Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Immunocytochemical detection of dendritic cell by S-100 protein in the chicken.

Positivity for S-100 protein in paraffin embedded chicken lymphoid tissue was found by using a polyclonal antibody against whole bovine S-100 protein. The S-100 protein-containing cells were observed in the locations which have been reported to contain avian dendritic cells such as the medulla of the bursal follicles, and the germinal centers and T-dependent areas in the spleen, Peyer's patches, caecal tonsil and Harderian gland. Positive cells were also found in the location where ellipsoid associated cell have been described, and between epithelial cells covering the Peyer's patches and the caecal tonsil, as well as between the cells lining the ducts within the Harderian gland. Macrophages were devoid of immunostaining. Our results confirm the location described elsewhere for chicken dendritic cells and indicate that S-100 protein can be considered as a cell marker for the identification of the chicken dendritic cell. Intraepithelial positive cells may be interdigitating dendritic cells in an unusual location (their function being the transport of the antigen from the epithelium to the diffuse lymphoid tissue), or cytotoxic T-lymphocytes which, in mammals, are immunoreactive for S-100 protein.     

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds

In Europe alone, each year 5500 people require a life‐saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large‐animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte‐ and cholangiocyte‐like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large‐animal model.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.     

Floral scent evolution in the section Pseudophrys: pollinator‐mediated selection or phylogenetic constraints?

• Sexually deceptive orchid species from the Mediterranean genus Ophrys usually interact with one or a few pollinator species by means of specific floral scents. In this study, we investigated the respective role of pollinator‐mediated selection and phylogenetic constraints in the evolution of floral scents in the section Pseudophrys.
• We built a phylogenetic tree of 19 Pseudophrys species based on three nuclear loci; we gathered a dataset on their pollination interactions from the literature and from our own field data; and we extracted and analysed their floral scents using solid phase microextraction and gas chromatography‐mass spectrometry. We then quantified the phylogenetic signal carried by floral scents and investigated the link between plant–pollinator interactions and floral scent composition using phylogenetic comparative methods.
• We confirmed the monophyly of the section Pseudophrys and demonstrated the existence of three main clades within this section. We found that floral scent composition is affected by both phylogenetic relationships among Ophrys species and pollination interactions, with some compounds (especially fatty acid esters) carrying a significant phylogenetic signal and some (especially alkenes and alkadienes) generating dissimilarities between closely related Pseudophrys pollinated by different insects.
• Our results show that in the section Pseudophrys, floral scents are shaped both by pollinator‐mediated selection and by phylogenetic constraints, but that the relative importance of these two evolutionary forces differ among compound classes, probably reflecting distinct selective pressures imposed upon behaviourally active and non‐active compounds.

     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes

The effect of maternal age on the incidence of chromosomal abnormalities was investigated on a large sample of 3,042 in vitro unfertilized human oocytes II obtained from 792 women aged 19-46 years and participating in an in vitro fertilization program for various indications. The chromosomal analysis combined a gradual fixation of oocytes and an adapted R-banding technique. A total of 1,397 interpretable karyotypes were obtained. Various types of numerical aberration were observed, involving conventional chromosome nondisjunction (3.5%), single-chromatid nondisjunction (5.9%), complex (0.8%) or extreme aneuploidy (0.5%), diploidy (5.4%), and set of single chromatids (3.8%). No significant difference was found in the mean age of women according to the various types of chromosomal abnormalities. A positive relationship was found between maternal age and the global rate of aneuploidy, in agreement with the findings of epidemiological studies. The incidence of both whole-chromosome nondisjunction and precocious chromatid separation were correlated to maternal aging but the most significant correlation was found between maternal aging and single-chromatid nondisjunction. The rate of diploidy was also correlated to a slight extent to maternal aging, whereas no correlation was found between maternal age and the rate of single-chromatid sets. These data reveal that single-chromatid malsegregation is an essential factor in the age-dependent occurrence of nondisjunction in human oocytes. Disturbance in sister-chromatid cohesion might be a causal mechanism predisposing to premature chromatid separation and subsequently to nondisjunction in female meiosis.