A novel role for cholecystokinin: regulation of mesenteric vascular resistance

The aim of this work was to characterize the vasoactive effect of cholecystokinin on mesenteric vasculature. The mesenteric vascular bed of 3-month-old Sprague-Dawley rats was isolated and perfused at constant flow and changes in perfusion pressure monitored. CCK peptides lacked any direct contractile or relaxing effect on the mesenteric smooth muscle. Transmural nerve stimulation (TNS, 200 mA, 0.2 ms, 8 and 16 Hz) elicited an increase in perfusion pressure reflecting contraction of the bed and CCK inhibited neurogenic contractions elicited by 8 and 16 Hz TNS. The inhibition of neurogenic contractions was blocked by the CCK2 receptor (CCK2R) antagonist, L-365,260 (10 and 100 nM), but not by the CCK1R antagonist, SR-27897. The inhibition of neurogenic contractions was reversed by the non-specific NOS inhibitor, L-NAME as well as by the specific nNOS inhibitor, S-methyl-L-thiocitrulline. In whole-mount segments of mesenteric arteries, CCK2R was detected in the adventitia, in nerve terminals, where it co-localized with synaptophysin and nNOS. CCK-8 immunoreactive fibers were also detected. These results suggest that CCK mediates vasodilatation of the mesenteric vascular bed through the release of NO via its presynaptic CCK2R. Our findings provide, for the first time, a neural mechanism by which CCK may increase mesenteric blood flow.     

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.     

Exposure of bovine oocytes to the endogenous metabolite 2-methoxyestradiol during in vitro maturation inhibits early embryonic development

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.     

Analysis of chromosome conservation in <Emphasis Type="Italic">Lemur catta</Emphasis> studied by chromosome paints and BAC/PAC probes

A panel of human chromosome painting probes and bacterial and P1 artificial chromosome (BAC/PAC) clones were used in fluorescence in situ hybridization (FISH) experiments to investigate the chromosome conservation of the ring-tailed lemur (Lemur catta, LCA) with respect to human. Whole chromosome paints specific for human chromosomes 7, 9, 11, 13, 14, 17, 18, 20, 21, and X were found to identify a single chromosome or an uninterrupted chromosomal region in LCA. A large set of partial chromosome paints and BAC/PAC probes were then used to refine the characterization of the rearrangements differentiating the two karyotypes. The results were also used to reconstruct the ancestral Lemuridae karyotype. Lemur catta, indeed, can be used as an outgroup, allowing symplesiomorphic (ancestral) rearrangements to be distinguished from apomorphic (derived) rearrangements in lemurs. Some LCA chromosomes are difficult to distinguish morphologically. The 'anchorage' of most LCA chromosomes to specific probes will contribute to the standardization of the karyotype of this species.     

Evolutionary history of chromosome 10 in primates

We have tracked the evolutionary history of chromosomes homologous to HSA10 (PHYL-10) in primates using appropriate panels of PCP, YAC, and BAC probes. This approach allowed us to delineate more precisely the PHYL-10 constitution in the ancestor of catarrhine, platyrrhine, and prosimians. The results suggest that (i) in the ancestor of prosimians PHYL-10 was organized in two separate PHYL-10p and PHYL-10q chromosomes; (ii) in the progenitor of New World monkeys PHYL-10p was a separate chromosome, while PHYL-10q was associated with a chromosome homologous to HSA16; (iii) in the ancestor of Old World monkeys PHYL-10 was a unique chromosome with a marker order corresponding to the orang form. We have also analyzed the cat, chosen as an outgroup for its very conserved karyotype. In agreement with published data our experiments show that the PHYL-10 in cat is structured in two blocks, PHYL-10p and PHYL-10q, both as part of larger chromosomes. The overall data indicate that, contrary to common opinion, PHYL-10p and PHYL-10q were distinct chromosomes in the primate ancestor. Analysis of the Saimiri sciureus (SSC) PHYL-10q marker order showed that it was isosequential with the Callithrix jacchus PHYL-10q, as well as with the PHYL-10q platyrrhine ancestral form. The SSC centromere, nevertheless, was located in a different chromosomal region, therefore suggesting that a centromeric repositioning event occurred in this species.     

Paraoxonase1-192 polymorphism modulates the effects of regular and acute exercise on paraoxonase1 activity

Regular exercise practise is a protective factor against coronary heart disease and enhances antioxidant systems, whereas acute exercise appears to be a major source of increased oxidative stress. Paraoxonase1 (PON1) is an antioxidant HDL-linked enzyme, whose activity toward paraoxon (PON1 activity) is strongly modulated by the PON1-192 polymorphism, comprising Q and R alleles for low and high PON1 activity, respectively. Another polymorphism at the PON1 locus, the PON1-55, modulates PON1 protein and activity levels. PON1 activity, lipid levels, and oxidized LDL concentration were determined in 17 healthy young volunteers before and after a 16-weeks aerobic exercise training period. Furthermore, PON1 activity was analyzed after a bout of exercise in both situations. We found that regular exercise was associated with a decrease in oxidized LDL levels, and an increase in PON1 activity in QQ subjects and with a decrease in PON1 activity in R carriers. A bout of exercise produced an increase in PON1 activity just after the bout of exercise, followed by a decrease in its activity. A recovery of the basal PON1 activity levels at 24 h was found in QQ subjects regardless of their training status and in trained R carriers, but not in untrained R carriers. These results suggest that the effects of regular and acute exercise on PON1 activity levels are modulated by PON1-192 polymorphism. Changes were less evident for the PON1-55 polymorphism.     

Heterozygous submicroscopic inversions involving olfactory receptor-gene clusters mediate the recurrent t(4;8)(p16;p23) translocation

The t(4;8)(p16;p23) translocation, in either the balanced form or the unbalanced form, has been reported several times. Taking into consideration the fact that this translocation may be undetected in routine cytogenetics, we find that it may be the most frequent translocation after t(11q;22q), which is the most common reciprocal translocation in humans. Case subjects with der(4) have the Wolf-Hirschhorn syndrome, whereas case subjects with der(8) show a milder spectrum of dysmorphic features. Two pairs of the many olfactory receptor (OR)-gene clusters are located close to each other, on both 4p16 and 8p23. Previously, we demonstrated that an inversion polymorphism of the OR region at 8p23 plays a crucial role in the generation of chromosomal imbalances through unusual meiotic exchanges. These findings prompted us to investigate whether OR-related inversion polymorphisms at 4p16 and 8p23 might also be involved in the origin of the t(4;8)(p16;p23) translocation. In seven case subjects (five of whom both represented de novo cases and were of maternal origin), including individuals with unbalanced and balanced translocations, we demonstrated that the breakpoints fell within the 4p and 8p OR-gene clusters. FISH experiments with appropriate bacterial-artificial-chromosome probes detected heterozygous submicroscopic inversions of both 4p and 8p regions in all the five mothers of the de novo case subjects. Heterozygous inversions on 4p16 and 8p23 were detected in 12.5% and 26% of control subjects, respectively, whereas 2.5% of them were scored as doubly heterozygous. These novel data emphasize the importance of segmental duplications and large-scale genomic polymorphisms in the evolution and pathology of the human genome.