The genetics of epilepsy in the Belgian tervuren and sheepdog

Idiopathic epilepsy is characterized by recurrent seizure activity without an identifiable underlying anatomic defect. Dogs experiencing repeated bouts of severe seizures are given therapeutic medication to control their frequency and severity. Idiopathic epilepsy has been reported in many dog breeds and was identified as the predominant health issue facing dog breeds in a recent survey by the American Kennel Club. A growing body of evidence supports a hereditary basis for idiopathic epilepsy, with a variety of genetic inheritance models proposed. In the Belgian tervuren and sheepdog, epilepsy is highly heritable with a polygenic mode of inheritance, though apparently influenced by a single autosomal recessive locus of large effect. In an effort to establish molecular linkage between the epileptic phenotype and the locus of large effect, we have screened genomic DNA from families of affected tervuren and sheepdogs with 100 widely dispersed, polymorphic canine microsatellite markers (0.595 average PIC value). Although not significant (LOD scores <3.0), three genomic regions have shown nominal linkage between markers and the epileptic phenotype. Additional related dogs are being screened with these and additional markers to increase the power to detect the presence of a linked locus.     

The promyelocytic leukemia protein protects p53 from Mdm2-mediated inhibition and degradation

The p53 protein is kept labile under normal conditions. This regulation is governed largely by its major negative regulator, Mdm2. In response to stress however, p53 accumulates and becomes activated. For this to occur, the inhibitory effects of Mdm2 have to be neutralized. Here we investigated the role of the promyelocytic leukemia protein (PML) in the activation of p53 in response to stress. We found that PML is critical for the accumulation of p53 in response to DNA damage under physiological conditions. PML protects p53 from Mdm2-mediated ubiquitination and degradation, and from inhibition of apoptosis. PML neutralizes the inhibitory effects of Mdm2 by prolonging the stress-induced phosphorylation of p53 on serine 20, a site of the checkpoint kinase 2 (Chk2). PML recruits Chk2 and p53 into the PML nuclear bodies and enhances p53/Chk2 interaction. Our results provide a novel mechanistic explanation for the cooperation between PML and p53 in response to DNA damage.     

An association between NQO1 genetic polymorphism and risk of bladder cancer

NAD(P)H:quinone oxidoreductase (NQO1) is a detoxification enzyme that plays a critical role in protecting cells against chemically induced oxidative stress, cytotoxicity, mutagenicity, and carcinogenicity. NQO1 protects cells from oxidative damage by preventing the generation of reactive oxygen species and reducing certain environmental carcinogens, such as nitroaromatic compounds, heterocyclic amines, and possible cigarette smoke condensate. A C-->T single nucleotide polymorphism in exon 6 was shown to reduce NQO1 enzyme activity, which may diminish the protection provided by NQO1. Therefore, we hypothesized that people with the variant allele genotypes of NQO1 are at higher risk for bladder cancer. In an ongoing case-control study, the NQO1 genotypes were successfully identified by polymerase chain reaction restriction fragment length polymorphism in 265 bladder cancer patients and 261 control subjects matched for age, sex, and ethnicity. The frequency of the variant NQO1 allele was 18% for controls and 21% for cases. The variant allele genotypes of NQO1 were associated with a higher risk of bladder cancer in Caucasians (odds ratio (OR)=1.51; 95% confidence interval (CI)=1.01-2.25). Further analysis in Caucasians showed an elevated bladder cancer risk in men (OR=1.75; 95% CI=1.08-2.85) but not in women (OR=1.16; 95% CI=0.57-2.37). In addition, the variant allele genotypes were associated with higher bladder cancer risk in ever smokers (OR=1.78; 95% CI=1.06-3.00), but not in never smokers (OR=1.19; 95% CI=0.65-2.20). These results suggest that the NQO1 genetic polymorphism modulates bladder cancer risk, especially in men and ever smokers.     

Elimination of high-light-inducible polypeptides related to eukaryotic chlorophyll a/b-binding proteins results in aberrant photoacclimation in Synechocystis PCC6803

The hli genes, present in cyanobacteria, algae and vascular plants, encode small proteins [high-light-inducible polypeptides (HLIPs)] with a single membrane-spanning alpha-helix related to the first and third helices of eukaryotic chlorophyll a/b-binding proteins. The HLIPs are present in low amounts in low light and they accumulate transiently at high light intensities. We are investigating the function of those polypeptides in a Synechocystis PCC6803 mutant lacking four of the five hli genes. Growth of the quadruple hli mutant was adversely affected by high light intensities. The most striking effect of the quadruple hli mutation was an alteration of cell pigmentation. Pigment changes associated with cell acclimation to increasing light intensity [i.e. decrease in light-harvesting pigments, accumulation of the carotenoid myxoxanthophyll and decrease in photosystem I (PSI)-associated chlorophylls] were strongly exacerbated in the quadruple hli mutant, resulting in yellowish cultures that bleached in high light and died as light intensities exceeded (>500 micromol photon m(-2) s(-1)). However, these pigment changes were not associated with an inhibition of photosynthesis, as probed by in vivo chlorophyll fluorescence, photoacoustic and O(2)-evolution measurements. On the contrary, the HLIP deficiency was accompanied by a stimulation of the photochemical activity, especially in high-light-grown cells. Western blot analyses revealed that the PSI reaction center level (PsaA/B) was noticeably reduced in the quadruple hli mutant relative to the wild type, whereas the abundance of the PSII reaction center protein D1 was comparatively little affected. The hli mutations did not enhance photoinhibition and photooxidation when cells were exposed over a short term to a very high light intensity. Together, the results of this study indicate that HLIPs are critical in the adaptation of the cyanobacterium to variations in light intensity. The data are consistent with the idea that HLIPs are involved, through a direct or indirect means, in nonphotochemical dissipation of absorbed light energy.     

Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

Deep desulfurization of extensively hydrodesulfurized middle distillate oil by Rhodococcus sp. strain ECRD-1

Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.     

Introduction

Photosynthesis Research -     

Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-(3)H(2)]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 +/- 0.008 min(-1) using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 +/- 0.00013 min(-1). An association rate constant of 7.3 x 10(6) M(-1) min(-1) was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B(max) = 23.87 +/- 1.15 pmol/mg protein, K(d) = 18.1 +/- 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC(50) in parentheses): azadirachtin (1.55 x 10(-8) M) > dihydroazadirachtin (3.16 x 10(-8) M) > dansyl dihydroazadirachtin (7.40 x 10(-8) M) > DNP-azadirachtin (7.50 x 10(-8) M) > biotin dihydroazadirachtin (1.27 x 10(-7) M) > 11-methoxy 22,23-dihydroazadirachtin (6.67 x 10(-7) M). [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:461-473 (1997).] Copyright 1997 Wiley-Liss, Inc.     

Period doubling of calcium spike firing in a model of a Purkinje cell dendrite

Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained current injection, the cell firing pattern can move from tonic firing of Ca²⁺ spikes to doublet firing and even to quadruplet firing or more complex firing. These firing patterns are not modified substantially if Na⁺ currents are blocked. We show that the experimental results can be viewed as a slow transition of the neuronal dynamics through a period-doubling bifurcation. To further support this conclusion and to understand the underlying mechanism that leads to doublet firing, we develop and study a simple, one-compartment model of Purkinje cell dendrite. The neuron can also exhibit quadruplet and chaotic firing patterns that are similar to the firing patterns that some of the Purkinje cells exhibit experimentally. The effects of parameters such as temperature, applied current, and potassium reversal potential in the model resemble their effects in experiments. The model dynamics involve three time scales. Ca²⁺- dependent K⁺ currents, with intermediate time scales, are responsible for the appearance of doublet firing, whereas a very slow hyperpolarizing current transfers the neuron from tonic to doublet firing. We use the fast-slow analysis to separate the effects of the three time scales. Fast-slow analysis of the neuronal dynamics, with the activation variable of the very slow, hyperpolarizing current considered as a parameter, reveals that the transitions occurs via a cascade of period-doubling bifurcations of the fast and intermediate subsystem as this slow variable increases. We carry out another analysis, with the Ca²⁺ concentration considered as a parameter, to investigate the conditions for the generation of doublet firing in systems with one effective variable with intermediate time scale, in which the rest state of the fast subsystem is terminated by a saddle-node bifurcation. We find that the scenario of period doubling in these systems can occur only if (1) the time scale of the intermediate variable (here, the decay rate of the calcium concentration) is slow enough in comparison with the interspike interval of the tonic firing at the transition but is not too slow and (2) there is a bistability of the fast subsystem of the spike-generating variables.