Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

Functional bitter taste receptors are expressed in brain cells

Hepatic stellate cells (HSC) store retinoids and upon activation differentiate into myofibroblast-like cells, a process whereby they lose their retinoid-containing lipid droplets. We reported earlier, activation of tissue factor (TF) in our MCT/LPS hepatotoxicity model. We now report the involvement of TF in the release of retinoid receptors RAR-α and RXR-α as accumulated lipid droplet during monocrotaline/lipopolysaccharide (MCT/LPS)-liver injury. Constitutive expression of RAR-α was observed in HSCs and endothelial cells of bile duct and portal vein, while expression of RXR-α was observed in certain pericentral hepatocytes and HSCs. Administration of sub-toxic doses of MCT or LPS strongly increased TF and RXR-α but not RAR-α expressions in HSCs and hepatocytes. However MCT/LPS co-treatment showed insoluble droplets containing RAR-α and RXR-α in the vicinity of the necrotic areas. Blocking TF with TF antisense oligonucleotides (TF-AS ODN) led to normal hepatocyte expression of RXR-α and upregulated the expression of RAR-α in HSCs. This study shows clear evidence of in vivo release of RAR-α and RXR-α as insoluble lipid droplets in liver injury. It is possible that these insoluble droplets of RAR-α and RXR-α could be used as markers for liver injury in general and activation of HSCs in particular. RXR-α appears to be a more sensitive than RAR-α as it was affected by even the subtoxic doses of MCT or LPS. The fact that TF-AS treatment not only down-regulated TF but also obliterated the release of RAR-α and RXR-α as insoluble lipid droplets in hepatocytes points towards TF being an important regulatory molecule for RAR-α and RXR-α.     

B family DNA polymerases asymmetrically recognize pyrimidines and purines

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.     

Baroreflex control of muscle sympathetic nerve activity as a mechanism for persistent sympathoexcitation following acute hypoxia in humans

This study tested the hypothesis that acute isocapnic hypoxia results in persistent resetting of the baroreflex to higher levels of muscle sympathetic nerve activity (MSNA), which outlasts the hypoxic stimulus. Cardiorespiratory measures were recorded in humans (26 ± 1 yr; n = 14; 3 women) during baseline, exposure to 20 min of isocapnic hypoxia, and for 5 min following termination of hypoxia. The spontaneous baroreflex threshold technique was used to determine the change in baroreflex function during and following 20 min of isocapnic hypoxia (oxyhemoglobin saturation = 80%). From the spontaneous baroreflex analysis, the linear regression between diastolic blood pressure (DBP) and sympathetic burst occurrence, the T50 (DBP with a 50% likelihood of a burst occurring), and DBP error signal (DBP minus the T50) provide indexes of baroreflex function. MSNA and DBP increased in hypoxia and remained elevated during posthypoxia relative to baseline (P < 0.05). The DBP error signal became progressively less negative (i.e., smaller difference between DBP and T50) in the hypoxia and posthypoxia periods (baseline: -3.9 ± 0.8 mmHg; hypoxia: -1.4 ± 0.6 mmHg; posthypoxia: 0.2 ± 0.6 mmHg; P < 0.05). Hypoxia caused no change in the slope of the baroreflex stimulus-response curve; however, there was a shift toward higher pressures that favored elevations in MSNA, which persisted posthypoxia. Our results indicate that there is a resetting of the baroreflex in hypoxia that outlasts the stimulus and provide further explanation for the complex control of MSNA following acute hypoxia.     

Protective Role of Cynodon dactylon in Ameliorating the Aluminium-Induced Neurotoxicity in Rat Brain Regions

Cynodon dactylon (Poaceae) is a creeping grass used as a traditional ayurvedic medicine in India. Aluminium-induced neurotoxicity is well known and different salts of aluminium have been reported to accelerate damage to biomolecules like lipids, proteins and nucleic acids. The objective of the present study was to investigate whether the aqueous extract of C. dactylon (AECD) could potentially prevent aluminium-induced neurotoxicity in the cerebral cortex, hippocampus and cerebellum of the rat brain. Male albino rats were administered with AlCl(3) at a dose of 4.2?mg/kg/day i.p. for 4?weeks. Experimental rats were given C. dactylon extract in two different doses of 300?mg and 750?mg/keg/day orally 1?h prior to the AlCl(3) administration for 4?weeks. At the end of the experiments, antioxidant status and activities of ATPases in cerebral cortex, hippocampus and cerebellum of rat brain were measured. Aluminium administration significantly decreased the level of GSH and the activities of SOD, GPx, GST, Na(+)/K(+) ATPase, and Mg(2+) ATPase and increased the level of lipid peroxidation (LPO) in all the brain regions when compared with control rats. Pre-treatment with AECD at a dose of 750?mg/kg b.w increased the antioxidant status and activities of membrane-bound enzymes (Na(+)/K(+) ATPase and Mg(2+) ATPase) and also decreased the level of LPO significantly, when compared with aluminium-induced rats. The results of this study indicated that AECD has potential to protect the various brain regions from aluminium-induced neurotoxicity.     

HPLC and GC analyses of in vitro-grown leaves of the cancer bush <Emphasis Type="Italic">Lessertia</Emphasis> (<Emphasis Type="Italic">Sutherlandia</Emphasis>) <Emphasis Type="Italic">frutescens</Emphasis> L. reveal higher yields of bioactive compounds

Lessertia frutescens L., commonly known as cancer-bush, is a medicinally reputed plant species indigenous to southern Africa. Field leaf extracts of this species are known to exhibit many curative properties. However, little is known about the bioactive compounds that are present in in vitro leaf extracts and seed extracts. The objective of this study was to verify the presence of and quantify l-canavanine, gamma amino butyric acid (GABA), arginine and d-pinitol in the seeds, field leaves and in vitro leaves of L. frutescens using gas and liquid chromatography. Methanolic extracts of in vitro leaves, field leaves and seeds were used. MRM chromatograms were recorded for l-canavanine and arginine using tandem mass spectrometry. GC chromatograms were recorded for GABA and d-pinitol using gas chromatography. d-Pinitol was found to be most abundant and was 14.75 and 18.17 mg/g in in vitro and field leaf extracts respectively, followed by GABA (7.29 and 3.48 mg/g), arginine (7.08 and 0.35 mg/g) and l-canavanine (0.55 and 0.08 mg/g). In the seed extracts, GABA content was found to be the highest (1.69 mg/g) followed by l-canavanine (0.37 mg/g), then d-pinitol (0.25 mg/g), and arginine (0.02 mg/g). In vitro leaves had higher quantities of all compounds, except for d-pinitol. This study therefore highlights the potential of bulking in vitro leaves for the extraction of the medicinal compounds, l-canavanine and GABA.     

Risky behaviour and HIV prevalence among Zambian men

The objective of this paper is to identify demographic, social and behavioural risk factors for HIV infection among men in Zambia. In particular, the role of alcohol, condom use and number of sex partners is highlighted as being significant in the prevalence of HIV. Multivariate logistic regressions were used to analyse the latest cross-sectional population-based demographic health survey for Zambia (2007). The survey included socioeconomic variables and HIV serostatus for consenting men (N = 4434). Risk for HIV was positively related to wealth status. Men who considered themselves to be at high risk of being HIV positive were most likely to be HIV positive. Respondents who, along with their sexual partner, were drunk during the last three times they had sexual intercourse were more likely to be HIV positive (adjusted odds ratio (AOR) 1.60; 95% confidence interval (CI) 1.00-2.56). Men with more than two sexual life partners and inconsistent condom use had a higher risk for being HIV positive (OR 1.89, 95% CI 1.45-2.46; and OR 1.49, 95% CI 1.10-2.02, respectively). HIV prevention programmes in Zambia should focus even more on these behavioural risk factors.     

Cdc42 regulates extracellular matrix remodeling in three dimensions

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Oleanane triterpenoids in the prevention and therapy of breast cancer: current evidence and future perspectives

Breast cancer is one of the most frequently diagnosed cancers and major cause of death in women in the world. Emerging evidence underscores the value of dietary and non-dietary phytochemicals, including triterpenoids, in the prevention and treatment of breast cancer. Oleanolic acid, an oleanane-type pentacyclic triterpenoid, is present in a large number of dietary and medicinal plants. Oleanolic acid and its derivatives exhibit several promising pharmacological activities, including antioxidant, anti-inflammatory, hepatoprotective, cardioprotective, antipruritic, spasmolytic, antiallergic, antimicrobial and antiviral effects. Numerous studies indicate that oleanolic acid and other oleanane triterpenoids modulate multiple intracellular signaling pathways and exert chemopreventive and antitumor activities in various in vitro and in vivo model systems. A series of novel synthetic oleanane triterpenoids have been prepared by chemical modifications of oleanolic acid and some of these compounds are considered to be the most potent anti-inflammatory and anticarcinogenic triterpenoids. Accumulating studies provide extensive evidence that synthetic oleanane derivatives inhibit proliferation and induce apoptosis of various cancer cells in vitro and demonstrate cancer preventive or antitumor efficacy in animal models of blood, breast, colon, connective tissue, liver, lung, pancreas, prostate and skin cancer. This review critically examines the potential role of oleanolic acid, oleanane triterpenoids and related synthetic compounds in the chemoprevention and treatment of mammary neoplasia. Both in vitro and in vivo studies on these agents and related molecular mechanisms are presented. Several challenges and future directions of research to translate already available impressive preclinical knowledge to clinical practice of breast cancer prevention and therapy are also presented.