Phylogenetic analysis of slippage-like sequence variation in the V4 rRNA expansion segment in tiger beetles (Cicindelidae)

Sequence variation in the middle part of the small-subunit rRNA was studied for representatives of the major groups in the family Cicindelidae (Coleoptera). All taxa exhibited a much expanded segment in variable region V4 compared to D. melanogaster. This expanded segment was not found in other groups of beetles, including three taxa in the closely related Carabidae. Secondary structure predictions indicate that the expanded segment folds into a single stem-loop structure in all taxa. Despite its structural conservation, the fragment differs strongly in primary sequence, even between closely related sister taxa. Several features of these sequences are consistent with slippage replication as the mechanism that has generated this sequence variation: the level of internal sequence repetition as measured by the relative simplicity factor (RSF), its variation in length between close relatives, and the strong nucleotide bias compared to the remainder of the gene. With few exceptions, there was also a correlation between sequence length and the level of sequence repetition, frequently interpreted as the result of slippage. Phylogenies inferred from the expansion segment were not consistent with existing hypotheses from other molecular data for the group. This indicates that DNA sequences in this region are not homologous throughout the entire Cicindelidae, but it leaves open the possibility that this expansion segment can be used for phylogeny reconstruction within subgroups. The implications of a phylogenetic approach to the understanding of slippage-like evolution are discussed.      

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

Comparison of airway and blood eosinophil function after in vivo antigen challenge.

Eosinophils (EOS) are important effector cells in allergic diseases and asthma. However, functional characteristics of the EOS have been derived primarily from studies of blood cells, and it is unlikely that such assessments reflect events occurring in tissues or airways. To establish more precisely the function of airway EOS, segmental Ag challenge was used to elicit and isolate large numbers of these cells. Airway, as well as blood, EOS were isolated from allergic patients 48 h after segmental Ag challenge. Both blood and bronchoalveolar lavage (BAL) EOS were fractionated over Percoll density gradients; by using this protocol, three density-distinct populations of pure (>90%) EOS were obtained from BAL fluid (1.100, 1.095, and 1.090 g/ml) and one from blood (1.100 g/ml). The functions of these various populations were compared by measuring superoxide generation, adherence to collagen and endothelial cell monolayers, cell surface receptors, and in vitro survival. BAL EOS of all three densities had greater superoxide generation and adherence with FMLP activation than did corresponding blood EOS. In contrast, blood and airway EOS responded similarly to PMA. BAL EOS also had increased expression of CD11b/CD18 and HLA-DR. The intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent marker indo-1/acetoxymethyl ester. FMLP caused a greater and more sustained increase in [Ca2+]i with BAL than blood EOS. EGTA blocked the sustained component of the [Ca2+]i response to FMLP. Our findings indicate that BAL EOS have an enhanced [Ca2+]i response to activation that may contribute to their functional up-regulation.