Inactivation of glucose-6-phosphate dehydrogenase in solution by low- and high-frequency ultrasound

Kinetics of inactivation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in 0.1 M phosphate buffer (pH 7.4) within temperature range from 36 to 50 degrees C was studied comparatively under conditions of exposure of enzyme solution to low-frequency (LF, 27 kHz, 60 W/cm2) or high-frequency (HF, 880 kHz, 1.0 W/cm2) ultrasound (USD). Inactivation of G6PDH was characterized by effective first-order rate constants: (k(in)) total (summarized) inactivation; (k(in)*) thermal inactivation; and (k(in)(usd)) ultrasonic inactivation. Dilution of enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k(in) > k(in)*. The rate constants increased upon increasing the temperature. The Arrhenius plots of the temperature dependencies of k(in) and k(in) (usd) have a salient point at 44 degrees C. The activation energy (Eact) of the total inactivation of G6PDH was higher than Eact for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: Eact in case of inactivation with low-frequency ultrasound (LF-USD) was higher than in case of inactivation with high-frequency ultrasound (HF-USD). The rate of the ultrasonic induced inactivation of this enzyme substantially decreased in the presence of low concentrations of traps of radicals HO. (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of the G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in enzyme solutions exposed to USD.     

Kinetics of Catalase Inactivation Induced by Ultrasonic Cavitation

Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4.0–11.0) within the temperature range from 36 to 55^o. Solutions of CAT were exposed to LF (20.8 kHz) ultrasound (specific power, 48–62 W/cm²). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s^–1) of total inactivation (k _in), thermal inactivation (*k _in), and ultrasonic inactivation (k _in(us)). In all cases, the following inequality was valid: k _in > *k _in. The value of k _in(us) increased with the ultrasound power (range, 48–62 W/cm²) and exhibited a strong dependence on the pH of the medium. On increasing initial concentration of CAT (0.4–4.0 nM), k _in(us) decreased. The three rate constants were minimum within the range pH 6.5–8.0; their values increased considerably at pH < 6.0 and pH > 9.0. At 36–55^o, the temperature dependence of k _in(us) was characterized by an activation energy (E _act) of 19.7 kcal/mol, whereas the value of E _act for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 g/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (O ), prevented sonication-induced CAT inactivation at 10% (k _in and *k _in increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.     

Flavonoids: Efficient protectors of glucose-6-phosphate dehydrogenase from ultrasonic cavitation-induced inactivation

Seven structurally diverse flavonoids have been shown to decrease glucose-6-phosphate dehydrogenase (G6PDH) inactivation in 0.1 M phosphate buffer (pH 7.4), induced by exposure to a high temperature (44°C), or by a low-frequency ultrasound (27 kHz, 60 Wt/cm²). The activity of the compounds was assessed by their ability to change effective first-order rate constants characterizing the total (thermal and ultrasonic), thermal, and ultrasonic inactivation of 2.5 nM G6PDH (k _in, k*_in, and k _in(us), respectively). The value dependences of these constants on flavonoid concentrations (0.01–50 μM) were obtained. Rank order of potency exhibited by the compounds in protecting G6PDH appeared as follows: hesperidin > morin > silibin > naringin = quercetin > kampferol ? astragalin. The data obtained confirm the crucial role of free radicals formed in the field of ultrasonic cavitation (HO^· and O ₂ ^·? in G6PDH inactivation in solutions.     

Thermal stability of immobilized α-chymotrypsin is additionally increased by chaotropic compounds

Summary Temperature dependence of the rate constant of irreversible thermal inactivation, k_in, of immobilized -chymotrypsin depends markedly on the number of covalent bonds between the enzyme and support. When the number of bonds is big enough (thirteen), the dependence is linear as presented in Arrhenius plot (log k_in versus reciprocal temperature). However, if the number of such bonds is moderate or small (six or two), the temperature dependence of k_in, has a pronounced zig-zag character. This difference in the inactivation behaviour is attributed to an ability of moderately or mildly attached -chymotrypsins to accomplish a transition into a less ordered, catalytically inactive conformation and to inability of rigidly bound enzyme to pass such a transition. Chaotropic salts additionally stabilize this loose conformation of mildly or moderately bound -chymotrypsins against irreversible thermal inactivation but are without effect on the stability of rigidly bound enzyme.     

Polymorphism of the Apolipoprotein E Gene and Risk of Myocardial Infarction

The apolipoprotein E (ApoE) gene polymorphism resulting from nucleotide substitutions in exon 4 was analyzed in Russian and Tatar patients with myocardial infarction (MI) from Bashkortostan. Alleles 2, 3, and 4 were identified by PCR. The genotype frequency distribution proved to be age-dependent in healthy Russians, genotype 2/3 increasing in frequency in subjects over 45. Russians who suffered MI under 45 had lower frequencies of genotype E3/3 (50.00% vs. 75.47% in controls of the same age, = 0.013, OR = 0.33) and allele 3 (72.12% vs. 85.85%, = 0.020, OR = 0.43) and a higher frequency of allele 4 (22.12% vs. 10.38%, = 0.030, OR = 2.45). Russians who suffered MI complicated by cardiogenic shock (CS) had a significantly higher frequency of genotype 3/4 and lower frequencies of genotype 3/3 and allele 3 as compared with MI patients without CS. In Tatars, genotype 4/4 occurred at a frequency of 14.29% in patients who suffered MI under 45, and was not detected in healthy subjects of the same age ( = 0.024, OR = 17.85). Thus, the ApoE polymorphism was associated with higher risk of MI in Russians and Tatars under 45.     

Stability of Glucose 6-Phosphate Dehydrogenase Complexed with Its Substrate or Cofactor in Aqueous and Micellar Environment

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), or cofactor, NADP⁺, has been studied within the range 20–40°C in three media: (a) 0.04 M NaOH–glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, k _in(s^–1). In the case of G6PDH–NADP⁺complexes, the values of k _inwere independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of k _infor the complex G6PDH–GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of k _incorresponded to the degree of hydration W ₀= 16.7; at W ₀> 16.7 and W ₀< 16.7, k _inincreased. Within the range 20–40°C, the values of k _inmeasured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of k _inwere characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6°C to 40°C. In all media studied, NADP⁺complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH–NADP⁺melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.     

Interaction between Viral and Cell Genes in Cervical Tumors

The results of the works carried out in the Laboratory of Molecular Biology of Viruses, CRC in the framework of the Human Genome program and devoted to the study of the activity of cell and viral genes in cervical cancer are summarized. DNA of human papillomaviruses persists in tumors both in episomal and integrated forms. Integration may occur in different regions of chromosomes. Viral transforming genes 6 and 7 are always present in tumor cells, while antibodies to these proteins are detected only in 30% of patients. Loss of heterozygosity is detected on the long and short arms of chromosome 6; some such cases are manifest already at the early stages of tumor progression, while others are typical of the late stages. A number of cell genes potentially involved in tumorigenesis are shown to be hypermethylated in CpG islands. Methylation of several genes at once is observed in 30% of tumors. Tumor progression is associated with increased expression of 16 ^ink4a, an inhibitor of cyclin-dependent kinases.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Rectification characteristics ofNitella membranes in respect to water permeability

Summary One of the membrane characteristics of plant cells, rectification, or the direction dependence of water permeability, was investigated inCharaceae internodes using the procedures we developed (Tazawa andKiyosawa 1973) for determining the endosmotic (k _pen) and exosmotic (k _pex) water permeabilities of the membranes (plasmalemma and tonoplast) in the transcellular osmosis system. Bothk _pen andk _pex were dependent on the osmotic pressure ( _o ) of the mannitol solution, which is the driving force for the transcellular osmosis. Thus, k_pen increased andk _pex decreased with _o . The rectification parameter, or the polarity (_p), defined ask _pen/k _pex tended to unity when _o approached zero.InNitella flexilis the specific resistances of the membranes to endosmosis and exosmosis,k _pen ^–1 andk _pex ^–1 , were linearly dependent on ₀. When the cell was partitioned into two equal halves,k _pen ^–1 =4.2×10⁴–1.1×10³₀,k _pex ^–1 =4.2×10⁴+2.9×10³₀, where the specific resistances are represented in cm^–1 sec atm. When _o is 0.1, 0.2, 0.3, 0.4, and 0.5 M mannitol eq., the rectification parameter is calculated as 1.3, 1.6, 1.9, 2.4, and 2.9, respectively. Essentially the same results were also obtained withChara australis.Results were discussed on the basis of changes in the hydration of the cytoplasm. Assuming that the driving force across the protoplasmic layer can be divided into two forces; one driving water across the plasmalemma and the other driving water across the tonoplast, we deduced that the cytoplasm on the endosmosis side is hydrated, while the cytoplasm on the exosmosis side is dehydrated. Analysis showed that changes in hydration depend on the rate of flow.This work was supported partly by a Research Grant from the Ministry of Education of Japan.     

Free energy dependence of the direct charge recombination from the primary and secondary quinones in reaction centers from Rhodobacter sphaeroides

The direct charge recombination rates from the primary quinone, k _AD (D⁺Q _A ^– DQ_A) and the secondary quinone, k _BD (D⁺Q _B ^– DQ_B), in reaction centers from Rhodobacter sphaeroides were measured as a function of the free energy differences for the processes, G _AD ⁰ and G _BD ⁰ , respectively. Measurements were performed at 21 °C on a series of mutant reaction centers that have a wide range of dimer midpoint potentials and consequently a large variation in G _AD ⁰ and G _BD ⁰ . As –G _AD ⁰ varied from 0.43 to 0.78 eV, k _AD varied from 4.6 to 28.6 s^–1. The corresponding values for the wild type are 0.52 eV and 8.9 s^–1. Observation of the direct charge recombination rate k _BD was achieved by substitution of the primary quinone with naphthoquinones in samples in which ubiquinone was present at the secondary quinone site, resulting specifically in an increase in the free energy of the D⁺Q _A ^– state relative to the D⁺Q_AQ _B ^– state. As –G _BD ⁰ varied from 0.37 to 0.67 eV, k _BD varied from 0.03 to 1.4 s^–1. The corresponding values for the wild type are 0.46 eV and 0.2 s^–1. A fit of the two sets of data to the Marcus theory for electron transfer yielded significantly different reorganization energies of 0.82 and 1.3 eV for k _AD and k _BD, respectively. In contrast, the fitted values for the coupling matrix element, or equivalently the maximum possible rate, were comparable (25 s^–1) for the two charge recombination processes. These results are in accord with Q_B having more interactions with dipoles, from both the surrounding protein and bound water molecules, than Q_A and with the primary determinant of the maximal rate being the quinone-donor distance.     

Thermal Stability of Glucose Oxidase from Penicillium adametzii

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70°C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (<15 g/ml) and pH > 5.5, the rate constants k _in, s^–1, for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of structures in stabilizing the protein globule. At a concentration of 15 g protein/ml, the activation energy E _Aof thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60°C had the following values: H= 78.4 kcal/mol, G= 25.5 kcal/mol, and S= 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Calcium-induced associations of the caseins: Thermodynamic linkage of calcium binding to colloidal stability of casein micelles

The caseins occur in milk as colloidal complexes of protein aggregates, calcium, and inorganic phosphate. As determined by electron microscopy, these particles are spherical and have approximately a 650 Å radius (casein micelles). In the absence of calcium, the protein aggregates themselves (submicelles) have been shown to result from mainly hydrophobic interactions. The fractional concentration of stable colloidal casein micelles can be obtained in a calcium caseinate solution by centrifugation at 1500g. Thus, the amount of stable colloid present with varying Ca²⁺ concentrations can be determined and then analyzed by application of equations derived from Wyman's Thermodynamic Linkage Theory. Ca²⁺-induced colloid stability profiles were obtained experimentally for model micelles consisting of only _s1- (a calcium insoluble casein) and the stabilizing protein -casein, eliminating the complications arising from - and minor casein forms. Two distinct genetic variants _s1-A andB were used. Analysis of _s1-A colloid stability profiles yielded a precipitation (salting-out) constantk ₁, as well as colloid stability (salting-in) parameterk ₂. No variations ofk ₁ ork ₂ were found with increasing amounts of -casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added -casein an association constant of 4 L/g could be calculated for the complexation of _s1-A and -casein. For the _s1-B and -casein micelles, an additional Ca²⁺-dependent colloidal destabilization parameter,k ₃, was added to the existingk ₁ andk ₂ parameters in order to fully describe this more complex system. Furthermore, the value ofk ₃ decreased with increasing concentration of -casein. These results were analyzed with respect to the specific deletion which occurs in _s1-caseinA in order to determine the sites responsible for these Ca²⁺-induced quaternary structural effects.     

Characterization of second site mutations show that fast proton transfer to QB − is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

The structural basis for proton coupled electron transfer to Q_B in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k _AB ⁽²⁾(Q_A ^–Q_B ^– + H⁺ Q_AQ_BH^–) by at least 10³ (pH 7.5) and a recombination rate k _BD (D⁺Q_AQ_B ^– DQ_AQ_B) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k _AB ⁽²⁾ on the free energy for electron transfer (G_et). k _AB ⁽²⁾ was independent of G_et in DN(L213) RCs, but dependent on G_et in native and all suppressor RCs. This shows that proton transfer limits the k _AB ⁽²⁾ reaction with a rate of 0.1s^–1 in DN(L213) RCs but is not rate limiting and at least 10⁸-fold faster in native and 10⁵-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near Q_B ^–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near Q_B.     

A comparative study of variation in codon 33 of the<Emphasis Type="Italic"> rpoS</Emphasis> gene in<Emphasis Type="Italic"> Escherichia coli</Emphasis> K12 stocks: implications for the synthesis of σ<Superscript>s</Superscript>

The Escherichia coli rpoS gene encodes an RNA polymerase sigma factor (sigma S or ^S) required for the expression of stationary-phase genes. In the first published rpoS sequence from E. coli K-12 codon 33 is given as CAG. However, several subsequent independent studies found the amber codon TAG at this position ( rpoSAm). Besides this amber codon, other codons such as TAT have also been found at this location in rpoS. Comparative genome analysis now leads us to propose TAG as the parental codon 33 in rpoS in E. coli K-12. Five different stocks of the strain W3110, which differ in the levels of ^S protein they express, were investigated. We sequenced the rpoS gene from these, and found a T at nucleotide position 97 in four out of the five stocks and a G at position 99 in three out of the five. W1485, a parental strain of W3110, and W3350, a derivative of W3110, are also rpoSAm mutants. Such rpoSAm mutants would be expected to show no RpoS activity. The retention of partial or intermediate ^S activity by suppressor-free rpoSAm mutants is therefore puzzling. We propose that a functional, N-terminally truncated, ^S (1–53^S) can be translated from a Secondary Translation Initiation Region (STIR) located downstream of the amber codon 33. It has recently been reported that a fragment of RpoS (1–53^S) that lacks the first 53 amino acids is functional when synthesized in vivo. Taken together, our results support the hypothesis that the original codon 33 of the rpoS gene in E. coli K-12 strains is the amber codon TAG.Communicated by W. Goebel     

Solution to a gene divergence problem under arbitrary stable nucleotide transition probabilities

Summary A nucleic acid chain L nucleotides in length, with the specific base sequence B₁B₂.B_L, each B_i being A, G, C, or T, is defined by the L-dimensional vector B = (B₁, B₂, , B_L), the k^th position in the chain being occupied by the base B_k. Let P_BB' be the twelve given constant non-negative transition probabilities that in a specified position the base B is replaced by the base B in a single step, and let P _BB' ^(XX) be the probability that the position goes from base B to B in X steps. An exact analytical expression for P _BB' ^(XX) is derived. Assuming that each base mutates independently of the others, an exact expression is derived for the probability P _BB' ^(XX) that the initial gene sequence B goes to a sequence B = (B₁, B₂, , B_L) after X = (X₁, X₂, , X_L) base replacements, where X_k is the number of single-step base replacements in the k^th position. The resulting equations allow a more precise accounting for the effects of Darwinian natural selection in molecular evolution than does the idealized but biologically less accurate assumption that each of the four nucleotides is equally likely to mutate to and be fixed as one of the other three. Illustrative applications of the theory to some problems in biological evolution are given.     

Unigenic and multigenicI region control of the immune responses of mice to the GAT10 and GLΦ-GLT terpolymers

Recombinant strains of mice with known alleles in theI region of theH-2 complex were used to map theH-2 linked immune response genes controlling responsiveness to random terpolymers GAT¹⁰ and GL. TheIr-GAT gene was mapped to either theIA orIB subregions. In contrast, data obtained in the GL-GLT system indicated multigenic control. The responsiveness of the B10.A(3R), B10.A(5R), and B10.S(9R) recombinants indicated that one immune response gene,IrGL-GLT _A, mapped to the right ofIB, i.e., in theIC subregion. The nonresponsiveness of the B10.A(1R), B10.A(2R), B10.M(17R), and AQR mice having responderIC ^d alleles butIA ^k-IB ^k nonresponder alleles and the positive response of a (C57BL/6 × A/J)F₁ hybrid derived from two nonresponder parental strains indicated the presence of a second gene inIA-IB subregions,Ir-GL-GLT _B. The interaction between these two genes, each present in a differentI subregion, controls the immune response.     

Cloned T cells recognize Trichinella spiralis antigen in association with an Ek βEk α restriction element

A method is described for the production of T-cell lines and clones specific for solubilized Trichinella spiralis antigens. hese T cells are antigen-specific and do not respond to challenge with a third party antigen (lysozyme). The proliferation responses of the cloned T cells are specifically inhibited by anti-I-E but not by anti I-A subregion monoclonal reagents. The inhibition patterns obtained are consistent with cis-gene complementation in B10.K cells involving the E^k -chain and the E^k -chain of the I-E molecule. Inhibition is obtained with an E^k -specific monoclonal antibody (H9-14.8) but not with an A^k -specific monoclonal antibody (10-2.16). Inhibition was also observed with Ia.7-specific (H40-242) or Ia.22-specific (17-3-3) monoclonal antibodies. The inhibition patterns were confirmed by antigen presentation experiments using recombinant inbred mice. Only B 10.K (E^k E^k spleen cells and not B 10.A(5R) (E^b E^k ) or B10.S(9R) (E^s E^k ) spleen cells could effectively present T. spiralis antigens. The role of hybrid Ia molecules in the immune response to T. spiralis is discussed.     

Rates of decay for the survival probability of a mutant gene

If qk is the extinction probability of a slightly supercritical branching process with offspring distribution P _kr : r = 0, 1, 2,..., then it is shown that if sup _r r ³ p _kr , < , inf ² _k > 0, and m _k 1, then 1 – q _k 2(m _k –1) _k ^–2, where m _k = _r rp _kr , ² _k = ^k _r r ² p _kr – m _k ². This provides a simple set of sufficient conditions for the validity of a conjecture of Ewens (1969) for the survival probability of a slightly advantageous mutant gene.Research supported in part by NSF grants DMS-8803639 and DMS-9007182