2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells

OBJECTIVE: RNase L is converted to an active form upon binding short 2',5'-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase-an RNA-protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B. METHODS: Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained. RESULTS: Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death. CONCLUSIONS: The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.     

Effects of the nonimmobilizer hexafluroethane on the model membrane dimyristoylphosphatidylcholine

BACKGROUND: Nonimmobilizers are agents that lack anesthetic properties, although their chemical structure is very similar to known anesthetics. The primary action site of both agents, whether at the membrane or target protein level, is still a matter of debate. However, increasing evidence points to the distinct modifications of the membrane physical properties that such agents induce. Such modification may play a role in the mechanism of anesthesia, and may therefore be related to the differences in their clinical behavior. METHODS: Molecular dynamics (MD) computer simulations have been used to investigate the distribution of a nonimmobilizer, hexafluroethane (HFE, C(2)F(6)), in a lipid membrane. The biologically relevant liquid-crystal phase of a hydrated dimyristoyl phosphatidyl choline (DMPC) bilayer was used as a membrane model. Two MD simulations corresponding to HFE mole fractions of 6% and 25% have been performed at room temperature and constant ambient pressure, for a duration of 2 nanoseconds each. RESULTS: The equilibrium configurations of HFE in the bilayer show that the nonimmobilizer molecules are evenly distributed along the lipid hydrocarbon chains with a slight preference for the bilayer center. This partitioning induces an expansion of the bilayer thickness and a lateral contraction of the membrane (decrease of the area per lipid). The presence of HFE has essentially no effect on the lipid acyl chain conformations in agreement with nuclear magnetic resonance (NMR) measurements of the chain order parameters. The partitioning of the nonimmobilizer does not influence the orientation of the lipid head-group dipole moment. CONCLUSIONS: The modifications induced by the presence of the nonimmobilizer HFE on a model membrane are distinct from those previously found for halothane (CF(3)CHBrCl), its anesthetic analogue, and appear to result from different distributions in the lipid bilayer. The results of the MD simulations show that (1) the changes in the average area per lipid and in the membrane thickness are opposite for the two agents and (2) HFE induces no change in the lipid head-group orientation, in contrast to halothane. These different effects (1) on the physical properties of the lipid bilayer and (2) on the electrostatic properties of the membrane-water interface may be linked to different clinical effects, and thus might contribute to the mechanism of general anesthesia.     

Leishmania donovani parasites interact with gamma/delta+ human peripheral blood T cells and induce susceptibility to NK cell-mediated lysis

We recently reported that Leishmania donovani infect the human T-cell line in vitro. To examine whether primary human T cells could be infected by this parasite, a direct interaction of the peripheral blood T cells with L. donovani was examined. The percentage of gamma/delta+ T cells was markedly increased when in vitro generated normal human T-cell blasts were cultured with L. donovani amastigotes. About 30% of the gamma/delta+ T cells in the parasite exposed T-cell blasts expressed parasite antigens intracellularly without detectable intracellular parasites. Parasite exposed T-cell blasts had a reduced surface expression of HLA-DR and were lysed by the sorted CD56+ cells. In contrast, neither L. donovani amastigotes nor T-cell blasts exposed to heat killed amastigotes and/or were sensitive to the NK cell-mediated lysis. Of interest is that about 10% CD3+ peripheral blood T cells in two out of three Indian Kala-azar patients tested expressed intracellular L. donovani antigens.     

Molecular phylogeny of leader proteinase gene of type A of <Emphasis Type="Italic">Foot-and-mouth disease virus</Emphasis> from India

Summary. We previously demonstrated the presence of three genotypes (IV, VI and VII) of type A (subtype A22) of Foot-and-mouth disease virus (FMDV) in India based on 1D gene sequence analysis. In the present study, the leader proteinase (L^pro) gene sequences of 35 type A FMDV field isolates sampled over a period of 24 years (1977–2000) have been analyzed. Maximum-likelihood (ML) phylogenetic analysis revealed four distinct genetic lineages (A–D), indicating high divergence in L gene of type A FMDV. Lineages A and D correspond to the earlier genotypes IV, and VII, respectively. The genotype VI isolates were divided into two separate lineages (B and C) in the tree with high confidence values (96–99%). One isolate (IND 21/90) showed incongruous grouping between the (1D and L) gene-based analyses, which could be due to intergenotypic recombination. The ML molecular clock hypothesis was easily rejected on the data set studied indicating the absence of clock-like evolution in the L gene. ML codon-substitution models identified positive selection in the amino acid site 194 of C-terminal extension of the Lb^pro with high posterior probability (>99%). The catalytic triad of the L^pro at C-51, H-148 and D-163 were conserved across all the Indian isolates studied. Finally, amino acid differences between the lineages have been discussed briefly.     

Mosaicism in a patient with Down syndrome reveals post-fertilization formation of a Robertsonian translocation and isochromosome

Metaphyseal chondrodysplasias (MCD) are skeletal disorders characterized by metaphyseal irregularities and, usually, by short stature. In MCD, wide heterogeneity exists with regard to clinical and radiological changes. We report on a patient with clinical and radiological findings of MCD who had coxa valga and normal height with metaphyseal involvement of the long bones. The short radii and ulnae showed a very severe change in their distal metaphyses, leading to mesomelic shortening confined to the upper limbs. Hematological, ophthalmological, and hearing examinations were normal. This type of MCD appears to represent a yet undescribed syndrome.     

The role of arbitrarily primed PCR in identifying the source of an outbreak of Legionnaires' disease.

An outbreak of community-acquired Legionnaires' disease (LD) occurred in Providence, R.I., in fall 1993. To find the outbreak source, exposures of 17 case patients were compared to those of 33 matched controls. Case patients were more likely than controls to have visited a section of downtown (area A) during the 2 weeks before illness (11 [65%] versus 9 [27%]; matched odds ratio, 6.5; P = 0.01). Water samples were cultured from 27 aerosol-producing devices within area A. Legionella pneumophila serogroup 1 isolates underwent monoclonal antibody (MAb) subtyping and arbitrarily primed PCR (AP-PCR). All four L. pneumophila serogroup 1 isolates available from case patients who visited area A had identical MAb and AP-PCR patterns. Among 14 environmental isolates, 5 had MAb patterns that matched the case patient isolates, but only 1 had a matching AP-PCR pattern. This investigation implicates a cooling tower in area A as the outbreak source and illustrates the usefulness of AP-PCR for identifying sources of LD outbreaks.     

CaO--P2O5--Na2O-based sintering additives for hydroxyapatite (HAp) ceramics

We have assessed the effect of CaO--P2O5--Na2O-based sintering additives on mechanical and biological properties of hydroxyapatite (HAp) ceramics. Five different compositions of sintering additives were selected and prepared by mixing of CaO, P2O5, and Na2CO3 powders. 2.5 wt% of each additive was combined with commercial HAp powder, separately, followed by ball milling, and sintering at 1250 degrees C and 1300 degrees C in a muffle furnace. Green and sintered densities of the compacts were analyzed for the influence of additives on densification of HAp. Phase analyses were carried out using an X-ray diffractometer. Vickers microhardness testing was used to evaluate hardness of sintered compacts of different compositions. A maximum microhardness of 4.6 (+/- 0.28) GPa was attained for a composition with 2.5 wt% addition of CaO:P2O5:Na2O in the ratio of 3:3:4. Results from mechanical property evaluation showed that some of these sintering additives improved failure strength of HAp under compressive loading. Maximum compressive strength was observed for samples with 2.5 wt% addition of CaO. Average failure strength for this set of samples was calculated to be 220 (+/- 50) MPa. Cytotoxicity, and cell attachment studies were carried out using a modified human osteoblast cell line called OPC-1. In vitro results showed that these compositions were non-toxic. Some sintering aids enhanced cell attachment and proliferation, which was revealed from SEM examination of the scaffolds seeded with OPC-1 cells.