The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar K_d as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The V_H and V_L genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical V_H genes but different V_L genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.     

Application of Thermostable Xylanase of Bacillus pumilus in Textile Processing

Desizing of cotton and micropoly fabrics was done using thermostable xylanase from Bacillus pumilus ASH. Micropoly fabric showed better desizing than cotton under same conditions. Violet scale readings from the TEGEWA test after enzymatic desizing for 90 min at pH 7.0 and at 60°C showed the readings falling in the range of 4–5, indicating good desizing efficiency. During bioscouring the weight loss values and liberation of reducing sugars were highest when EDTA was used along with xylanase. The weight loss value of 1.5% was observed for dry cotton fabric after 1 h in case of agitated system at pH 7.0 and at an optimal enzyme dosage of 5 IU/g. The weight loss values and the liberation of reducing sugars were higher in case of cotton fabrics. Wetting time of fabrics was lowered significantly after 60 min of bioscouring using xylanase. Increase in temperature or concentration of surfactant led to further reduction in the wetting time. The whiteness values of fabrics after bioscouring were 0.9% higher than the chemically scoured fabrics indicating good efficacy of xylanase during the scouring process.     

Rhizosphere: its structure,bacterial diversity and significance

Sustainable agricultural practices are the answer to multifaceted problems that have resulted due to prolonged and indiscriminate use of chemical based agronomic tools to improve crop productions for the last many decades. The hunt for suitable ecofriendly options to replace the chemical fertilizers and pesticides has thus been aggravated. Owing to their versatile and unmatchable capacities microbial agents offer an attractive and feasible option to develop the biological tools to replace/supplement the chemicals. Exploring the microorganisms that reside in close proximity to the plant is thus a justified move in the direction to achieve this target. One of the most lucrative options is to look into the rhizosphere. Rhizosphere may be defined as the narrow zone of soil that surrounds and get influenced by the roots of the plants. It is rich in nutrients compared to the bulk soil and hence exhibit intense biological and chemical activities. A wide range of macro and microorganisms including bacteria, fungi, virus, protozoa, algae, nematodes and microarthropods co-exist in rhizosphere and show a variety of interactions between themselves as well as with the plant. Plant friendly bacteria residing in rhizosphere which exert beneficial affect on it are called as plant growth promoting rhizobacteria (PGPR). Here we review the structure and bacterial diversity of the rhizosphere. The major points discussed here are: (1) structure and composition of the rhizosphere (2) range of bacteria found in rhizosphere and their interactions with the plant with a particular emphasis on PGPR (3) mechanisms of plant growth promotion by the PGPR (4) rhizosphere competence.     

Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.     

Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Annual wormwood (Artemisia annua L.) produces an array of complex terpenoids including artemisinin, a compound of current interest in the treatment of drug-resistant malaria. However, this promising antimalarial compound remains expensive and is hardly available on the global scale. Synthesis of artemisinin has not been proved to be feasible commercially. Therefore, increase in yield of naturally occurring artemisinin is an important area of investigation. The effects of inoculation by two arbuscular mycorrhizal (AM) fungi, Glomus macrocarpum and Glomus fasciculatum, either alone or supplemented with P-fertilizer, on artemisinin concentration in A. annua were studied. The concentration of artemisinin was determined by reverse-phase high-performance liquid chromatography with UV detection. The two fungi significantly increased concentration of artemisinin in the herb. Although there was significant increase in concentration of artemisinin in nonmycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in artemisinin concentration may not be entirely attributed to enhanced P-nutrition and improved growth. A strong positive linear correlation was observed between glandular trichome density on leaves and artemisinin concentration. Mycorrhizal plants possessed higher foliar glandular trichome (site for artemisinin biosynthesis and sequestration) density compared to nonmycorrhizal plants. Glandular trichome density was not influenced by P-fertilizer application. The study suggests a potential role of AM fungi in improving the concentration of artemisinin in A. annua.     

Verotoxic Escherichia coli (STEC) from beef and its products

In the present investigation, out of 27 (24.10%) strains of Escherichia coli isolated from 112 beef samples comprising raw meat (45), kabab (36) and kofta (31), 9 (33.33%) belonging to 7 different serotypes were verotoxic as tested by vero cell cytotoxic assay. Serotype O145 was the predominant STEC in raw meat. Interestingly, one STEC-O157 strain was also detected. All the STEC strains were positive for Stx genes by polymerase chain reaction showing stx2 (77.78%) to be most predominant followed by stx1 (22.22%). Phenotypic enterohaemolysin production on washed sheep blood agar supplemented with CaCl2 revealed 6 (66.67%) STEC strains to be positive. Presence of STEC in cooked beef products, viz., kabab and kofta appeared to be a matter of concern and potential threat to public health.     

Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.     

F-box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress

F-box proteins constitute a large family in eukaryotes and are characterized by a conserved F-box motif (approximately 40 amino acids). As components of the Skp1p-cullin-F-box complex, F-box proteins are critical for the controlled degradation of cellular proteins. We have identified 687 potential F-box proteins in rice (Oryza sativa), the model monocotyledonous plant, by a reiterative database search. Computational analysis revealed the presence of several other functional domains, including leucine-rich repeats, kelch repeats, F-box associated domain, domain of unknown function, and tubby domain in F-box proteins. Based upon their domain composition, they have been classified into 10 subfamilies. Several putative novel conserved motifs have been identified in F-box proteins, which do not contain any other known functional domain. An analysis of a complete set of F-box proteins in rice is presented, including classification, chromosomal location, conserved motifs, and phylogenetic relationship. It appears that the expansion of F-box family in rice, in large part, might have occurred due to localized gene duplications. Furthermore, comprehensive digital expression analysis of F-box protein-encoding genes has been complemented with microarray analysis. The results reveal specific and/or overlapping expression of rice F-box protein-encoding genes during floral transition as well as panicle and seed development. At least 43 F-box protein-encoding genes have been found to be differentially expressed in rice seedlings subjected to different abiotic stress conditions. The expression of several F-box protein-encoding genes is also influenced by light. The structure and function of F-box proteins in plants is discussed in light of these results and the published information. These data will be useful for prioritization of F-box proteins for functional validation in rice.     

Ameliorative Role of Pre-Sowing Proline Treatment in <i>Coriandrum sativum</i> L. Seedlings under Mercury Toxicity

Heavy metal toxicity is one of the major ecosystem concerns globally in present time and is also responsible for significant threat to agronomic crops. The current work was conducted to investigate the possible ameliorative role of proline in Coriandrum sativum L. seedlings treated with mercury (Hg). The seedlings were exposed to different concentrations of Hg (0, 0.1, 0.3 and 0.5 mM) for 20 days. The effects of pre-sowing treatment with proline were studied on C. sativum seedlings in terms of pigment (chlorophylls, carotenoids and anthocyanins), malondialdehyde (MDA), antioxidant compound (glutathione, total phenolic compounds, ascorbic acid) and osmolytes (proline, glycine betaine). Additionally, activities of antioxidant enzymes, namely catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were also studied. A strong decline of photosynthetic pigment concentrations was observed in leaves of C. sativum under Hg toxicity. Treatment of seeds with proline reduced the loss of photosynthetic pigments, counteract Hg-triggered oxidative stress, likely preserving the functionality of antioxidant apparatus under Hg stress. The increment of total polyphenols and glycine betaine also contributed in ameliorating Hg toxicity, suggesting the use of exogenous proline as a potential method to enhance the plant tolerance against heavy metal stress.