Validation of the GROMOS force-field parameter set 45A3 against nuclear magnetic resonance data of hen egg lysozyme

The quality of molecular dynamics (MD) simulations of proteins depends critically on the biomolecular force field that is used. Such force fields are defined by force-field parameter sets, which are generally determined and improved through calibration of properties of small molecules against experimental or theoretical data. By application to large molecules such as proteins, a new force-field parameter set can be validated. We report two 3.5 ns molecular dynamics simulations of hen egg white lysozyme in water applying the widely used GROMOS force-field parameter set 43A1 and a new set 45A3. The two MD ensembles are evaluated against NMR spectroscopic data NOE atom–atom distance bounds, ³J_NH and ³J coupling constants, and ¹5N relaxation data. It is shown that the two sets reproduce structural properties about equally well. The 45A3 ensemble fulfills the atom–atom distance bounds derived from NMR spectroscopy slightly less well than the 43A1 ensemble, with most of the NOE distance violations in both ensembles involving residues located in loops or flexible regions of the protein. Convergence patterns are very similar in both simulations atom-positional root-mean-square differences (RMSD) with respect to the X-ray and NMR model structures and NOE inter-proton distances converge within 1.0–1.5 ns while backbone ³J_HN-coupling constants and ¹H– ¹5N order parameters take slightly longer, 1.0–2.0 ns. As expected, side-chain ³J-coupling constants and ¹H– ¹5N order parameters do not reach full convergence for all residues in the time period simulated. This is particularly noticeable for side chains which display rare structural transitions. When comparing each simulation trajectory with an older and a newer set of experimental NOE data on lysozyme, it is found that the newer, larger, set of experimental data agrees as well with each of the simulations. In other words, the experimental data converged towards the theoretical result.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

Comparison of the ability of three Aspergillus strains to form aflatoxins on bakery products and on nutrient agar

The growth of Aspergillus parasiticus NRRL 2999, A. parasiticus NRRL 3000 and A. flavus NRRL 3251 on whole wheat bread and on cake (Rührkuchen) was compared and the formation of the aflatoxins B₁, B₂, G₁, G₂ and M₁ on these substrates and, for purpose of comparison, on malt extract agar was determined. On cake the moulds grew better than on bread and formed the highest yields of aflatoxins. Malt extract agar was the most unfavourable substrate for toxin production. The ratio M₁/B₁ on bread and cake was in the order of 0.1–0.4 and was higher than the data reported for grains. The highest yields of aflatoxin B₁ (1.0 g/g) were produced by A. flavus NRRL 3251 on cake.     

Thermodynamic and kinetic considerations of Q-cycle mechanisms and the oxidant-induced reduction of cytochromesb

In coenzyme Q-cycles, it is proposed that one electron from the quinol reduces the Rieske iron sulfur center (E _m280 mV) and the remaining electron on the semiquinone reduces cytochromeb _T (E _m–60 mV). TheE _mfor the two-electron oxidation of the quinol is 60 mV and therefore the reduction of cytochromeb _T by quinol is not favorable. As the stability constant for the dismutation of the semiquinone decreases, the calculatedE _mfor the Q/QH couple is lowered to values below theE _mof cytochromeb _T. Contemporary coenzyme Q-cycles are based on the belief that the lower value for theE _mof the Q/QH couple compared to theE _mfor cytochromeb _T means that the semiquinone is a spontaneous reducing agent for theb-cytochrome. The analysis in the paper shows that this is not necessarily so and that neither binding sites nor ionization of the semiquinoneper se alters this situation. For a Q-cycle mechanism to function,ad hoc provisions must be made to drive the otherwise unfavorable reduction of cytochromeb _T by the semiquinone or for the simultaneous transfer of both electrons to cytochromeb _T and cytochromec ₁ (or the iron sulfur protein). Q-cycle mechanisms with these additional provisions can explain the observation thus far accumulated. A linear path which is functionally altered by conformational changes may also explain the data.     

Phlorizin inhibits hexose transport across the plasmalemma of Riccia fluitans

The effect of phlorizin has been tested on hexose transport and hexose-induced changes of electrical potential (_m) and conductance (g _m) across the plasmalemma of rhizoid and thallus cells of the aquatic liverwort Riccia fluitans. The decrease of _m (depolarization) and g _m induced by 1 mM 3-oxymethyl-D-glucose (3-OMG) is substantially inhibited by simultaneous addition of 2 mM phlorizin, whereas no significant response was observed when phlorizin was added alone or several minutes after the sugar. Current-voltage data show that the 3-OMG-generated electrical inward current of 0.016 A m^-2 drops to 0.010 A m^-2 when phlorizin is present. Uptake as well as efflux of [¹⁴C]-3-OMG is strongly and reversibly inhibited by phlorizin between 0.2 and 5 mM. The results are consistent with our hypothesis that the hexose carrier has one binding site with competitive inhibition of glucose uptake by phlorizin (k _i=0.08 mM). The electrical data indicate that phlorizin affects an _m step of the carrier transport cycle.Abbreviation 3-OMG 3-oxymethyl-D-glucose     

Modelling based method for pharmacokinetic hypotheses test

The aim of this work is to propose methods to test mechanism of synergy of toxic agents in bees. A synergy between prochloraz, an imidazole fungicide, and deltamethrin, a pyrethroid insecticide, was demonstrated experimentally. The hypothesis is that prochloraz modifies the penetration or the metabolism of deltamethrin. This hypothesis is tested using a pharmacokinetic box model. A previous experimental work showed that bee instantaneous mortalities were higher, from the time t ₁ to the time t ₂ after spraying, in groups sprayed with deltamethrin at dose D ₀ in the presence of prochloraz (+P) than in those sprayed with deltamethrin alone at a dose time as high (). We postulate that accrued mortality is proportional to the cumulated internal deltamethrin (ID ₂). ID ₂ of treatment (+P) had to be greater than ID ₂ of treatment () during the period from t ₁ to t ₂ so that the hypothesis would be consistent with the experimental data. The limit, for which the hypothesis is conceivable, is the ID ₂₍₎ = ID _2(+P ) curve. We study, in particular, the asymptotic behaviour of the limit curve when different parameters of the kinetic model tend to 0 or . These limits allow to verify quickly and easily whether a mechanism is conceivable or not As the limits are calculated with algebraic values, the test can be used for other synergies.     

Structural comparison of the genes of two HLA-DR supertypic groups: The loci encoding DRw52 and DRw53 are not truly allelic

The organization and sequence of the HLA-DR chain genes are compared in the two supertypic groups, DRw52 and DRw53, which together account for more than 80% of HLA-DR alleles. From the structural data, we conclude that these two groups represent distinct lineages which have followed different patterns of evolution. The fine structure of the chain locus encoding the DRw53 specificity corresponds most closely to the DR II pseudogene in the DRw52 haplotypes. Concomitantly, the DR I locus in DRw53 haplotypes is more closely related to both of the two expressed DR loci of theDRw5 haplotypes (DR I and DR III). These two loci are the result of a recent duplication. This leads to the proposal that both expressed DR chain genes in the DRw52 haplotypes (DR I and DR III) are derived from a single precursor locus, while the two loci expressed in the DRw53 haplotypes are derived from distinct ancestral loci. The genes encoding DRw52 and DRw53 are therefore not true alleles of the same original locus. A scheme is proposed that accounts for the evolution of DR specificities within the DRw52 and DRw53 groups of haplotypes. It is evident that the differentHLA-DR alleles are not structurally equidistant and that one must take into consideration different degrees of heterozygosity or mismatch among the DR alleles.     

Co-occurrence of aflatoxin B<Subscript>1</Subscript> and cyclopiazonic acid in sour lime (<Emphasis Type="Italic">Citrus aurantifolia Swingle</Emphasis>) during post-harvest pathogenesis by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

During hot and humid seasons, extensive rot of sour lime was observed to be caused by Aspergillus flavus. In view of this, investigations were undertaken to obtain data on the production of various toxins by A. flavus during post harvest pathogenesis of sour lime. Sixty percent of the pathogenic A. flavus isolates were detected to be aflatoxin B₁ producers in sour lime tissue. It was also noted that thirty three percent of aflatoxigenic A. flavus isolates had the potential to coproduce cyclopiazonic acid (CPA). Such aflatoxigenic isolates produced quantitatively more CPA (ranging from 250.0 to 2501.3 g/kg) than aflatoxin B₁ (ranging from 141.3 to 811.7 g/kg) in the affected sour lime. This study demonstrates for the first time that sour lime are a favourable substrate for aflatoxin B₁ and cyclopiazonic acid production by A. flavus isolates. This is of great concern to the health of consumers.     

The effect of antifoam agents on mass transfer in bioreactors

The influence of antifoam agents on the liquid-phase mass transfer coefficient in stirred tank and bubble column bioreactors is studied. A physical model based on a surface-renewal concept and additional data in 40-dm³ bubble column bioreactor are presented. Comparisons between the physical model and the data indicate that the model predicts the maximum influence of antifoam agents on the liquid-phase mass transfer coefficient.List of Symbols a 1/m specific surface area - D m²/s diffusivity - D _c m bubble column diameter - d _vs m bubble diameter - g m/s² gravitational acceleration - k _L m/s liquid-phase mass transfer coefficient - k _LO m/s liquid-phase mass transfer coefficient for clean surface - N 1/s impeller speed - Sc Schmidt number (v/D) - U _sg m/s superficial gas velocity Greek Letters W/kg energy dissipation rate per unit mass - _g gas hold-up - Pa s viscosity - v m²/s kinematic viscosity - kg/m³ density - N/m surface tension     

Genetic polymorphism of human plasma α1-B-glycoprotein: phenotyping by immunoblotting or by a simple method of 2-D electrophoresis

Summary Genetic polymorphism of human plasma (serum) ₁B-glycoprotein (₁B) was observed using one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 of plasma samples followed by Western blotting with specific antiserum to ₁B. A simple method of two-dimensional agarose gel electrophoresis (pH 5.4) — horizontal PAGE (pH 9.0) of plasma samples, followed by general protein staining, was reported as an alternative method for ₁B typing. The there different phenotypes of ₁B observed (designated 1-1, 1-2, and 2-2) were apparently identical to those reported by Altland et al. (1983), who used double one-dimensional electrophoresis. Family data supported the hypothesis that the three ₁B phenotypes are determined by two codominant alleles at an autosomal locus, designated A1B. Allele frequencies in a Swedish population were: A1B ^*1, 0.937; A1B ^*2, 0.063; PIC, 0.111. For clues on linkage relationships of human A/B, the previously known linkages of A1B in pigs and horses, including the one between A1B and the gene that determines susceptibility to malignant hyperthermia in pigs were discussed.     

A protein backbone psi and phi angle dependence of 2J(N(i),C alpha(i-1)): the new NMR experiment and quantum chemical calculations

A new pulse sequence exploiting double- and zero-quantum evolution of two-spin ¹⁵N–¹³C coherence is proposed for the accurate measurements of ²J _{N( i ),C( i - 1)} coupling constants. Application of the new experiment is presented for ¹³C,¹⁵N-labeled ubiquitin sample. The density functional theory calculations of ²J _{N( i ),C( i - 1)} coupling constants have been performed to study their dependence on both (i - 1) and (i - 1) angle in model peptides, and the results exhibit a good correlation with experimental data.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7563-7This revised version was published online in April 2005. In the previous version, the name of the last author, Joanna Sadlej, was missing.     

Electrical membrane potential and resistance in photoautotrophic suspension cells of Chenopodium rubrum L.

On photoautotrophically grown, suspension-cultured cells of Chenopodium rubrum L. the electrical potential difference V _mand the electrical resistance across plasmalemma and tonoplast have been measured using one or two intracellular micro-electrodes. In a mineral test-medium of 5.8 mM ionic strength V _mvalues between 100 and 250 mV, 40% thereof between 170 and 200 mV, and a mean value (±S.E.M.) of 180.6±3.4 mV have been recorded. The average membrane input resistance R _mwas 269±36 M, corresponding to an average membrane resistivity r _mof 3.0 m². V _mand r _mare sensitive to light, temperature, and addition of cyanide, suggesting the presence of an electrogenic hyperpolarizing ion pump, and are ascribed essentially to the plasmalemma. A hexose-specific saturable electrogenic membrane channel is identified through a decrease of V _mand r _mupon addition of hexoses. The hexoseconcentration-dependent depolarization V _msaturates at 92 mV and returns half-saturating concentrations (apparent k _mvalues) of 0.16 mM galactose, 0.28 mM glucose, and 0.48 mM fructose. The magnitude of V _mand r _mwell agrees with pertinent data from mesophyll cells in situ (where only V _mdata are available) and from photoautotrophic lower plant cells. However, V _mis markedly higher than reported for heterotrophically grown suspension cells of different higher plants (with which r _mdata have not been reported so far). It is concluded from the present study and a companion paper on water transport (Büchner et al., Planta, in press) that photoautotrophically grown Chenopodium suspension cells closely resemble mesophyll cells as to cell membrane transport properties.Abbreviations V _m membrane potential(mV) - R _o input resistance () - R _m membrane input resistance () - r _m specific resistance (resistivity) of the membrane (m²)     

Identifying populations useful for improving parents of a single cross based on net transfer of alleles

Summary Theory and methods for identifying populations (P _y ) with the highest frequency of favorable dominant alleles not present in an elite single cross (I ₁× I ₂) have been developed recently. During selection, new favorable alleles can be transferred from P _y to either I ₁ or I ₂ only at the risk of losing favorable alleles already present in the single cross. A net improvement (NI) statistic, which estimates the relative number of favorable alleles that can be gained from P _y minus the relative number of favorable alleles that can be lost from I ₁ or I ₂, is presented. NI is calculated as maximum [(I ₁×P _y –I ₁×I ₂)/2,(I ₂×P _y –I ₁×I ₂)/2]. Because I ₁ × I ₂ is constant in an experiment, the method reduces to choosing P _y populations with the best mean performance in combination with either I ₁ or I ₂. For a set of maize (Zea mays L.) grain yield data, NI was highly correlated to three other statistics proposed for choosing populations, namely: (1) minimally biased estimate (l ) of the relative number of favorable dominant alleles present in P _y but not in I ₁ and I ₂; (2) minimum upper bound on l ; and (3) predicted performance of the three-way cross [P _y (I ₁× I ₂)]. While l estimates potential improvement likely to be achieved only through long-term recurrent selection, NI is probably a better predictor of short-term improvement in single-cross performance.A contribution from Lifaco Genetics, a subsidiary of Groupe Limagrain     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue in ₁-bungarotoxin (gb ₁-Bgt), the A chain of ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp in ₁-Bgt. The Trp of ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain of ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure of ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain in ₁-Bgt.     

A comparison of signal detection between an echolocating dolphin and an optimal receiver

Summary An electronic simulated target apparatus was used in a two-experiment study to compare the target detection performance of an echolocating bottlenose dolphin with an optimal receiver. Random Gaussian noise with a relatively flat spectrum from 20 to 160 kHz was used as a masking source. Experiment I was conducted to establish a technique for estimating the echo energy-to-noise ratio,E _e /N, at the dolphin's threshold of detection. Dolphins typically vary the amplitude of their emitted signal over a large range making it difficult to estimateE _e /N. In the first part of experiment I, the simulated echo was a double click, the pulses separated by 200 s, with each pulse being a replica of the dolphin's transmitted signal. A staircase psychophysical procedure was used to obtain the detection threshold, and the echo energy-to-noise ratio based on the highest amplitude click emitted per trial, (E _e /N)_max, was determined at each reversal point. The second echo type consisted of one of the animal's echolocation clicks, previously measured, digitized and stored in an erasable programmable read-only memory (EPROM). The electronic target simulator was modified so that every time the dolphin emitted an echolocation signal, the EPROM was triggered to produce two pulses separated by 200 s. On any trial, the EPROM signal was played back at a fixed amplitude, regardless of the amplitude of the dolphin's emitted signal. TheE _e /N obtained with the EPROM signal at threshold was found to be 2.9 dB lower than (E _e /N)_max obtained with the normal phantom target. Therefore an estimate ofE _e /N can be obtained by subtracting 2.9 dB from (E _e /N)_max.Experiment II was conducted to obtain isosensitivity data that could be plotted in an ROC (receiver operating characteristic) format. The response bias of the dolphin was manipulated by varying the food reinforcement payoff matrix. In terms of the ratio of correct detections to correct rejections, the payoff matrix was varied over four values: 11, 41, 81, and 14. A modified method of constants procedure was used to obtain the dolphin target detection performance data. Each session consisted of two 20-trial blocks in which a strong echo was used in the first block and a weak echo in the second block. The energy-to-noise ratio required by an optimal detector to approximate the dolphin's performance was obtained by determining the appropriate detection sensitivity,d, that best fitted the dolphin's data plotted in an ROC format. The results of experiment II indicated that the dolphin required approximately 7.4 dB higherE _e /N than an optimal detector to detect the phantom target.Abbreviations response bias - d detection sensitivity - E _e echo energy flux density - EPROM erasable programmable read-only memory - N noise spectral density - p(t) instantaneous acoustic pressure - P(Y/SN) probability of detection - P(Y/N) probability of false alarm - ROC receiver-operating-characteristics - SE source energy flux density     

Polynucleotides. XI. Thermodynamics of (A) N -2(U) infinity from the dependence of T m N on oligomer length

The variation in the helix-coil transition temperature, T_m^N, with oligomer length, N, for the system ((I)) has been examined. The results for N = 4-13, measured in 0.2M Na+, have been analyzed in terms of the expression of Blake (1972): ((II)) where c_m is the free oligomer concentration at T_m^N, and V_r^f is the thermodynamic free volume available to a helical base-triplet residue. The correlation coefficient for the fit to expression (II) of data obtained over a 50° temperature range is 0.997 when ΔH_r = ?12.6 kcal/mole of base-triplets (independent of oligomer length (N ? 4) or temperature), the value previously obtained from both calorimetry of (A)_∞·2(U)_∞ and (A)₄ concentration dependence of T_m. It is found that V_r^f = 8.0 × 10^?4 1/mole (± 30%) or 1.33 ų per helical base-triplet, and is constant with temperature. A maximum value for V_r^f of 21.0 × 10^?4 1/M (± 1.3%), equivalent to 3.54 ų per helical basetriplet is obtained by the same treatment of the helix-coil transition data for the three-stranded helix formed by adenosine (N = 1) and 2(U)_∞ obtained by Davies and Davidson (1971).     

Use of recombinant substitution lines in the construction of RFLP-based genetic maps of chromosomes 6A and 6B of tetraploid wheat (Triticum turgidum L.)

RFLP-based genetic maps of chromosomes 6A and 6B of Triticum turgidum have been constructed using data obtained by the study of Triticum turgidum var durum cv Langdon-T. t. var dicoccoides recombinant substitution lines (RSLs) supplemented with data obtained from F₃ families derived from Langdon dicoccoides 6A and 6B disomic substitution lines. The average RFLP frequencies detected for the two chromosomes in a test of 45 DNA clones with six restriction enzymes were 56% and 53%, respectively, and a subset of 32 clones gave frequencies of 75% and 72%, respectively. Seventeen loci were mapped in 6A and 18 in 6B. With the possible exception of 5 loci in the centromeric region of 6A, all of the mapped 6A and 6B loci are located in the same arm as are homologous loci in hexaploid wheat, and the linear order of the loci is the same in the two chromosomes, except possibly close to the centromere. Major differences in genetic distances exist between homologous loci located in the proximal regions of the 6AL and 6BL linkage groups, however, the distances being much larger in the former than in the latter. The 6B maps that were constructed using data from both the RSL and the F₂ populations and using data from the RSL population alone closely resemble one another, indicating that the 6B RSL population, composed of 85 lines, can be reliably used for genetic mapping. Additional studies must be conducted before the utility of the 6A RSL population, composed of 66 lines, can be adequately assessed.     

Marker-based estimates of identity by descent and alikeness in state among maize inbreds

Molecular markers are useful for determining relationships and similarity among inbreds, especially if the proportion of marker loci with alleles common to inbreds i and j is partitioned into: (1) the probability that marker alleles are identical by descent (_Mf_ij); and (2) the conditional probability that marker alleles are alike in state, given that they are not identical by descent ( _ij). Our objectives were to: develop a method, based on tabular analysis of restriction fragment length polymorphism marker data, for estimating _Mf_ij, _ij, and the parental contribution to inbred progeny; validate the accuracy of the method with a simulated data set; and compare the pedigree-based coefficient of coancestry (f_ij) and _Mf_ij among a set of maize (Zea mays L.) inbreds. Banding patterns for 73 probeenzyme combinations were determined among 13 inbreds. Iterative estimation of _Mf_ij, _ij, and the parental contribution to progeny was performed with procedures similar to a tabular analysis of pedigree data. Deviations of _Mf_ij from pedigree-based f_ij ranged from 0.002 to 0.288, indicating large effects of selection and/or drift during inbreeding for some inbreds. Differences between marker-based estimates and expected values of parental contribution to inbred progeny were as large as 0.205. Results for a simulated set of inbreds indicated that tabular analysis of marker data provides more accurate estimates of _Mf_ij and _ij than other methods described in the literature. Tabular analysis requires the availability of marker data for all the progenitors of each inbred. When marker data are not available for the parents of a given inbred, _Mf_ij and _ij may still be calculated if parental contributions to the inbred are assumed equal to their expectations.     

Temperature functions of thermal death in yeasts and their relation to the maximum temperature for growth

Summary Parameters of thermal death were determined in 10 strains of yeast species whose maximum temperatures for growth (T _max) ranged from 22 to 49°C. Arrhenius plots of the specific thermal death rates (k _d) formed a positional sequence at the level of the experimental points that corrresponded in all but one case to the sequence of the respective T _max values. Extrapolated k _d values at higher or lower temperatures no longer formed this sequence.The correlation of the temperature functions with T _max could be characterized in terms of a new activation parameter, for which the name thermal death activation constant is introduced. It has the following form: T.D.A. – S where H and S are respectively the apparent heat and entropy of activation of thermal death and n is the number of degrees above T _max (expressed in °K) at which the T.D.A. constant exists.Seven mesophilic yeasts had a T.D.A. constant between 72 and 79 calxmol^-1 degree^-1 at n values between 1 and 4°. This suggested that the destructive process that limits k _d in these strains is of the same species as one that contributes to the establishment of T _max. Two psychrophilic yeasts apparently had a similar T.D.A. constant but at a high n value (about 12.5°C) which suggested that in these strains T _max is governed by a destructive process unrelated to the one that underlies thermal death. The strain of the nearly thermophilic Hansenula angusta (T _max 49°C) did not fit in either group.The significance of the T.D.A. constant is discussed and expressions for H and S in terms of bond activation parameters are proposed.     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.