The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Exudates as a fallback food for Callimico goeldii

Fallback foods have been defined as resources for which a species has evolved specific masticatory and digestive adaptations, and are consumed principally when preferred foods are scarce. In the present field investigation, we examine fungi, fruit, and exudate consumption in one group of Callimico goeldii in order to determine the importance of exudates as a fallback food for this species. Based on a total of 1,198 hr of quantitative behavioral data collected between mid-November 2002-August 2003, we found that pod exudates of Parkia velutina accounted for 19% of callimico feeding time in the dry season. This resource was not consumed in the wet season when fruits and fungi were the most common items in the diet. In the dry season of 2005 (July), the same callimico study group did not consume Parkia pod exudates. Instead, the group ate exudates obtained from holes gouged in tree trunks by pygmy marmosets and exudates resulting from natural weathering and insect damage on trunks, roots, and lianas. Pod exudates are reported to contain greater amounts of readily available energy than do trunk and root exudates, and were consumed throughout all periods of the day, particularly in the late afternoon. Trunk and root exudates were consumed principally in the morning. We propose that digestive adaptations of the hindgut, which enable callimicos to exploit fungi (a resource high in structural carbohydrates) year-round, predispose them to efficiently exploit and process exudates as fallback foods when other resources, such as ripe fruits, are scarce.     

Cholinergic Surface Antigen Chol-1 Is Present in a Subclass of VIP-Containing Rat Cortical Synaptosomes

The colocalization of vasoactive intestinal polypeptide (VIP) with the cholinergic specific surface antigen Chol-1 was investigated in synaptosomes derived from the rat cerebral cortex. Immunoaffinity purification of cortical synaptosomes using antisera to Chol-1 resulted in the copurification of VIP and cholinergic nerve terminals. VIP was purified with a yield of 75% of that of choline acetyltransferase (ChAT). These results suggest that approximately 53% of the cortical cholinergic terminals contain VIP, whereas 75% of the cortical VIP content is present in these cholinergic terminals. Both hypotonic lysis and depolarization of the nerve terminals resulted in the differential release of VIP and acetylcholine (ACh), indicating the different compartmentalization in the same nerve terminal. Complement-mediated lysis of cholinergic nerve terminals, using antisera to Chol-1, resulted in the release of 64% of the ChAT, 71% of ACh, and 27% of the VIP. The application of our method enables quantifying and mapping, with a fast, efficient, and specific technique, the coexisting peptides in cholinergic neurons of distinct brain areas.     

Putative Cholinergic-Specific Gangliosides in Guinea Pig Forebrain

The nature of the cholinergic-specific antigen Chol-1 recognized by an antiserum raised against Torpedo cholinergic electromotor synaptosomal plasma membranes was investigated in guinea pig forebrain to establish whether it has a gangliosidic nature in guinea pig as in Torpedo. Gangliosides extracted from guinea pig forebrain and extensively purified to eliminate peptide contaminants were effective in inhibiting the selective lysis of the cholinergic subpopulation of cortical synaptosomes induced by the antiserum. Neuraminidase, protease, alkali, and heat treatment did not impair the inhibitory activity of gangliosides. Whereas the antiserum recognized many gangliosides from Torpedo electric organ, the immunostaining of guinea pig forebrain gangliosides separated on TLC showed only two immunopositive bands migrating close to GT1b and GQ. After affinity purification on Torpedo electric organ gangliosides the activity of the antiserum in inducing complement-mediated lysis was increased and it still recognized the two ganglioside bands on TLC. These results strongly suggest the existence of two polysialogangliosides bearing antigenic determinants specific for the cholinergic neurons.     

Dual hypocretin receptor antagonism is more effective for sleep promotion than antagonism of either receptor alone

The hypocretin (orexin) system is involved in sleep/wake regulation, and antagonists of both hypocretin receptor type 1 (HCRTR1) and/or HCRTR2 are considered to be potential hypnotic medications. It is currently unclear whether blockade of either or both receptors is more effective for promoting sleep with minimal side effects. Accordingly, we compared the properties of selective HCRTR1 (SB-408124 and SB-334867) and HCRTR2 (EMPA) antagonists with that of the dual HCRTR1/R2 antagonist almorexant in the rat. All 4 antagonists bound to their respective receptors with high affinity and selectivity in vitro. Since in vivo pharmacokinetic experiments revealed poor brain penetration for SB-408124, SB-334867 was selected for subsequent in vivo studies. When injected in the mid-active phase, SB-334867 produced small increases in rapid-eye-movement (REM) and non-REM (NR) sleep. EMPA produced a significant increase in NR only at the highest dose studied. In contrast, almorexant decreased NR latency and increased both NR and REM proportionally throughout the subsequent 6 h without rebound wakefulness. The increased NR was due to a greater number of NR bouts; NR bout duration was unchanged. At the highest dose tested (100 mg/kg), almorexant fragmented sleep architecture by increasing the number of waking and REM bouts. No evidence of cataplexy was observed. HCRTR1 occupancy by almorexant declined 4-6 h post-administration while HCRTR2 occupancy was still elevated after 12 h, revealing a complex relationship between occupancy of HCRT receptors and sleep promotion. We conclude that dual HCRTR1/R2 blockade is more effective in promoting sleep than blockade of either HCRTR alone. In contrast to GABA receptor agonists which induce sleep by generalized inhibition, HCRTR antagonists seem to facilitate sleep by reducing waking "drive".     

Discovery of benzoylisoindolines as a novel class of potent, selective and orally active GlyT1 inhibitors

Benzoylisoindolines were discovered as a novel structural class of GlyT1 inhibitors. SAR studies and subsequent lead optimization efforts focused primarily on addressing hERG liability and on improving in vivo efficacy resulted in the identification of potent GlyT1 inhibitors displaying excellent selectivity and in vivo PD and PK profiles.     

Purification,biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties

Background

A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection.

Methods

Peptides from Coffea canephora seeds were extracted in Tris–HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses.

Results

The purified peptide of 9 kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9 kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP₁ exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP₁ interfered in a dose-dependent manner with glucose-stimulated, H⁺-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP₁ family, represented here by Cc-LTP₁, was also able to inhibit mammalian α-amylase activity in vitro.

Conclusions and general significance

In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.     

Discovery of benzoylpiperazines as a novel class of potent and selective GlyT1 inhibitors

Screening of the Roche compound library led to the identification of the benzoylpiperazine 7 as a structurally novel GlyT1 inhibitor. The SAR which was developed in this series resulted in the discovery of highly potent compounds displaying excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo efficacy after oral administration.