Substrate specificity of avian influenza H5N1 neuraminidase.

AIM: To characterise neuraminidase (NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus. METHODS: Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography (HPLC) analysis of the enzymatic product. RESULTS: The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked (P < 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin (HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid. CONCLUSION: H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.

Proteome analyses of cellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone, a novel antibiotic candidate.

The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC) values ranged from 31.25-62.5 µg/ml, and the minimal bactericidal concentration (MBC) was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5-125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml) affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.

Enhancement of viable Campylobacter detection by chemotactic stimuli.

The effects of chemotactic stimuli on motility ability of viable Campylobacter to pass through a 0.45 microm pore size filter in viscous condition were investigated. Reference strains including C. jejuni ATCC 33291, C. coli MUMT 18407, C. lari ATCC 43675, and C. upsaliensis DMST 19055 were used. The initial numbers of artificially-inoculated viable cells per g of chicken meat were approximately 10 to 10(4). Constituents of mucin plus bile (1:1), varieties of amino acids, and sodium salts were added into a soft-agar-coated membrane filter and incubated at both 37 degrees C and 42 degrees C for 24h. The drop plate method was used to determine numbers of viable Campylobacter at 6, 12, 18, and 24h. After 6h, constituents of mucin plus bile at the concentrations of 1, 5, and 10% demonstrated significant increases in numbers of viable cells (p<0.05). The numbers of the organisms at 42 degrees C were higher than those at 37 degrees C. In contrast, no significant difference in cell numbers was observed by adding amino acids or sodium salts. In addition, the role of starvation on chemotactic responses was also studied. Starved cells showed lower chemotactic response than non-starved cells. This method permitted rapid detection of viable thermophilic Campylobacter.

A novel method and simple apparatus for the detection of thermophilic Campylobacter spp. in chicken meat products.

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.

Analysis of gyrA mutations related to quinolone resistance in Escherichia coli isolates originating from pet, human, vegetable and ice in Bangkok and vicinity.

Escherichia coli was used to investigate quinolone resistance and mutations in gyrA gene of E. coli isolated from pet (dog and cat), human (pet's owner), vegetable and edible ice in Bangkok and vicinity. Susceptibility test for nalidixic acid (NA) showed similar percent resistance among the sample sources but a lower ciprofloxacin (CIP) resistance was found particularly in human source. Mutations within quinolone resistance determining region of gyrA gene analyzed using non-radioactive single-strand conformation polymorphism (SSCP) and sequencing showed 10 different SSCP patterns. E. coli isolates from pet, vegetable and ice showed more variety of patterns than strains isolated from human. Four out of 10 SSCP patterns were identified as having mutations in amino acids positions 83 (Ser to Leu) and position 87 (Asp to Asn). These mutations were observed only in NA-resistant strains and combined mutations were observed only in E. coli isolated from humans and pets. As only 24% of NA- and CIP-resistant E coli isolates contained gyrA mutations, other quinolone resistant mechanisms may be involved. Nevertheless, gyrA mutations may be used to monitor nalidixid acid resistance in E. coli.

Allele-specific loss of heterozygosity at the DAL-1/4.1B (EPB41L3) tumor-suppressor gene locus in the absence of mutation.

DAL-1/4.1B (EPB41L3)is a member of the protein 4.1 superfamily, which encompasses structural proteins that play important roles in membrane processes via interactions with actin, spectrin, and the cytoplasmic domains of integral membrane proteins. DAL-1/4.1B localizes within chromosomal region 18p11.3, which is affected by loss of heterozygosity (LOH) in various adult tumors. Reintroduction of this protein into DAL-1/4.1B-null lung and breast tumor cell lines significantly reduced the number of cells, providing functional evidence that this protein possesses a growth suppressor function not confined to a single cell type. For characterization of the mutational mechanisms responsible for loss of DAL-1/4.1B function in tumors, the exon-intron structure of DAL-1/4.1B was examined for mutations in 15 normal/tumor pairs of non-small cell lung carcinoma by single-strand conformation polymorphism analysis. These studies revealed that small intragenic mutations are uncommon in DAL-1/4.1B. Furthermore, LOH analysis on 129 informative early-stage breast tumors utilizing a new intragenic C/T single-nucleotide polymorphism in exon 14 revealed that LOH resulted in preferential retention of the C-containing allele, suggesting that allele-specific loss is occurring. These studies indicate that mechanisms such as imprinting or monoallelic expression in combination with loss of heterozygosity may be responsible for loss of the DAL-1/4.1B protein in early breast disease.