Implication of a critical residue (Glu175) in structure and function of bacterial luciferase

Structural properties of a bacterial luciferase mutant, evolved by random mutagenesis, have been investigated. Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. By random mutagenesis, one of the mutants generated by a single mutation on LuxA at position 175 (E175G) resulted in the "slow decay" Xenorhabdus luminescens luciferase was converted into a luciferase with a significantly more rapid decay rate [Hosseinkhani, S., Szittner, R. and Meighen, E.A. (2005) Biochemical Journal 385, 575-580]. A single mutation (E175G), in a loop that connects alpha helix 5 and beta sheet 5 brought about changes in the kinetic and structural properties of the enzyme. Enhancement of tryptophan fluorescence was observed with a lower degree of fluorescence quenching by acrylamide upon mutation. Near- and far-UV circular dichroism spectra of the native and mutant forms suggested formation of an intermediate structure, further supported by 8-anilino-1-naphthalene-sulphonic acid (ANS) fluorescence which indicated lower exposure of hydrophobic residues as a result of mutation. Fluorescence quenching studies utilizing acrylamide indicated a more accessible fluor for the native form. Thus, the E175G point mutation appears to change the enzymatic decay rate by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by Fourier transform IR spectroscopy. Overall, the mutation caused structural changes that go beyond the simple change in orientation of Glu175.     

Association of PNPLA3 rs738409 polymorphism with liver steatosis but not with cirrhosis in patients with HBV infection: Systematic review with meta‐analysis

Background

Hepatitis B virus (HBV) infection is a worldwide health issue and is well known for being the main cause of developing secondary liver complications such as cirrhosis and hepatocellular carcinoma (HCC). The PNPLA3 rs738409 polymorphism has been investigated conclusively with occurrence risk of steatosis and cirrhosis. Therefore, performing a meta‐analysis of the available studies with the aim of clarifying the association between rs738409 and occurrence risk of steatosis and cirrhosis among HBV‐infected patients would be helpful.

Methods

Chronic HBV infection was defined as the persistence of HBsAg for more than 6 months. To gather sufficient data for this meta‐analysis, reliable databases were conclusively searched using appropriate keywords. Only studies that satisfied the inclusion criteria were enrolled in the present study.

Results

This meta‐analysis pooled four studies with 1135 cases of chronic hepatitis B (CHB) to evaluate the impact of PNPLA3 SNP on liver steatosis and also pooled five studies with 3713 cases of CHB to evaluate the impact of PNPLA3 SNP on cirrhosis. The association of rs738409 with each complication was investigated. The rs738409 was found to be associated with steatosis in recessive [p = 4.57 × 10^–6, odds ratio (OR) = 2.85], dominant (p = 4.35 × 10^‐6, OR = 1.84), co‐dominant (p = 6.18 × 10^‐8; OR = 3.74) and allelic (p = 9.79 × 10^‐9; OR = 1.78) models. No association was found between rs738409 and cirrhosis development in recessive (p = 0.99, OR = 1.00), dominant (p = 0.30, OR = 0.92), co‐dominant (p = 0.74; OR = 0.96) and allelic (p = 0.45; OR = 0.96) models.

Conclusions

Although the PNPLA3 rs738409 G allele has been associated with the risk of steatosis in CHB patients, no association between this polymorphism and the risk of cirrhosis was seen.     

The Impact of Dietary and Metabolic Risk Factors on Cardiovascular Diseases and Type 2 Diabetes Mortality in Brazil

Background

Trends in food availability and metabolic risk factors in Brazil suggest a shift toward unhealthy dietary patterns and increased cardiometabolic disease risk, yet little is known about the impact of dietary and metabolic risk factors on cardiometabolic mortality in Brazil.

Methods

Based on data from Global Burden of Disease (GBD) Study, we used comparative risk assessment to estimate the burden of 11 dietary and 4 metabolic risk factors on mortality due to cardiovascular diseases and diabetes in Brazil in 2010. Information on national diets and metabolic risks were obtained from the Brazilian Household Budget Survey, the Food and Agriculture Organization database, and large observational studies including Brazilian adults. Relative risks for each risk factor were obtained from meta-analyses of randomized trials or prospective cohort studies; and disease-specific mortality from the GBD 2010 database. We quantified uncertainty using probabilistic simulation analyses, incorporating uncertainty in dietary and metabolic data and relative risks by age and sex. Robustness of findings was evaluated by sensitivity to varying feasible optimal levels of each risk factor.

Results

In 2010, high systolic blood pressure (SBP) and suboptimal diet were the largest contributors to cardiometabolic deaths in Brazil, responsible for 214,263 deaths (95% uncertainty interval [UI]: 195,073 to 233,936) and 202,949 deaths (95% UI: 194,322 to 211,747), respectively. Among individual dietary factors, low intakes of fruits and whole grains and high intakes of sodium were the largest contributors to cardiometabolic deaths. For premature cardiometabolic deaths (before age 70 years, representing 40% of cardiometabolic deaths), the leading risk factors were suboptimal diet (104,169 deaths; 95% UI: 99,964 to 108,002), high SBP (98,923 deaths; 95%UI: 92,912 to 104,609) and high body-mass index (BMI) (42,643 deaths; 95%UI: 40,161 to 45,111).

Conclusion

suboptimal diet, high SBP, and high BMI are major causes of cardiometabolic death in Brazil, informing priorities for policy initiatives.     

Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O₂ chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (f_V) maximally at 100 μM (EC₅₀ = 24.5 μM). Hypoxia elevated f_V from 93.8 to 145.3 breaths min^-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced f_V at 50 μM (EC₅₀ = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O₂-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O₂ sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.     

HHEX gene polymorphisms and type 2 diabetes mellitus: A case-control report from Iran

Genome-wide association studies indicated that hematopoietically-expressed homeobox (HHEX) gene is a remarkable candidate for type 2 diabetes (T2D) mellitus susceptibility in spite of the fact that the results are ambiguous in some cases. So, this study aimed to evaluate the possible correlation between HHEX gene polymorphisms and T2D development in a sample of the Iranian population. The rs1111875G/A, rs7923837A/G, and rs5015480C/T HHEX gene polymorphisms were genotyped in 250 cases and 250 matched (age and sex) healthy controls using tetra-amplification-refractory mutation system-polymerase chain reaction method. The finding revealed the all measured inheritance models of rs1111875G/A and of rs5015480C/T variants dramatically increase the risk of T2D while another polymorphism (rs7923837A/G) was not associated with risk/protective role in T2D. The results indicated that rs1111875G/A and rs5015480C/T may contribute to the enhancement of T2D risk in a sample of the southeast Iranian population.     

Molecular characterization and expression of cyclic nucleotide gated ion channels 19 and 20 in Arabidopsis thaliana for their potential role in salt stress

Cyclic nucleotide gated ion channels (CNGCs) in plants have very important role in signaling and development. The study reports role of CNGC19 and CNGC20 in salt stress in A. thaliana. In-silico, genome wide analysis showed that CNGC19 and CNGC20 are related to salt stress with maximum expression after 6 h in A. thaliana. The position of inserted T-DNA was determined (in-vivo) through TAIL-PCR for activation tagged mutants of CNGC19 and CNGC20 under salt stress. The expression of AtCNGC19 and AtCNGC20 after cloning under 35S promoter of expression vectors pBCH1 and pEarleyGate100 was determined in A. thaliana by real-time PCR analysis. Genome wide analysis showed that AtCNGC11 had lowest and AtCNGC20 highest molecular weight as well as number of amino acid residues. In-vivo expression of AtCNGC19 and AtCNGC20 was enhanced through T-DNA insertion and 35S promoter in over-expressed plants under high salt concentration. AtCNGC19 was activated twice in control and about five times under 150 mM NaCl stress level, and expression value was also higher than AtCNGC20. Phenotypically, over-expressed plants and calli were healthier while knock-out plants and calli showed retarded growth under salinity stress. The study provides new insight for the role of AtCNGC19 and AtCNGC20 under salt stress regulation in A. thaliana and will be helpful for improvement of crop plants for salt stress to combat food shortage and security.     

mDia1 Targets v-Src to the Cell Periphery and Facilitates Cell Transformation,Tumorigenesis, and Invasion

The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.The small GTPase Rho functions as a molecular switch in cell morphogenesis through remodeling of the actin cytoskeleton (3, 14). Rho cycles between the inactive GDP-bound form and the active GTP-bound form. This process is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) specific to Rho; the former group catalyzes the exchange of GDP to GTP (50), and the latter accelerates the hydrolysis of bound GTP (24). When Rho is activated in fibroblasts, actin stress fibers are formed. Rho proteins are frequently overexpressed in human cancers, such as cancers of the colon and breast and lung and testicular germ cell tumors (34). A positive correlation between the expression level of RhoA and disease progression was also reported in breast cancer and testicular germ cell tumors. RhoC, on the other hand, has been repeatedly identified as a gene positively associated with metastasis (4, 21, 40). The clinical significance of Rho in cancer is further implicated by a discovery that a RhoA GAP named Dlc-1 (deleted liver cancer 1) functions as a tumor suppressor in humans (47). Thus, it was known for some time that heterozygous deletions on chromosome 8p22 are common in human tumors, such as cancers of the breast, prostate, lung (5, 22), and especially liver (15). Recent studies have revealed a strong association of deletion of DLC-1 in this region with clinical cancers, and complementary in vitro experiments showed that DLC-1 functions as a potent tumor suppressor, depletion of which causes RhoA hyperactivation and results in tumorigenesis in harmony with other oncogenes, such as Myc and Ras (47). Importantly, heterozygous deletions in chromosome 8p22 are found to be nearly as common as that of TP53 in clinical cancers, indicating the significant importance of DLC-1 and Rho signaling in clinical tumors (18). Consistent with these findings, there are several reports on the requirement of Rho activity in cell transformation in vitro. For example, coexpression of Raf and dominant active RhoA facilitates focus formation, and expression of dominant-negative RhoA suppresses oncogenic Ras-induced focus formation in NIH 3T3 cells (30). In addition, active forms of Rho GEFs, such as Dbl and Ect2, have potent transforming activities in cultured cells in vitro (31). Thus, there are ample in vitro and clinical data indicating the involvement of Rho signaling in oncogenesis.Cell transformation often leads to a change in cell morphology. This morphological change associates with a change in the organization of actin filaments. Nontransformed cells often have thick bundled actin fibers known as stress fibers. When transformed by some oncogenes, such as Ras and v-Src, the actin stress fibers disappear and the cells dramatically alter their shape to the round refractile cell body (49). Alternatively, actin dot structures called podosomes are often formed. This remodeling of the cytoskeleton is believed to contribute to several aspects of the transformed phenotype, including adhesion-independent cell growth and increased migration abilities. Such actin remodeling associated with oncogenesis appears at odds with the requirement of Rho signaling in oncogenesis, because Rho activation leads to formation of actin fibers. Thus, there is a paradox of why transformed cells require Rho signaling yet show dissolution of actin cytoskeleton (27).Among many Rho effectors, two effector molecules, named mDia (44) and ROCK (11), have important roles in actin cytoskeleton remodeling (27). mDia produces straight actin filaments by catalyzing actin nucleation and polymerization, and ROCK activates myosin to cross-link actin filaments for induction of actomyosin bundles and contractility. Further, mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action (42). Thus, actin remodeling inside the cell can be determined primarily by the balance between mDia and ROCK activities. Of the two, the involvement of ROCK in tumors has been widely examined by the use of its small molecule inhibitors, such as Y-27632 (26, 43), and the Rho-ROCK pathway has been strongly implicated in cancer migration and tumor metastasis and invasion. On the other hand, the role of ROCK in oncogenesis remains ambiguous. While its requirement in Ras-induced cell transformation was indicated by the use of Y-27632, examination in Ras-transformed cells revealed that the majority of ROCK is sequestered in an inactive pool by sustained extracellular signal regulated-kinase (ERK)-mitogen-activated protein (MAP) kinase activity under active Ras (33), which might be one of the mechanisms for dissolution of stress fibers found in Ras transformants. Thus, how Rho signaling contributes to oncogenesis remains an open question.Study of Rho effectors other than ROCK has been hampered by the absence of available inhibitors. Recently we generated mDia1 knockout mice (36). Here, we used mouse embryonic fibroblast (MEF) cells derived from these mice and analyzed the involvement of mDia1 and its mechanism of action in v-Src-induced cell transformation and tumorigenesis. v-Src is the oldest widely studied oncogene, yet it remains unknown where in the cell it exerts its oncogenic potential. It was previously reported that temperature-sensitive (ts) v-Src accumulates in the perinuclear region at the restrictive temperature and migrates to the periphery upon a temperature shift in a manner dependent on the actin cytoskeleton and Rho (6, 37). However, the underlying mechanism of this v-Src targeting has not been fully elucidated, and whether this targeting is required for v-Src-induced oncogenesis remains to be shown. Using mDia1-deficient MEF cells, we have addressed these questions. Here we have shown that actin filaments produced by mDia1 are a prerequisite for v-Src targeting, and this v-Src targeting is critical for its role in cell transformation and tumorigenesis. Our results further show that the Rho-mDia1 pathway functions as a link between oncogenesis and invasion.