Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium

Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.     

Comparative analysis of the expression patterns of <Emphasis Type="Italic">Wnts</Emphasis> during chick limb development

Wnts control a number of processes during limb development—from initiating outgrowth and controlling patterning, to regulating cell differentiation in a number of tissues. We analyzed the expression pattern of various Wnts (4, 5a, 5b, 6, 11, and 14) in whole mount in situ hybridization during chick wing development. From HH stage 26, expression of Wnt 4 is observed in the central elbow region and wrist-forming regions, and during later stages, expression is seen in the joint-forming regions of the whole limb. Wnt 5a is expressed throughout the limb mesenchyme during early limb developmental stages, and later, at HH stage 23, it becomes predominantly confined to the distal tip, leaving low expression levels proximally. At HH stage 29, expression at the distal tip is restricted to the interdigital regions, and at day 8, expression is seen in the region surrounding the phalanges. Wnt 5b expression is first observed in the AER at HH stage 20 and later in the dorsal and ventral mesenchyme surrounding the cartilage elements of the limb. Expression of Wnt 6 is observed from HH stage 17 until day 8 in the dorsal and ventral ectoderm and also in the dorsoventral limb boundaries. Expression of Wnt 11 is observed in the proximal dorsal mesenchyme of the limb from HH stage 23 onward and later in the dorsal and ventral subectodermal mesenchyme and in the regions adjacent to the digits at day 8. Weak expression of Wnt 14 is observed at the proximal mesenchyme of the limb at HH stage 23; later, it extends as a transverse strip surrounding the cartilage elements as well as in the interdigital mesenchyme.This paper is dedicated to Professor Dr. W. Zenker on the occasion of his 80th birthday.     

Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production

RNA editing of the hepatitis delta virus (HDV) antigenome at the amber/W site by the host RNA adenosine deaminase ADAR1 is a critical step in the HDV replication cycle. Editing is required for production of the viral protein hepatitis delta antigen long form (HDAg-L), which is necessary for viral particle production but can inhibit HDV RNA replication. The RNA secondary structural features in ADAR1 substrates are not completely defined, but base pairing in the 20-nucleotide (nt) region 3' of editing sites is thought to be important. The 25-nt region 3' of the HDV amber/W site in HDV genotype I RNA consists of a conserved secondary structure that is mostly base paired but also has asymmetric internal loops and single-base bulges. To understand the effect of this 3' region on the HDV replication cycle, mutations that either increase or decrease base pairing in this region were created and the effects of these changes on amber/W site editing, RNA replication, and virus production were studied. Increased base pairing, particularly in the region 15 to 25 nt 3' of the editing site, significantly increased editing; disruption of base pairing in this region had little effect. Increased editing resulted in a dramatic inhibition of HDV RNA synthesis, mostly due to excess HDAg-L production. Although virus production at early times was unaffected by this reduced RNA replication, at later times it was significantly reduced. Therefore, it appears that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efficiency but for optimal viral replication and secretion.     

Carboxylated glycans mediate colitis through activation of NF-kappa B

The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn's disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4(+)CD45RB(high) T cell transfer to Rag1(-/-) mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4(+) T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-kappaB p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.     

A novel medium for the enhanced cell growth and production of prodigiosin from <Emphasis Type="Italic">Serratia marcescens</Emphasis> isolated from soil

Background

Prodigiosin produced by Serratia marcescens is a promising drug owing to its reported characteristics of having antifungal, immunosuppressive and antiproliferative activity. From an industrial point of view the necessity to obtain a suitable medium to simultaneously enhance the growth of Serratia marcescens and the pigment production was the aim of this work. The usage of individual fatty acid as substrate in industries would be cost-effective in the long run and this paved the way for us to try the effect of different fatty acid-containing seeds and oils of peanut, sesame and coconut as source of substrate.

Results

The addition of sugars only showed slight enhancement of prodigiosin production in nutrient broth but not in fatty acid containing seed medium. The powdered peanut broth had supported better growth of Serratia marcescens and higher yield of prodigiosin when compared with the existing nutrient broth and peptone glycerol broth. A block in prodigiosin production was seen above 30°C in nutrient broth, but the fatty acid seed medium used by us supported prodigiosin production upto 42°C though the yields were lower than what was obtained at 28°C. From the results, the fatty acid form of carbon source has a role to play in enhanced cell growth and prodigiosin production.

Conclusion

We conclude by reporting that the powdered and sieved peanut seed of different quality grades were consistent in yielding a fourty fold increase in prodigiosin production over the existing media. A literature survey on the composition of the different media components in nutrient broth, peptone glycerol broth and the fatty acid containing seeds and oils enabled us to propose that the saturated form of fatty acid has a role to play in enhanced cell growth and prodigiosin production. This work has also enabled us to report that the temperature related block of prodigiosin biosynthesis varies with different media and the powdered peanut broth supports prodigiosin production at higher temperatures. The medium suggested in this work is best suitable from an industrial point of view in being economically feasible, in terms of the higher prodigiosin yield and the extraction of prodigiosin described in this paper is simple with minimal wastage.

     

N -Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth

In this study we show that embryonic neurite growth-promoting protein amphoterin binds to carboxylated N -glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand-binding V-domain of the receptor contains two potential N -glycosylation sites, we hypothesized that N -glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti-carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N -glycans with non-sialic acid carboxylate groups, but not the HNK-1 (3-sulfoglucuronyl beta1-3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE-transfected neuroblastoma cells. This outgrowth requires full-length, signalling-competent RAGE, as cells expressing cytoplasmic domain-deleted RAGE are unresponsive. These results indicate that carboxylated N -glycans on RAGE play an important functional role in amphoterin-RAGE-mediated signalling.     

Long-Term Environmental Correlates of Invasion by Lantana camara (Verbenaceae) in a Seasonally Dry Tropical Forest

Invasive species, local plant communities and invaded ecosystems change over space and time. Quantifying this change may lead to a better understanding of the ecology and the effective management of invasive species. We used data on density of the highly invasive shrub Lantana camara (lantana) for the period 1990–2008 from a 50 ha permanent plot in a seasonally dry tropical forest of Mudumalai in southern India. We used a cumulative link mixed-effects regression approach to model the transition of lantana from one qualitative density state to another as a function of biotic factors such as indicators of competition from local species (lantana itself, perennial grasses, invasive Chromolaena odorata, the native shrub Helicteres isora and basal area of native trees) and abiotic factors such as fire frequency, inter-annual variability of rainfall and relative soil moisture. The density of lantana increased substantially during the study period. Lantana density was negatively associated with the density of H. isora, positively associated with basal area of native trees, but not affected by the presence of grasses or other invasive species. In the absence of fire, lantana density increased with increasing rainfall. When fires occurred, transitions to higher densities occurred at low rainfall values. In drier regions, lantana changed from low to high density as rainfall increased while in wetter regions of the plot, lantana persisted in the dense category irrespective of rainfall. Lantana seems to effectively utilize resources distributed in space and time to its advantage, thus outcompeting local species and maintaining a population that is not yet self-limiting. High-risk areas and years could potentially be identified based on inferences from this study for facilitating management of lantana in tropical dry forests.     

RhoGAP18B Isoforms Act on Distinct Rho-Family GTPases and Regulate Behavioral Responses to Alcohol via Cofilin

Responses to the effects of ethanol are highly conserved across organisms, with reduced responses to the sedating effects of ethanol being predictive of increased risk for human alcohol dependence. Previously, we described that regulators of actin dynamics, such as the Rho-family GTPases Rac1, Rho1, and Cdc42, alter Drosophila’s sensitivity to ethanol-induced sedation. The GTPase activating protein RhoGAP18B also affects sensitivity to ethanol. To better understand how different RhoGAP18B isoforms affect ethanol sedation, we examined them for their effects on cell shape, GTP-loading of Rho-family GTPase, activation of the actin-severing cofilin, and actin filamentation. Our results suggest that the RhoGAP18B-PA isoform acts on Cdc42, while PC and PD act via Rac1 and Rho1 to activate cofilin. In vivo, a loss-of-function mutation in the cofilin-encoding gene twinstar leads to reduced ethanol-sensitivity and acts in concert with RhoGAP18B. Different RhoGAP18B isoforms, therefore, act on distinct subsets of Rho-family GTPases to modulate cofilin activity, actin dynamics, and ethanol-induced behaviors.     

MHC class II‐assortative mate choice in European badgers (Meles meles)

The major histocompatibility complex (MHC) plays a crucial role in the immune system, and in some species, it is a target by which individuals choose mates to optimize the fitness of their offspring, potentially mediated by olfactory cues. Under the genetic compatibility hypothesis, individuals are predicted to choose mates with compatible MHC alleles, to increase the fitness of their offspring. Studies of MHC‐based mate choice in wild mammals are under‐represented currently, and few investigate more than one class of MHC genes. We investigated mate choice based on the compatibility of MHC class I and II genes in a wild population of European badgers (Meles meles). We also investigated mate choice based on microsatellite‐derived pairwise relatedness, to attempt to distinguish MHC‐specific effects from genomewide effects. We found MHC‐assortative mating, based on MHC class II, but not class I genes. Parent pairs had smaller MHC class II DRB amino acid distances and smaller functional distances than expected from random pairings. When we separated the analyses into within‐group and neighbouring‐group parent pairs, only neighbouring‐group pairs showed MHC‐assortative mating, due to similarity at MHC class II loci. Our randomizations showed no evidence of genomewide‐based inbreeding, based on 35 microsatellite loci; MHC class II similarity was therefore the apparent target of mate choice. We propose that MHC‐assortative mate choice may be a local adaptation to endemic pathogens, and this assortative mate choice may have contributed to the low MHC genetic diversity in this population.     

International regulatory requirements for Leptospira vaccine potency testing. Roundtable: Current requirements and opportunity for harmonization

Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests.