Doubled haploid plants following colchicine treatment of microspore-derived embryos of oilseed rape (<Emphasis Type="Italic">Brassica napus L.</Emphasis>)

An efficient method for producing doubled haploid plants of oilseed rape (Brassica napus L.) was established using in vitro colchicine treatment of haploid embryos. Haploid embryos in the cotyledonary stage were treated with one of four colchicine concentrations (125, 250, 500 and 1,000 mg/L); for one of three treatment durations (12, 24 and 36 h) at one of the two temperatures (8 and 25°C) and were compared to control embryos (without colchicine treatment). The number of chromosomes, seed recovery, size and density of leaf stomata, and pollen grain size from regenerated plants were determined. No doubled haploid plants were regenerated from control embryos; however, the doubled haploid plants were regenerated from colchicine-treated embryos. A high doubling efficiency, 64.29 and 66.66% of regenerated plants, was obtained from 250 mg/L colchicine treatment for 24 h and 500 mg/L colchicine treatment for 36 h, respectively, at 8°C. Following 500 mg/L colchicine treatment for 36 h, a few plants regenerated (9 plants). At the higher colchicine concentration (1,000 mg/L), no plant regenerated. These results indicate that the colchicine treatment of embryos derived from microspores can induce efficient chromosome doubling for the production of doubled haploid lines of oilseed rape.     

An assessment of the role of domain F and pest sequences in estrogen receptor half-life and bioactivity

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3–4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [³⁵S]methionine/[³⁵]Scysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.     

Population statistics and biological traits of Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae) affected by LC30 of thiamethoxam and pirimicarb

Although selective pesticides are recommended to use in integrated pest management (IPM) programmes, they could adversely affect biological agents. So, the aim of the current study was to investigate the LC₃₀ of pirimicarb and thiamethoxam on Hippodamia variegata. Two pesticides, pirimicarb and thiamethoxam, when applied at concentrations of 1522.8 and 251.3?mg (ai)/L, respectively, on the third instar larvae of the insect, produced 30% mortality. Also, results showed that neither pirimicarb nor thiamethoxam affect stage age distribution (c_x), fecundity (eggs/female) and adult development time. These two pesticides extended preadult duration significantly (p?<?0.0001, F?=?31.22, df?=?122). Also, survival rate (l_x) and age-specific reproductive value (v_x) decreased and some changes in age-specific life expectancy (e_x) happened. Generally, more adverse effects were found in the population treated by thiamethoxam. The results showed that pirimicarb and thiamethoxam have potential to harm the predatory ladybird in IPM programme, though in sublethal doses.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Probing the interaction of silver nanoparticles with tau protein and neuroblastoma cell line as nervous system models

Interestingly pharmaceutical sciences are using nanoparticles (NPs) to design and develop nanomaterials-based drugs. However, up to recently, it has not been well realized that NPs themselves may impose risks to the biological systems. In this study, the interaction of silver nanoparticles (AgNPs) with tau protein and SH-SY5Y neuroblastoma cell line, as potential nervous system models, was examined with a range of techniques including intrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and acridine orange/ethidium bromide (AO/EB) dual staining method. Fluorescence study showed that AgNPs with a diameter of around 10–20 nm spontaneously form a static complex with tau protein via hydrogen bonds and van der Waals interactions. CD experiment revealed that AgNPs did not change the random coil structure of tau protein. Moreover, AgNPs showed to induce SH-SY5Y neuroblastoma cell mortality through fragmentation of DNA which is a key feature of apoptosis. In conclusion, AgNPs may induce slight changes on the tau protein structure. Also, the concentration of AgNPs is the main factor which influences their cytotoxicity. Since, all adverse effects of NPs are not well detected, so probably additional more specific testing would be needed.     

Investigation of cytocrom c oxidase gene subunits expression on the Multiple sclerosis

INTRODUCTION:

Multiple sclerosis (MS) is an autoimmune inflamatory disease, which affects the (Central Nervous System) and leads to the destruction of myelin and atrophy of the axons. Genetic factors, in addition to environmental ones, seem to play a role in MS. Numerous studies have reported mitochondrial defects including a reduction in cytochrome c oxidase (COX) complex function related to the reduction of mitochondrial genes expression in the cortex tissue of patients with MS have been reported.

MATERIALS AND METHODS:

This study aimed to assess COX5B and COX2 genes expression in MS patients and compare it with normal subjects. We determine expression levels of genes COX5B and COX2, and also gene reference ß-actine using real–time polymerase chain reaction (RT-PCR) method. Data were obtained and obtained and standardized with the gene reference and were analyzed using independent sample t-test with SPSS and Excel programs.

RESULT AND DISCUSSION:

The resultshowed COX5B gene expression reduced significant in MS patients compared to normal subjects (P < −0.05) whereas, there was no significant difference in the COX2 gene expression between normal subjects and patients. Thus, it can be claimed that down-regulation of mitochondrial electron transport chain genes supported the hypothesis that hypoxia-like tissue injury in MS may be due to mitochondrial genes, different expression impairment.     

Optimization of the Emerson–Trinder enzymatic reaction by response surface methodology

The Emerson–Trinder reaction has been optimized in this work using an initial rate spectrophotometric method and response surface methodology (RSM). In this investigation, the variation range of critical variables along with the fixed parameters were selected based on a preliminary ‘one at a time’ (OVAT) procedure for the subsequent RSM chemometric analysis as follows: pH (6–10), buffer concentration (50–250?mM), 4-aminoantipyrine (4-AAP) concentration (1–5?mM), temperature (25–45°C). The optimum values of fixed parameters were: 4-fluorophenol (4-FP, 30?mM), horseradish peroxidase (HRP) enzyme activity (0.12?U?mL^?1), and the fixed concentration of the H₂O₂ in the chemometric experiments was 11.4 µM. The non-linear nature of the experimental response of the reaction system was explained by a second-order polynomial equation, which revealed the impact of the experimental factors, their interactions and also their optimum values. The results of the reported RSM analysis proved to be quite appropriate for the design and optimization of this reaction, as illustrated by the relatively high value of the determination coefficient (R²=96.7%) for the fitting of quadratic model, along with the satisfactory results generated by the analysis of variance (ANOVA). All the evaluated analytical characteristics of this method: typical reaction progress curves, resulting linear calibration curve, within-day precisions at low and at high levels, and the upper and lower detection limits were, also, reported. In addition, to check the quality of the optimization and validity of the model, the assay of H₂O₂, in pooled serum matrix and in cosmetic samples, was performed.     

New diprenylated dihyrochalcones from leaves of Artocarpus elasticus

Two new diprenylated dihydrochalcones, elastichalcone A 1 and elastichalcone B 2 and three known compounds were isolated from the leaves of Artocarpus elasticus. Their structures were determined by various spectroscopic techniques (UV, IR, MS, 1D-NMR and 2D-NMR). Elastichalcone B 2 and a known compound exhibited good free radical scavenging activity with IC₅₀ values of 11.30 and 11.89 μg/ml, respectively.     

Linkage to HIV,TB and Non-Communicable Disease Care from a Mobile Testing Unit in Cape Town,South Africa

Background

HIV counseling and testing may serve as an entry point for non-communicable disease screening.

Objectives

To determine the yield of newly-diagnosed HIV, tuberculosis (TB) symptoms, diabetes and hypertension, and to assess CD4 count testing, linkage to care as well as correlates of linkage and barriers to care from a mobile testing unit.

Methods

A mobile unit provided screening for HIV, TB symptoms, diabetes and hypertension in Cape Town, South Africa between March 2010 and September 2011. The yield of newly-diagnosed cases of these conditions was measured and clients were followed-up between January and November 2011 to assess linkage. Linkage to care was defined as accessing care within one, three or six months post-HIV diagnosis (dependent on CD4 count) and one month post-diagnosis for other conditions. Clinical and socio-demographic correlates of linkage to care were evaluated using Poisson regression and barriers to care were determined.

Results

Of 9,806 clients screened, the yield of new diagnoses was: HIV (5.5%), TB suspects (10.1%), diabetes (0.8%) and hypertension (58.1%). Linkage to care for HIV-infected clients, TB suspects, diabetics and hypertensives was: 51.3%, 56.7%, 74.1% and 50.0%. Only disclosure of HIV-positive status to family members or partners (RR=2.6, 95% CI: 1.04-6.3, p=0.04) was independently associated with linkage to HIV care. The main barrier to care reported by all groups was lack of time to access a clinic.

Conclusion

Screening for HIV, TB symptoms and hypertension at mobile units in South Africa has a high yield but inadequate linkage. After-hours and weekend clinics may overcome a major barrier to accessing care.