EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

Transpiration and drought resistance of Douglas-fir seedlings exposed to excess ammonium

 In a pot trial growth and transpiration of 3-year-old Douglas-fir seedlings on an acid, sandy soil was examined at a deficient (30 kg N ha^{–  1} year^{–  1}) and an excessive level (120 kg N ha^{–  1} year^{–  1}) of NH₄ application. Dissolved ammonium sulphate was applied to the pots weekly for two growing seasons. In half of the pots a complete set of other nutrients was applied in optimal proportions to the applied nitrogen. Water supply was optimal and transpiration was recorded. At the end of the second treatment season irrigation was stopped for 2 weeks during dry and sunny weather. Both high application of NH₄ and additional nutrients increased shoot growth and transpiration demand in the first treatment year. The root system was smaller at higher N level and this reduced water uptake accordingly. In the second year the combination of high NH₄ ⁺ and additional nutrients affected root functioning predominantly due to salinity effects and this seriously decreased water uptake capacity and shoot water potentials, finally resulting in tree death. Without addition of other nutrients the high NH₄ ⁺ application resulted in a high degree of soil acidification, which damaged the roots, that showed a decrease in water uptake capacity. At the low NH₄ supply level soil acidification was lower, and root functioning was not affected, and the trees recovered quickly from the imposed drought. Higher needle K and P status depressed transpiration rates at the low NH₄ application rate. Received: 9 January 1995 / Accepted: 18 September 1995     

Multiple xenobiotic-inducible P450s are involved in alkoxycoumarin and alkoxyresorufin metabolism in higher plants

O-Dealkylation of two series of fluorescent 7-alkoxy-coumarins and 7-alkoxyphenoxazones by plant cytochrome P450s was investigated in Helianthus tuberosus tuber tissues treated with prototype P450 inducers, environmental pollutants or agrochemicals. Methoxy-, ethoxy-, propoxy-and butoxycoumarins and methoxy- and ethoxyresorufins were metabolized by fplant microsomes. Dealkylation of pentoxy- and benzyloxyresorufins was not detected. All dealkylating activities were enhanced by aging plant tissues in the presence of xenobiotics, in some cases up to 20-fold relative to the activities detected in control tissues. Increases in total P450 in the same tissues never exceeded 3-fold. The isozymes induced by prototype P450 inducers clearly differed from those in mammalian liver. That multiple P450s with overlapping substrate specificities were involved in the metabolism of both alkoxycoumarins and alkoxyresorufins was demonstrated by (1) the differential induction of the activities in response to exposure to xenobiotics, (2) the differential inhibition of the activities by clotrimazole, paclobutrazole and tetcyclacis in aminopyrine and benzo(a)pyrene-treated tissues, and(3) the selective inhibition observed with antibodies raised against purified ethoxycoumarin deethylase fractions. Our results suggest that the measurement of the dealkylation of such fluorescent substrates in plants might be useful to monitor environmental pollution.     

Skeletal muscle: A paradigm for testing principles of bioenergetics

Muscular activity converts chemical energy into useful work and metabolism restores muscle to its original state. This essay explores the organization and interactions of the regulatory system(s) which allow this energy balance to occur. The term energy balance is used in a biochemical rather than a thermodynamic sense—concerned not with deductions from the physical principles of thermodynamics, but rather with those enzymatic processes which nature evolved and which operate at remarkably fixed stoichiometry. Energy balance is a statement of conservation of energy put into biochemical observables.³¹P NMR spectroscopy is one of the most useful techniques for investigating these questions quantitatively under physiological conditionsin vivo. The author (1) describes the rules or principles of biochemical energy balance; (2) discusses sample results from human muscle to demonstrate its use in studying this class of questions; (3) presents a simple model of integrated cellular respiration to demonstrate its sufficiency to account for the main observations.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.     

Interaction of force transmission and sarcomere assembly at the muscle-tendon junctions of carp (Cyprinus carpio): ultrastructure and distribution of titin (connectin) and α-actinin

At muscle-tendon junctions of red and of white axial muscle fibres of carp, new sarcomeres are found adjacent to existing sarcomeres along the bundles of actin filaments that connect the myofibrils with the junctional sarcolemma. As the filament bundles that transmit force to the junction originate proximal to new sarcomeres, they probably relieve these new sarcomeres from premature loading. In red fibres, these filament bundles are long (up to 20 m) and dense, permitting light-microscopical immunohistochemistry (double reactions: anti-titin or anti--actinin and phalloidin). New sarcomeres have clear I bands; their A band lengths are similar to those of older sarcomeres and the thick filaments lie in register. T tubules are found at the distal side of new sarcomeres but terminal Z lines are absent. The late addition of -actinin suggests that -actinin mainly has a stabilizing role in sarcomere formation. The presence of titin in the terminal fibre protrusions is in agreement with its supposed role in sarcomere formation, viz. the integration of thin and thick filaments. The absence of a terminal Z line from sarcomeres with well-registered A bands suggests that this structure is not essential for the anchorage of connective (titin) filaments.     

Cortical hypometabolism in injured brain: New correlations with the noradrenergic and serotonergic systems and with behavioral deficits

Changes with time after injury in behavioral deficits, as determined by the Morris swim test, and the in vivo specific binding of HEAT, a selective ₁-adrenoreceptor ligand, were compared with the time-course of development of cortical hypometabolism in rats with focal freezing lesions. In our trauma model, cortical hypometabolism was detectable in the lesioned hemisphere at 4 hr, became maximal (50% of normal) at 3 days and diminished towards normal on days 5 and 10 post-injury. Progressive impairment of acquisition of the Morris water maze task was demonstrated up to day 3 post-lesion with improvement thereafter. On day 3 the latency to reach criterion was 60% longer in lesioned animals than in corresponding sham-operated ones. An increase in the volume of distribution of HEAT, limited to cortical areas of the lesioned hemisphere, was demonstrable at 4 hr post-lesion and reached its maximum on day 3 (200% of normal) with subsequent return toward normal on days 5 and 10. Several types of drugs were shown previously to modify the cortical hypometabolism associated with cerebral injury. The present data indicate that the same drugs also modify the in vivo binding of HEAT and the behavioral deficits induced by brain lesions. Ibuprofen, a non-steroidal anti-inflammatory drug, p-chlorophenylalanine, an inhibitor of serotonin synthesis, ketanserin, a specific 5HT₂-receptor antagonist, and prazosin, an ₁-adrenergic receptor blocker all normalized the in vivo binding of HEAT in the cortical areas of the lesioned hemisphere. All groups of animals treated with these drugs also showed subtle, but statistically highly significant improvements in latency to locate the platform in the Morris water maze. Taken together these results show good correlation between behavioral deficits, changes in ₁-noradrenergic receptor binding and cortical hypometabolism in injured brain. This supports the hypothesis that post-injury cortical hypometabolism is a reflection of cortical functional depression in which both the serotonergic and noradrenergic neurotransmitter systems play a role, compatible with their inhibitory effects in the cortex and their postulated involvement in cortical information processing.Special issue dedicated to Dr. Leon S. Wolfe.     

Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.     

Synthesis and bioassay of isoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent,selective inhibitors of juvenile hormone esterase

Four 3-alkylthio-1,1,1-trifluoro-2-propanones with juvenile hormone-like side chains were prepared from citronellol and homogeraniol. These substrates were designed as possible transition-state analogs for the juvenile hormone (JH)-specific esterases present in insects. These four isoprenoid trifluoromethyl ketones were assayed in vitro with JH esterase and general esterases from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera, Noctuidae), and with eel acetylcholinesterase and bovine chymotrypsin. JH esterase inhibition I₅₀ values were in the nanomolar range for all four compounds, while the other esterases had I₅₀'S which were 10³ to 10⁵ higher. The high selectivity of these inhibitors is believed to be due to their similarity in size and functionality to natural JH III. Treatment of T. ni larvae in vivo with solutions of the most active analog, 3-[(E)-4,8-dimethyl-3,7-nonadienylthio]-1,1,1-trifluoro-2-propanone (DNTFP) causes a dose-dependent delay in pupation and a concurrent selective inhibition of JH esterase. These data support the hypothesis that the reduction in in vivo JH titer in larval T. ni is due, in part, to hydrolysis of the hormone by selective esterases. DNTFP appears to be competing with JH for the active site of JH esterase.     

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.