Hyperproliferation and p53 status of lens epithelial cells derived from alphaB-crystallin knockout mice

alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of alphaB-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative alphaB-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative alphaB-/- cells. However, the reentry of hyperproliferative alphaB-/- cells into S phase and mitosis after DNA damage by gamma-irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from alphaB-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative alphaB-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative alphaB-/- cells were labeled with anti-beta-tubulin antibodies. Examination of the hyperproliferative alphaB-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone alphaB-crystallin protects cells from acquiring impaired p53 protein and genomic instability.     

Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.     

Lethal and sublethal effects of single and double applications of Bacillus thuringiensis variety kurstaki on spruce budworm (Lepidoptera: Tortricidae) larvae

We conducted laboratory experiments to examine the effects of single versus double exposures of spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae) female larvae to various concentrations of a Bacillus thuringiensis variety kurstaki (Btk) commercial formulation (Foray 48B). Our main objective was to document the vulnerability to Btk and the sublethal responses of fifth-instar larvae that survived from a first ingestion of Btk during their fourth stadium and to compare them with insects treated either during their fifth or fourth stadium only. As reported in the literature, fifth-instar larvae were more vulnerable than fourth-instar larvae, but only at low and medium concentrations. Fifth-instar larvae that had survived Btk ingestion during their fourth stadium were more vulnerable to a high concentration of Btk and had a shorter feeding inhibition period than those that had not been exposed during their fourth stadium. Compared with a single treatment at the fourth stadium, a double exposure to Btk further reduced the population by 20-30%, depending on the concentration applied. The second treatment also induced another feeding inhibition period and increased larval development time by 14%. The impact of the different treatments on pupal weight depended on whether treated insects exhibited supernumerary instars. In the absence of developmental polymorphism, a higher concentration, a late, or a double exposure to Btk significantly reduced pupal weight.     

Synergistic interactions between volicitin,jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays

Plants display differential responses following mechanical damage and insect herbivory. Both caterpillar attack and the application of caterpillar oral secretions (OS) to wounded leaves stimulates volatile emission above mechanical damage alone. Volicitin ( N- 17-hydroxylinolenoyl- l -glutamine), present in beet armyworm (BAW, Spodoptera exigua ) OS, is a powerful elicitor of volatiles in excised maize seedlings ( Zea mays cv. Delprim). We consider some of the mechanistic differences between wounding and insect herbivory in maize by examining the activity of volicitin, changes in jasmonic acid (JA) levels, and volatile emission from both intact plant and excised leaf bioassays. Compared to mechanical damage alone, volicitin stimulated increases in both JA levels and sesquiterpene volatiles when applied to intact plants. In a bioassay comparison, excised leaves were more sensitive and produced far greater volatile responses than intact plants following applications of both volicitin and JA. In the excised leaf bioassay, volicitin applications (10–500 pmol) to wounded leaves resulted in dose dependent JA increases and a direct positive relationship between JA and sesquiterpene volatile emission. Interestingly, volicitin-induced JA levels did not differ between intact and excised bioassays, suggesting a possible interaction of JA with other regulatory signals in excised plants. In addition to JA, insect herbivory is known to stimulate the production of ethylene. Significant increases in ethylene were induced only by BAW herbivory and not by either wounding or volicitin treatments. Using intact plant bioassays, ethylene (at 1 µl l⁻¹ or less) greatly promoted volatile emission induced by volicitin and JA but not mechanical damage alone. For intact plants, wounding, elicitor-induced JA and insect-induced ethylene appear to be important interacting components in the stimulation of insect-induced volatile emission.     

The extended interactions and Gla domain of blood coagulation factor Xa

The serine protease factor Xa (FXa) is inhibited by ecotin with picomolar affinity. The structure of the tetrameric complex of ecotin variant M84R (M84R) with FXa has been determined to 2.8 A. Substrate directed induced fit of the binding interactions at the S2 and S4 pockets modulates the discrimination of the protease. Specifically, the Tyr at position 99 of FXa changes its conformation with respect to incoming ligand, changing the size of the S2 and S4 pockets. The role of residue 192 in substrate and inhibitor recognition is also examined. Gln 192 from FXa forms a hydrogen bond with the P2 carbonyl group of ecotin. This confirms previous biochemical and structural analyses on thrombin and activated protein C, which suggested that residue 192 may play a more general role in mediating the interactions between coagulation proteases and their inhibitors. The structure of ecotin M84R-FXa (M84R-FXa) also reveals the structure of the Gla domain in the presence of Mg(2+). The first 11 residues of the domain assume a novel conformation and likely represent an intermediate folding state of the domain.     

Autophagy: a barrier or an adaptive response to cancer

Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity. The involvement of proteins with properties of tumor suppressor and oncogenic properties at different steps of the pathway implies that autophagy must be considered in tumor progression. Autophagy as a stress response mechanism protects cancer cells from low nutrient supply or therapeutic insults. Autophagy is also involved in the elimination of cancer cells by triggering a non-apoptotic cell death program, suggesting a negative role in tumor development. These two aspects of autophagy will be discussed in this review.     

Heat-shock protein 70 (Hsp70) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (genus: Tegula)

Although previous studies have demonstrated that heat-shock protein 70 (Hsp70) can be induced by environmental stress, little is known about natural variation in this response over short time scales. We examined how Hsp70 levels varied over days to weeks in two intertidal snail species of the genus Tegula: Sampling was conducted both under naturally changing environmental conditions and in different vertical zones on a rocky shore. The subtidal to low-intertidal T. brunnea was transplanted into shaded and unshaded mid-intertidal cages to assess temporal variation in Hsps under conditions of increased stress. For comparison, the low to mid-intertidal T. funebralis was transplanted into mid-intertidal cages, within this species' natural zone of occurrence. Snails were sampled every 3 to 4 days for one month, and endogenous levels of two Hsp70-kDa family members (Hsp72 and Hsp74) were quantified using solid-phase immunochemistry. Following periods of midday low tides, levels of Hsps increased greatly in transplanted T. brunnea but not in T. funebralis. Levels of Hsps increased less in T. brunnea transplanted to shaded cages than to unshaded cages, suggesting that prolonged emersion and reduction in feeding time per se are factors that are only mildly stressful. Upregulated levels of Hsps returned to base levels within days. In unmanipulated snails collected from their natural zones, Hsp levels showed little change with thermal variation, indicating that these species did not experience thermally stressful conditions during this study. However, under common conditions in the mid-intertidal zone, Hsp70 levels reflected the different thermal sensitivities of the physiological systems of these two species.