Structure of the O-acetylserine sulfhydrylase isoenzyme CysM from Escherichia coli

The enzyme O-acetylserine sulfhydrylase participates in the biosynthesis of l-cysteine in bacteria and plants. The structure of isoenzyme B (CysM) from Escherichia coli was established in a hexagonal crystal form at 2.7 A resolution (wild-type) and in a merohedrally twinned tetragonal crystal form at 2.1 A resolution (surface mutant). Structural superpositions revealed the variations with respect to isoenzyme A (CysK) and explained the different substrate specificities. A geometric model of the reaction catalyzed by CysM is proposed. Both isoenzymes are used for the production of l-amino acid derivatives as building blocks for the synthesis of peptides and peptidomimetic drugs. Since the structure of CysM revealed a remarkable main chain variation at the active center, it constitutes a further starting point for engineering mutants with novel substrate specificities.     

The effect of leptin on mouse trophoblast cell invasion

The hormone leptin is produced by adipose tissue and can function as a signal of nutritional status to the reproductive system. The expression of leptin receptor and, in some species, leptin, in the placenta suggests a role for leptin in placental development, but this role has not been elucidated. Leptin is required at the time of embryo implantation in the leptin-deficient ob/ ob mouse and has been shown to upregulate expression of matrix metalloproteinases (MMPs), enzymes involved in trophoblast invasion, in cultured human trophoblast cells. This led us to the hypothesis that leptin promotes the invasiveness of trophoblast cells crucial to placental development. We found that leptin stimulated mouse trophoblast cell invasion through a matrigel-coated insert on Day 10, but not Day 18 of pregnancy. Optimal stimulation occurred at a concentration of 50 ng/ml leptin, similar to the peak plasma leptin concentration during pregnancy in the mouse. Leptin treatment did not stimulate proliferation of mouse trophoblast cells in primary culture. Leptin stimulation of invasion was prevented by 25 muM GM6001, an inhibitor of MMP activity. Our results suggest that leptin may play a role in the establishment of the placenta during early pregnancy and that this function is dependent on MMP activity. This effect of leptin may represent one mechanism by which body condition affects placental development.     

Influence of O(3)/O(2)-pneumoperitoneum as an oxidative stressor on duration of anaesthesia, loss of different reflexes and cytokine mRNA expression

We analysed the effect of intraperitoneal insufflated ozonized oxygen on the anaesthetic strength generated by tribromoethanol, ketamine/xylazine, chloral hydrate, pentobarbital, and urethane in male Wistar rats. High dosages of anaesthetic drugs normally used for deep surgical anaesthesia were injected. The ozonized oxygen gas mixture was given five times daily on five consecutive days at 0.8 mg ozone/kg body weight before anaesthesia. The reflexes were measured 15, 30, 60, 90, 120, 180, and 240 min after injection of the anaesthetic drug. The sleeping time and the loss and regain of six different reflexes on noxious and non-aversive stimuli were recorded during the 4 h of observation. O(3)/O(2)-pneumoperitoneum (O(3)/O(2)-PP) reduced the sleeping time induced by tribromoethanol and ketamine/xylazine and increased it for chloral hydrate and pentobarbital. In accordance to the changes in the duration of anaesthesia, the O(3)/O(2)-PP induced significant changes in the loss of different reflexes. Additionally, the modulatory effect of the anaesthetic drugs on splenic cytokine mRNA expression was further influenced by O(3)/O(2)-PP. Thus, the influence of an oxidative stressor on anaesthetic potency and on the resting immune system has to be taken into account for experimental designs in which surgical anaesthesia is necessary for small laboratory animals.     

Gonadal hormones masculinize and defeminize reproductive behaviors during puberty in the male Syrian hamster

Three experiments were conducted to test whether testicular hormones secreted during puberty masculinize and defeminize the expression of adult reproductive behavior. Experiment 1 tested the hypothesis that gonadal hormones during puberty masculinize behavioral responses to testosterone (T) in adulthood. Male hamsters were castrated either before puberty (noTduringP) or after puberty (TduringP). All males were implanted with a 2.5-mg T pellet 6 weeks following castration and tested once for masculine reproductive behavior 7 days after the onset of T replacement. TduringP males displayed significantly more mounts, intromissions, and ejaculations than noTduringP males. Experiment 2 tested the hypothesis that gonadal hormones during puberty defeminize behavioral responses to estrogen (EB) and progesterone (P). Eight weeks following castration, noTduringP and TduringP males were primed with EB and P and tested for lordosis behavior with a stud male. Behavioral responses of males were compared to that of ovariectomized (OVX) and hormone primed females. NoTduringP males and OVX females displayed significantly shorter lordosis latencies than TduringP males. Experiment 3 investigated whether prolonged T treatment or sexual experience could reverse the deficits in masculine behavior caused by the absence of T during puberty. Extending the T treatment from 7 to 17 days did not ameliorate the deficits in masculine behavior caused by absence of T during puberty. Similarly, when the level of sexual experience was increased from one to three tests, the deficits in masculine behavior persisted. These studies demonstrate that gonadal hormones during puberty further masculinize and defeminize neural circuits and behavioral responsiveness to steroid hormones in adulthood.     

Analysis of volatiles from black pine (Pinus nigra): significance of wounding and egg deposition by a herbivorous sawfly

The composition of headspace volatiles of black pine (Pinus nigra) was analysed by coupled gas chromatography-mass spectrometry (GC-MS). It has been shown in a previous study that egg deposition of the sawfly Diprion pini on P. sylvestris induces a quantitative change of the pine volatile blend . Chrysonotomyia ruforum, an egg parasitoid of D. pini, is known to be attracted by volatiles from egg-carrying P. sylvestris, but not by odour from egg-laden P. nigra. Therefore, the present study focused on the question whether also P. nigra as another host plant of this sawfly responds to egg deposition by change of its volatile blend. The headspace of untreated, egg-carrying, and artificially wounded P. nigra twigs were compared. The artificial damage inflicted to the twigs mimicked the damage by the sawfly female prior to egg deposition. Thirty five mainly terpenoid compounds that were identified in more than 50% of the egg-carrying P. nigra twigs could also be detected in the headspace of untreated and artificially wounded twigs. Quantitative differences of the blends of volatiles from differently treated P. nigra twigs were compared by multivariate data analyses. PLS-DA (projection to latent structures-discriminant analysis) revealed that blends of volatiles from differently treated P. nigra differed significantly. When comparing volatiles from egg-carrying and artificially wounded P. nigra with respective P. sylvestris samples, qualitative and quantitative differences were detected. The differences in volatile composition of P. nigra and P. sylvestris are discussed with special respect to the egg parasitoid's response to odours of egg-carrying pine twigs.     

Dufour gland of the digger wasp<Emphasis Type="Italic"> Liris niger:</Emphasis> structure and developmental and biochemical aspects

The tubiform Dufour gland in the digger wasp species Liris niger is about 1.0 mm long ( 0.15 mm). An alternating arrangement of longitudinal and circumferential bundles of striated muscle fibers surrounds the gland. The Dufour gland, together with the venom gland, enters the sting base and terminates in the sting. The glandular epithelium is monolayered. Glands about 3 day after imaginal ecdysis have an empty lumen but a thick lining epithelium. The gland cells are characterized by a well-developed vesicular smooth endoplasmic reticulum, sparse rough ER and numerous free ribosomes. They also exhibit several electron-lucent vesicles and autophagic vacuoles. Secretion of electron-dense material via the gland cuticle into the gland lumen is apparent. Glands more than 20 days after imaginal ecdysis display a large lumen and a thin epithelium. The cells show signs of degeneration with numerous cytolytic inclusions. Dufour gland liquid contains numerous polypeptides of molecular weights ranging from 14 to about 200 kDa. In addition the secretion consists predominantly of straight-chain hydrocarbons, accompanied by small amounts of esters. The major hydrocarbons are pentadecane and (Z)-8-heptadecene. Dufour gland secretion may have several functions: (1) the polypeptides might be involved in the gluing process of the eggs, while (2) the hydrocarbon oils may function as lubricants for the lancets and (3) might soften the secretion, thus allowing easier application of the glue. The lipophilic volatile material (4) might also be involved in pheromonal signaling.     

Gene therapy in Parkinson’s disease

Gene therapy in Parkinsons disease appears to be at the brink of the clinical study phase. Future gene therapy protocols will be based on a substantial amount of preclinical data regarding the use of ex vivo and in vivo genetic modifications with the help of viral or non-viral vectors. To date, the supplementation of neurotrophic factors and substitution for the dopaminergic deficit have formed the focus of trials to achieve relief in animal models of Parkinsons disease. Newer approaches include attempts to influence detrimental cell signalling pathways and to inhibit overactive basal ganglia structures. Nevertheless, current models of Parkinsons disease do not mirror all aspects of the human disease, and important issues with respect to long-term protein expression, choice of target structures and transgenes and safety remain to be solved. Here, we thoroughly review available animal data of gene transfer in models of Parkinsons disease.     

Structural basis for the function of Clostridium difficile toxin B

Toxin B is a member of the family of large clostridial cytotoxins which are of great medical importance. Its catalytic fragment was crystallized in the presence of UDP-glucose and Mn2+. The structure was determined at 2.2 A resolution, showing that toxin B belongs to the glycosyltransferase type A family. However, toxin B contains as many as 309 residues in addition to the common chainfold, which most likely contribute to the target specificity. A superposition with other glycosyltransferases shows the expected positions of the acceptor oxygen atom during glucosyl transfer and indicates further that the reaction proceeds probably along a single-displacement pathway. The C1' donor carbon atom position is defined by the bound UDP and glucose. It assigns the surface area of toxin B that forms the interface to the target protein during the modifying reaction. A docking attempt brought the known acceptor atom, Thr37 O(gamma1) of the switch I region of the RhoA:GDP target structure, near the expected position. The relative orientation of the two proteins was consistent with both being attached to a membrane. Sequence comparisons between toxin B variants revealed that the highest exchange rate occurs around the active center at the putative docking interface, presumably due to a continuous hit-and-evasion struggle between Clostridia and their eukaryotic hosts.