Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copperATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (Wnd) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The fx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell, Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multivesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of fx mice.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.

Integrable variant of the one-dimensional Hubbard model

A new integrable model which is a variant of the one-dimensional Hubbard model is proposed. The integrability of the model is verified by presenting the associated quantum R-matrix which satisfies the Yang-Baxter equation. We argue that the new model possesses the SO(4) algebra symmetry, which contains a representation of the eta-pairing SU(2) algebra and a spin SU(2) algebra. Additionally, the algebraic Bethe ansatz is studied by means of the quantum inverse scattering method. The spectrum of the Hamiltonian, eigenvectors, as well as the Bethe ansatz equations, are discussed. (C) 2002 American Institute of Physics.

Constitutive expression of a phenylalanine ammonia-lyase gene from Stylosanthes humilis in transgenic tobacco leads to enhanced disease resistance but impaired plant growth

Transgenic tobacco plants expressing a phenylalanine ammonia-lyase cDNA (ShPAL), isolated from Stylosanthes humilis, under the control of the 35S promoter of the cauliflower mosaic virus were produced to test the effect of high level PAL expression on disease resistance. The transgenic plants showed up to eightfold PAL activity and were slowed in growth and flowering relative to non-transgenic controls which have segregated out the transgene. The expression of the ShPAL transgene and elevated PAL levels were correlated and stably inherited. In T-1 and T-2 tobacco plants with increased PAL activity, lesion expansion was significantly reduced by up to 55% on stems inoculated with the Oomycete pathogen Phytophthora parasitica pv. nicotianae, Lesion area was significantly reduced by up to 50% on leaves inoculated with the fungal pathogen Cercospora nicotianae. This study provides further evidence that PAL has a role in plant defence. (C) 2002 Elsevier Science Ltd. All rights reserved.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Complex language functions and subcortical mechanisms: evidence from Huntington's disease and patients with non-thalamic subcortical lesions

The neuropathological changes associated with Huntington's disease (HD) are most marked in the head of the caudate nucleus and, to a lesser extent, in the putamen and globus pallidus, suggesting that at least part of the language impairments found in patients with HD may result from non-thalamic subcortical (NTS) pathology. The present study aimed to test the hypothesis that a signature profile of impaired language functions is found in patients who have sustained damage to the non-thalamic subcortex, either focally induced or resulting from neurodegenerative pathology. The language abilities of a group of patients with Huntington's disease (n=13) were compared with those of an age- and education-matched group of patients with chronic NTS lesions following stroke (n=13) and a non-neurologically impaired control group (n=13). The three groups were compared on language tasks that assessed both primary and more complex language abilities. The primary language battery consisted of The Western Aphasia Battery and The Boston Naming Test, whilst the more complex cognitive-linguistic battery employed selected subtests from The Test of Language Competence-Expanded, The Test of Word Knowledge and The Word Test-Revised. On many of the tests of primary language function from the Western Aphasia Battery, both the HD and NTS participants performed in a similar manner to the control participants. The language performances of the HD participants were significantly more impaired (p<0.05 using modified Bonferroni adjustments) than the control group, however, on various lexico-semantic tasks (e. g. the Boston Naming Test and providing definitions), on both single-word and sentence-level generative tasks (e. g. category fluency and formulating sentences), and on tasks which required interpretation of ambiguous, figurative and inferential meaning. The difficulties that patients with HD experienced with tasks assessing complex language abilities were strikingly similar, both qualitatively and quantitatively, to the language profile produced by NTS participants. The results provide evidence to suggest that a signature language profile is associated with damage to the non-thalamic subcortex resulting from either focal neurological insult or a degenerative disease.

Regulatory interactions in Arabidopsis thaliana acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) contains catalytic and regulatory subunits, the latter being required for sensitivity to feedback regulation by leucine, valine and isoleucine. The regulatory subunit of Arabidopsis thaliana AHAS possesses a sequence repeat and we have suggested preciously that one repeat binds leucine while the second binds valine or isoleucine, with synergy between the two sites. We have mutated four residues in each repeat, based on a model of the regulatory subunit. The data confirm that there are separate leucine and valine/isoleucine sites, and suggest a complex pathway for regulatory signal transmission to the catalytic subunit. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Occult Axillary Lymph Node Metastases in Breast Cancer Do Matter: Results of 10-Year Survival Analysis

Axillary lymph node status is one of the most powerful prognostic factors for patients with breast cancer and is often critical in stratifying patients into adjuvant treatment regimens. In 203 apparently node-negative cases of breast cancer, a combination of immunohistochemical staining and step-sectioning identified occult metastases in 25% of cases. Ten-year follow-up information is available for these patients. Histologic features of the primary tumor and immunohistochemical staining for estrogen receptor, progesterone receptor, Her-2, and p53 were also evaluated. With multivariate analysis, both occult metastases and higher histologic grade of the primary tumor were independent predictors of disease-free survival. Histologic grade was the only significant independent predictor of overall survival. Estrogen receptor, progesterone receptor, Her-2, and p53 status did not predict the presence of metastases or survival when all tumor types were considered together. Metastases >0.5 mm significantly predicted a poorer disease-free survival when invasive ductal carcinomas were considered alone. Histologic grade was significantly associated with disease-free survival in the premenopausal and perimenopausal patients but not in the postmenopausal patients. The presence of occult metastases approached significance for overall survival in the premenopausal and perimenopausal patients but not in the postmenopausal patients.