Long-term suppression of pituitary-gonadal function with three-month depot of leuprorelin acetate in a girl with central precocious puberty

OBJECTIVE: The efficacy of a 3-month depot preparation of the GnRH agonist leuprorelin acetate in central precocious puberty was studied. METHODS: Treatment with a 3-month depot of leuprorelin acetate was performed subcutaneously in a 7.3-year-old girl with central precocious puberty. RESULTS: During treatment the hormonal suppression was constant and complete as demonstrated by suppressed GnRH stimulation tests and prepubertal estradiol plasma levels. The size and volume of the uterus and ovaries returned to the normal range. The rate of bone maturation was significantly reduced with a ratio deltaBA/deltaCA of 0.58 for 3 treatment years. Thus, the effects of treatment were comparable to those reported for treatment with 1-month depot of GnRH agonists. CONCLUSION: Three-month depots have the advantage of a prolonged injection interval which is more convenient for the patients and reduces costs by necessitating fewer visits to the physician and being approximately 10% cheaper than the 1-month depot. We suggest that comparative and randomized studies be performed to make 3-month depots of GnRH agonists available for routine use in children with central precocious puberty.     

Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes

Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem-loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.     

The consequences of acute cold exposure on protein oxidation and proteasome activity in short-tailed field voles,microtus agrestis

During cold exposure, animals upregulate their metabolism and food intake, potentially exposing them to elevated reactive oxygen species (ROS) production and oxidative damage. We investigated whether acute cold (7 +/- 3 degrees C) exposure (1, 10, or 100 h duration) affected protein oxidation and proteasome activity, when compared to warm controls (22 +/- 3 degrees C), in a small mammal model, the short-tailed field vole Microtus agrestis. Protein carbonyls and the chymotrypsin-like proteasome activity were measured in plasma, heart, liver, kidney, small intestine (duodenum), skeletal muscle (gastrocnemius), and brown adipose tissue (BAT). Trypsin-like and peptidyl-glutamyl-like proteasome activities were determined in BAT, liver, and skeletal muscle. Resting metabolic rate increased significantly with duration of cold exposure. In skeletal muscle (SM) and liver, protein carbonyl levels also increased with duration of cold exposure, but this pattern was not repeated in BAT where protein carbonyls were not significantly elevated. Chymotrpsin-like proteasome activity did not differ significantly in any tissue. However, trypsin-like activity in SM and peptidyl-glutamyl-like activity in both skeletal muscle and liver, were reduced during the early phase of cold exposure (1-10 h), correlated with the increased carbonyl levels in these tissues. In contrast there was no reduction in proteasome activity in BAT during the early phase of cold exposure and peptidyl-glutamyl-like activity was significantly increased, correlated with the lack of accumulation of protein carbonyls in this tissue. The upregulation of proteasome activity in BAT may protect this tissue from accumulated oxidative damage to proteins. This protection may be a very important factor in sustaining uncoupled respiration, which underpins nonshivering thermogenesis at cold temperatures.     

Mechanism of nucleocapsid protein catalyzed structural isomerization of the dimerization initiation site of HIV-1

Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.     

Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat

The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1). Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.     

Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Modeling temporal and spatial colony-site dynamics in a long-lived seabird

We studied the determinants of colony site dynamics in Audouin's gull, Larus audouinii, breeding in a small archipelago of the western Mediterranean. Data on island occupation were available for a series of 25 years, since first colonization of the archipelago in 1973. Group behavior was studied in relation to the components of dispersal: permanence or abandonment (extinction) on an island previously occupied and permanence or occupation (colonization) of another island. Generalized Linear Mixed Models (GLMMs) were used to identify the relative contribution of each explanatory variable to the probability of colony abandonment. Gulls showed a low probability (3%) of abandoning one of the islands (Grossa I.), especially when the colony was increasing in numbers from time t_i-1 to t_i. However, the probability of abandoning Grossa increased up to 31% when the colony was declining. The probability of island abandonment was very high for all other islands (range 66–99%) when the colony was declining, but much lower (range 36–82%) when it was increasing. Hence, we suggest that island abandonment by Audouin's gull is at least a two-step process. The first step (dispersal of a portion of the colony) probably takes place at random, as an evolutionary load typical of a species evolved in unstable habitats. The second step, a further loss of breeding pairs, seems to feedback on the first loss of members of the colony (public information), likely perceived as a loss of colony quality. Colonization of islands by gulls abandoning Grossa I. was marginally and negatively affected by the density of breeding yellow-legged gulls, a predatory species. Results apply to conservation ecology since they highlight the need to protect not only occupied patches but also those empty at present.     

Ancistrolikokine D,a 5,8'-coupled naphthylisoquinoline alkaloid,and related natural products from Ancistrocladus likoko

A new naphthylisoquinoline alkaloid, ancistrolikokine D, and the likewise 5,8'-coupled alkaloid ancistroealaine A, as well as two further, biosynthetically related, but nitrogen-free natural products, ancistronaphthoic acid B and cis-isoshinanolone, have been isolated from Ancistrocladus likoko J. LEACUTE;ONARD (Ancistrocladaceae). The 5,8'-coupling of the new alkaloids and of the alkaloids isolated earlier hints at a close phylogenetic relationship of A. likoko to other Central African Ancistrocladus species. The compounds show moderate activities against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei rhodesiense.     

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.