Detection of allogeneic Qa/TL and Ly specificities on murine tumor cells with IgD in tumor-regressor serum

Summary Serum from C3H/He mice, which show regression of MM2 tumor cells after transplantation and removal (regressor serum, RS) contains non-gammaglobulin components that cross-react with various tumor cells of mice [22, 23]. In addition to tumor cells, various allogeneic lymphocytes are also susceptible to an RS-dependent lymphocyte-mediated cytotoxic reaction. To identify tumor cell surface antigens that cause the cross-reactive host response, the serum components were analyzed by absorption of RS with allogeneic lymphocytes. RS components were found to recognize allogeneic lymphocyte antigens including Qa-2 and Ly6.2. Specificity for the Qa-2 antigen was further tested using Qa-2-congenic mice. The expression of Qa-2 antigen was detected on the surfaces of MM2 and other tumor cells derived from H-2^k mice (seven among nine cell lines tested) by a membrane immunofluorescence method using a Qa-2-specific mAb. Physical characteristics of the Qa-2-specific component in RS were determined and found to differ from those of regular IgGs but to be similar to those of IgDs. Using an enzyme-linked immunosorbent assay with an IgD-specific mAb and Qa-2-lacZ fusion protein, the existence of IgD in RS with specificity for Qa-2 was confirmed. These results suggest that the RS component with Qa-2 specificity is an IgD, the specificity and physiological role of which are unknown.     

Changes in caspase expression in Alzheimer's disease: comparison with development and aging

Changes in caspase-2 and -3 protein levels in Alzheimer's disease (AD) brains were assessed in comparison with their expression in development and aging. The protein levels of caspase-2 and -3 were significantly increased in AD brains, caspase-2 in the particulate fraction and caspase-3 in both the cytosolic and particulate fractions, compared with controls. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that caspase-3 levels were high from E19 to 2 weeks, while caspase-2 remained high from 4 to 96 weeks, indicating that the expression of caspase-2 and -3 is differentially regulated during development and aging. These results suggest that caspase-2 and -3 are upregulated in AD and that the changes in AD are different from those observed in senescent brains.     

Is tissue culture a viable system with which to examine environmental and hormonal regulation of cold acclimation in woody plants?

In vitro-grown saskatoon berry (Amelanchier alnifolia Nutt.) plantlets were exposed to various hormonal treatments, dormancy-inducing and cold acclimation conditions to determine if this in vitro system would be viable for dormancy/hardiness studies in woody plants. Low temperature induced significant hardiness levels in plantlets to ?27°C after 6 weeks at 4°C but did not approach liquid nitrogen levels of fully hardened, field-grown buds. Control plantlets were consistently killed at ?5°C throughout this period. Significant hardiness was attained under both short and long day/low temperature conditions; however, hardiness was reduced under continuous light or dark treatments. A pre-exposure to the typical short photoperiod regime of woody plants did not significantly increase the rate of acclimation in these plantlets. The presence/absence of phytohormones in the media have a pronounced influence on the ability of plantlets to cold acclimate. Hormone-free media increased hardiness to ?10.5°C after 2 weeks in treatment. Addition of abscisic acid (ABA) increased cold hardiness levels (?12°C) while addition of benzylaminopurine (BAP) to this hormone-free media decreased hardiness to ?5.3°C. A combination of BAP and ABA treatments produced LT₅₀ values intermediate between individual applications of either hormone. Conversely, α-naphthaleneacetic acid (NAA) could not counteract the ABA-induced hardiness. ABA treatments alone were not able to harden plantlets to the extent attained under low temperature acclimation conditions. Further, ABA could not maintain the hardiness levels of cold-acclimating treatments and plantlets de-acclimated to ?9°C in BAP + ABA media. Subculturing in itself significantly elevated cold hardiness in plantlets to ?9°C on BAP + NAA media within 3 days after subculture and thereafter plantlets dehardened to ?5°C. While tissue culture has value in specific cases, caution should be taken when using tissue-cultured plantlets as a system to evaluate environmental regulation of cold acclimation in woody plants, in part, due to the influence of phytohormones in the media.     

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.     

Increase in phospholipase C-delta1 protein levels in aluminum-treated rat brains

The effect of administration of aluminum to rats on the level of three phospholipase C (PLC) isozymes (beta1, gamma1, and delta1) was assessed in a variety of brain tissues. After exposure to aluminum, a statistically significant increase in malondialdehyde, an index of lipid peroxidation, was observed. In addition, there was a significant reduction in the catalytic activity of low molecular weight phosphotyrosine phosphatase, which loses its activity during oxidative stress. This suggests that oxidative stress is induced in brain tissues exposed to aluminum. The protein level of PLC-delta1, but not that of PLC-beta1 or -gamma1, was significantly increased in brains where oxidative stress had been induced. The total PLC activity in aluminum-treated rat brains was significantly higher than that in control brains. These results suggest that PLC-delta1 protein levels in brain tissues are increased by the induction of oxidative stress, giving an explanation for its up-regulation in Alzheimer's disease.     

A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma

We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.     

NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Effects of hyperoxia and acrylonitrile on the phospholipase C isozyme protein levels in rat heart and brain

We previously showed that hyperoxia exerts oxidative stress on the rat cerebral cortex, and the protein levels of phospholipase C (PLC) -beta1 and -delta1, but not PLC-gamma1, were changed. Acrylonitrile (ACN) appears to induce astrocytomas through induction of oxidative stress on the rat brain selectively. This study compared hyperoxia or ACN treatments of rats with respect to lipid peroxidation and PLC levels in the heart and cerebral cortex. Treatment of rats with ACN promoted lipid peroxidation in the heart and cerebral cortex, the percent increase above control being greater in the cortex than heart. Hyperoxia did not cause significant increases in lipid peroxidation in the cerebral cortex or heart. In the ACN-treated cerebral cortex, significant increases in the PLC-beta1 and -delta1 in the cytosol, and PLC-gamma1 in the cytosolic and particulate fractions, and lysate were observed. In the rat heart, in which PLC-beta1 could not be detected, PLC-gamma1 and -delta1 were increased and decreased in the cytosolic and particulate fractions, respectively, by hyperoxia. In addition, the expression level of PLC-gamma1 was decreased in the lysate by the treatment. In the heart treated with ACN, there was no change in the level of PLC-gamma1, while PLC-delta1 was elevated in all fractions. These findings suggested that the expression levels of PLC isozymes are altered by hyperoxia and ACN, but there are apparent differences in these altered levels between the different levels of oxidative stress, and between the organs.