A dynamic model for nitrogen-stressed lettuce

A previously developed dynamic lettuce model, designed to predict growth and nitrate content under the normal range of glasshouse environmental conditions, has been extended to cover high nitrogen-stress situations. Under severe shortage of nitrogen, lettuce has been observed to grow at a very slow rate, as well as to have abnormally low water content, low reduced-nitrogen content and negligible nitrate content. The new model mimics these observations by adding to the original model a storage compartment for 'excess' carbon. The resulting model has three compartments: (1) 'vacuole', where the soluble non-structural material is stored, and the nitrate : carbon ratio may vary as needed to maintain a constant osmotic potential; (2) 'structure', a metabolically active compartment with fixed chemical composition; and (3) 'excess-carbon', which serves as a long-term storage of 'waterless' carbohydrates. Simulations with the model illustrate its ability to predict the effect of light, temperature and nitrogen in the nutrient solution on the long-term growth and composition of lettuce. They also illustrate the effects of plant size, and the associated relative growth rate, on the characteristic times of transient responses resulting from step changes in the environment.     

Contribution of the Atm protein to maintaining cellular homeostasis evidenced by continuous activation of the AP-1 pathway in Atm-deficient brains

Maintenance of genome stability is essential for keeping cellular homeostasis. The DNA damage response is a central component in maintaining genome integrity. Among of the most cytotoxic DNA lesions are double strand breaks (DSBs) caused by ionizing radiation or radiomimetic chemicals. ATM is missing or inactivated in patients with ataxia-telangiectasia. Ataxia-telangiectasia patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Additional features are immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. Disruption of the mouse Atm locus creates a murine model of ataxia-telangiectasia that exhibits most of the clinical features of the human disease but very mild neuronal abnormality. The ATM protein is a multifunctional protein kinase, which serves as a master regulator of cellular responses to DSBs. There is growing evidence that ATM may be involved in addition to the DSB response in other processes that maintain processes in cellular homeostasis. For example, mounting evidence points to increased oxidative stress in the absence of ATM. Here we report that the AP-1 pathway is constantly active in the brains of Atm-deficient mice not treated with DNA damaging agents. A canonical activation (increased phosphorylation of mitogen-activated protein kinase kinase-4, c-Jun N-terminal kinase, and c-Jun) of the AP-1 pathway was found in Atm-deficient cerebra, whereas induction of the AP-1 pathway in Atm-deficient cerebella is likely to mediate elevated expression of c-Fos and c-Jun. Although Atm(+/+) mice are capable of responding to ionizing radiation by activating stress responses such as the AP-1 pathway, Atm-deficient mice display higher basal AP-1 activity but gradually lose their ability to activate AP-1 DNA-binding activity in response to ionizing radiation. Our results further demonstrate that inactivation of the ATM gene results in a state of constant stress.     

c-Abl tyrosine kinase selectively regulates p73 nuclear matrix association

p73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73 alpha nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73 alpha but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73 alpha is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation.     

Energy metabolism in uncoupling protein 3 gene knockout mice

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.     

Thrombin stimulates the expression of PDGF in lung epithelial cells

Several growth factors, including platelet-derived growth factor (PDGF), have been implicated in the mechanism of lung and airway remodeling. In the present study, we evaluated whether thrombin may promote lung and airway remodeling by increasing PDGF production from lung and airway epithelial cells. Conditioned medium (CM) was prepared by treating epithelial cells with increasing concentrations of thrombin; before use in the assays, CM was treated with hirudin until complete inhibition of thrombin activity. CM from epithelial cells stimulated the proliferation of lung fibroblasts and bronchial smooth muscle cells. Anti-PDGF antibody significantly inhibited this CM proliferative activity, implicating PDGF in this effect. Enzyme immunoassay and RT-PCR demonstrated that thrombin induced the secretion and expression of PDGF from bronchial and alveolar epithelial cells. RT-PCR showed that epithelial cells express the thrombin receptors protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist peptide was also found to induce PDGF secretion from epithelial cells, suggesting that the cellular effect of thrombin occurs via a PAR-1-mediated mechanism. Overall, this study showed for the first time that thrombin may play an important role in the process of lung and airway remodeling by stimulating the expression of PDGF via its cellular receptor, PAR-1.     

Production and purification of SV40 major capsid protein (VP1) in Escherichia coli strains deficient for the GroELS chaperone machine

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.