A modified bioassay for microbial mosquito larvicides.

The proposed bioassay-the grid assay-eliminates the influence of cannibalism while allowing recycling to take place. The LC50 values of the grid assay were intermediate to those of the group and the individual assay. The grid assay thus reflects the true toxicity values of the microbial pesticides being tested. Viable count studies of the microbial pesticides employed, viz., Bacillus thuringiensis H-14 and Bacillus sphaericus 2362, performed along with the assay support the results of the bioassay.

Differential cell behaviour on polylysine and gelatin in primary culture of mouse mammary adenocarcinoma.

Cells dissociated from spontaneous and transplanted tumours of C3HJax mammary gland have been cultured on polylysine and gelatin substrates. The isolated cells proliferated to form monolayers with high degree of organoid structure as indicated by formation of alveolar cavities. Differences were observed in the cell attachment, growth pattern, number and size of alveolar cavities, cells which lined the cavity and cell morphology on polylysine and gelatin substrates as compared to conventional cell culture plastic surface. On polylysine more than 90% cells attached rapidly, within 15-45 min after plating, with or without serum and formed confluent monolayers marked by presence of large and small alveolar cavities. Multiple interacting cell types took part in organization of the cavity. Cells lining the cavity constantly proliferated and rearranged to expand it. On gelatin, 60-70% cells attached over a period of 6-24 hr in presence of serum and formed confluent monolayers dominated by small alveolar cavities. Cells forming the cavities were epithelial in nature and cavities once formed did not increase in size. Upon subculture, the cell morphology on these substrates was strikingly different. On polylysine, the predominant cell type had numerous irregular microvilli whereas on gelatin, cells had smoother boundaries with a few stunted cytoplasmic extensions. The cell attachment on conventional surface was low, 40-50%. When seeded at high cell density, formation of alveolar cavities was suppressed and at low cell density, cultures were marked by contact inhibition of cells and failure to attain confluence. These results suggest differential behaviour and interaction of mammary tumour epithelium with the substrates used.

Isolation & characterization of angiogenic factors from cultured cells & conditioned media.

Angiogenic factors were isolated by ion exchange chromatography from three established cell lines viz., HEp 2, HeLa and CHO, a primary culture of mouse mammary adenocarcinoma and from conditioned media of HEp2 and cultured mammary adenocarcinoma cells. The angiogenic activity was assayed by chicken chorioallantoic membrane assay. The angiogenic factor eluted at 0.5 M, exhibited lambda max at 258 +/- 1 nm, contained protein and nucleic acid. The cellular angiogenic factor showed a ratio of 1:1 for protein and nucleic acid whereas the secreted angiogenic factor had 3-5 parts of protein to 1 part of nucleic acid. The angiogenic factor from HEp2 and CHO cells did not bind to heparin-agarose. Microheterogeneity of the angiogenic factors was established by SDS-PAGE. Antiserum raised against the cellular angiogenic factor from HEp2 cells, showed a titre of 1:1600 by ELISA. The angiogenic factor was directly localized on whole cells by ELISA. Cellular as well as secreted angiogenic factors crossreacted with the antibody. Neutralizing effect of the antiserum on induction of angiogenesis was detected on chicken chorioallantoic membrane.

Production & formulation of Bacillus thuringiensis var. israelensis & B. sphaericus 1593.

Three fermentation media each for bulk growth of B. thuringiensis var. israelensis and B. sphaericus 1593 were formulated using defatted groundnut cake (Arachis hypogea) as the first nitrogen source and gram flour (Cicer arientinum), soy bean (Glycine max) and defatted milk powder as the second nitrogen source. Medium containing gram flour showed highest toxicity (14.45 micrograms/l) in case of B. thuringiensis var. israelensis whereas medium containing milk powder was found to be highly toxic with B. sphaericus 1593 (51.39 micrograms/l). Sustained release floating pellet formulations of B. thuringiensis var. israelensis and B. sphaericus 1593 exhibited toxicity of 77 per cent and above for 42 days at a dose of 500 micrograms/l for 4th instar larvae of Culex pipiens quinquefasciatus Say.

Growth & toxicity of Bacillus thuringiensis var israelensis.

Chemically defined media containing glutamic acid, glutamine and aspartic acid at a 20 mM concentration individually supported abundant growth and sporulation of Bacillus thuringiensis var israelensis. The parasporal crystals produced in these media were toxic to fourth instar larvae of Culex pipiens quinquefasciatus Say. The medium containing 20 mM of glutamine induced early sporulation of B. thuringiensis var israelensis. An LC50 value of 3.92 micrograms/l was obtained. Calcium alginate and agar agar immobilized formulations of Bacillus thuringiensis var israelensis exhibited toxicity of 90 per cent and above to C. pipiens quinquefasciatus Say larvae, over a period of 90 days.

Isolation & characterization of tumour angiogenesis factor from solid tumours & body fluids from cancer patients.

Tumour angiogenesis factor (TAF) was isolated from malignant solid tumours (10) and from pleural and peritoneal fluids (10) collected from cancer patients. Normal tissues and body fluids from individuals with no clinical history of cancer did not show any detectable levels of TAF. Also, no angiogenic activity was detectable in the benign tumour samples studied (2). The TAFs isolated were all ribonucleoproteins. Molecular weight determination by SDS-PAGE (9%) of the TAFs isolated by DEAE cellulose chromatography of tumour extracts showed them to be 18,000 dalton (D) ribonucleoproteins, while the TAFs isolated by immunoaffinity chromatography (using immobilized anti-TAF IgG) of solid tumour extracts and body fluids had a molecular weight of 38,000 D. The TAFs isolated by both the methods were found to be angiogenic by the chick chorioallantoic membrane and the mouse intradermal assays. Immunoaffinity chromatography could be used for the one-step purification of TAF from solid tumour extracts as well as from body fluids.

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation in Bacillus thuringiensis var. thuringiensis.

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis in Bacillus thuringiensis var. thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,214 C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.