Persistence of Bacillus thuringiensis parasporal crystal insecticidal activity in soil

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai (H-serotype 7), strain HD137, streptomycin-resistant mutant were added to acidic (pH 5.0) natural and autoclaved soil and incubated at ?0.10 MPa, 25°C. Populations of B. thuringiensis in both soil treatments showed exponential rates of mortality which were represented by linear regression, the loss of viability being greater in natural than autoclaved soil. In natural soil, parasporal crystal insecticidal activity was lost at a complex, nonexponential rate. The initial, rapid decrease of activity gradually slowed, and the level of activity stabilized at 10% of the original inoculum level after 250 days incubation, until the cessation of sampling at >2 years. In autoclaved soil no significant (P > 0.2) loss of parasporal crystal insecticidal activity was detected over the same period, which suggested that soil microorganisms were responsible for the loss of crystal insecticidal activity in the natural, nonsterilized soil. The rate of loss of crystal activity in natural soil correlated well with assay data reported in the literature using Galleria mellonella, which measures the combined activity of spore and crystal. In autoclaved soil correlation was poor, probably due to variability in the bioassay data.     

Bluelight-induced,flavin-mediated transport of redox equivalents across artificial bilayer membranes

Summary This paper continues our studies of physico-chemical properties of vesicle-bound flavins. Based on previous results, an advanced model system was designed in order to study the mechanisms underlying bluelight-induced redox transport across artificial membranes. The lumen of single-shelled vesicles was charged with cytochromec, and amphiphilic flavin (AF1 3, AF1 10) was bound to the membrane. Upon bluelight irradiation redox equivalents are translocated from exogeneous 1e ^–(EDTA)-and 2e ^–(BH₃CN^–) donors across the membrane finally reducing the trapped cytochromec both under aerobic and anaerobic conditions. The mechanisms involved are explored and evidence for the involvement of various redox states of oxygen, dihydroflavin and flavosemiquinone is presented.     

Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

β-decay,Bremsstrahlen, and the origin of molecular chirality

Summary A brief review is presented of the Vester-Ulbricht -decay Bremsstrahlen hypothesis for the origin of optical activity, and of subsequent experiments designed to test it. Certain of our experiments along these lines, begun in 1974 and involving the irradiation of racemic and optically active amino acids in a 61.7 KCi⁹⁰Sr–⁹⁰Y Bremsstrahlen source, have now been completed and are described. After 10.89 years of irradiation with a total Bremsstrahlen dose of 2.5×10⁹ rads, crystallinedl-leucine, norleucine, and norvaline suffered 47.2, 33.6, and 27.4% radiolysis, respectively, but showed no evidence whatsoever of asymmetric degradation.d- andl-Leucine underwent about 48% radiolysis and showed 2.4–2.9% radioracemization. Other samples in solution were too severely degraded to analyze. Probable intrinsic reasons for the failure of the Vester-Ulbricht mechanism to afford asymmetric radiolysis in the present and related experiments involving -decay Bremsstrahlen are enumerated.A portion of this material was presented at the 7th International Conference on the Origins of Life, Mainz, FRG, July 10–15, 1983     

Regulation of respiration and ATP synthesis in higher organisms: Hypothesis

The present view on the regulation of respiration and ATP synthesis in higher organisms implies only Michaelis-Menten type kinetics and respiratory control as regulatory principles. Recent experimental observations, suggesting further regulatory mechanisms at respiratory chain complexes, are reviewed. A new hypothesis is presented implying regulation of respiration and ATP synthesis in higher organisms mainly via allosteric modification of respiratory chain complexes, in particular of cytochromec oxidase. The allosteric effectors, e.g., metabolites, cofactors, ions, hormones, and the membrane potential are suggested to change the activity and the coupling degree of cytochromec oxidase by binding to specific sites at nuclear coded subunits. Recent results on the structure and activity of cytochromec oxidase, supporting the hypothesis, are reviewed.Dedicated to Professor Dr. Carl Martius on the occasion of his 80th birthday.     

Thermodynamics of the charge recombination in photosystem II

The temperature dependence of the electric field-induced chlorophyll luminescence in photosystem II was studied in Tris-washed, osmotically swollen spinach chloroplasts (blebs). The system II reaction centers were brought in the state Z⁺P⁺-Q_A ^-Q_B ^- by preillumination and the charge recombination to the state Z⁺PQ_AQ_B ^- was measured at various temperatures and electrical field strengths. It was found that the activation enthalpy of this back reaction was 0.16 eV in the absence of an electrical field and diminished with increasing field strength. It is argued that this energy is the enthalpy difference between the states IQ_A ^- and I^-Q_A and accounts for about half of the free energy difference between these states. The redox state of Q_B does not influence this free energy difference within 150 s after the photoreduction of Q_A. The consequences for the interpretation of thermodynamic properties of Q_A are discussed.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - I intermediary electron acceptor - Mops 3-(N-morpholino)propanesulphonic acid - P (P₆₈₀) primary electron donor - PS II photosystem II - Q_A and Q_B first and second quinone electron acceptors - Tricine N-tris(hydroxymethyl)methylglycine - Tris tris-(hydroxymethyl)aminomethane - Z secondary electron donor Dedicated to Professor L.N.M. Duysens on the occasion of his retirement     

Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer

A newly developed modulation fluorometer is described which operates with 1 sec light pulses from a light-emitting diode (LED) at 100 KHz. Special amplification circuits assure a highly selective recording of pulse fluorescence signals against a vast background of non-modulated light. The system tolerates ratios of up to 1:10⁷ between measuring light and actinic light. Thus it is possible to measure the dark fluorescence yield and record the kinetics of light-induced changes. A high time resolution allows the recording of the rapid relaxation kinetic following a saturating single turnover flash. Examples of system performance are given. It is shown that following a flash the reoxidation kinetics of photosystem II acceptors are slowed down not only by the inhibitor DCMU, but by a number of other treatments as well. From a light intensity dependency of the induction kinetics the existence of two saturated intermediate levels (I₁ and I₂) is apparent, which indicates the removal of three distinct types of fluorescence quenching in the overall fluorescence rise from F₀ to F_max.Abbreviations Q_A and Q_B consecutive electron acceptors of photosystem II - PS II photosystem II - P 680 reaction center chlorophyll of photosystem II - F₀ minimum fluorescence yield following dark adaptation - F_max maximum fluorescence yield - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea - DCCD N,N-dicyclohexylcarbodiimide - PQ plastoquinone - DAD diaminodurene Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized -galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show supressed respiration of TCA cycle compounds; -galactosidase was not synthesized because the mutant lacked a functional PTS. Cyclic adenosine-3, 5-monophosphate did not reverse the repression by glucose of SEA or -galactosidase synthesis in glucose-grown S. aureus 196E. An active PTS appears to be necessary to demonstrate glucose (catabolite) repression in S. aureus.Abbreviations SEA staphylococcal enterotoxin A - SEB staphylococcal enterotoxin B - SEC staphylococcal enterotoxin C - PTS phosphoenolpyruvate phosphotransferase system - CAS casamino acids salts medium - TCA tricarboxylic acid cycle     

Fed-batch culture technology

The use of fed-batch procedures offers distinct advantages over other modes of operation of bioreactors, and is a widely researched technique. These advantages are discussed; some uses of fed-batch procedures and the associated methods of modelling and control are reviewed.     

Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.