GABA and 5-HT chitosan nanoparticles enhanced liver cell proliferation and neuronal survival in partially hepatectomised rats: GABAB and 5-HT2A receptors functional

Nanoparticulate drug delivery systems provide wide opportunities for solving problems associated with drug stability or disease states and create great expectations in the area of drug delivery (Bosselmann & Williams, 2012). Nanotechnology, in a simple way, explains the technology that deals with one billionth of a meter scale (Ochekpe, et al., 2009). Fewer side effects, poor bioavailability, absorption at intestine, solubility, specific delivery to site of action with good pharmacological efficiency, slow release, degradation of drug and effective therapeutic outcome, are the major challenges faced by most of the drug delivery systems. To a great extent, biopolymer coated drug delivery systems coupled with nanotechnology alleviate the major drawbacks of the common delivery methods. Chitosan, deacetylated chitin, is a copolymer of β-(1, 4) linked glucosamine (deacetylated unit) and N- acetyl glucosamine (acetylated unit) (Radhakumary et al., 2005). Chitosan is biodegradable, non-toxic and bio compatible. Owing to the removal of acetyl moieties that are present in the amine functional groups of chitin, chitosan is readily soluble in aqueous acidic solution. The solubilisation occurs through the protonation of amino groups on the C-2 position of D-glucosamine residues whereby polysaccharide is converted into polycation in acidic media. Chitosan interacts with many active compounds due to the presence of amine group in it. The presence of this active amine group in chitosan was exploited for the interaction with the active molecules in the present study. Nanoparticles of chitosan coupled drugs are utilized for drug delivery in eye, brain, liver, cancer tissues, treatment of spinal cord injury and infections (Sharma et al., 2007; Li, et a., 2009; Paolicelli et al., 2009; Cho et al., 2010). To deliver drugs directly to the intended site of action and to improve pharmacological efficiency by minimizing undesired side effects elsewhere in the body and decrease the long-term use of many drugs, polymeric drug delivery systems can be used (Thatte et al., 2005).

Development of mechanical methods for cell-tray propagation and field transplanting of dwarf napiergrass (Pennisetum purpureum Schumach.)

Since dwarf napiergrass (Pennisetum purpureum Schumach.) must be propagated vegetatively due to lack of viable seeds, root splitting and stem cuttings are generally used to obtain true-to-type plant populations. These ordinary methods are laborious and costly, and are the greatest barriers for expanding the cultivation area of this crop. The objectives of this research were to develop nursery production of dwarf napiergrass in cell trays and to compare the efficiency of mechanical versus manual methods for cell-tray propagation and field transplanting. After defoliation of herbage either by a sickle (manually) or hand-mowing machine, every potential aerial tiller bud was cut to a single one for transplanting into cell trays as stem cuttings and placed in a glasshouse over winter. The following June, nursery plants were trimmed to a 25–cm length and transplanted in an experimental field (sandy soil) with 20,000 plants ha^(−1) either by shovel (manually) or Welsh onion planter. Labour time was recorded for each process. The manual defoliation of plants required 44% more labour time for preparing the stem cuttings (0.73 person-min. stemcutting^(−1)) compared to using hand-mowing machinery (0.51 person-min. stem-cutting^(−1)). In contrast, labour time for transplanting required an extra 0.30 person-min. m^(−2) (14%) using the machinery compared to manual transplanting, possibly due to the limited plot size for machinery operation. The transplanting method had no significant effect on plant establishment or plant growth, except for herbage yield 110 days after planting. Defoliation of herbage by machinery, production using a cell-tray nursery and mechanical transplanting reduced the labour intensity of dwarf napiergrass propagation.

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies

The L-glutamate transporter GLT-1 is an abundant CNS membrane protein of the excitatory amino acid transporter (EAAT) family which controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using RT-PCR, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N- and C-termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected HEK-293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (V5, HA or FLAG) into the second extracellular domain of each isoform allowed co-immunoprecipitation and tr-FRET studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms are able to combine to form homomeric and heteromeric assemblies, each of which are expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Cell cycle profiles and expression of p21CIP1 and P27KIP1 during myocyte development

The ability of the cardiac myocyte to divide ceases shortly after birth. Thus, following severe injury, e.g., during myocardial infarction, the mature heart is unable to regenerate new tissue to replace the dead or damaged tissue. The identification of the molecules controlling the cessation of myocyte cell division may lead to therapeutic strategies which aim to re-populate the damaged myocardial area. Hence, we have determined the cell cycle profile, expressions and activities of the cyclin-dependent kinase inhibitors (CDKIs), p21CIP1 and p27KIP1, during rat ventricular myocyte development. Fluorescent activated cell sorting (FACS) analyses showed the percentage of S phase myocytes to be decreased significantly throughout development, concomitant with a significant increase in the percentage of G0/G1 and G2/M phase cells. The expression of p21CIP1 and p27KIP1 increased significantly throughout cardiac development and complexed differentially with a number of cyclins and CDKs. Furthermore, an adult myocyte extract reduced neonatal myocyte CDK2 kinase activity significantly (>30%, p<0.05) whereas immunodepletion of p21CIP1 from adult lysates restored CDK2 kinase activity. Thus, p21CIP1 and p27KIP1 may be important for the withdrawal of cardiac myocytes from the cell cycle and for maintaining the G0/G1 and G2/M phase blockades.

Role of rpoS in the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure-sensitive and took up more PI than the parent strains with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure-sensitive, was unaffected in membrane resealing implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

Synthetic modified N-POMC(1-28) controls in vivo proliferation and blocks apoptosis in rat adrenal cortex

The identity of the pro-opiomelanocortin (POMC)-derived mitogen in the adrenal cortex has been historically controversial. We have used well-established in vivo models, viz., hypophysectomized (Hyp) or dexamethasone (Dex)-treated rats, to study the effect of the synthetic modified peptide N-terminal POMC (N-POMC(1-28)) on DNA synthesis in the adrenal cortex, as assessed by BrdU incorporation and compared with adrenocorticotropic hormone (ACTH). We evaluated the importance of disulfide bridges on proliferation by employing N-POMC(1-28) without disulfide bridges and with methionines replacing cysteines. Acute administration of synthetic modified N-POMC(1-28) distinctly increased DNA synthesis in the zona glomerulosa and zona fasciculata, but not in the zona reticularis in Hyp rats, whereas in Dex-treated rats, this peptide was effective in all adrenal zones. ACTH administration led to an increase of BrdU-positive cells in all adrenal zones irrespective of the depletion of Hyp or Dex-POMC peptides. The use of the ACTH antagonist, ACTH(7-38), confirmed the direct participation of ACTH in proliferation. Two different approaches to measure apoptosis revealed that both peptides similarly exerted a protective effect on all adrenocortical zones, blocking the apoptotic cell death induced by hypophysectomy. Thus, ACTH(1-39) and N-POMC(1-28) have similar actions suggesting that the disulfide bridges are important but not essential. Both peptides seem to be important factors determining adrenocortical cell survival throughout the adrenal cortex, reinforcing the idea that each zone can be renewed from within itself.

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+](i)) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G alpha(q/11)-coupled M-1, M-3 and M-5 receptors and intracellular calcium stores, whereas G alpha(i/o)-protein coupled M-2 receptor activity mediated neuronal differentiation. (C) 2008 Elsevier Inc. All rights reserved.

The novel ruthenium-gamma-linolenic complex [Ru(2)(aGLA)(4)Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru(2)(aGLA)(4)Cl] according to elemental analyses data, referred to as Ru(2)GLA. The electronic spectra of Ru(2)GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru(2)GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and gamma-linolenic acid (GLA). The properties of Ru(2)GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru(2)GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru(2)GLA enters the cells and ICP-AES elemental analysis found all increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru(2)GLA (22 +/- 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru(2)GLA (44 +/- 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 +/- 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru(2)GLA exposed cells. The EC(50) for Ru(2)GLA decreased with increasing time of exposure from 285 mu M at 24h, 211 mu M at 48 h to 81 mu M at 72 h. In conclusion, Ru(2)GLA is a novel drug with anti proliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright (C) 2009 John Wiley & Sons, Ltd.

Evaluation of Cell Proliferation and Apoptosis in Placentas of Rats with Severe Diabetes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Canine Subcutaneous Mast Cell Tumors: Cellular Proliferation and KIT Expression as Prognostic Indices

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers-including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)-as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.

Stress hormones increase cell proliferation and regulates interleukin-6 secretion in human oral squamous cell carcinoma cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proliferation and apoptotic rates and increased frequency of p63-positive cells in the prostate acinar epithelium of alloxan-induced diabetic rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Boron deficiency inhibits petiole and peduncle cell development and reduces growth of cotton

The effect of boron (B) on cotton growth and fruit shedding may be due not only to physiological or biochemical effects, but also to vascular tissue malformation. This experiment investigated petiole and floral peduncle anatomical alterations and growth of cotton supplied with deficient and sufficient B in nutrient solution. Cotton (Gossypium hirsutum cv. 'Delta Opal') plants were grown in solutions containing 0, 1.5, 3.0, 4.5, and 6.0 mu mol L-1 of B from 22 to 36 d after plant emergence (DAPE). From 36 to 51 DAPE, B was omitted from the nutrient solution. Petioles from young leaves and floral bud peduncles (first position of the first sympodial) were sampled and the cross-section anatomy observed under an optical microscope. The number of vascular bundles of the petiole was decreased in B-deficient plants and the xylem was disorganized. Phloem elements in the peduncle vascular cylinder of B-deficient plants did not show clear differentiation. The few xylem elements that were formed were also disorganized. Modifications caused by B deficiency may have impaired B and photosynthate translocation into new cotton growth. Boron accumulation in the shoot of B-deficient plants suggested that there was some B translocation within the plant. It could be inferred that cotton growth would be impaired by the decrease in carbohydrate translocation rather than by B deficiency in the tissue alone.