Determination of effective diffusivity of substrate in biological films and particles

Summary Particle supported biofilms of uniform thickness were generated in an aerobic fluidized-bed reactor with phenol as the carbon source. A method was developed for determining the effective diffusivities of oxygen and phenol using trypan blue, a vital stain as the tracer. The effective diffusivities of oxygen and phenol were found to be 2.72×10^–6 cm²/s and 1.12×10^–6 cm²/s respectively.Nomenclature C_i initial solute concentration in bulk, g/cm³ - C_t solute concentration in bulk at time t, g/cm³ - C bulk solute concentration at equilibrium, g/cm³ - D molecular diffusivity, cm²/s - D effective diffusivity, cm²/s - D_o D_p D_tb molecular diffusivity of oxygen, phenol and trypan blue, cm²/s - D_o, D_p, D_tb effective diffusivity of oxygen, phenol and trypan blue, cm²/s - D_s molecular diffusivity of substrate, cm²/s - D_s effective diffusivity of substrate, cm²/s - K partition coefficient - M_t amount of solute in the particle at time t, g - M amount of solute in the particle at equilibrium, g - r particle radius, cm - r _bp radius of the particle with biofilm, cm - S substrate concentration, g/cm³ - S_b substrate concentration in bulk, g/cm³ - S_i initial substrate concentration, g/cm³ - V₁ solute molar volume, cm³/g mol Greek Symbols bf porosity of the biofilm - tortuosity factor     

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.     

Spectral position control of dye absorption band maximum in an orienting matrix

The continuous control of the maximum position of the dye absorption band (the zero of the derivative dD ()/d of the cell's optical density D ()) in a nematic matrix is demonstrated experimentally, as a result of changing the angle between the optical axis of a planar-oriented sample and the plane of polarization of absorbed light incident normal to the optical axis. The theory proposed describes quantitatively the experimental dependence (). The rotation of the polarizer with given frequency results in the spectral position modulation of the solute band maximum () within (=0°)–(90°)=700 cm^–1.     

In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls

Saturable and reversible in vitro binding of [¹⁴C]riboflavin was found to occur on subcellular, sedimentable particles from maize coleoptiles and Cucurbita hypocotyls. The K_D was ca. 6 M, the pH optimum was near 6.0, and the number of binding sites amounted to 0.1–0.5 M on a fresh-weight basis. When the reducing agent dithionite was present, riboflavin binding increased-the K_D was 2.5 M, and the pH optimum above 8.0. The binding was specific: flavin mononucleotide (FMN) and flavin adenosine-dinucleotide (FAD) bound less tightly to these sites than riboflavin and another major soluble flavin, the previously described riboflavin-analog FX, occurring in grass coleoptiles. These flavin-binding sites were localized on vesicles derived from plasmalemma and endoplasmic reticulum by analyzing sucrose and metrizamide density gradients and marker enzymes.Abbreviations CCO cytochrome-c oxidase - CCR NADH-cytochrome-c oxidoreductase - ER endoplasmic reticulum - FAD flavin-adenosinedinucleotide - FMN flavin mononucleotide - MOPS N-morpholino-3-propansulfonic acid - NADH reduced -nicotinamide dinucleotide - nKP n thousand times g pellet - NPA l-naphthylphthalamic acid - PM plasma membrane, plasmalemma - RBF riboflavin - IAA indoleacetic acid - BA benzoic acid     

Gravitational sedimentation can solve the problem of cellular récycling of yeast in continuous alcoholic fermentation

Adjustments in the geometry of the separation zone of an inclined parallel plate sedimenter, previously developed, permitted an extensive increase in the volumetric clarification rate of broth containing yeast (S. cerevisiae). The prototype, having an internal capacity of 1340 ml, was fed with fermentation broth containing 18.8% v/v cells, while 16.4 ml/min of clarified broth containing 0.3% v/v cells was removed in the overflow. The underflow, containing 23.8% v/v cells, was recycled to the fermenter at a rate of 60.6 ml/min. These results demonstrated the viability of using exclusively gravitational sedimentation for cellular recycling in continuous alcoholic fermentation. Without a doubt, this system represents the simplest technological alternative among those thus far proposed for continuous alcoholic fermentation. The low cost of installation, maintenance and operation permitted projection of its application for any scale of production.List of Symbols A Cross sectional area of the sedimentation zone - b Distance between two parallel plates, height of the triangle or diameter of the circle (for rectangular, triangular or circular cross sections of the sedimentation zone, respectively) - b Mean distance travelled by the cells during sedimentation within the sedimentation zone with each cross sectional geometry - B _f Biomass content of the fermentation broth - B _o Biomass content of the overflow - B _u Biomass content of the underflow - Eff. Sedimentation efficiency - f Factor corresponding to the clarification velocity obtained with a certain cross sectional geometry relative to that obtained with the rectangular sedimentation zone geometry - g Gravitational acceleration - H Length of the plates - Q _a Clarification rate - Q _f Feed rate - Q _o Overflow rate - Q _u Underflow rate - rect Indicates a rectangular cross section - S Total sedimentation area (horizontal projection of the internal contour of the sedimentation zone - tr Indicates a triangular cross section - _s Linear settling velocity of one cell in the broth - _v Linear clarification velocity of the broth in a vertical sedimenter = _s - Linear clarification velocity of the broth in an inclined sedimenter of slope - Slope of the sedimentation zone relative to the horizontal - Porosity factor = 1 — (volume fraction of cells) - _cell Cell density - _m Density of the medium - _broth Broth viscosity     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg^-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg^-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl₂. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 M chloramphenicol, 10 M 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits (49 kDa), (39 kDa) and (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxy-glutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of and but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric -structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the -subunits.Non-standard abbreviations DTT dithiothreitol - PAGE polyaccrylamide gel electrophoresis - VIS visible     

Identification of a complete set of isogenic wheat/rye D-genome substitution lines by means of Giemsa C-banding

Summary A complete set of isogenic wheat/rye D-genome substitutions were produced by crossing an inbred line of spring rye Secale cereale L. cv. Prolific to a tetraploid wheat, the A-and B-genomes of which had previously been extracted from hexaploid wheat, Triticum aestivum L. em Thell. cv. Thatcher. After chromosome doubling, the derived hexaploid triticale (x Triticosecale Wittmack) was backcrossed to 6x Thatcher and selection for wheat/rye substitution lines was carried out in BCF₃ to BCF₆ families by using Giemsa C-banding. Five fertile disomic wheat/rye D-genome substitution lines were obtained and their chromosomal constitution was determined to be 1D/1R, 2D/2R, 7D/4R, 6D/6R, 7D/7R. The two remaining 3R and 5R substitutions are at the moment in a monosomic condition. Another 1D/7R substitution was detected but this plant was very weak and sterile, indicating that only substitutions between homoeologous chromosomes result in fertile, vigorous plants. Furthermore, many rye telocentrics as well as rye-rye and rye-wheat translocations were selected. Since all lines selected in this program share the same genetic background of Thatcher wheat, genetic heterogeneity is excluded. The material is very useful, therefore, for analyzing the effects of different rye chromosomes or chromosome segments in an otherwise homozygous background.Contribution No. 797     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

Performance,kinetics, and substrate utilization in a continuous yeast fermentation with cell recycle by ultrafiltration membranes

Summary A continuous single stage yeast fermentation with cell recycle by ultrafiltration membranes was operated at various recycle ratios. Cell concentration was increased 10.6 times, and ethanol concentration and fermentor productivity both 5.3 times with 97% recycle as compared to no recycle. Both specific growth rate and specific ethanol productivity followed the exponential ethanol inhibition form (specific productivity was constant up to 37.5 g/l of ethanol before decreasing), similar to that obtained without recycle, but with greater inhibition constants most likely due to toxins retained in the system at hight recycle ratios.By analyzing steady state data, the fractions of substrate used for cell growth, ethanol formation, and what which were wasted were accounted for. Yeast metabolism varied from mostly aerobic at low recycle ratios to mostly anaerobic at high recycle ratios at a constant dissolved oxygen concentration of 0.8 mg/kg. By increasing the cell recycle ratio, wasted substrate was reduced. When applied to ethanol fermentation, the familiar terminology of substrate used for Maintenance must be used with caution: it is not the same as the wasted substrate reported here.A general method for determining the best recycle ratio is presented; a balance among fermentor productivity, specific productivity, and wasted substrate needs to be made in recycle systems to approach an optimal design.Nomenclature B Bleed flow rate, l/h - C _T Concentration of toxins, arbitrary units - D Dilution rate, h^-1 - F Filtrate or permeate flow rate, removed from system, l/h - F _o Total feed flow rate to system, l/h - K _s Monod form constant, g/l - P Product (ethanol) concentration, g/l - P _o Ethanol concentration in feed, g/l - PP} Adjusted product concentration, g/l - PD Fermentor productivity, g/l-h - R Recycle ratio, F/F _o - S Substrate concentration in fermentor, g/l - S _o Substrate concentration in feed, g/l - V Working volume of fermentor, l - V _MB Viability based on methylene blue test - X Cell concentration, g dry cell/l - X _o Cell concentration in feed, g/l - Y _ATP Cellular yield from ATP, g cells/mol ATP - Y _ATPS Yield of ATP from substrate, mole ATP/mole glucose - Y _G True growth yield or maximum yield of cells from substrate, g cell/g glucose - Y _P Maximum theoretical yield of ethanol from glucose, 0.511 g ethanol/g glucose - Y _P/S Experimental yield of product from substrate, g ethanol/g glucose - Y _x/s Experimental yield of cells from substrate, g cell/g glucose - S _NP/X Non-product associated substrate utilization, g glucose/g cell - k ₁, k₂, k₃, k₄ Constants - k ₁ ^APP , k ₂ ^APP Apparent k ₁, k₃ - k ₁ ^TRUE True k ₁ - m Maintenance coefficient, g glucose/g cell-h - m ^* Coefficient of substrate not used for growth nor for ethanol formation, g glucose/g cell-h - Specific growth rate, g cells/g cells-h, reported as h^-1 - _m Maximum specific growth rate, h^-1 - v Specific productivity, g ethanol/g cell-h, reported as h^-1 - v _m Maximum specific productivity, h^-1     

Hormonal regulation of hepatic glycogenolysis inAmphibolurus nuchalis,the western netted dragon: an in vitro study

Summary In mammals hepatic glycogenolysis is controlled by several hormones using cyclicAMP, Ca²⁺ and/or diacylglycerol as intracellular messengers. In contrast, in teleost fish, lungfish and amphibians fewer hormones promote hepatic glycogenolysis, and cyclicAMP is the sole intra-cellular messenger. This suggests that the -adrenergic mechanism became associated with the liver after amphibians separated from the vertebrate line. Reptiles separated later, and the aim of this study is to elucidate the hormonal control of hepatic glycogenolysis in a reptile,Amphibolurus nuchalis, and especially to determine which adrenergic receptor system is operative.InA. nuchalis liver pieces cultured in vitro, adrenaline and glucagon stimulated glycogen breakdown and glucose release, glycogen phosphorylase activity and accumulation of cyclicAMP in the tissue. Neurohypophysial peptides did not affect these parameters. These actions of adrenaline were completely blocked by the -adrenergic antagonist, propranolol and slightly reduced by the -adrenergic antagonist, phentolamine. Removal of Ca²⁺ from the medium and addition of the Ca²⁺ chelator, EGTA, did not block the actions of adrenaline, and the Ca²⁺ ionophore A23187 did not mimic these actions.The -adrenegic ligand [¹²⁵I]-iodocyanopindolol (ICP) bound specifically to an isolated membrane preparation fromA. nuchalis liver with a calculated K_D of 100 pM and a Bmax of 37.6 fmol·mg protein^–1. The adrenergic ligands propranolol, isoprenaline, adrenaline, noradrenaline, phenylephrine and phentolamine displaced ICP with K_D's of 20 nM, 1 M, 4.5 M, 32 M, 35 M and 500 M, respectively. The ₂-adrenergic ligand yohimbine did not bind specifically to the membrane, but at 1 nM and 100 pM, specific binding of the ₁-adrenergic ligand prazosin was 45% of total with a mean of 11.3 fmoles·mg protein^–1 specifically bound.These findings indicate that the glycogenolytic actions of adrenaline are mediated primarily via -adrenergic receptors inA. nuchalis, but that -adrenergic receptors may also play some role in the control of hepatic metabolism.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Uncoupling and isotope effects in γ-butyrobetaine hydroxylation

Replacement of unlabeled -butyrobetaine with -[2,3,4-²H₆]butyrobetaine has a profound effect on the stoichiometry between decarboxylation of 2-oxoglutarate and hydroxylation in the reaction catalyzed by human -butyrobetaine hydroxylase. The ratios between decarboxylation and hydroxylation are 1.16 with Unlabeled and 7.48 with deuterated -butyrobetaine as substrate. From these ratios an internal isotope effect of 41 has been calculated. DV in the overall reaction measured as 2- oxoglutarate decarboxylation is 2.5 and D_V/K is 1.0. For -butyrobetaine hydroxylase fromPseudomonas sp. AK 1, 2-oxoglutarate decarboxylation exceeds hydroxylation with 10% when deuterated -butyrobetaine is used. No excess was found with unlabeled substrate and no internal isotope effect could be calculated. D_V for the bacterial enzyme is 6.     

Chromosome banding in amphibia

A cytogenetic study performed on a population of the South American leptodactylid frog Eleutherodactylus maussi revealed multiple sex chromosomes of the X₁X₁X₂X₂/X₁X₂Y (=XXAA/XXA^Y) type. The diploid chromosome number is 2n=36 in all females and 2n=35 in most males. The multiple sex chromosomes originated by a centric fusion between the original Y chromosome and a large autosome. In male meiosis the X₁X₂Y (=XXA^Y) multiple sex chromosomes form a classical trivalent configuration. E. maussi is the first species discovered in the class Amphibia that is distinguished by a system of multiple sex chromosomes. Only one single male was found in the population with 2n=36 chromosomes and lacking the Y-autosomal fusion. This karyotype (XYAA) is interpreted as the ancestral condition, preceding the occurrence of the Y-autosome fusion.by H.C. Macgregor     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Photoperiodism and thermoperiodism in the predatory mite Amblyseius potentillae are probably based on the same mechanism

Summary A comparative study was made of the photoperiodic and thermoperiodic induction of diapause in the phytoseiid mite Amblyseius potentillae. Sensitivity to thermoperiod was found to be highest during the protonymphal and deutonymphal stages, with some sensitivity still being present in the young adult. Summation of both photoperiodic and thermoperiodic cycles was shown to take place, which demonstrated the presence of a photoperiodic counter as well as a thermoperiodic counter in these mites. Vitamin A appeared to be necessary for some early step in the physiological mechanism of diapause induction and not just for the expression of the diapause response. The light sensitivity threshold for photoperiodic induction of diapause was found to be extremely low, viz. less than 0.02 W/cm². Moreover, the light sensitivity threshold appeared to be strongly temperature dependent in A. potentillae. Experiments in which the mites experienced various sequences of short-day photoperiods and short-day thermoperiods, applied either concurrently or in succession, showed that the information collected by the photoperiodic counter and the thermoperiodic counter is integrated into one induction sum. These results strongly suggest that photoperiodic and thermoperiodic induction of diapause in these mites is based on the same physiological mechanism.Abbreviations DD continuous darkness - LL continuous light - LD light-dark cycle (e.g. LD 16:8 is a cycle of 16 h of light and 8 h of darkness) - TC thermoperiodic cycle (e.g. TC 16:8 (27°: 15°) is a thermoperiod with a 16 h thermophase of 27 °C and an 18 h cryophase of 15°C)     

Interpretation of the gas residence time distributions in large stirred tank reactors

The behaviour of dispersed gas in large aerated stirred tank reactors is modelled by means of a Markov-process, which distinguishes between small recirculation bubbles with stagnant gas content, large rising bubbles with active gas content and exchange of stagnant and active gas contents, the gas exchange region at the impeller. The measurements of the gas residence time distributions (RTDs) in an 1.5 m³ aerated stirred tank reactor with water and Penicillium chrysogenum cultivation medium are interpreted by this model.List of Symbols CPR CO₂ production rate - OTR oxygen transfer rate - PRS pseudo random signal - RTD residence time distribution - V gas volume - recirculation coefficient - mean gas residence time Indices act active gas - ex gas exchange - stagn stagnant gas - tot total gas Dedicated to the 65th birthday of Professor Fritz Wagner.The authors thank Hoechst AG for the strain and the medium components, the GBF for the support of the experiments and H.M. Rüffer thanks the Verband der Chemischen Industrie for a Fond-der-Chemie scholarship.