Manami Tanaka

Some molecular insights into schistosome evolution

Robust phylogenies based on molecular data for species within the genus Schistosoma have been generated in recent years. The considerable progress made in understanding the relationships between many of the 19 recognised species of Schistosoma is reviewed with particular attention being given to the detection and analysis of parasite variation as shown by studies on ribosomal RNA genes, mitochondrial DNA and RAPDs. For the most part, molecular phylogenies agree with observations based on morphological or life-history characteristics. It is clear that the parasites of man do not form a monophyletic group and that close relationships exist between parasites within species groups, especially in the S. haematobium group of species. The S. japonicum group appears to be the most divergent of the species groups and yet little DNA sequence variation has been observed between various isolates of S: japonicum. Some of the less studied schistosomes have yet to be examined at the molecular level and may prove to be interesting links between the species groups as has recently been shown with S. hippopotami. The power of molecular approaches for the analysis of schistosomes at the population and individual level is now apparent, especially for S. mansoni. Important questions remain concerning the maintenance of parasite diversity and how schistosomes respond to selection pressures imposed either during natural progression through the life-cycle or through drug treatment or vaccination. Gene discovery and gene mapping projects are leading to a better understanding of the schistosome genome and can be expected to contribute significantly to future comparative evolutionary studies.

Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo.

We have identified a novel human septin family gene Bradeion, which is specifically expressed in human colorectal cancer and malignant melanoma. In order to analyze the implications of tumor-specific gene expression, ribozymes and its derivatives were specifically designed and transfected into various colorectal adenocarcinoma cell lines for Bradeion inactivation. We constructed ribozyme expression plasmids controlled by a human tRNAVal promoter, and both hammerhead ribozyme and its allosteric derivative maxizyme were used for two different forms of Bradeion mRNA. The sequence-specific cleavage of Bradeion mRNA resulted in significant growth inhibition and G2 arrest in human cancer cell lines, detected by flow cytometry analysis. In addition, in vivo mice studies demonstrated marked tumor growth suppression by the Bradeion-specific ribozymes. Thus, the tumor-specific and selective marker Bradeion also provides valuable tools as a potential target for colorectal cancer therapy.

Yeast artificial chromosome (YAC) -based genome mapping of Schistosoma mansoni

Schistosoma mansoni has 7 pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 × 108 bp. We initiated the molecular genetic approach for the detailed characterization and understanding of the evolutionary biology of schistosomes. We have constructed a yeast artificial chromosome (YAC)-library with partially digested parasite genomic DNA, and the chromosome location of each insert was detected by fluorescent in situ hybridization (FISH). The library contains > 2283 clones with an average insert size of 358 kb, which represents a 2.6-fold coverage of the genome (> 7.2 × 108 bp). 100 randomly selected YAC clones were localized by FISH and found to be distributed widely among all chromosomes. The assembly of 14 YACs distributed almost the whole region of chromosome 3. Generated expressed sequenced tags (ESTs) derived from a unidirectional cDNA library were also used for further characterization of the YAC inserts. These results indicate that an extensive contig assembly of the entire chromosomes and a reasonably detailed gene map should be feasible in the near future.

Rapid diagnosis of Treponema pallidum in serum using latex piezoelectric immunoassay

We developed a conventional immunosensor of Treponema pallidum (TP) using a quartz crystal microbalance (QCM). A QCM is able to measure an agglutination of TP due to TP antigen-immobilized latex particles within 10 min.

Conventional diagnosis of Treponema pallidum in serum using latex piezoelectric immunoassay

We developed a conventional immunosensor for Treponema pallidum (TP) to combine quartz crystal microbalance (QCM) with the agglutination reaction of immunized latex beads. TP induced an immunoreaction due to treponemal antigen-immobilized latex particles. We succeeded in measuring the concentration of TP in the human serum within 10 min by QCM method.

The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTP. At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease.

The p53 gene expression and its developmental regulation in schistosomes

We have studied the gene expression, especially of the oncoproteins, and its regulation in schistosomes. Schistosomes have a complex life cycle with a defined dimorphic lifestyle. The parasite are so far unique in biology in expressing oncogene products in their adult stage. In order to characterize the expression and developmental regulation, a lambda gt 11 cDNA library and lambda EMBL4 genomic DNA library of each growth stage of Schistosoma mansoni and S. japonicum was constructed, and was screened with various monoclonal antibodies against oncogene products. One positive plaque reacted to anti-p53 antibody (Ab-2, Oncogene Science, Inc.) was further analyzed. This fusion protein was about 120 KDa in molecular weights, and expressed as 1.4 Kb RNA in the adult stage. P53 gene is well-known as the negative regulator of the cell cycle, and the mutations in the gene are turning out to be the most common genetic alterations in human cancers. The comparison of the gene structure among species and stages were being conducted. Chromosome structures, C-band formation, and the results of in situ hybridization using the phage probe would be discussed.

Characterization of a Schistosoma mansoni homologue of the gene encoding the breast basic conserved protein 1/L13 ribosomal protein

The Schistosoma mansoni gene sequence encoding the breast basic conserved protein 1/ribosomal protein L13 has been isolated from an adult worm cDNA library using the Expressed Sequence Tag strategy. The cDNA codes for a putative protein of 184 amino acids which is approximately 55% identical to other eukaryotic L13 ribosomal proteins. A PCR amplified genomic fragment containing the coding region of the gene was seen to possess only a single large intron interrupting the open reading frame. Studies of gene expression by RT-PCR showed the transcript is expressed in distinct stages of the parasite life cycle. The cDNA was also hybridized with an ordered cosmid library of S. mansoni and the identified cosmids were mapped to chromosomes 3 and W by chromosomal in situ suppression hybridization.

The cloning and sequencing of ribosomal protein S18 of parasitic protozoa, Entamoeba histolytica

A cDNA clone encoding ribosomal protein S18 has been isolated from parasitic protozoa, Entamoeba histolytica. The cDNA insert was 495 nucleotides long and an open reading frame (468 nucleotides including the stop codon) coded for 156 amino acid protein which was highly homologous with human, plant and yeast counterparts. PROSITE analysis of the ribosomal protein S18 in eukaryote revealed homology with prokaryotic ribosomal protein S13 signature, which is known to be involved in the initiation of translation.

Mini-review: the ultrasonographical and serological chanbges and their improvement after praziquantel treatment Schistosoma japonicum infected patients in Leyete, Philippines

We have identified the specific ultrasonographical (US) changes in Schistosoma japonicum infected patients with the serological changes in general liver function markers. The US examination with the following haematological and biochemical serum analysis was performed on 102 patients in Schistosomiasis Hospital, Leyte, Philippines. The US liver images were classified into 4 patterns according to the development of periportal fibrosis and the patterns of echogenic bands. Among various haematological and biochemical serum parameters of liver damage. The serum levels of total bile acid (TBA) and procollagen-III-peptide (P-III-P) correlated well with the development of hepatic fibrosis and the portal hypertension. These patients were subsequently treated with praziquantel (3 x 20 mg/kg), and the improvement of the thickening of the portal vein wall and the intensity of the echogenic band formation was detected 6 months after treatment. The significant US changes could not be detected in the patients with severe hepatic fibrosis caused in the long term infection. The results revealed that the US examination with the serum TBa level would provider a sensitive tool to monitor the severity of the infection and also the improvement occurred shortly after praziquantel treatment.