Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.     

Inheritance of resistance to bacterial blight in common bean

Summary Inheritance of resistance to common bacterial blight in the trifoliate leaf, plant canopy, and pods was controlled by a single major gene. Additive followed by dominance effects were more important than epistatic interactions. Narrow-sense heritability values ranged from 0.18 to 0.87 for trifoliate leaf, from 0.26 to 0.76 for canopy, and from 0.11 to 0.36 for pods. Observed gains from selection for resistance were higher than expected gains. Implications of these results in breeding for resistance are discussed.     

Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin.

Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

Relationship between respiratory pauses and heart rate during sleep in normal premature and full-term newborns

To investigate the relationship between central respiratory pauses and heart rate, we performed polygraphic recordings in 23 normal newborns (35 to 41 weeks conceptional age). We monitored the electroencephalogram, rapid eye movements, movements of the upper and lower limbs, chin and diaphragmatic electromyogram, electrocardiogram, thoracic and abdominal respiratory movements, air flow and transcutaneous PO2. Heart rate changes were analysed by computer measurement of R-R intervals and by cardiotachography. Respiratory pauses occurring after body movements and those not preceded by movements were studied separately. We analysed 1128 respiratory pauses greater than 3 s duration. No respiratory pause lasted more than 12 s. Independently of age, sleep state and respiratory pause duration, heart rate was significantly lower at the onset of respiratory pause, compared to control periods (selected away from the pause: 10 s before its onset and 20 s after its end). Heart rate slowed still further through the respiratory pause and reverted toward the baseline level after its end. When no movements preceded the respiratory pause, heart rate just before the pause was lower compared to control periods. These findings suggest the existence of simultaneous central commands responsible for both respiratory pause and heart rate deceleration.     

Development of a fructose based medium for biosynthesis of cyclosporin-A byBeauveria nivea

Summary A new fructose based medium was developed to growBeauveria nivea ATCC 34921 for the production of Cyclosporin A (CyA). Two different sugars and three different nitrogen sources were used for CyA production. Different pH levels in the broth revealed that the best pH for CyA production was 5.5. Maximum concentration of 170 mg CyA/L of broth was obtained with the new medium in 8 days corresponding to yields of 14.8 mg CyA/g dry weight biomass and 5.67 mg CyA/g fructose supplied.     

Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers

The 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis binds irreversibly to Aedes albopictus cells, Choristoneura fumiferana cells, and erythrocytes. The binding to cells increased with both toxin concentration and time and when the cells were first preincubated with unlabeled toxin. Binding data indicated a two- to threefold increase in the rate of binding after the amount of the membrane-bound toxin reached approximately 3.5 fmol/3 x 10(5) A. albopictus cells or 3.3. fmol/2 x 10(5) C. fumiferana cells. When this level of bound toxin was reached, the toxins also began forming aggregates at the cell membrane. The toxin aggregates were extracted with 10% Triton X-100 and separated from the monomers with a 5 to 20% sucrose density gradient. The toxin aggregates isolated from A. albopictus and C. fumiferana cell membranes were ca. 400 kilodaltons, while those isolated from human erythrocytes were significantly smaller. The proportion of the toxin found in aggregate form increased rapidly with the amount of toxin bound; however, the molecular size of the aggregates remained constant. Eleven monoclonal antibodies raised against the native form of the toxin blocked 80 to 97% of the toxin binding to cells. The epitope of one of these monoclonal antibodies was mapped to a domain which included the cysteine, suggesting the importance of the domain around this amino acid to binding. Toxin binding and cell lysis were also inhibited by treating the toxin with HgCl2, further indicating the importance of the C-terminal hydrophobic cysteine-containing domain in cytolytic activity of the 25-kilodalton protein.     

The effect of histidine on the structure and antitumor activity of metal-5-halouracil complexes

The ternary complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) ions with 5-halouracils, viz., 5-fluorouracil (5FU), 5-chlorouracil (5ClU), and 5-bromouracil (5BrU), and the biologically important ligand L-histidine (HISD) have been synthesized and characterized by elemental analysis, conductance measurements, infrared spectra, electronic spectra, and magnetic moment (room temperature) measurements. On the basis of these studies, the structures of the complexes have been proposed. All these ternary complexes were screened for their antitumor activity against Dalton's lymphoma in C3H/He mice. It was found that only Mn(II)-5BrU-HISD, Co(II)-5BrU-HISD, Cu(II)-5ClU-HISD, Cu(II)-5BrU-HISD, Zn(II)-5FU-HISD, and Zn(II)-5BrU-HISD complexes have significant antitumor activity with T/C greater than 125% (where T and C represent mean lifespan of treated mice and control mice respectively). The Mn(II)-5FU-HISD, Co(II)-5FU-HISD, Co(II)-5ClU-HISD, Ni(II)-5ClU-HISD, Ni(II)-5BrU-HISD, and Zn(II)-5ClU-HISD complexes are also effective antitumor agents, with T/C greater than 115%. The complexes that showed effective antitumor action in vivo were also found to inhibit 3H-thymidine incorporation (DNA replication) in Dalton's lymphoma cells in vitro.     

Male reproductive success in harem troops of hanuman langurs (Presbytis entellus)

Based on cross-sectional and longitudinal data collected in 1967–1988 by various observers, male reproductive success was studied in the Hanuman langurs of Jodhpur, India. The harem-structured social organization ensures a high degree of paternity certainty. Births occur throughout the year, with significant peaks and minima in March and November, respectively (n =398).The interbirth interval averages 16.7 months (n = 114).The duration of harem residencies varies between 3 days and ≥ 74.0 months, with a mean of 26.5 (n = 64). Harem holder replacements occur during all months of the year. No male achieves residency in more than one troop, suggesting that residency is associated with a distinct peak in the resource holding potential of a given male. Reproductive success among males varies considerably. Male mortality is high due to migration and intrasexual competition, leading to an adult sex ratio of 1:4.9. It is estimated that one-quarter of all adult males will never gain harem residency. Conceptions achieved outside harem residencies are so rare (4.7%) that a viable low-risk strategy, opting for longevity instead of harem residency, is unlikely. Tenure length has a stronger influence on male reproductive success than harem size because interbirth intervals are significantly shorter in small harems than in larger ones. It is assumed that females in one-male breeding structures compete for sperm and that such competition is more intense in larger harems.     

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.