These data suggest that the origin of myo1-associated phenotypes is not due to deficient chitinase expression and may instead be due to a deregulation of cell wall metabolism in these cells.

Flow cytometry analysis of cell cycle in myosin II-deficient yeast.

OBJECTIVE: To determine whether cell cycle changes can be detected in myosin II-deficient cells using flow cytometry techniques. BACKGROUND: Although the primary role of myosin II (Myo1p) in the yeast Saccharomyces cerevisiae is in cytokinesis we have reported that this conventional myosin also appears to influence the regulation of cell wall metabolism as indicated by increases in the expression of chitin metabolizing enzymes in a null mutant of the MYO1 gene. The expression of these enzymes is known to be regulated in the cell cycle suggesting that cell cycle changes may alter their expression. METHODS: Flow cytometry was employed to assess the nuclear DNA content of logarithmic yeast cell cultures as a means of determining changes in the cell cycle of Myo1p-deficient cells. RESULTS: Significant changes were observed in the Myo1p-deficient strain suggesting that these cells are arrested in G2/M-phase of the cell cycle. CONCLUSIONS: Based on the results of this preliminary study, we propose a model in which the increased activity of chitin metabolizing enzymes may be explained by a mitotic arrest in these cells.

Sustained response to interferon in a patient with common variable immunodeficiency and hepatitis C.

Hepatitis C infection progresses faster in patients with hypogammaglobulinemia, often leading to death from liver failure within 10 yr of exposure. Response to interferon treatment has been poor in these patients. We report the case of a patient with common variable immunodeficiency and hepatitis C in whom a sustained biochemical and viral remission was obtained after treatment with interferon.

In vivo phosphorylation of type II myosin in Saccharomyces cerevisiae.

Phosphorylation of the myosin heavy chain has been shown to be a key regulatory mechanism of several non-muscle myosins. In this study we present evidence demonstrating that the yeast type II myosin heavy chain is phosphorylated in vivo. Phosphorylation of serine residues was confirmed by direct metabolic labeling with [32P] and by indirect immunostaining of phosphoserine with a specific monoclonal antibody. Loss of immunoreactivity in a targeted deletion of the 26 amino acid carboxyl terminal segment of the type II myosin heavy chain suggests that the phosphorylation occurs at one or more serine residues located between residues 1903 and 1928.

Identification of genes that encode Fasciola-specific arc 2 antigens.

Previous studies have shown that serum from humans with fascioliasis had precipitating antibodies that identified a line of precipitation as specific for Fasciola. This Fasciola-specific band was designated arc 2. The specificity of a Fasciola-specific anti-arc 2 antiserum, evaluated by Western blot analysis, reveals a mosaic of antigens with molecular weights in the range of 10-43 kD. In the current study, the antibodies reacting with a subset of antigens in the 10-21-kD range were eluted from nitrocellulose and used to probe an F. hepatica cDNA library. Four clones were purified and amplified by the polymerase chain reaction. The DNA hybridizations among these clones revealed different groups of clones derived from different mRNAs. The finding of multiple genes supports the observation that F. hepatica arc 2 is a mosaic of antigens. The studies show that molecular cloning technology can be used to identify potential genes, each of which encode distinct antigens that may be used for the specific immunodiagnosis of fascioliasis.

Prevalence and distribution of introns in non-ribosomal protein genes of yeast.

Relatively few genes in the yeast Saccharomyces cerevisiae are known to contain intervening sequences. As a group, yeast ribosomal protein genes exhibit a higher prevalence of introns when compared to non-ribosomal protein genes. In an effort to quantify this bias we have estimated the prevalence of intron sequences among non-ribosomal protein genes by assessing the number of prp2-sensitive mRNAs in an in vitro translation assay. These results, combined with an updated survey of the GenBank DNA database, support an estimate of 2.5% for intron-containing non-ribosomal protein genes. Furthermore, our observations reveal an intriguing distinction between the distributions of ribosomal protein and non-ribosomal protein intron lengths, suggestive of distinct, gene class-specific evolutionary pressures.

Hepatitis C viral RNA amplification by the polymerase chain reaction allows detection of false positive HCV ELISAs among patients with non-A, non-B hepatitis.

Serum from patients which tested positive for hepatitis C virus (HCV) by Enzyme Linked Immunosorbent Assay (ELISA) were analyzed for the presence of HCV RNA by nested Polymerase Chain Reaction (PCR) and for anti-HCV antibodies by Recombinant Immunoblot Assay (RIBA II). Total RNA was extracted from whole blood by a new procedure and subjected to reverse transcription of HCV RNA employing primers to the conserved 5' non-coding region of the HCV genome. PCR performed on these samples uncovered several false positive ELISAs. Reciprocal confirmation between PCR and RIBA II results was observed. These results substantiate this variation of the HCV PCR assay as a reliable alternative for routine confirmation of HCV serological tests.

Sequence analysis of a Fasciola hepatica glutathione S-transferase cDNA clone.

A lambda gt11 Fasciola hepatica cDNA library was previously constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding F. hepatica glutathione S-transferase (FhGST) (GenBank Accession Number M93434) was cloned by screening the library with a rabbit anti-FhGST antiserum. The FhGSTs are suspected to be early developmentally expressed-proteins that act as potent immunogens and may be useful as candidate vaccines against the parasite. The cDNA was sequenced and contains 627 translated bases encoding a 24,211-Dalton polypeptide with 209 amino acids and an isoelectric point (pI) of 5.324. This polypeptide has significant homology with 26-kD GSTs from Schistosoma mansoni (57.42%) and S. japonicum (57.14%). In addition, we have identified the predicted T cell epitopes in the FhGST protein molecule.

Calcium stimulates molecular and cellular events during the yeast-to-mycelium transition in Sporothrix schenckii.

Calcium ions (Ca2+) have been identified as mediators of proliferative and morphogenetic processes in many eukaryotic cells. The effects of these ions on the cellular and macromolecular processes that accompany the dimorphic transition from the yeast-to-mycelial form of Sporothrix schenckii have been studied. Ca2+ were found to stimulate germ tube formation and growth in these cells at an optimal concentration of 1.0 mM. Studies concerning the effects of this cation on the molecular processes that precede germ tube formation revealed that the earliest molecular event which was stimulated by 1.0 mM Ca2+ was RNA synthesis. An increased incorporation of radioactivity into RNA in the presence of 1.0 mM Ca2+ was first observed at 0-3 h, and subsequently at all other times tested, following inoculation. A stimulation in rRNA and tRNA synthesis was detected in the presence of 1.0 mM Ca2+. The incorporation of radioactivity into proteins was stimulated 3-5 h following induction in the presence of Ca2+ suggesting a specific effect of Ca2+ on protein synthesis. This increased incorporation takes place prior to the start of DNA synthesis. Incorporation of radioactivity into DNA was also stimulated in the presence of Ca2+, 6 and 9 h after inoculation. This stimulation resulted in nuclear division taking place with a shorter lag period and proceeding with increased kinetics. The results reported here are evidence that Ca2+ plays a role in the control of the early molecular and cellular processes that accompany the yeast-to-mycelium transition in S. schenckii and offer an explanation of how Ca2+ can control the expression of the dimorphic potential of this fungus.

Fasciola hepatica: molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein.

Immunization of mice with an antigenic polypeptide from Fasciola hepatica adult worms and having an apparent molecular mass of 12,000 Da (Fh12) has been shown to reduce the worm burden from challenge infection with Schistosoma mansoni by more than 50%. Moreover, mice infected with S. mansoni develop antibodies to Fh12 after 5-6 weeks of infection, indicating that this Fasciola-derived antigen is a cross-reactive, cross-protective protein. A lambda gt11 F. hepatica cDNA library was constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding a cross-reactive polypeptide (Fh15) was cloned by screening the F. hepatica lambda gt11 library with a monospecific, polyclonal rabbit antiserum against pure, native Fh12. The cDNA was sequenced and the predicted amino acid sequence revealed an open reading frame encoding a 132-amino-acid protein with a predicted molecular weight of 14,700 Da. This protein has significant homology to a 14-kDa S. mansoni fatty acid-binding protein. Comparison of the protective-inducing activity of recombinant Fh15 with that of purified Fh12 against schistosomes and Fasciola is warranted.