Free energy calculations and molecular dynamics simulations of wild-type and variants of the DNA-EcoRI complex.

Molecular dynamics simulations and free energy calculations of the wild-type EcoRI-DNA complex and several variants have been performed in aqueous solvent. In general, he theoretical estimations of the free energy differences (DeltaDeltaA) qualitatively agree well with the corresponding experimental data. The modifications which were experimentally found unfavorable compared to the wild-type complex were also found to be so in theoretical estimates. The mutant where the amino group of the base Ade(6) was replaced by a hydrogen atom eliminating one H-bond between the DNA and the protein, was experimentally found to be more stable than the wild-type complex. It was speculated that the modification also caused a structural relaxation in the DNA making DeltaDeltaA favorable. Our theoretical estimate yields a positive DeltaDeltaA in this case, but the difference is small, and no significant local structural relaxation was observed. The major H-bonds between the DNA and the protein in the wild-type complex are found to be maintained in the different mutants although the specific and non-specific interaction energies between the interacting the DNA bases and the protein residues are different in different mutants. The interaction pattern of the other nearby nucleotides are significantly influenced by each modification. Thus, the alteration of the non-specific interactions may also play an indirect role in determining the specificity of the complex. The interaction of the Gua(4) of the DNA with the protein is found to be most sensitive to any alteration in the recognition site. Because Gua(4) is the nucleotide closest to the scissile bond, this extra sensitivity seems to play an important role in altering the functional activity of the complex.     

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.     

Amino acid volume and hydropathy of a transmembrane site determine glycine and anesthetic sensitivity of glycine receptors.

Two specific amino acid residues in transmembrane segments (TM) 2 and 3 are critical for the enhancement of glycine receptor (GlyR) function by volatile anesthetics. To determine which physicochemical characteristics of these sites determine their roles in anesthetic actions, an extensive series of single amino acid mutations at amino acid residue 288 (Ala-288) in TM3 of the alpha1 GlyR subunit was tested for modulation by volatile anesthetics. The mutations changed the apparent affinities of receptors for glycine; replacements with larger volumes and less hydropathy exhibited higher affinities for glycine. Potentiation by anesthetics was reduced by specific mutations at Ala-288. The molecular volume of the substituents was negatively correlated with the extent of potentiation by isoflurane, enflurane, and 1-chloro-1,2,2-trifluorocyclobutane, whereas there was no correlation between anesthetic enhancement and polarity, hydropathy, or hydrophilicity of substituents. In contrast to anesthetics, no correlation was found between the effects of the nonanesthetics 1,2-dichlorohexafluorocyclobutane or 2, 3-dichlorooctafluorobutane and any physicochemical property of the substituent. These results suggest that the molecular volume and hydropathy of the amino acid at position 288 in TM3 regulate glycine and anesthetic sensitivity of the GlyR and that this residue might represent one determinant of an anesthetic binding site.     

Risk Factors for High Endoparasitic Burden and the Efficiency of a Single Anthelmintic Treatment of Danish Horses

A questionnaire survey regarding endoparasite control practices in Danish horse herds was carried out in 1995. The participating veterinarians and herd owners were sampled using convenience and purposive sampling. In the analysis of risk factors for development of a high endoparasitic burden (>200 eggs per gram faeces) 903 horses were sampled and the analysis of the efficiency of a single anthelmintic treatment was based on 605 horses. The following factors had a significant effect on the endoparasitic burden: herd type, age of the horses, use of pasture rotation, anthelmintic treatment of horses visiting the herd, use of an adviser in the planning of endoparasite control and advice regarding pasture rotation. An interaction between pasture rotation and advice regarding pasture rotation was found, but due to high collinearity this was not reported. The factors influencing significantly on the reduction of the faecal egg count after a single anthelmintic treatment were the type of herd, the age of the horse, the drug used, and the anthelmintic-resistance-status of the herd. A negative effect of permanent pastures was observed. If pasture hygiene was performed on the advice of the veterinarian, the effect of a single anthelmintic treatment was less compared to a single anthelmintic treatment without any advice. An interaction between the treatment group and the resistance-status of the herd was found. Additional factors, normally accounted for, when endoparasites and anthelmintic resistance is discussed, were investigated, but not found significant in this study.     

Isolation and characterization of highly polymorphic microsatellite loci in the 2‐Spot Ladybird,Adalia bipunctata

Contrary to theoretical predictions, female 2‐spot ladybirds (Adalia bipunctata) mate many more times than necessary to maintain high fertilisation success and may gain through the acquisition of material or genetic benefits. In order to investigate this mating system in detail, microsatellite markers have been isolated using a modified enrichment technique. Thirty‐nine loci were successfully amplified by polymerase chain reaction (PCR), of which only two were monomorphic. Detailed characterization of ten loci revealed very high levels of polymorphism. These markers are likely to be invaluable tools with which to study population genetics and patterns of paternity in this species.     

Vers une approche physiopathologique des troubles de l’éjaculation

Seminal emission and sperm expulsion are under the control of both the sympathetic and parasympathetic outflows and also of the somatic innervation conveyed by the pudendal nerve. The 2 phases of ejaculation are reflexive with the reflexes handled at the thoraco-lumbar and sacral levels of the spinal cord. Such a spinal organization remains widely unknown. The role of various peripheral neurotransmitters has been evidenced including norepinephrine and acetylcholine and also peptidergic, purinergic i.e. ATP and nitric oxide. Stimulation of the seminal tract afferents play a crucial in the onset of ejaculatory mechanisms. Except for the dorsal nerve of the penis, there is a lack of information concerning these afferents. Several supraspinal centers i.e. hypothalamus, medial amygdala, pons and nucleus paragigantocellularis exert descending and ascending inhibitory and excitatory influences on spinal nuclei controlling emission and expulsion of sperm. Central neurotransmission responsible for this supraspinal control could involve serotonin, oxytocin and norepinephrine. In the light of the available anatomical and neurophysiological data, pathophysiological aspects of ejaculatory disorders are futher discussed. Premature ejaculation could be related to a periheral and central hypersentivity. Most of the other ejaculation abnormalities are likely mainly related to an impairment of the central mechanisms.