Tools for the study of protein quality control systems: use of truncated homoserine trans-succinylase as a model substrate for ATP-dependent proteolysis in Escherichia coli

Protein quality control, mediated by chaperones and ATP-dependent proteases, is essential for maintaining balanced growth and for regulating critical processes. To study these systems it is necessary to have model substrate proteins. However, most cellular proteins are stable and the few unstable proteins are usually regulatory and present in low concentrations, making them unsuitable for studies, especially in vivo. We present HTS(Delta1-6), a truncated homoserine trans-succinylase (HTS) which is unstable, can be expressed at high levels and has an enzymatic, measurable, activity. This protein can serve as a good model substrate for Escherichia coli ATP-dependent proteolysis.     

Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.     

Solid cancer incidence in atomic bomb survivors: 1958-1998

This is the second general report on radiation effects on the incidence of solid cancers (cancers other than malignancies of the blood or blood-forming organs) among members of the Life Span Study (LSS) cohort of Hiroshima and Nagasaki atomic bomb survivors. The analyses were based on 17,448 first primary cancers (including non-melanoma skin cancer) diagnosed from 1958 through 1998 among 105,427 cohort members with individual dose estimates who were alive and not known to have had cancer prior to 1958. Radiation-associated relative risks and excess rates were considered for all solid cancers as a group, for 19 specific cancer sites or groups of sites, and for five histology groups. Poisson regression methods were used to investigate the magnitude of the radiation-associated risks, the shape of the dose response, how these risks vary with gender, age at exposure, and attained age, and the evidence for inter-site variation in the levels and patterns of the excess risk. For all solid cancers as a group, it was estimated that about 850 (about 11%) of the cases among cohort members with colon doses in excess of 0.005 Gy were associated with atomic bomb radiation exposure. The data were consistent with a linear dose response over the 0- to 2-Gy range, while there was some flattening of the dose response at higher doses. Furthermore, there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15 Gy or less. The excess risks for all solid cancers as a group and many individual sites exhibit significant variation with gender, attained age, and age at exposure. It was estimated that, at age 70 after exposure at age 30, solid cancer rates increase by about 35% per Gy (90% CI 28%; 43%) for men and 58% per Gy (43%; 69%) for women. For all solid cancers as a group, the excess relative risk (ERR per Gy) decreases by about 17% per decade increase in age at exposure (90% CI 7%; 25%) after allowing for attained-age effects, while the ERR decreased in proportion to attained age to the power 1.65 (90% CI 2.1; 1.2) after allowing for age at exposure. Despite the decline in the ERR with attained age, excess absolute rates appeared to increase throughout the study period, providing further evidence that radiation-associated increases in cancer rates persist throughout life regardless of age at exposure. For all solid cancers as a group, women had somewhat higher excess absolute rates than men (F:M ratio 1.4; 90% CI 1.1; 1.8), but this difference disappears when the analysis was restricted to non-gender-specific cancers. Significant radiation-associated increases in risk were seen for most sites, including oral cavity, esophagus, stomach, colon, liver, lung, non-melanoma skin, breast, ovary, bladder, nervous system and thyroid. Although there was no indication of a statistically significant dose response for cancers of the pancreas, prostate and kidney, the excess relative risks for these sites were also consistent with that for all solid cancers as a group. Dose-response estimates for cancers of the rectum, gallbladder and uterus were not statistically significant, and there were suggestions that the risks for these sites may be lower than those for all solid cancers combined. However, there was emerging evidence from the present data that exposure as a child may increase risks of cancer of the body of the uterus. Elevated risks were seen for all of the five broadly classified histological groups considered, including squamous cell carcinoma, adenocarcinoma, other epithelial cancers, sarcomas and other non-epithelial cancers. Although the data were limited, there was a significant radiation-associated increase in the risk of cancer occurring in adolescence and young adulthood. In view of the persisting increase in solid cancer risks, the LSS should continue to provide important new information on radiation exposure and solid cancer risks for at least another 15 to 20 years.     

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.     

Skp2-mediated p27(Kip1) degradation during S/G2 phase progression of adipocyte hyperplasia

p27(Kip1), an important regulator of Cdk2 activity and G1/S transition, is tightly regulated in a cell-type and condition-specific manner to integrate mitogenic and differentiation signals governing cell cycle progression. We show that p27 protein levels progressively declined from mid-G1 through late-G2 phase as density-arrested 3T3-L1 preadipocytes synchronously reentered the cell cycle during early stages of adipocyte differentiation. This dramatic fall in p27 protein accumulation was due, at least in part, to a decrease in protein stability. Specific inhibitors of the 26S proteasome were shown to completely block the decrease in p27 protein levels throughout G1, increase the abundance of ubiquitylated p27 protein, and inhibit G1/S transition resulting in G1 arrest. It is further demonstrated that p27 was phosphorylated on threonine 187 during S phase progression by Cdk2 and that phosphorylated p27 was polyubiquitylated and degraded. Furthermore, we demonstrate that Skp2 and Cks1 dramatically increased during S/G2 phase progression concomitantly with the maximal fall in p27 protein. Complete knockdown of Skp2 with RNA interference partially prevented p27 degradation equivalent to that observed with Cdk2 blockade suggesting that the SCF(Skp2) E3 ligase and other proteasome-dependent mechanisms contribute to p27 degradation during preadipocyte replication. Interestingly, Skp2-mediated p27 degradation was not essential for G1/S or S/G2 transition as preadipocytes shifted from quiescence to proliferation during adipocyte hyperplasia. Finally, evidence is presented suggesting that elevated p27 protein in the absence of Skp2 was neutralized by sequestration of p27 protein into Cyclin D1/Cdk4 complexes.     

Impact of combining planting date and chemical control to reduce larval densities of stem-infesting pests of sunflower in the central plains

The guild of stem-infesting insect pests of sunflower, Helianthus annuus L., within the central Plains is a concern to producers chiefly due to losses caused by plant lodging from the sunflower stem weevil, Cylindrocopturus adspersus (LeConte) (Coleoptera: Curculionidae), and Dectes texanus texanus LeConte (Coleoptera: Cerambycidae). The incidence of a root boring moth, Pelochrista womonana (Kearfott) (Lepidoptera: Tortricidae), also has increased. Experiments were conducted in three locations in Colorado and Kansas during 2001-2003 to investigate the potential of combining planting date and foliar and seed treatment insecticide applications to lower insect stalk densities of these three pests. The impact of these strategies on weevil larval parasitoids also was studied. Eight sunflower stem weevil larval parasitoid species were identified. All were Hymenoptera and included the following (relative composition in parentheses): Nealiolus curculionis (Fitch) (42.6%), Nealiolus collaris (Brues) (3.2%) (Braconidae), Quadrastichus ainsliei Gahan (4.2%) (Eulophidae), Eurytoma tylodermatis Ashmead (13.1%) (Eurytomidae), Neocatolaccus tylodermae (Ashmead) (33.7%), Chlorocytus sp. (1.6%), Pteromalus sp. (0.5%) (Pteromalidae), and Eupelmus sp. (1.0%) (Eupelmidae). The results from this 3-yr study revealed that chemical control was often reliable in protecting the sunflower crop from stem pests and was relatively insensitive to application timing. Although results in some cases were mixed, overall, delayed planting can be a reliable and effective management tool for growers in the central Plains to use in reducing stem-infesting pest densities in sunflower stalks. Chemical control and planting date were compatible with natural mortality contributed by C. adspersus larval parasitoids.     

The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance

UbC is one of two stress-inducible polyubiquitin genes in mammals and is thought to supplement the constitutive UbA genes in maintaining cellular ubiquitin (Ub) levels during episodes of cellular stress. We have generated mice harboring a targeted disruption of the UbC gene. UbC(-/-) embryos die between embryonic days 12.5 and 14.5 in utero, most likely owing to a severe defect in liver cell proliferation. Mouse embryonic fibroblasts from UbC(-/-) embryos exhibit reduced growth rates, premature senescence, increased apoptosis and delayed cell-cycle progression, with slightly, but significantly, decreased steady-state Ub levels. UbC(-/-) fibroblasts are hypersensitive to proteasome inhibitors and heat shock, and unable to adequately increase Ub levels in response to these cellular stresses. Most, but not all of the UbC(-/-) phenotypes can be rescued by providing additional Ub from a poly hemagglutinin-tagged Ub minigene expressed from the Hprt locus. We propose that UbC is regulated by a process that senses Ub pool dynamics. These data establish that UbC constitutes an essential source of Ub during cell proliferation and stress that cannot be compensated by other Ub genes.     

Brief communication: new reconstruction of the Taung endocast

Earlier reconstructions of the Taung endocast, from the juvenile type specimen for Australopithecus africanus, were achieved without benefit of the advanced computer technology that is available today and before morphological differences were identified that distinguish endocasts of Paranthropus from those of A. africanus. Here, we reconstruct and measure a relatively complete virtual endocast of Taung and provide a new cranial capacity estimate of 382 cm(3) and a projected adult capacity of 406 cm(3), which are smaller than previous estimates. Linear measurements and ratios were also obtained from an endocast of Sts 5 and five Paranthropus endocasts and compared with those of Taung. A number of previously unrecognized foramina, processes, and canals are identified in the bony material that adheres to the base of the Taung endocast. The newly reconstructed virtual endocast of Taung displays a number of shape features that sort it more closely with gracile than robust australopithecines, including squared-off frontal lobes in dorsal view, and the shape of the tips of its temporal poles. The Taung endocast also shares some features with Paranthropus endocasts, while other characteristics such as small temporal lobes may be due to its juvenile status. Just how much of Taung's unique morphology is due to its juvenile status may eventually be clarified by comparing its endocast with those from other juvenile australopithecines such as the 3.3-million-year-old juvenile from Dikika, Ethiopia.