Structural and functional insights of Wilson disease copper-transporting ATPase

Wilson disease is an autosomal recessive disorder of copper metabolism. The gene for this disorder has been cloned and identified to encode a copper-transporting ATPase (ATP7B), a member of a large family of cation transporters, the P-type ATPases. In addition to the core elements common to all P-type ATPases, the Wilson copper-transporting ATPase has a large cytoplasmic N-terminus comprised six heavy metal associated (HMA) domains, each of which contains the copper-binding sequence motif GMT/HCXXC. Extensive studies addressing the functional, regulatory, and structural aspects of heavy metal transport by heavy metal transporters in general, have offered great insights into copper transport by Wilson copper-transporting ATPase. The findings from these studies have been used together with homology modeling of the Wilson disease copper-transporting ATPases based on the X-ray structure of the sarcoplasmic reticulum (SR) calcium-ATPase, to present a hypothetical model of the mechanism of copper transport by copper-transporting ATPases.     

Ultraviolet light sensitivity,unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of ³H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m²), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.     

Low growth temperature-induced changes to pigment composition and photosynthesis in Zea mays genotypes differing in chilling sensitivity

The effects on pigment composition and photosynthesis of low temperature during growth were examined in the third leaf of three chilling-tolerant and three chilling-sensitive genotypes of Zea mays L. The plants were grown under a controlled environment at 24 or 14 °C at a photon flux density (PFD) of 200 or 600 μ mol m^–2 s^–1. At 24 °C, the two classes of genotypes showed little differences in their photosynthetic activity and their composition of pigments. At 14 °C, photosynthetic activity was considerably reduced but the chilling-tolerant genotypes displayed higher photosynthetic rates than the chilling-sensitive ones. Plants grown at 14 °C showed a reduced chlorophyll (Chl) a + b content and a reduced Chl a / b ratio but an increased ratio of total carotenoids to Chl a + b . These changes in pigment composition in plants grown at low temperature were generally more pronounced in the chilling-sensitive genotypes than in the tolerant ones, particularly at high PFD. Furthermore, at 14 °C, all the genotypes showed increased ratios of lutein, neoxanthin and xanthophyll-cycle carotenoids to Chl a + b but a reduced ratio of β -carotene to Chl a + b , especially at high PFD. At 14 °C, the chilling-tolerant genotypes, when compared with the sensitive ones, were characterized by higher contents of β -carotene and neoxanthin, a lower content of xanthophyll-cycle carotenoids, a lower ratio of xanthophylls to β -carotene, and less of their xanthophyll-cycle carotenoid pool in the form of zeaxanthin. These differences between the two classes of genotypes were more pronounced at high PFD than at low PFD. The results are discussed in terms of the relationship that may exist in maize between pigment composition and the capacity to form an efficient photosynthetic apparatus at low growth temperature.     

Design, synthesis, and biological evaluation of novel water-soluble N-mustards as potential anticancer agents

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xenografts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa′ at the maximum tolerable dose with relatively low toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa′ in rats was also investigated. The current studies suggest that this agent is a promising candidate for preclinical studies.     

Early development and pathogenesis of Lobatostoma manteri Rohde (Trematoda: Aspidogastrea)

The prosobranch Planaxis sulcatus is reported as a new natural host of Lobatostoma manteri at Heron Island, Great Barrier Reef. Planaxis sulcatus and Cerithium moniliferum were experimentally infected with large numbers of eggs. The larvae hatch in the stomach and migrate immediately along the ducts of the digestive gland into the digestive follicles. The larvae feed on the secretion and probably epithelial cells of the follicles. The acetabulum is used for adhesion to the epithelium and contributes to its erosion. In heavily infected snails, the digestive follicles disappear gradually and the larvae live in cavities lined by a flattened epithelium, parts of which show secretory activity. In snails dissected 47–49 and 65–66 days after infection, the cavities are fused, forming several large spaces which communicate with each other; only small parts of the epithelium are still secretory. Concentrations of amoebocytes occur in the walls of the digestive gland and in the wall between digestive gland and stomach of infected Planaxis. Some young worms were found in the stomach of Planaxis. No tissue reactions were seen around the stomach except in the wall between digestive gland and stomach. In Cerithium with 65–67 days old infection, the cavities contain much detritus and disintegrating cells, the epithelium is practically non-secretory and surrounded by loose connective tissue. In larvae with a body length of approximately 0·5–0·6 mm, the acetabulum begins to divide into alveoli and its anterior end grows forward; the anterior alveoli gradually increase in size and new alveoli are formed in the posterior undivided zone. In two specimens of approximately 1·3 mm body length, the whole adhesive disk was divided into half the number of alveoli usually found in adults. Allometric shifts during growth of the worms are analysed.     

Microbial nitrate respiration--genes, enzymes and environmental distribution

Nitrate is a key node in the network of the assimilatory and respiratory nitrogen pathways. As one of the ‘fixed’ forms of nitrogen, nitrate plays an essential role in both nature and industry. For bacteria, it is both a nitrogen source and an electron acceptor. In agriculture and wastewater treatment, nitrate respiration by microorganisms is an important issue with respect to economics, greenhouse gas emission and public health. Several microbial processes compete for nitrate: denitrification, dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation. In this review we provide an up to date overview of the organisms, genes and enzymes involved in nitrate respiration. We also address the molecular detection of these processes in nature. We show that despite rapid progress in the experimental and genomic analyses of pure cultures, knowledge on the mechanism of nitrate reduction in natural ecosystems is still largely lacking.     

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.     

Temperature acclimation in a biochemical model of photosynthesis: a reanalysis of data from 36 species

The Farquhar et al. model of C(3) photosynthesis is frequently used to study the effect of global changes on the biosphere. Its two main parameters representing photosynthetic capacity, V(cmax) and J(max), have been observed to acclimate to plant growth temperature for single species, but a general formulation has never been derived. Here, we present a reanalysis of data from 36 plant species to quantify the temperature dependence of V(cmax) and J(max) with a focus on plant growth temperature, i.e. the plants' average ambient temperature during the preceding month. The temperature dependence of V(cmax) and J(max) within each data set was described very well by a modified Arrhenius function that accounts for a decrease of V(cmax) and J(max) at high temperatures. Three parameters were optimized: base rate, activation energy and entropy term. An effect of plant growth temperature on base rate and activation energy could not be observed, but it significantly affected the entropy term. This caused the optimum temperature of V(cmax) and J(max) to increase by 0.44 degrees C and 0.33 degrees C per 1 degrees C increase of growth temperature. While the base rate of V(cmax) and J(max) seemed not to be affected, the ratio J(max) : V(cmax) at 25 degrees C significantly decreased with increasing growth temperature. This moderate temperature acclimation is sufficient to double-modelled photosynthesis at 40 degrees C, if plants are grown at 25 degrees C instead of 17 degrees C.