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.     

Intensive genetic assessment of the mating system and reproductive success in a semi-closed population of the mottled sculpin,Cottus bairdi

Most genetic surveys of parentage in nature sample only a small fraction of the breeding population. Here we apply microsatellite markers to deduce the genetic mating system and assess the reproductive success of females and males in an extensively collected, semi-closed stream population of the mottled sculpin fish, Cottus bairdi. In this species, males guard nest rocks where females deposit the eggs for fertilization. The potential exists for both males and females to mate with multiple partners and for males to provide parental care to genetically unrelated offspring. Four hundred and fifty-five adults and subadults, as well as 1,259 offspring from 23 nests, were genotyped at five polymorphic microsatellite loci. Multilocus maternal genotypes, deduced via genetic analyses of embryos, were reconstructed for more than 90% of the analysed nests, thus allowing both male and female reproductive success to be estimated accurately. There was no genetic evidence for cuckoldry, but one nest probably represents a takeover event. Successful males spawned with a mean of 2.8 partners, whereas each female apparently deposited her entire clutch of eggs in a single nest (mean fecundity = 66 eggs/female). On average, genetically deduced sires and dams were captured 1.6 and 9.3 metres from their respective nests, indicating little movement by breeders during the spawning season. Based on a 'genetic mark-recapture' estimate, the total number of potentially breeding adults (c. 570) was an order-of-magnitude larger than genetically based estimates of the effective number of breeders (c. 54). In addition, significantly fewer eggs per female were deposited in single than in multidam nests. Not only were perceived high-quality males spawning with multiple partners, but they were receiving more eggs from each female.     

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.     

Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.     

CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load

The causal relationships among CD4 cell depletion, HIV replication, and immune activation are not well understood. HIV-2 infection, "nature's experiment" with inherently attenuated HIV disease, provides additional insights into this issue. We report the finding that in HIV-2 and HIV-1 patients with a comparable degree of CD4 depletion the imbalance in the relative sizes of the naive and memory T cell populations and the up-regulation of CD4 and CD8 cell activation markers (HLA-DR, CD38, CD69, Fas molecules) are similar, even though the viral load in the plasma of HIV-2-infected patients is two orders of magnitude lower than in HIV-1 patients and HIV-2 patients are known to have slower rates of CD4 T cell decline and a better clinical prognosis. Moreover, we found a similar increase in the frequency of cycling CD4 T cells (Ki67+), which was in strong correlation with the expression of activation markers. Finally, the level of T cell anergy, as assessed by the proliferative responses to CD3 stimulation and to a panel of microbial Ags, proved to be comparable in HIV-1 and HIV-2 patients with a similar degree of CD4 depletion despite large differences in viral load. Our data are consistent with a direct causal relationship between immune activation and CD4 cell depletion in HIV disease and an only indirect relation of these parameters to the virus replication rate. Invoking the concept of proximal immune activation and virus transmission, which links efficient transmission of virus to local cell activation and proliferation in response to Ags and inflammation, we propose an integrative interpretation of the data and suggest that strongly elevated immune activation induces CD4 cell depletion and not vice versa, with potential implications for the choice of treatment strategies.     

Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.     

Comparison of the emulsion characteristics of Rhodococcus erythropolis and Escherichia coli SOXC-5 cells expressing biodesulfurization genes

Biodesulfurization of fuel oils is a two-phase (oil/water) process which may offer an interesting alternative to conventional hydrodesulfurization due to the mild operating conditions and reaction specificity afforded by the biocatalyst. For biodesulfurization to realize commercial success, a variety of process considerations must be addressed including reaction rate, emulsion formation and breakage, biocatalyst recovery, and both gas and liquid mass transport. This study evaluates emulsion formation and breakage using two biocatalysts with differing hydrophobic characteristics. A Gram-positive (Rhodococcus erythropolis) biocatalyst, expressing the complete 4S desulfurization pathway, and a Gram-negative biocatalyst (Escherichia coli), expressing only the gene for conversion of dibenzothiophene (DBT) to DBT sulfone, are compared relative to their ability to convert DBT and the ease of phase separation as well as biocatalyst recovery following desulfurization.