Plasma concentrations of 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in Rhesus monkeys after administration by various routes

A method is described for the estimation of 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ by double antibody radioimmunoassay. Plasma samples obtained from animals treated with 9-methylene-16,16-dimethyl-PGE₂,1-adamantanamine salt were extracted with diethyl ether to recover the prostaglandin. The validation of sample preparation and assay procedure are presented. Rhesus females were treated by several routes of administration and the samples assayed for drug content. Maximum blood levels were probably reached 30 minutes following subcutaneous injection and within 30 seconds of an intravenous injection. Results of the acute intravenous injection indicate an initial half-life of approximately one minute in peripheral circulation. Continuous intravenous infusion at 3 increasing doses of this compound resulted in a stepwise increase in plasma drug concentrations. Vaginal administration of 9-methylene-16,16-dimethyl-PGE₂,1-adamantanamine salt in suppositories produced a dose dependent increase in plasma drug concentration. Higher plasma drug concentrations were produced when the prostaglandin was delivered in H-15 base suppositories than in E-76 base suppositories.     

Serum FSH, LH and testosterone in the male rhesus following prostaglandin injection.

Adult male rhesus were treated with PGE2, PGF2 alpha or the 13,14-dihydro-15-keto metabolite of PGE2 in a randomized crossover design. Serum concentrations of FSH, LH and testosterone were determined and compared to the respective values in the same uninjected animals. No significant changes were noted in controls or following the metabolite injection. FSH increased gradually for 4 hours after metabolite treatment. In contrast, injection of PGF2 alpha was followed by an abrupt (within 15 minutes) increase in LH and testosterone. FSH increased gradually in 2 of 3 treated animals. Injection of PGE2 was followed by a similar abrupt increase in LH concentration. This was not always associated with a significant increase in testosterone or FSH. These results demonstrate that injections of PGE2 or PGF2 alpha can change serum gonadotropin and testosterone concentrations in male rhesus monkeys, and that the effects of these two prostaglandins are qualitatively different.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.     

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> studies

Background  

The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats.     

COOH-terminal 26-amino acid residues of progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo

Transgenic mice (hGAS) that overexpress human progastrin are more susceptible than wild-type mice (FVB/N) to the induction of colonic aberrant crypt foci (ACF) and adenomas by the chemical carcinogen azoxymethane. We have previously shown significantly increased levels of colonic mitosis in hGAS compared with FVB/N mice after gamma-radiation. To investigate whether the effects of progastrin observed in hGAS colon require the presence of other forms of circulating gastrin, we have crossed hGAS (hg(+/+)) with gastrin knockout (G(-/-)) mice to generate mice that express progastrin and no murine gastrin (G(-/-)hg(+/+)). After azoxymethane, G(-/-)hg(+/+) mice developed significantly more ACF than control G(-/-)hg(-/-) mice (which do not express any forms of gastrin). G(-/-)hg(+/+) mice also exhibited significantly increased colonic mitosis both before and after exposure to 8 Gray Gy gamma-radiation or 50 mg/kg azoxymethane compared with G(-/-)hg(-/-). Treatment of G(-/-)hg(-/-) mice with synthetic progastrin (residues 21-101 of human preprogastrin) or G17 extended at its COOH terminus corresponding to the COOH-terminal 26-amino-acid residues of human preprogastrin (residues 76-101, G17-CFP) resulted in continued colonic epithelial mitosis after gamma-radiation, whereas glycine-extended gastrin-17 and the COOH-terminal tryptic fragment of progastrin [human preprogastrin-(96-101)] had no effect. Immunoneutralization with an antibody against G17-CFP before gamma-radiation significantly decreased colonic mitosis in G(-/-)hg(+/+) mice to levels similar to G(-/-)hg(-/-). We conclude that progastrin does not require the presence of other forms of gastrin to exert proliferative effects on colonic epithelia and that the portion of the peptide responsible for these effects is contained within amino acid residues 76-101 of human preprogastrin.     

RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics

Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER-Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).