GRK2 negatively regulates glycogen synthesis in mouse liver FL83B cells

G-protein-coupled receptor (GPCR) kinases (GRKs) are serine/threonine kinases that desensitize agonist-occupied classical GPCRs. Although the insulin receptor (IR) is a tyrosine kinase receptor, the IR also couples to G-proteins and utilizes G-protein signaling components. The present study was designed to test the hypothesis that GRK2 negatively regulates IR signaling. FL83B cells, derived from mouse liver, were treated with insulin and membrane translocation of GRK2 was determined using immunofluoresecence and Western blotting. Insulin caused an increase in the translocation of GRK-2 from cytosol to the plasma membrane. To determine the role of GRK2 in IR signaling, GRK2 was selectively down-regulated ( approximately by 90%) in FL83B cells using a small interfering RNA technique. Basal as well as insulin-induced glycogen synthesis (measured by d-[U-(14)C]glucose incorporation) was increased in GRK2-deficient cells compared with control cells. Similarly, GRK2 deficiency increased the basal and insulin-stimulated phosphorylation of Ser(21) in glycogen synthase kinase-3alpha. Insulin-induced tyrosine phosphorylation of the IR was similar in control and GRK2-deficient cells. Basal and insulin-stimulated phosphorylation of Tyr(612) in insulin receptor subunit 1 was significantly increased while phosphorylation of Ser(307) was decreased in GRK2-deficient FL83B cells compared with control cells. Chronic insulin treatment (24 h) in control cells caused an increase in GRK2 (56%) and a decrease in IR (50%) expression associated with the absence of an increase in glycogen synthesis, suggesting impairment of IR function. However, chronic insulin treatment (24 h) did not decrease IR expression or impair IR effects on glycogen synthesis in GRK2-deficient cells. We conclude that (i) GRK2 negatively regulates basal and insulin-stimulated glycogen synthesis via a post-IR signaling mechanism, and (ii) GRK2 may contribute to reduced IR expression and function during chronic insulin exposure.     

The first report on the induction of hairy roots in Trapa natans,a unique aquatic plant with photosynthesizing roots

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation.

     

Association of single nucleotide polymorphisms of CACNA1A gene in migraine

INTRODUCTION:

Migraine is a chronic, neurovascular polygenic disease where genetic and environmental factors are involved in its etiology. Dysfunction of neuronal ion transportation can provide a model for predisposition for common forms of migraine. Mutations in genes encoding ion channels disturb the rhythmic function of exposed tissue that may also explain the episodic nature of migraine. Our aim was to study the single nucleotide polymorphisms of CACNA1A gene in migraine patients.

MATERIALS AND METHODS:

The subjects were the patients of migraine, in the age range of 18-80 years, diagnosed by a Neurologist, as per the diagnostic criteria of International Headache Society (IHS) Classification 2004 after excluding other causes of headache by clinical examination and relevant investigations.The controls were the age and sex matched healthy persons from the same population excluding the relatives of patients. Only those patients and the controls, who voluntarily participated in the study, were taken and their blood samples were taken for the study. Deoxyribonucleic acid (DNA) extraction was performed according to the manufacturer''s protocol for Qiagen DNA extraction kits (Qiagen, Hilden, NRW, Germany). DNA content was quantified by spectrophotometric absorption (Nanodrop Spectrophotometer, BioLab, Scoresby, VIC, Australia). Polymerase chain reaction was performed using an iCycler Thermal Cycler (Bio.Rad, Hercules, CA, USA). The polymorphic analysis of CACNA1A gene was carried out by two methods: Restriction fragment length polymorphism and sequencing.

RESULTS:

The study included a total of 25 patients of migraine, diagnosed on out-patient department basis as per IHS Classification 2004 and compared with age and sex matched 25 healthy controls. Most of the patients 23 (92%) were below the age of 50 years. 20 of the patients (80%) were females and 5 (20%) were males. The polymorphic analysis of CACNA1A gene revealed the presence of only the wild form of the gene for the codon E993V in both case and control groups.

CONCLUSION:

In our study, we could not find any polymorphism of CACNA1A gene in the selected patients. Instead the wild type of genotype was found in both patients and controls. This negative result presented here, implies that if the CACNA1A gene is involved in typical migraine (with and without aura), its contribution is very modest and therefore difficult to discern. Nevertheless, there are other genes that could be considered potential candidates for typical migraine susceptibility for which further research is needed.     

用微核试验研究小枣的抗突变作用

目的:应用微核试验(Micronucleus test,MCNT)技术研究小枣汁对环磷酰胺(CP)的抗突变作用的影响.方法:使用蚕豆根尖细胞微核技术进行微核率检测.结果:结果表明,不同浓度的小枣汁对环磷酰胺诱发的蚕豆根尖细胞微核有明显的抑制作用,1号-阳性和阴性对照试验组的微核率分别为52‰、30‰、26‰、16‰、10‰、9‰、70‰、7‰,小枣汁使CP诱发的微核率从52.0±5.3‰降低至9.7±2.5(P<0.001).结论:说明小枣汁对CP诱发的遗传损伤具有明显的修复、保护以及抗突变作用.     

Small numbers of residual tumor cells at the site of primary inoculation are critical for anti-tumor immunity following challenge at a secondary location

Luciferase-transduced B16 murine melanoma cells (luc-B16) inoculated in ear skin do not form tumors but prevent tumor formation by luc-B16 cells injected into the footpad. To determine the requirements for such immunity, we followed the fate of luc-B16 cells following ear injection. Surprisingly, small numbers of viable luc-B16 cells were detected in tumor-free mouse skin for up to 60 days post-inoculation. After 1 week, the number of Foxp3⁺CD4⁺CD25⁺ T cells (along with foxp3 mRNA expression) increased rapidly in the injected ear skin. Residual tumor cells in ears were reduced in mice treated with anti-CD25 mAb and in CD4-deficient mice, but increased in CD8-deficient mice. Strikingly, the loss of luc-B16 cells in the ear skin, either spontaneously or following amputation of the injected ear, resulted in significantly enhanced tumor formation by parental and luciferase-expressing B16 cells after footpad injection. These studies suggest that small numbers of tumor cells (possibly regulated by CD4⁺CD25⁺ regulatory T cells expressing Foxp3) are required for effective host anti-tumor responses at alternate inoculation sites.     

Hydrolysis of 2,4-dinitrophenylphosphate promoted by hydroxoaquatetraaminecobalt(III) ions

The hydrolysis of 2,4-dinitrophenylphosphate (DNPP) to orthophosphate and 2,4-dinitrophenolate (DNP) is accelerated in the presence of excess tn₂Co(H₂O)_2³⁺ or trpnCo(H₂O)_2³⁺ at rates which maximize at pHs close to those at which the hydroxoaquatetraaminecobalt(III) complex concentrations peak (tn₂, pH 6.4; trpn, pH 6.0; tn = trimethylenediamine; trpn = 3,3′,3″-triaminotripropylamine). For dilute DNPP solutions (10⁻⁴ M) the hydrolysis rates (25°C, 0.50 M NaClO₄) increase with increasing Co/DNPP ratio in ways that are qualitatively as well as quantitatively different for the two systems (trpn: steady increase moving toward rate saturation, higher rates; tn₂: ‘S’-shaped curve with very low rates at low ratios, lower rates compared to trpn for comparable ratios). For the trpn system the results are interpreted on the basis of pre-equilibrium formation of the 1:1 monodentate-DNPP cobalt complex by substitution of the labile water on cobalt, and rate-determining attack by the cis-coordinated hydroxide on the phosphorus center to affect hydrolysis. For the tn₂ system the main path to hydrolysis is through a 2:1 cobalt to DNPP complex in which attack by a cis-coordinated hydroxide is again involved. The more complex rate behavior and the slower hydrolysis rates observed for tn₂ system result from the formation of cis and trans isomers in which trans arrangements of coordinated DNPP and hydroxide leave the latter unavailable to participate in intramolecular hydrolysis. Computer fitting of the observed rate data provides values of equilibrium and rate constants for the two systems. Detailed mechanistic schemes are proposed. For the trpn system at pH 6.0 and a 25:1 cobalt to DNPP ratio (5 × 10⁻⁵ M DNPP) the observed acceleration over hydrolysis in the absence of the cobalt complex is 3 × 10³; the calculated specific rate constant for hydrolysis in the reactive 1:1 complex (k 0.2 s⁻¹) represents an acceleration over the unpromoted rate of 3 × 10⁴.     

The Fraction of Cancer Attributable to Ways of Life,Infections, Occupation,and Environmental Agents in Brazil in 2020

Many human cancers develop as a result of exposure to risk factors related to the environment and ways of life. The aim of this study was to estimate attributable fractions of 25 types of cancers resulting from exposure to modifiable risk factors in Brazil. The prevalence of exposure to selected risk factors among adults was obtained from population-based surveys conducted from 2000 to 2008. Risk estimates were based on data drawn from meta-analyses or large, high quality studies. Population-attributable fractions (PAF) for a combination of risk factors, as well as the number of preventable deaths and cancer cases, were calculated for 2020. The known preventable risk factors studied will account for 34% of cancer cases among men and 35% among women in 2020, and for 46% and 39% deaths, respectively. The highest attributable fractions were estimated for tobacco smoking, infections, low consumption of fruits and vegetables, excess weight, reproductive factors, and physical inactivity. This is the first study to systematically estimate the fraction of cancer attributable to potentially modifiable risk factors in Brazil. Strategies for primary prevention of tobacco smoking and control of infection and the promotion of a healthy diet and physical activity should be the main priorities in policies for cancer prevention in the country.     

TP53 mutations as biomarkers for cancer epidemiology in Latin America: current knowledge and perspectives

Due to particular social and economical development, and to the impact of globalization of lifestyles, Latin America shows a superposition of cancers that are frequent in low resource countries (gastric, oesophageal squamous cell and cervical cancers) and high resource countries (cancers of breast, colon and rectum, lung and prostate). Latin America thus offers opportunities for investigating the impact on changing lifestyle patterns on the occurrence of cancer. At the molecular level, mutations in the tumor suppressor gene TP53 are common in many cancers and their distribution can be informative of the nature of the mutagenic mechanisms, thus giving clues to cancer etiology and molecular pathogenesis. However most of the data available are derived from studies in industrialized countries. In this review, we discuss current trends on cancer occurrence in Latin American countries, and we review the literature available on TP53 mutations and polymorphisms in patients from Latin America. Overall, a total of 285 mutations have been described in 1213 patients in 20 publications, representing 1.5% of the total number of mutations reported world-wide. Except for hematological cancers, TP53 mutation frequencies are similar to those reported in other regions of the world. The only tumor site presenting significant differences in mutation pattern as compared to other parts of the world is colon and rectum. However, this difference is based on a single study with 35 patients. Recently, a characteristic TP53 mutation at codon 337 (R337H) has been identified in the germline of children with adrenocortical carcinoma in Southern Brazil. Further and better focused analyses of TP53 mutation patterns in the context of epidemiological studies, should help to improve our understanding of cancer etiology in order to develop appropriate health policies and public health programs in Latin America.     

Modifications and deletions of helices within the hairpin ribozyme-substrate complex: an active ribozyme lacking helix 1

Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.