Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

Leukemia inhibitory factor receptor signaling negatively modulates nerve growth factor-induced neurite outgrowth in PC12 cells and sympathetic neurons

Nerve growth factor (NGF) is required for the development of sympathetic neurons and subsets of sensory neurons. Our current knowledge on the molecular mechanisms underlying the biological functions of NGF is in part based on the studies with PC12 rat pheochromocytoma cells, which differentiate into sympathetic neuron-like cells upon NGF treatment. Here we report that the expression of leukemia inhibitory factor receptor (LIFR), one of the signaling molecules shared by several neuropoietic cytokines of the interleukin-6 family, is specifically up-regulated in PC12 cells following treatment with NGF. Attenuation of LIFR signaling through stable transfection of antisense- or dominant negative-LIFR constructs enhances NGF-induced neurite extension in PC12 cells. On the contrary, overexpression of LIFR retards the growth of neurites. More importantly, whereas NGF-induced Rac1 activity is enhanced in antisense-LIFR and dominant negative-LIFR expressing PC12 cells, it is reduced in LIFR expressing PC12 cells. Following combined treatment with NGF and ciliary neurotrophic factor, sympathetic neurons exhibit attenuated neurite growth and branching. On the other hand, in sympathetic neurons lacking LIFR, neurite growth and branching is enhanced when compared with wild type controls. Taken together, our findings demonstrate that LIFR expression can be specifically induced by NGF and, besides its known function in cell survival and phenotype development, activated LIFR signaling can exert negative regulatory effects on neurite extension and branching of sympathetic neurons.     

Unbound form of tomato inhibitor-II reveals interdomain flexibility and conformational variability in the reactive site loops

The Potato II (Pot II) family of proteinase inhibitors plays important roles in the constitutive and inducible defense of plants against predation by a wide range of pests. The structural basis of inhibition by a multidomain Pot II family inhibitor was revealed recently by the structure of the ternary complex between the two-headed tomato inhibitor-II (TI-II) and two molecules of subtilisin Carlsberg. Here we report the 2.15-A resolution crystal structure of the unbound form of TI-II that reveals significant conformational flexibility in the absence of bound proteinase molecules. The four independent copies of unbound TI-II in the asymmetric unit of the unit cell display a range of different conformations when compared with the bound form of the inhibitor, most strikingly in the orientations of the inhibitory domains and in the conformations of the reactive site loops. One of the two linker segments (residues 74 to 79) between the two domains as well as the adjacent beta-strand in Domain I (residues 80-85) is well ordered in all four copies of the unbound inhibitor, even though this region appeared to be disordered in the structure of the ternary complex. Conformational flexibility seen in the reactive site loops of unbound TI-II suggests a mechanism by which the inhibitor can balance the need for tight binding with the need for broad inhibitory function.     

Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli

Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited. In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography. Far UV circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand. Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made available for biochemical and biophysical characterizations.     

p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.     

Hypertension and its related factors in Taiwanese elderly people

BACKGROUND: Our study used data collected in Chung-Hsing Village in May 1998 to evaluate the prevalence of hypertension and its correlates in Taiwanese elderly people. METHODS: All of individuals aged 65 and over were recruited as study subjects. A total of 1,093 persons, out of 1,774 registered residents, were contacted by face-to-face interview The response rate was 61.6 percent. However, only 586 respondents had blood tests and completed questionnaires. Analysis in this study was based on these 586 subjects. In order to study the significant correlates of hypertension, the t-test, chi-square analysis, and multivariate logistic regression were used. RESULTS: Our results showed that 66 percent were men and 34 percent were women. The mean age was 73.1 +/- 5.3 years. The proportions of hypertension were 53.09 percent in men and 56.06 percent in women (p > 0.05). After controlling the other covariates, the multivariate logistic regression analysis showed that the significant related factors of hypertension were obesity (OR = 1.88, 95 percent CI = 1.06-3.34, p < 0.05) and renalfunction impairment (OR = 1.69, 95 percent CI = 1.02-2.80, p < 0.05). CONCLUSIONS: The prevalence of hypertension was high in elderly people. Hypertension is significantly associated with obesity and renalfunction impairment in elderly people.     

Helicobacter pylori in familial clusters based on antibody profile

Studies have shown a high prevalence of Helicobacter pylori infection in close communities and that intrafamilial spread during early childhood may be a route of transmission. A total of 72 household members from 21 families were enrolled in this study. Sera from individuals showed 50/72 (69.4%) seropositive for IgG against H. pylori by ELISA. Western blots showed diversity in the protein profiles with molecular masses ranging from approximately 8 to 130 kDa. Cohen's kappa statistical analysis of the blot patterns showed that nine families demonstrated similar profiles (100%), while 4 other families showed varying similarities (17-50%). The results support the hypothesis of intrafamilial transmission of H. pylori. Furthermore, serological studies can be used as an effective approach to determine the familial status in relation to H. pylori infection.     

Adhesion of flowing monocytes to hypoxia-reoxygenation-exposed endothelial cells: role of Rac1, ROS,and VCAM-1

Production ofreactive oxygen species (ROS) by ischemic tissue afterischemia-reperfusion (I/RP) is an important factor that contributes to tissue injury. The small GTPase Rac1 mediates the oxidative burst, and ROS act on signaling pathways involved in expression of inflammatory genes. Because there is evidence implicating monocytes in the pathogenesis of I/RP injury, our objective was todetermine the molecular mechanisms that regulate adhesive interactions between monocytes and hypoxia-reoxygenation (H/RO)-exposed cultured endothelial cells (ECs). When U937 cells were perfused over human umbilical vein ECs at 1 dyn/cm², H (1 h at 1%O₂)/RO (13 h) significantly increased the fluxes of rollingand stably adherent U937 cells. Either EC treatment with theantioxidant pyrrolidine dithiocarbamate (PDTC) or infection withAdRac1N17, which results in expression of the dominant-negative form ofRac1, abolished H/RO-induced ROS production, attenuated rolling, andabolished stable adhesion of U937 cells to H/RO-exposed ECs. Infectionwith AdRac1N17 also abolished H/RO-induced upregulation of vascularcell adhesion molecule (VCAM)-1. In turn, blocking VCAM-1 abolishedU937 cell stable adhesion and slightly increased rolling. We concludedthat the Rac1-dependent ROS partially regulate rolling and exclusivelyregulate stable adhesion of monocytic cells to ECs after H/RO and thatstable adhesion, but not rolling, is mediated by ROS-induced expressionof VCAM-1.

     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.