Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.     

Morphology of cultured human epidermal melanocytes observed by atomic force microscopy

The objective of this study was to image the surface structure of cultured human epidermal melanocytes using atomic force microscopy (AFM). Epidermis obtained from human foreskins was treated with 0.5% dispase. Cell suspensions of the epidermis were prepared and seeded in six-well plates, in which sheets of mica had been placed. Samples for AFM were fixed on mica and scanning AFM images were captured by contacting and tapping modes operated under normal atmospheric pressure and temperature. Human epidermal melanocytes exhibited rounded, oval, triangular or quadrangular perikarya from which eight to 10 thick dendrites arose. These dendrites first bifurcated near the soma and then divided profusely into daughter branches, which spread out in all directions. We observed string-like long thin projections, growth cones and shorter thicker projections, which arose from the dendritic shafts, in which groups of melanosomes were arrayed. In addition to such structures, the most striking feature was the presence of filopodia arising from the melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes. The termini of dendrites formed unbranched terminal protrusions (approximately 1,500-2,000 nm wide) consisting of two to three melanosomes wrapped in an arc, with their filopodia extending outwards. The tips of these structures also appeared to be squeezed and finally pinched off by the melanocyte to form a pouch filled with numerous melanosomes. We conclude that secondary and tertiary branches and subordinate branches might take part in transferring melanosomes into keratinocytes in addition to the transfer through the tips of the dendritic shafts. The melanin granules were expelled by exocytosis.     

Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts

adeA and adeB genes homologous to Saccharomyces cerevisiae ADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD(-), sC(-)), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzae adeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.     

Chemical composition of the essential oils of two Alpinia species from Hainan Island, China

The essential oils of two Alpinia species, ie. A. hainanensis and A. katsumadai, from Hainan Island, China were analyzed by using GC-MS. The major constituents in the leaf oil of A. hainanensis were ocimene (27.4%), beta-pinene (10.1%), 9-octadecenoic acid (6.5%), n-hexadecanoic acid (5.8%), 9,12-octadecadienoic acid (5.4%), and terpinen (4.3%). The oil constituents obtained from the flowers of A. hainanensis were ocimene (39.8%), beta-pinene (17.7%), terpinene (5.5%), p-menth-1-en-ol (4.9%), caryophyllene (4.9%), and phellandrene (4.4%). In A. katsumadai, the major constituents in the leaf oil were p-menth-1-en-ol (22.0%), terpinen (19.0%), 4-carene (9.1%), 1,8-cineole (8.3%), and camphor (5.6%). The major constituents in the flower oil were p-menth-1-en-ol (21.3%), 1,8-cineole (20.2%), terpinen (12.6%), phellandrene (7.0%), 4-carene (6.4%), and beta-pinene (5.2%).     

Labeling neurons in vivo for morphological and functional studies

Increasingly sophisticated strategies for labeling cells in vivo are providing unprecedented opportunities to study neurons in living animals. Transgenic expression of genetically encoded reporters enables us to monitor changes in neuronal activity in response to sensory stimuli, and the labeling of single neurons with fluorescent proteins allows the dynamics of neuronal connectivity to be observed in transgenic animals over periods ranging from minutes to months. Advances in transient labeling techniques such as viral infection and electroporation provide a rapid means by which to analyze neuronal gene function in vivo. These new approaches to labeling, manipulating and imaging neurons in intact organisms are transforming the way in which the nervous system is studied.     

Influence of dietary NaCl on L-arginine transport in the renal medulla

Previous work demonstrated that l-arginine, the substrate for nitric oxide (NO) synthase, is carried into inner medullary collecting duct (IMCD) cells via system y+, that the major system y+ gene product in IMCD is the cationic amino acid transporter 1 (CAT1), and that blockade of l-arginine uptake in the renal medulla decreases NO and leads to systemic hypertension. The present study determined the influence of dietary sodium intake on l-arginine uptake in IMCD, on CAT1 immunoreactive protein in the renal medulla, and on the hypertensive response to blockade of l-arginine uptake in the renal medulla. Transport studies in bulk-isolated IMCD demonstrated that l-arginine uptake by IMCD was significantly greater (663 +/- 100 pmol x mg(-1) x min(-1), n = 6) in rats exposed to a low-sodium diet (0.4% NaCl) compared with rats on a normal (1% NaCl, 519 +/- 78 pmol x mg(-1) x min(-1), n = 6) or high-sodium diet (4.0% NaCl, 302 +/- 27 pmol x mg(-1) x min(-1), n = 6). Immunoblotting experiments demonstrated that CAT1 immunoreactive protein was significantly decreased by approximately 30% in rats maintained on a high-NaCl diet (n = 5) compared with rats on a low-NaCl diet (n = 5). In contrast to the l-arginine transport and immunoblotting data, in vivo blockade of l-arginine uptake led to hypertension of equal magnitude in rats maintained on a low- or high-NaCl diet. These results indicate that sodium loading leads to a decrease in immunoreactive CAT1 protein in the rat renal medulla, resulting in decreased l-arginine uptake capacity. The decrease in l-arginine uptake capacity, however, does not alter the blood pressure response to l-arginine uptake inhibition in the renal medulla.     

Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF

Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O(2)) possibly by binding to the NPY Y(3) receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10(-7) M NPY or 10(-7) g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by approximately 70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y(3)-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.     

P2X7 receptor-mediated apoptosis of human cervical epithelial cells

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X₇ receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 µM in hECE cells and 1 µM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-, it had only a mild effect on caspase-8. Both BzATP and TNF- activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was 0.5 µM, which is in the range of values that suffice to activate the P2X₇ receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X₇ receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway. cervix; epithelium; ATP; 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate