Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX)

Summary The changes in cell wall strength of Hansenula polymorpha have been investigated in continuous cultures with respect to the recovery of methanol oxidase (MOX). Cultures grown on several substrate mixtures that enable induction of MOX have been compared with cultures grown on methanol as the sole inducer. The effects of dilution rate (D) on lysis properties have been studied. The cell wall strength was consistently influenced by growth media and D. Media containing glycerol/methanol showed the slowest lysis kinetics, with a large fraction of non-degradable cell wall material. In continuous cultures grown on a mixture of glucose and methanol both the resistance to zymolyase and the mean cell wall thickness increased at D<0.1 h^–1. The yield of MOX by zymolyase lysis is reproducible and up to 100% higher than that of the standard ultrasonic treatment. The lysis kinetics indicated that zymolyase punctures the cell wall; since the release rate of MOX is lower than that of protein, the cell contents will leak through. At D-values>0.2 h^–1, both protein and MOX release rates increase, reflecting a change in lysis mechanism due to the increased fraction of thin daughter cells. Kinetic analysis of zymolyase lysis using both physical and enzymatic methods provides information for achieving optimal recovery of MOX.Abbreviations and symbols C _MOX MOX activity [MOX units·g X^–1] - D dilution rate [h^–1] - MOX methanol oxidase - k_c decay rate constant of A 610 nm [min^–1] - k_d decay constant of MOX activity [min^–1] - k_dis dissociation rate constant [min^–1] - k_MOX release rate constant of MOX activity [min^–1] - k_p release rate constant of protein [min^–1] - R recovery efficiency of enzyme [-] - St stability of enzyme [-]     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Crab: snail size-structured interactions and salt marsh predation gradients

We studied size-structured predator-prey interactions between blue crabs (Callinectes sapidus) and marsh periwinkles (Littoraria irrorata) with a combination of field studies, laboratory experiments and individual-based modeling. Size distributions of Littoraria differed among years at the same sites in a salt marsh and could largely be explained by dominance of strong cohorts in the population. At a given site, abundance increased with elevation above tidal datum. Size-selective predation by blue crabs does not appear to be an important regulator of snail size distributions but may have a major effect on local abundance. Laboratory studies indicated that predator-prey interactions between Callinectes and Littoraria are strongly size-dependent. Crabs were generally effective at feeding on periwinkles at size ratios greater than approximately 6 (crab width: snail length). At lower size ratios crabs were far less effective at manipulating the snails, which often survived but with damaged shells. An individual-based model which incorporated information about incidence of snail shell scarring (resulting from non-lethal interactions) and snail density, predicted reduced predation rates and smaller average crab size with distance from the low tide refugium for crabs.     

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

An amino acid, lethal to New Hampshire chickens (LD₅₀, 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2(S),3(R)-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.     

Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency-Induced Hyperammonemia in the Rat

Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia- and glutamine-related metabolism was studied during liver insufficiency-induced hyperammonemia by measuring plasma flow and venous-arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra-organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval-shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval-shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, gamma-aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval-shunted and liver-ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency-induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and gamma-aminobutyric acid could be related to altered ammonia metabolism.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Dual Energy-Limited Chemostats

The absence of nitrification in soils rich in organic matter has often been reported. Therefore, competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h⁻¹. Ammonium limitation of A. globiformis was achieved by increasing the glucose concentration in the reservoir stepwise from 0 to 5 mM while maintaining the ammonium concentration at 2 mM. The numbers of N. europaea and N. winogradskyi cells decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations for both dilution rates. Critical carbon-to-nitrogen ratios of 11.6 and 9.6 were determined for the dilution rates of 0.004 and 0.01 h⁻¹, respectively. Below these critical values, coexistence of the competing species was found in steady-state situations. Although the numbers were strongly reduced, the nitrifying bacteria were not fully outcompeted by the heterotrophic bacteria above the critical carbon-to-nitrogen ratios. Nitrifying bacteria could probably maintain themselves in the system above the critical carbon-to-nitrogen ratios because they are attached to the glass wall of the culture vessels. The numbers of N. europaea decreased more than did those of N. winogradskyi. This was assumed to be due to heterotrophic growth of the latter species on organic substrates excreted by the heterotrophic bacteria.     

Development ofDentalium following removal of D-quadrant blastomeres at successive cleavage stages

Summary Each primary micromere and macromere of the D-quadrant ofDentalium was deleted, through the mesentoblast stage, to investigate the way in which the polar lobe cytoplasm exerts its influence on development.-D and -1D embryos form an apical tuft but no posttrochal structures.-2D embryos form an apical tuft and a reduced posttrochal region without a shell. -3D and -4D are externally similar to control embryos. -1d embryos and -1c embryos have an apical tuft with a reduced number of cilia. Embryos in which both 1c and 1d are deleted lack the apical tuft.-2d embryos lack shell and most other posstrochal structures. -3d and-4d embryos appear externally equivalent to controls.The polar lobe cytoplasm exerts its influence sequentially, and as inIlyanassa the maximal effect is at the third quartet stage.