Serum-free hybridoma culture: ethical, scientific and safety considerations

Despite considerable progress in the development of cell culture techniques, including the development of the serum- and protein-free media that now routinely support hybridoma and mammalian cell growth, fetal bovine serum (FBS) supplemented media are still commonly used: a practice that raises ethical, scientific and safety concerns. The use of FBS in hybridoma culture media is examined here, with regards to the development and production of monoclonal antibodies (mAbs), and it is our recommendation that researchers adopt serum-free cell culture methods to reduce animal use in this area.     

Slow aggregation of lysozyme in alkaline pH monitored in real time employing the fluorescence anisotropy of covalently labelled dansyl probe

The onset of hen egg white lysozyme aggregation on exposure to alkaline pH of 12.2 and subsequent slow growth of soluble lysozyme aggregates (at 298 K) was directly monitored by steady-state and time-resolved fluorescence anisotropy of covalently attached dansyl probe over a period of 24 h. The rotational correlation time accounting for tumbling of lysozyme in solution (40 microM) increased from approximately 3.6 ns (in pH 7) to approximately 40ns on exposure to pH 12.2 over a period of 6 h and remained stable thereafter. The growth of aggregates was strongly concentration dependent, irreversible after 60 min and inhibited by the presence of 0.9 M l-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine.HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of L-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.     

Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ～55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.     

Dysfunction of the ubiquitin ligase ube3a may be associated with synaptic pathophysiology in a mouse model of huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.     

Excessive zinc in diet induces leptin resistance in Wistar rat through increased uptake of nutrients at intestinal level

ProjectThe ob gene has either been found to be mutant defective resulting in a deficiency of its product leptin or leptin has been found to be resistant to its receptors in obese human and rodents. The factors inducing leptin resistance have not been identified. Since excessive bioavailability of Zn has been implicated in obesity, we investigated if its excess in diet induces leptin resistance.ProcedureFor the investigations, three groups of Wistar rats were included in this study and they were fed on equicalories semi synthetic basal diet containing 20 mg, 40 mg or 80 mg Zn/kg diet for 120 days. There after they were sacrificed for hormonal status and intestinal investigations.ResultsThe data of this study revealed that the food intake, gain in body weight, serum leptin, glucose, insulin, cortisol increased with increased Zn concentration in diet. TEM study showed a positive correlation between Zn concentration in diet and number of microvilli/unit surface area of the mucosal epithelial cells of the intestine.ConclusionThe results of this study suggest that excessive bioavailability of Zn induces leptin resistance through increased uptake of nutrients at intestinal level, leading to the growth of the fat cells which aggravated the leptin synthesis and its release in the blood stream. In spite of its higher circulating level, it was unable to reduce the food intake and gain in body weight in Zn treated rats equivalent to the control group.     

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.^1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.^4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.^1,2The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically¹ or analytically,^2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,^1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.^1,2,7In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N₃) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein – strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.     

Water saving traits co-map with a major terminal drought tolerance quantitative trait locus in pearl millet [Pennisetum glaucum (L.) R. Br.]

Low transpiration rates in pearl millet under fully irrigated conditions decrease plant water use at vegetative stage and then increase the water availability during grain filling and finally the terminal drought tolerance. Hundred and thirteen recombinant inbred lines developed from a cross between H77/833-2 and PRLT2/89-33 (terminal drought-sensitive?×?tolerant genotype) were evaluated to map transpiration rate (Tr, a proxy for canopy conductance), organ weights, leaf area and thickness and to study their interactions. Transpiration rate was increased by two H77/833-2 and two PRLT2/89-33 alleles on linkage group (LG) 2, whose importance depended on the vapor pressure deficit. The two H77/833-2 and one PRLT2/89-33 alleles co-mapped to a previously identified major terminal drought tolerance quantitative trait locus (QTL), although in a much smaller genetic interval. The other Tr allele from H77/833-2 also enhanced biomass dry weight and co-located with a formerly identified stover and tillering QTL. Leaf characteristics were linked to two loci on LG7. Plant water use was increased and decreased by different loci combinations for Tr, tillering and leaf characteristics, whose respective importance depended on the environmental conditions. Therefore, different alleles influence plant water use and have close interactions with one another and with the environment, so that different ideotypes for plant water use exist or could be designed from specific allele combinations conferring particular physiological characteristics for specific adaptation to a range of terminal drought conditions.     

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

Growth and photosynthetic pigments responses of two varieties of Catharanthus roseus to triadimefon treatment

Triadimefon, potential fungicide cum plant-growth retardant was used in this study to investigate its effect on the growth and the photosynthetic pigment contents of two varieties of Catharanthus roseus (L.) G. Don. The plants of both varieties were subjected to 15 mg l(-1) triadimefon treatment by soil drenching 30, 45, 60, and 75 days after planting (DAP). Plants were uprooted on 90 DAP, and morphological parameters, like plant height, number of leaves, leaf area, root length and fresh and dry weights were determined. The photosynthetic pigments, like chlorophylls a and b, total chlorophyll, carotenoids, floral pigment, anthocyanin, were extracted and estimated. It was observed that plant height, number of leaves and leaf area were decreased and that root length, fresh and dry weights were increased under triadimefon treatment. The photosynthetic and floral pigments were increased under triadimefon treatment in both varieties. The results suggest that the application of this plant-growth retardant (triadimefon) has favourable effects on the reduction of plant height; it can thus be used for replacing manual hand pruning and for improving floral and vegetation colour in bedding plants like C. roseus.