Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Appraisal of Transdermal Water-in-Oil Nanoemulgel of Selegiline HCl for the Effective Management of Parkinson’s Disease: Pharmacodynamic,Pharmacokinetic, and Biochemical Investigations

In the present study, the potential of transdermal nanoemulsion gel of selegiline hydrochloride for the treatment of Parkinson’s disease was investigated. Water-in-oil nanoemulsions were developed by comparing low- and high-energy methods and were subjected to thermodynamic stability tests, in vitro permeation, and characterization studies. In vitro studies indicated that components of nanoemulsion acted as permeation enhancers with highest flux of 3.531 ± 1.94 μg/cm²/h from nanoemulsion SB6 containing 0.5 mg selegiline hydrochloride, 3% distilled water, 21% S _mix (Span 85, Tween 80, PEG 400), and 76% isopropyl myristate by weight. SB6 with the least droplet size of 183.4 ± 0.35 nm, polydispersity index of 0.42 ± 0.06 with pH of 5.9 ± 0.32 and viscosity of 22.42 ± 0.14 cps was converted to nanoemulsion gel NEGS4 (viscosity = 22,200 ± 400 cps) by addition of Viscup160® for ease of application and evaluated for permeation, safety, and pharmacokinetic profile in Wistar rats. It provided enhancement ratio 3.69 times greater than conventional gel. NEGS4 showed 6.56 and 5.53 times increase in bioavailability in comparison to tablet and conventional gel, respectively, along with sustained effect. Therefore, the developed water-in-oil nanoemulsion gel promises to be an effective vehicle for transdermal delivery of selegiline hydrochloride.     

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Synthetic Antimicrobial Peptide Polybia MP-1 (Mastoparan) Inhibits Growth of Antibiotic Resistant Pseudomonas aeruginosa Isolates From Mastitic Cow Milk

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) offer a potent and effective alternative for treatment of antibiotic resistant microbes. Mastoparans or...     

Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.     

Impacts of an Invasive Non-Native Annual Weed,Impatiens glandulifera,on Above- and Below-Ground Invertebrate Communities in the United Kingdom

Vegetation community composition and the above- and below-ground invertebrate communities are linked intrinsically, though few studies have assessed the impact of non-native plants on both these parts of the community together. We evaluated the differences in the above- (foliage- and ground-dwelling) and below-ground invertebrate communities in nine uninvaded plots and nine plots invaded by the annual invasive species Impatiens glandulifera, in the UK during 2007 and 2008. Over 139,000 invertebrates were identified into distinct taxa and categorised into functional feeding groups. The impact of I. glandulifera on the vegetation and invertebrate community composition was evaluated using multivariate statistics including principal response curves (PRC) and redundancy analysis (RDA). In the foliage-dwelling community, all functional feeding groups were less abundant in the invaded plots, and the species richness of Coleoptera and Heteroptera was significantly reduced. In the ground-dwelling community, herbivores, detritivores, and predators were all significantly less abundant in the invaded plots. In contrast, these functional groups in the below-ground community appeared to be largely unaffected, and even positively associated with the presence of I. glandulifera. Although the cover of I. glandulifera decreased in the invaded plots in the second year of the study, only the below-ground invertebrate community showed a significant response. These results indicate that the above- and below-ground invertebrate communities respond differently to the presence of I. glandulifera, and these community shifts can potentially lead to a habitat less biologically diverse than surrounding native communities; which could have negative impacts on higher trophic levels and ecosystem functioning.     

Role of hydrophobicity in bacterial adherence to carbon nanostructures and biofilm formation

The role of cell and surface hydrophobicity in the adherence of the waterborne bacterium Mycobacterium smegmatis to nanostructures and biofilm formation was investigated. Carbon nanostructures (CNs) were synthesized using a flame reactor and deposited on stainless steel grids and foils, and on silicon wafers that had different initial surface hydrophobicities. Surface hydrophobicity was measured as the contact angle of water droplets. The surfaces were incubated in suspensions of isogenic hydrophobic and hydrophilic strains of M. smegmatis and temporal measurements of the numbers of adherent cells were made. The hydrophobic, rough mutant of M. smegmatis adhered more readily and formed denser biofilms on all surfaces compared to its hydrophilic, smooth parent. Biofilm formation led to alterations in the hydrophobicity of the substratum surfaces, demonstrating that bacterial cells attached to CNs are capable of modifying the surface characteristics.     

Arbuscular Mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress

Photosynthesis Research - Drought stress (DS) is amongst one of the abiotic factors affecting plant growth by limiting productivity of crops by inhibiting photosynthesis. Damage due to DS and its...