Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1beta and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes. We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-gamma strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-gamma up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1beta. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-gamma and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.     

Unique in vivo associations with SmgGDS and RhoGDI and different guanine nucleotide exchange activities exhibited by RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14)

We compared the in vivo characteristics of hemagglutinin (HA)-tagged RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14) stably expressed in Chinese hamster ovary (CHO) cells. Proteins co-precipitating with these HA-tagged GTPases were identified by peptide sequencing or by Western blotting. Dominant negative RhoA(Asn-19) co-precipitates with the guanine nucleotide exchange factor (GEF) SmgGDS but does not detectably interact with other expressed GEFs, such as Ost or Dbl. SmgGDS co-precipitates minimally with wild-type RhoA and does not detectably associate with RhoA(Val-14). The guanine nucleotide dissociation inhibitor RhoGDI co-precipitates with RhoA, and to a lesser extent with RhoA(Val-14), but does not detectably co-precipitate with RhoA(Asn-19). Wild-type RhoA is predominantly in the [(32)P]GDP-bound form, RhoA(Val-14) is predominantly in the [(32)P]GTP-bound form, and negligible levels of [(32)P]GDP or [(32)P]GTP are bound to RhoA(Asn-19) in (32)P-labeled cells. Immunofluorescence analyses indicate that HA-RhoA(Asn-19) is excluded from the nucleus and cell junctions. Microinjection of SmgGDS cDNA into CHO cells stably expressing HA-RhoA causes HA-RhoA to be excluded from the nucleus and cell junctions, similar to the distribution of RhoA(Asn-19). Our findings indicate that the expression of RhoA(Asn-19) may specifically inhibit signaling pathways that rely upon the SmgGDS-dependent activation of RhoA.     

Efficacy of pyramiding greenbug (Homoptera: Aphididae) resistance genes in wheat

Durable resistance to greenbug, Schizaphis graminum (Rondani), in wheat is a goal of wheat improvement teams, and one that has been complicated by the regular occurrence of damaging biotypes. Simulation modeling studies suggest that pyramiding resistance genes, i.e., combining more than one resistance gene in a single cultivar or hybrid, may provide more durable resistance than sequential releases of single genes. We examined this theory by pyramiding resistance genes in wheat and testing a series of greenbug biotypes. Resistance genes Gb2, Gb3, and Gb6, and pyramided genes Gb2/Gb3, Gb2/Gb6, and Gb3/Gb6 were tested for effectiveness against biotypes E, F, G, H, and I. By comparing reactions of plants with pyramided genes to those with single resistance genes, we found that pyramiding provided no additional protection over that conferred by the single resistance genes. Based on the results of this test, we concluded that the sequential release of single resistance genes, combined with careful monitoring of greenbug population biotypes, is the most effective gene deployment strategy for greenbug resistance in wheat.     

Distribution of benthic algae in the upper Illinois River basin in relation to geology and land use

• 1 Benthic‐algal distributions in the upper Illinois River basin, IL, U.S.A., were examined in relation to geology, land use, water chemistry and stream habitat using (detrended) (canonical) correspondence analysis, autecological metrics and indicator‐species analysis in order to identify the major environmental gradients influencing community variation.
• 2 Ionic composition and major nutrient [i.e. nitrogen (N) and phosphorus (P)] concentration of surface waters, salinity (Na‐Cl type), substratum type and physiognomic form of dominant species were primary factors contributing to variation in benthic‐algal assemblages of the basin. Basin geology was a significant contributing factor, but the explained variance associated with this factor was less than that related to land use.
• 3 Proportions of algal biomass consisting of cyanophytes, filamentous chlorophytes, halophilic diatoms and diatoms which utilize nitrogen heterotrophically were greater in eutrophic river segments than in less nutrient‐enriched segments. Composition of the benthic flora indicated meso‐eutrophic or eutrophic conditions throughout the basin; there were few diatoms indicative of hypertrophic waters. Shifts in diatom‐assemblage structure in response to nutrient loading provided an incomplete representation of the community‐response curve.
• 4 A weighted‐averages regression model based on total P and benthic‐algal abundances (all divisions included) yielded a highly significant correlation (r² = 0.83) between species‐inferred [WA_(tol)] and observed total P, with systematic bias (increased deviation of residuals) occurring only at concentrations greater than ～ 1.0 mg L^?1 total P. This result indicates that total P regression and calibration models can be predictable for a river basin receiving excessive loadings of phosphorus.

     

Effects of soil and water content on methyl bromide oxidation by the ammonia-oxidizing bacterium Nitrosomonas europaea

Little information exists on the potential of NH(3)-oxidizing bacteria to cooxidize halogenated hydrocarbons in soil. A study was conducted to examine the cooxidation of methyl bromide (MeBr) by an NH(3)-oxidizing bacterium, Nitrosomonas europaea, under soil conditions. Soil and its water content modified the availability of NH(4)(+) and MeBr and influenced the relative rates of substrate (NH(3)) and cosubstrate (MeBr) oxidations. These observations highlight the complexity associated with characterizing soil cooxidative activities when soil and water interact to differentially affect substrate and cosubstrate availabilities.     

New insights into methyl bromide cooxidation by Nitrosomonas europaea obtained by experimenting with moderately low density cell suspensions

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions ( approximately 6 x 10(7) cells ml(-1)) of the NH(3)-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH(4)(+) and 0.44, 0. 22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH(4)(+), 18% of the NH(4)(+), and 35% of the NH(4)(+), respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH(4)(+)-MeBr combinations examined (10 to 20 micromol mg [dry weight] of cells(-1)). Approximately 90% of the NH(3)-dependent O(2) uptake activity and the NO(2)(-)-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO(2)(-) production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO(2)(-) accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO(2)(-)-producing and MeBr-oxidizing activities could not be attributed directly to NH(4)(+) or NH(3) limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO(2)(-) and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH(3)-oxidizing activity.     

Phosphinic analog of methionine inhibits growth of leucosis cell L1210 and transforms to phosphinic analog of S-adenosylmethionine

A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.