A spectrophotometric method for calmodulin assay

The activity of calmodulin as an activator of cAMP phosphodiesterase was assayed. AMP was hydrolyzed by 5'-nucleotidase, and the adenosine formed was measured by both liquid scintillation counting and spectrophotometry at 265 nm. Calmodulin activities measured by the two methods were equivalent, indicating that spectrophotometric assay of calmodulin can be used in place of the isotopic method.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.     

Influence of the food plants on the degree of parasitism of larvae ofHeliothis armigera byCotesia kazak

Influence of the food plants ofHeliothis armigera (Hb.) on the degree of parasitism by exotic parasiteCotesia kazak Telenga (Hymenoptera: Braconidae) was studied in cages in the laboratory on 7 food plants such as cotton (Gossypium hirsutum L.), tomato (Lycopersicon esculentum Mill), okra [Abelmoschus esculentus (L.) Moench], Dolichos (dolichos lablab L.), pigeonpea [Cajanus cajan (L.) Millsp.], Cowpea (Vigna unquiculata (L.) and chickpea (Cicer arietium L.). To determine the preference of the parasite 2 test methods were employed. In single plant choice test cotton was most preferred. Next in order of preference were tomato and okra. Dolichos, pigeonpea, cowpea and chickpea were least preferred. In multiple choice test, however, cotton and okra were preferred followed by tomato. Parasites were seen visiting these plants very frequently and high parasitism was recorded on these plants. Chick pea, pigeon pea, cowpea and Dolichos were the least preferred food plants. There appears to be some difference in fecundity as affected by some food plants. Exposure on okra, cotton and tomato resulted in higher cocoon production as compared to pigeonpea, Dolichos, cowpea and chickpea. There was, however, no difference in sex-ratio and longevity of the progeny as affected by food plants. This exotic parasite should be released first in crops such as cotton, okra and tomato on whichH. armigera is a very serious pest in India and elsewhere. Contribution No. 140/86 of the Indian institute of Horticultural Research, Bangalore-560 089     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Design of liposome to improve encapsulation efficiency of gelonin and its effect on immunoreactivity and ribosome inactivating property

Gelonin, purified from the seeds of Gelonium multiflorum, using cation-exchange and gel-filtration chromatography was characterised for its purity, homogeneity and molecular weight by reverse-phase HPLC (RP-HPLC) and SDS-PAGE analysis. The HPLC purified gelonin was used for entrapment studies in the liposomes. Liposomes were prepared by reverse phase evaporation (REV) technique using three different types of lipid composition in the same molar ratio. The method resulted in 75–80% entrapment efficiency of gelonin in the liposomes. Entrapped and unentrapped gelonin was characterized for physico-chemical, immunochemical and biological properties. The immunoreactivity of entrapped gelonin was fully preserved but the ribosome-inactivating property was slightly inhibited. The method involved mild conditions, highly reproducible and the liposomes produced appeared to be stable for several months. It has important implications in the development of cell type specific cytotoxic agents where a chemical cross-linking is involved which significantly inhibits both immunoreactivity and ribosome-inactivating ability of the toxin.     

Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neo^R) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neo^R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo^R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo^R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.     

Inhibition of red light-induced seed germination by indole-3-acetic acid inHygrophila auriculata

Seed germination inHygrophila auriculata (Schumach.) Haines was found to be under phytochrome control. Exogenous indole-3-acetic acid (IAA) application at concentrations greater than 1×10^–6 M inhibited germination in the dark, as well as in the light. Red light-induced radicle growth, prior to radicle protrusion through seed coverings and measured as an angle formed by the radicle with the seed axis, was found to be inhibited by IAA. Delay in application of IAA to red light-irradiated seeds resulted in a gradual increase in percent germination, which probably corresponded to the time-course of Pfr action. It is suggested that exogenously applied IAA probably reimposes dormancy in red light-induced seeds ofHygrophila auriculata.     

The occurrence of long chain polyprenols in leaves of plants of Rosaceae family and their isolation by time-extended liquid chromatography.

The long chain polyprenols composed of 30 and more isoprene units from leaves of plants belonging to the genera Potentilla and Rosa have been described. They occur in the form of fatty acid esters. The composition of polyprenol mixture was species dependent and its content reached ca. 0.5% wet weight. Large scale preparation of individual polyprenols from a natural polyprenol mixture was performed using time-extended liquid chromatography on the hydrophobic gel Lipidex-5000.     

Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.