Partial Characterization of bermuda, carabao, cogon, and talahib grass pollen extracts

@#<p style="text-align: justify;"><strong>BACKGROUND</strong>: Grass pollen grains are important causes of respiratory allergies. The Philippines has a different grass flora compared to that of western countries, so pollen extracts have to be processed for use in the diagnosis and treatment of respiratory allergies. The local pollen extracts available in clinical practice have not yet been characterized, which is important in improving extract quality.<br /><strong>OBJECTIVE:</strong> This study aims to perform physicochemical characterization through protein content determination and gradient sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from four grasses: Cynodon dactylon (bermuda grass), Axonopus compressus (carabao grass), Imperata cylindrica (cogon), and Saccharum spontaneum (talahib) and immunologic characterization by identifying its IgE-binding component through immunoblot.<br /><strong>METHODS</strong>: This is a descriptive study. The pollen grains were processed into allergen extracts and protein contents were determined. The extracts were separated by gradient SDS-PAGE and subjected to immunoblotting. Bands were visualized using Fluorchem C2 aided with Alpha View Software.<br /><strong>RESULTS:</strong> Total protein in the pollen extracts ranged from 281.3-968.61 µg/ml. Protein bands of bermuda were in the 14.4-66.3 kDa range, carabao grass at 3.5-66.3 kDa, cogon at 3.5-200 kDA, and talahib at 21.5-66.3 kDa. A single IgE-binding protein band was seen on immunoblot at 55.4 kDa using a single serum sample.<br /><strong>CONCLUSION</strong>: Protein contents of the allergen extracts vary. The molecular weights of the different protein bands seem to correspond to known groups of grass pollen allergens. There was only one IgE-binding protein band seen on preliminary immunoblot.</p>

Allelic frequencies of single nucleotide polymorphisms (SNPs) in the N-acetyltransferase 2 (NAT2) gene of Filipinos

OBJECTIVES:The present study aims to determine the frequency of occurrence of NAT2*4, NAT2*5A, NAT2*6B, NAT2*7A and NAT2*14A alleles by PCR-RFLP among Filipino volunteers. These alleles correspond to substitutions in the following sites: C341T, G590A, G857A and G191A, respectively, of the NAT2 gene. The presence of specific SNP combination was also used to deduce acetylation status and estimate genotype frequency and describe them in comparison with other populations based on literature. METHODS: Genomic DNA from peripheral blood lymphocytes from 129 healthy Filipino volunteers was used to amplify the NAT2 gene segment. The RFLP analysis was done by restricting the expected PCR product with Kpn1, Taq1, BamH1 and Msp1/Al1, respectively, to detect the 4 alleles: NAT2*4, NAT2*5A, NAT2*6B, NAT2*7A and NAT2*14A. RESULTS:The calculated allelic frequencies in Hardy-Weinberg equilibrium of NAT2*5A (C481T), NAT2*6B (G590A), NAT2*7A (G857A) and NAT2*14A (G191A) were 0.058, 0.097, 0.182 and 0.046, respectively. NAT2*4 had an allele frequency of 0.617. Nine genotypes were determined: NAT2*4/*4, NAT2*4/*5A, NAT2*4/*6B, NAT2*4/*7A, NAT2*4/*14A, NAT2*5A/*7A, NAT2*6B/*7A, NAT2*6B/*14A and NAT2*7A/*14A. From these genotypes, acetylator phenotypes were deduced. A trimodal pattern of distribution was established: rapid, intermediate and slow acetylators with the following percentages, 47.3%, 41.1 % and 11.6%. Among the slow acetylator SNPs determined, NAT2*7A was found as the most frequent allele and NAT2*14A was found as the least frequent allele. CONCLUSION:The study showed the mutation profile and observed genotypic similarities and differences of Filipinos with other Asian populations and Americans and other Caucasians based on literature. The results also suggest a trimodal pattern of distribution of acetylators and lesser number of slow acetylators among Filipino populations, a characteristic similar to other Asian populations but significantly different from Americans and other Caucasians. The occurrence of NAT2*7A and NAT2*14A can be further sequenced to verify the observed genotype.

Association of cholesteryl ester transfer protein (CETP) Taq1B polymorphism with response to simvastatin treatment in hypercholesterolemic Filipino patients

Although statins reduced cardiovascular mortality, these drugs did not prevent myocardial infarction in some patients. Previous studies showed that genetic variation in cholesteryl ester transfer protein (CETP) gene was linked to this response. The identified gene is characterized by two different variants: B1 and B2 alleles identified by the presence and absence, respectively, of a restriction site for the enzyme Taq1 in intron 1. The present study identified the variation in Taq1B of the gene using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) in 130 patients. An association study of Taq1B with the response of 24 middle-aged dyslipidemic patients to simvastatin treatment for 8 weeks was also done. The overall allele frequencies of B1 and B2 alleles were 0.548 and 0.462, respectively. The genotype frequencies were in Hardy-Weinberg equilibrium. The distinguishing feature of individuals with B1B1 genotype when treated with simvastatin was their rapid increase in high density lipoprotein (HDL) observed after 2 weeks which continued till the 8th week treatment. The expected HDL elevation among individuals with B1B2 genotype was observed only after the 8th week simvastatin treatment.

Antigen sandwich ELISA predicts RT-PCR detection of dengue virus genome in infected culture fluids of Aedes albopictus C6/36 cells.

Antigen detection by sandwich ELISA was evaluated to predict RT-PCR detection of dengue viral genome in infected culture fluid of Aedes albopictus clone C6/36 cells. Serum specimens collected from dengue patients within 5 days from onset of fever in 2 hospitals in Metro Manila, Philippines, were inoculated into C6/36 cells, and incubated at 28 degrees C. A total of 282 infected culture fluid specimens were harvested and examined by sandwich ELISA and RT-PCR to detect dengue viral antigen and genome, respectively. In the sandwich ELISA, the P/N ratio was calculated by dividing optical density (OD) of a given test specimen by the OD of the standard negative specimen. Samples with a P/N ratio > or = 4.001 were positive for viral genome detection by RT-PCR. The sensitivity and specificity of antigen sandwich ELISA with RT-PCR as the standard, were 90.4% and 100%, respectively. Although antigen sandwich ELISA is less sensitive than RT-PCR, its usefulness lies in its capability to screen a large number of samples at a minimum cost, especially during an outbreak. Samples that meet a set cutoff value can undergo confirmation by RT-PCR for further epidemiological studies.