Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose CI 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.     

Changes in extracellular levels of dopamine metabolites in somatosensory cortex after peripheral denervation

This study examined the effects of a nerve transection on monoamine release from primary somatosensory cortex. The technique of microdialysis was employed to sample extracellular levels of norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in the barrel field of freely moving rats following the surgical transection of the contralateral infraorbital nerve. Microdialysates obtained 3, 4, and 5 days after deafferentation were analyzed using high-performance liquid chromatography with electrochemical detection. We found a significant increase in the release of the dopamine metabolites, DOPAC and HVA from the deafferented cortex. Three days after deafferentation the release of DOPAC was three-fold higher in the deafferented than in the control animals, and remained about 100% higher in the next two days in this group of animals. The release of HVA showed a gradual increase following the deafferentation procedure, since a 92% larger value on day 3 increased to a 338% difference on day 5. On the other hand, the release rate of NE and the levels of the serotonin metabolite 5-HIAA were not significantly affected by the deafferentation procedure. These results are discussed in the context of the possible participation of dopamine in the reorganization of the deafferented somatosensory cortex.     

Social Learning of a Novel Foraging Skill by White-throated Magpie-jays (Calocitta formosa,Corvidae): a Field Experiment

The cooperatively breeding white-throated magpie-jay (Calocitta formosa) uses a variety of foraging tactics to find, harvest, and process food. Members of territorial groups forage together and may gain information about how to acquire food by observing each other. A field experiment was performed to determine whether a novel skill, door opening to gain access to food, was more rapidly acquired by members of groups in which a trained individual performed the skill. A higher proportion of jays in groups with behavioural ‘models’ (trained birds that opened doors in the presence of group members) acquired the door-opening skill than those groups without models. Young birds acquired the behaviour more frequently than older individuals. Aggressive behaviour at feeders may have affected the spread of the behaviour by reducing the likelihood that individuals performed the behaviour in the presence of other group members but may also have encouraged subordinate individuals to attempt door opening rather than ‘scrounge’.     

Six things Popper would like biologists not to ignore: In memoriam, Karl Raimund Popper, 1902–1994

To honour the memory of Sir Karl Popper, I put forward six elements of his philosophy which might be of particular interest to biologists and to philosophers of biology and which I think Popper would like them not to ignore, even if they disagree with him. They are: the primacy of problems; the criticizability of metaphysics (and thus the dubiousness of materialism); how downward causation might be real; how norms should matter to scientists; why dogmatism should be avoided; how genuine science is recognizable. I preface these six things with a brief discussion of Popper's early (but later recanted) mistakes concerning biology.     

Measurement and modelling of photosynthetic response of pearl millet to soil phosphorus addition

There have been no studies of the effects of soil P deficiency on pearl millet (Pennisetum glaucum (L.) R. Br.) photosynthesis, despite the fact that P deficiency is the major constraint to pearl millet production in most regions of West Africa. Because current photosynthesis-based crop simulation models do not explicitly take into account P deficiency effects on leaf photosynthesis, they cannot predict millet growth without extensive calibration. We studied the effects of soil addition on leaf P content, photosynthetic rate (A), and whole-plant dry matter production (DM) of non-water-stressed, 28 d pearl millet plants grown in pots containing 6.00 kg of a P-deficient soil. As soil P addition increased from 0 to 155.2 mg P kg^–1 soil, leaf P content increased from 0.65 to 7.0 g kg^–1. Both A and DM had maximal values near 51.7 mg P kg^–1 soil, which corresponded to a leaf P content of 3.2 g kg^–1. Within this range of soil P addition, the slope of A plotted against stomatal conductance (g_s) tripled, and mean leaf internal CO₂ concentration ([CO₂]_i) decreased from 260 to 92 L L^–1, thus indicating that P deficiency limited A through metabolic dysfunction rather than stomatal regulation. Light response curves of A, which changed markedly with P leaf content, were modelled as a single substrate, Michaelis-Menten reaction, using quantum flux as the substrate for each level of soil P addition. An Eadie-Hofstee plot of light response data revealed that both K_M, which is mathematically equivalent to quantum efficiency, and V_max, which is the light-saturated rate of photosynthesis, increased sharply from leaf P contents of 0.6 to 3 g kg^–1, with peak values between 4 and 5 g P kg^–1. Polynomial equations relating K_M and V_max, to leaf P content offered a simple and attractive way of modelling photosynthetic light response for plants of different P status, but this approach is somewhat complicated by the decrease of leaf P content with ontogeny.     

Effect of concentration and environmental form of tetradecenyl succinic acid on its mineralization in soil

Tetradecenyl succinic acid (TSA) is the major component of a detergent builder (C12-C14 alkenyl succinic acid), which is inherently biodegradable. ¹⁴C-TSA was dosed as a component of sewage sludge into a soil with a history of sludge amendment at final added concentrations of 1.5 and 30 mg (kg soil)^-1. In addition, it was dosed to the soil in an aqueous solution to a final added concentration of 30 mg (kg soil)^-1. Dose and form were found to have a pronouced effect on the mineralization kinetics. When dosed in a realistic form and concentration (i.e. 1.5 mg (kg soil)^-1 as a component of sludge), TSA was mineralized at its highest rate and to its greatest extent, and the mineralization half-life was 2.4 days. When dosed at 30 mg (kg soil)^-1 as a component of sludge, mineralization began immediately, and the half-life was 23 days. In contrast, when dosed at this concentration in aqueous solution, the onset of mineralization was preceded by a 13 day lag period and the mineralization half-life was 69 days. Primary biodegradation and mineralization rates of TSA were very similar. Approximately, half the radioactivity was evolved as ¹⁴CO₂, while the remaining radioactivity became non-extractable, having presumably been incorporated into biomass or natural soil organic matter (humics). This study demonstrated that TSA is effectively removed from sludge-amended soils as a result of biodegradation. Furthermore, it showed the effect that dose form and concentration have on the biodegradation kinetics and the importance of dosing a chemical not only at a relevant concentration but also in the environmental form in which it enters the soil environment.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Genetic conflict and evolution of mammalian X-chromosome inactivation

The existence of parentally imprinted gene expression in the somatic tissues of mammals and plants can be explained by a theory of intragenomic genetic conflict, which is a logical extension of classical parent-offspring conflict theory. This theory unites conceptually the phenomena of autosomal imprinting and X-chromosome inactivation. We argue that recent experimental studies of X-chromosome inactivation and andro-genetic development address previously published predictions of the conflict theory, and we discuss possible explanations for the occurrence of random X-inactivation in the somatic tissues of eutherians. © 1995 Wiley-Liss, Inc.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Rescue of lethal subunits into functional K+ channels.

In a chimeric, voltage-dependent K+ channel (CHM), the valine at position 369 and the leucine at position 374 interact within the pore or P-region to regulate ion permeation and block. Here we show that the point mutation, CHM V369L, abolished channel function whereas previous experiments showed that CHM V369 and CHM V369I are functional. Coinjection of "lethal" CHM V369L cRNA with CHM L374V cRNA but not CHM cRNA generated functional heteromultimers. The whole-cell Rb+/K+ conductance ratio was 2.98 +/- 0.43 for CHM L374V and was reduced to 0.87 +/- 0.04 for the coexpressed CHM V369L and CHM L374V subunits. When single-channel currents were recorded, a single class of CHM V369L/CHM L374V heteromultimers was identified. This class was readily distinguishable from CHM L374V homomultimers by K+ conductance, gating, and blockade by internal tetraethylammonium. Coinjection experiments at various RNA ratios suggest that the CHM V369L/CHM L374V heteromultime, assuming it to be a tetramer, was composed of three CHM L374V subunits and one CHM V369L subunit. It appears that in the critical P-region of CHM position 369 may tolerate only one leucine.