Reduced folate carrier-1 G80a gene polymorphism is associated with neuroblastoma's development.

Neuroblastoma is a malignant embryonal tumor of neural crest cells that give rise to the sympathetic nervous system, responsible for 10-70% of all cases of childhood cancer. Because of its early appearance, it has been suggested that risk factors active in the prenatal can be associated with the pathogenesis of neuroblastoma. The aim of this study was to investigate whether the genetic polymorphisms MTHFR C677T and A1298C, MTR A2756G, TYMS 2R/3R and SLC19A1 G80A, involved in folate metabolism, increase the risk of neuroblastoma in Brazilian children. This study comprised 31 Brazilian children (0-14 years old) diagnosed with neuroblastoma compared with 92 controls. Investigation of polymorphisms MTHFR C677T, MTR A2756G and SLC19A1 A80G was performed using PCR-RFLP, the TYMS 2R/3R using PCR and MTHFR A1298C using AS-PCR. The SLC19A1 A80A genotype was significantly associated with the development of neuroblastoma, compared with the control group (Williams G-Test = 0.0286; OR = 5.1667; 95% CI = 1.4481-18.4338; p = 0.0175). When analyzed together, the 80AG+AA genotypes showed a trend toward association (OR = 3.3033; 95% CI = 1.0586-10.3080; p = 0.0563). Our results suggest that individuals carriers of genotype AA for the SLC19A1 gene present risk for the development of neuroblastoma and possibly have difficulty in absorption of folic acid by the cells, and this may adversely affect the metabolism of folate causing genomic instability and promoting the development of cancer. This is the first retrospective/prospective study to examine the relationship between polymorphisms of folate pathway genes and risk of neuroblastoma.

Boophilus microplus cathepsin L-like (BmCL1) cysteine protease: specificity study using a peptide phage display library.

The tick Rhipicephalus (Boophilus) microplus is one of the most important bovine ectoparasites, a disease vector responsible for losses in meat and milk productions. A cysteine protease similar to cathepsin L, named BmCL1, was previously identified in R. microplus gut, suggesting a role of the enzyme in meal digestion. In this work, BmCL1 was successfully expressed in Pichia pastoris system, yielding 54.8 mg/L of culture and its activity was analyzed by synthetic substrates and against a R. microplus cysteine protease inhibitor, Bmcystatin. After rBmCl1 biochemical characterization it was used in a selection of a peptide phage library to determine rBmCL1 substrate preference. Obtained sequenced clones showed that rBmCL1 has preference for Leu or Arg at P(1) position. The preference for Leu at position P(1) and the activation of BmCL1 after a Leu amino acid residue suggest possible self activation.

Biochemical characterization of a Kunitz type inhibitor similar to dendrotoxins produced by Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) hemocytes.

A novel chymotrypsin inhibitor identified in fat body and hemocyte cDNA libraries of Boophilus microplus was named BmCI (B. microplus Chymotrypsin Inhibitor) (Genbank EU636772). The putative BmCI amino acid sequence presented a 22-residue-signal peptide and 58-residue-mature protein. BmCI amino acid sequence analysis allowed its classification as a Kunitz-BPTI inhibitor with six cysteine residues, a theoretical pI of 7.8, and the presence of Tyr at P1 position in the putative reactive site, suggesting inhibitory activity toward chymotrypsin. In this work, we reported the biochemical characterization of BmCI. The recombinant BmCI expressed in yeast Pichia pastoris was purified by size exclusion and reverse phase chromatographies. rBmCI expression yield was of 1mgL(-1) of culture. Purified rBmCI confirmed its chymotrypsin inhibitory activity with a low K(i) (6.2pM). The BmCI gene expression analysis by semi-quantitative RT-PCR indicated its transcription in the hemocytes, salivary gland and ovary. The cytotoxic activity of purified rBmCI was demonstrated in BALB/c 3T3 mouse fibroblasts. As assessed by the MTT reduction assay, rBMCI induced a dose-dependent decrease in 3T3 fibroblasts viability (IC(50)=8microM). Moreover, flow cytometry analysis revealed that rBmCI is able to induce apoptosis, whereas no effect was observed on cell cycle progression. In conclusion, we demonstrated that rBmCI is cytotoxic against mammalian cells and obtained evidence that this growth inhibition is caused by an apoptosis-inducing activity.

BmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae.

BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. The inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K(i) 0.4 and 0.3nM) and subtilisin A (K(i) 1.4nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K(i) 50nM) and BmSI-6 (K(i) 2.2nM). The BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi.

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus.

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.