Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1.     

Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background

Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods

Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results

Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion

We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.     

Plasma adiponectin is increased in mice selectively bred for high wheel-running activity, but not by wheel running per sé.

Mice selectively bred for high wheel-running activity (S) have decreased fat content compared to mice from randomly bred control (C) lines. We explored whether this difference was associated with alterations in levels of circulating hormones involved in regulation of food intake and energy balance, and whether alterations were caused by the presence of a running wheel. Plasma levels of leptin, adiponectin, and corticosterone as well as body composition were analyzed in male S mice housed with (+) and without (-) access to running wheels at ages of 10 and 18 months. These levels were compared to those found in C+ mice. Plasma corticosterone did not differ among groups. While plasma leptin levels tended to be lower in S+ mice as compared to S- or C+ mice, these differences were largely attributable to differences in fat content. Adiponectin levels were increased in S mice (+60%) compared to C mice, irrespective of wheel access. High levels of this hormone may be a trait co-segregated in mice bred for high wheel-running activity.     

Tumor specific phage particles promote tumor regression in a mouse melanoma model

Within cancer research, phage display libraries have been widely used for the identification of tumor targeting peptides and antibodies. Additionally, phages are known to be highly immunogenic; therefore we evaluated the immunotherapeutic potential of tumor specific phages to treat established solid tumors in a mouse model of melanoma. We developed two tumor specific phages, one derived from a peptide phage display library and one Fab expressing phage with known specificity, for the treatment of mice bearing palpable B16-F10 or B16/A2K^b tumors. Therapy in B16-F10 tumor bearing mice with tumor specific phages was superior to treatment with non-tumor specific phages and lead to delayed tumor growth and increased survival. In B16/A2K^b tumor bearing mice, therapy with tumor specific phages resulted in complete tumor regression and long-term survival in 50% of the mice. Histological analysis of tumors undergoing treatment with tumor specific phages revealed that phage administration induced a massive infiltration of polymorphonuclear neutrophils. Furthermore, phages induced secretion of IL-12 (p70) and IFN-γ as measured in mouse splenocyte culture supernatants. These results demonstrate a novel, immunotherapeutic cancer treatment showing that tumor specific phages can promote regression of established tumors by recruitment of inflammatory cells and induction of Th1 cytokines.     

Dopamine-beta-hydroxylase in postural tachycardia syndrome

Norepinephrine is frequently elevated in postural tachycardia syndrome (POTS), a syndrome of heterogeneous etiology characterized by a >30 beats/min increase in heart rate with standing. Norepinephrine is synthesized from dopamine by dopamine-beta-hydroxylase (DBH). The results of a preliminary study suggested that the T allele frequency of the DBH -1021C-->T polymorphism is elevated in POTS. This allele correlates with low DBH activity and might predict reduced serum DBH activity in patients with POTS. To test the hypothesis that low DBH activity and the underlying -1021C-->T polymorphism are associated with increased susceptibility to POTS, we measured serum DBH activity in POTS and determined its relationship to the DBH genotype and plasma norepinephrine. Serum DBH was similar for 83 normal volunteers and 42 patients with POTS: median (range) = 22.5 (0.5-94.2) and 19.6 (0.1-68.8) nmol.min(-1).ml(-1), respectively (P = 0.282). The genotype frequencies for 254 control and 157 POTS patients were not different between groups ( approximately 63% CC genotype and approximately 5% TT genotype, P = 0.319). The T allele associated with lower serum DBH in both groups [control serum DBH = 15.7 (SD 12.3) and 35.1 nmol.min(-1).ml(-1) (SD 18.6) for T carriers and noncarriers, respectively; POTS serum DBH = 8.2 (SD 5.6) and 28.5 nmol.min(-1).ml(-1) (SD 14.7) for T carriers and noncarriers, respectively]. High DBH in POTS was linked to elevated plasma levels of norepinephrine. Although DBH activity and genotype are unlikely to be primary determinants of susceptibility to POTS, differences in DBH activity in POTS may reflect differences in the level of sympathetic activation.     

Design, synthesis, and biological evaluation of pyrazinones containing novel P1 needles as inhibitors of TF/VIIa

Herein is described the design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors were designed to explore replacement and variation of the P1 amidine described previously [J. Med. Chem.2003, 46, 4050]. The P1 needle replacements were selected based upon their reduced basicity compared to the parent phenyl amidine (pKa approximately 12). A contributing factor towards the oral bioavailability of a compound is the ionization state of the compound in the intestinal tract. The desired outcome of the study was to identify an orally bioavailable TF-VIIa inhibitor.     

Endobrevin/VAMP-8 is the primary v-SNARE for the platelet release reaction

Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8-/- platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8-/- mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Leptin levels and body composition of mice selectively bred for high voluntary locomotor activity

Selective breeding produced four replicate lines of high-runner (HR) mice that run on wheels for approximately 2.7 times more revolutions per day than four unselected control lines. Previous studies found that HR mice of both sexes have lower body fat (isotope dilution at 15 wk of age) and that males (females not studied) have smaller retroperitoneal fat pads (17 wk). HR mice also exhibit elevated plasma corticosterone and insulin-stimulated glucose uptake by some hindlimb muscles but apparently do not differ in circulating insulin or glucose levels (males at 18 wk). Given their lower body fat and higher activity levels, we hypothesized that HR mice would have lower circulating leptin levels than controls. Female mice were given wheel access for 6 d at 7 wk of age, as part of the routine wheel testing for the selective breeding protocol, and then were killed after one additional week without wheels to reduce possible acute effects of activity on leptin. As hypothesized, serum leptin levels were significantly lower in HR mice. ANCOVA indicated that leptin was strongly positively correlated with both total body fat (measured by ether extraction) and body mass change from weaning, but HR mice still had significantly lower adjusted leptin levels (ANCOVA). Within HR lines but not within control lines, individual variation in leptin levels was negatively correlated with amount or speed of wheel running measured a week before being killed. Growth from weaning to euthanasia and body dry mass were lower in HR mice than in controls, but absolute dry masses of the ventricles, liver, gut, and uterus plus ovaries did not significantly differ, nor did percentage of the total dry mass as fat. HR mice offer a novel model for studying the causes and consequences of physiologically relevant variations in serum leptin.