Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes.

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and alphav integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.

Weak influence of near-surface layer on solar deep convection zone revealed by comprehensive simulation from base to surface.

The solar convection zone is filled with turbulent convection in highly stratified plasma. Several theoretical and observational studies suggest that the numerical calculations overestimate the convection velocity. Since all deep convection zone calculations exclude the solar surface due to substantial temporal and spatial scale separations, the solar surface, which drives the thermal convection with efficient radiative cooling, has been thought to be the key to solve this discrepancy. Thanks to the recent development in massive supercomputers, we are successful in performing the comprehensive calculation covering the whole solar convection zone. We compare the results with and without the solar surface in the local domain and without the surface in the full sphere. The calculations do not include the rotation and the magnetic field. The surface region has an unexpectedly weak influence on the deep convection zone. We find that just including the solar surface cannot solve the problem.

Asteroseismic detection of latitudinal differential rotation in 13 Sun-like stars.

The differentially rotating outer layers of stars are thought to play a role in driving their magnetic activity, but the underlying mechanisms that generate and sustain differential rotation are poorly understood. We report the measurement using asteroseismology of latitudinal differential rotation in the convection zones of 40 Sun-like stars. For the most significant detections, the stars' equators rotate approximately twice as fast as their midlatitudes. The latitudinal shear inferred from asteroseismology is much larger than predictions from numerical simulations.

High-resolution structure of the conger eel galectin, congerin I, in lactose-liganded and ligand-free forms: emergence of a new structure class by accelerated evolution.

BACKGROUND: Congerin I is a member of the galectin (animal beta-galactoside-binding lectin) family and is found in the skin mucus of conger eel. The galectin family proteins perform a variety of biological activities. Because of its histological localization and activity against marine bacteria and starfish embryos, congerin I is thought to take part in the eels' biological defense system against parasites. RESULTS: The crystal structure of congerin I has been determined in both lactose-liganded and ligand-free forms to 1. 5 A and 1.6 A resolution, respectively. The protein is a homodimer of 15 kDa subunits. Congerin I has a beta-sheet topology that is markedly different from those of known relatives. One of the beta-strands is exchanged between two identical subunits. This strand swap might increase the dimer stability. Of the known galectin complexes, congerin I forms the most extensive interaction with lactose molecules. Most of these interactions are substituted by similar interactions with water molecules, including a pi-electron hydrogen bond, in the ligand-free form. This observation indicates an increased affinity of congerin I for the ligand. CONCLUSIONS: The genes for congerin I and an isoform, congerin II, are known to have evolved under positive selection pressure. The strand swap and the modification in the carbohydrate-binding site might enhance the cross-linking activity, and should be the most apparent consequence of positive selection. The protein has been adapted to functioning in skin mucus that is in direct contact with surrounding environments by an enhancement in cross-linking activity. The structure of congerin I demonstrates the emergence of a new structure class by accelerated evolution under selection pressure.

Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression.

The melanoma-associated antigen ME491 is expressed strongly during the early stages of tumor progression. The ME491 gene was molecularly cloned by means of DNA-mediated gene transfer followed by screening a lambda genomic library with human repetitive Alu sequences as a probe. The cloned DNA, after transfection into mouse L-cells, generated a protein with characteristics that were indistinguishable in Western blot analysis from the ME491 antigen expressed by human melanoma cells. Repeat-free subfragments of the cloned DNA were used for further studies. By Northern blot analysis, the subfragments detected a single 1.2-kilobase mRNA in the transformants and various human melanoma cell lines. ME491 complementary DNA clones were then obtained by probing a melanoma complementary DNA library with the genomic subfragments. Nucleotide sequence analysis of the cloned complementary DNA indicated that the ME491 antigen consists of 237 amino acids (Mr 25,475) with four transmembrane regions and three putative N-glycosylation sites. No significant structural homology was observed with other proteins thus far reported. We observed that the amounts of mRNA varied greatly with different melanoma cell lines. Southern blot analysis revealed no amplification or rearrangement of the ME491 gene in the human melanoma cell lines tested, including both high and low expressors of this antigen. The ME491 gene has been mapped to chromosome region 12p12----12q13 by somatic cell hybrid analysis and more narrowly localized to 12q12----12q14 by in situ hybridization.

In vitro properties of human melanoma cells metastatic in nude mice.

We have developed a human melanoma metastasis model in nude mice. In this model, a human variant cell line (451-LU) was obtained that spontaneously metastasized in nude mice. This variant cell line was selected from the lung of a nude mouse after several in vivo passages of human melanoma WM164 cells previously isolated from a melanoma metastasis of a patient. The WM164 cells were not competent for metastasis in nude mice prior to this selection. We compared the phenotypes of the parental nonmetastatic cell line and the metastatic variant with respect to growth at clonal seeding densities in protein-free medium (growth factor independence), in vitro invasion through reconstructed basement membranes, secretion of proteolytic enzymes, expression of tumor-associated antigens, and chromosomal abnormalities. Metastatic 451-LU cells showed significantly increased growth factor independence when grown at clonal seeding densities as compared to the parental cells. In in vitro chemoinvasion assays, metastatic 451-LU cells were significantly more invasive than the parental cells. The metastatic variant secreted collagenase and tissue type plasminogen activator at levels 10- and 3-fold higher than the parental WM164 cells, respectively. Polyclonal antibodies to tissue type plasminogen activator significantly inhibited invasion through reconstructed basement membranes. In metastatic 451-LU cells, expression of nerve growth factor receptor was elevated, both at the protein and transcriptional level. Metastatic cells were aneuploid with a mode of 97 chromosomes, whereas the parental nonmetastatic cells had a mode of 52 chromosomes. Our studies suggest that metastatic melanoma cell variants selected in vivo show increased independence of exogenous growth factors when grown at clonal cell densities, enhanced invasiveness in vitro, greater secretion of proteolytic enzymes, and increased chromosome mode as compared to the nonmetastatic parental cells. The data further suggest that melanoma cells isolated from metastatic lesions and maintained in vitro have an unstable invasive phenotype but that metastatic variant cells can readily be selected.

CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis.

The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycineaspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin beta1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta1 antibodies. Finally, we show that OPN binding by CD44 variants/beta1-containing integrins promotes cell spreading, motility, and chemotactic behavior.

Neurogenic control of parenchymal arterioles in the cerebral cortex.

Central neural vasomotor mechanisms act on the parenchymal vasculature of the brain to regulate regional cerebral blood flow (rCBF). Among the diverse components of the local neural circuits of the cerebral cortex, many may contribute to the regulation of rCBF. For example, the cholinergic vasodilative system that originates in the basal forebrain acts on the neocortex and hippocampus. Notably, rCBF is reduced in the elderly and patients with dementia. The vasodilatory response, independent of changes in blood pressure and glucose metabolism in the brain, occurs in the parenchymal arterioles to produce a significant increase in cortical rCBF. Recent studies illuminate the physiological role of the cholinergic vasodilator system related to neurovascular coupling, neuroprotection, and promotion of the secretion of nerve growth factor. In this review, cellular mechanisms and species differences in the neurogenic control of vascular systems, as well as benefits of the cholinergic vasodilatory systems against cerebral ischemia- and age-dependent impairment of neurovascular plasticity, are further discussed.

Large-scale magnetic fields at high Reynolds numbers in magnetohydrodynamic simulations.

The 11-year solar magnetic cycle shows a high degree of coherence in spite of the turbulent nature of the solar convection zone. It has been found in recent high-resolution magnetohydrodynamics simulations that the maintenance of a large-scale coherent magnetic field is difficult with small viscosity and magnetic diffusivity (≲10 (12) square centimenters per second). We reproduced previous findings that indicate a reduction of the energy in the large-scale magnetic field for lower diffusivities and demonstrate the recovery of the global-scale magnetic field using unprecedentedly high resolution. We found an efficient small-scale dynamo that suppresses small-scale flows, which mimics the properties of large diffusivity. As a result, the global-scale magnetic field is maintained even in the regime of small diffusivities-that is, large Reynolds numbers.

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells.

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.

Molecular cloning of the murine homologue of CD63/ME491 and detection of its strong expression in the kidney and activated macrophages.

The mouse homologue of CD63/ME491 (Mu-CD63) was molecularly cloned and analyzed. Mu-CD63 exhibited a strikingly high similarity to CD63/ME491 and the rat homologue. Northern blot analysis revealed that Mu-CD63 mRNA was expressed strongly in the kidney of adult mice, especially in the glomerulus fraction, implying the possibility that Mu-CD63 plays an important role in maintaining normal renal function. Activated macrophages and splenocytes exhibited strong expression of Mu-CD63 mRNA, whereas cultured thymocytes barely expressed the mRNA irrespective of cell activation. Taken together, the present results suggest that Mu-CD63 expression is associated with differentiation and/or development of certain cell types, but not necessarily with cell proliferation.

Unusually large numbers of electrons for the oxidation of polyphenolic antioxidants.

Reaction mechanisms of polyphenolic antioxidants were studied using electrochemical methods (flow column electrolysis and cyclic voltammetry). In flow column electrolysis, the numbers (ns) of electrons involved in the oxidation of catechols (chlorogenic acid and caffeic acid) became larger than two (i.e. the number of -OH moieties) at pH > 7; the n-values finally reached ca. 4 at pH 10. Other polyphenols including catechin, ellagic acid, and curcumin exhibited higher n-values than the numbers of -OH moieties in the whole pH range studied (4 < pH < 10). Such unusually large n-values for polyphenols were found to correlate to their irreversible behavior in cyclic voltammetry. A digital simulation analysis of the voltammograms of chlorogenic acid clearly showed that the electrode reaction at higher pHs can be elucidated in terms of a quasi-reversible electron transfer followed by a chemical reaction and also suggested that the chemical reaction is of second order to the concentration of chlorogenic acid, i.e. a dimerization reaction. In a similar manner, polyphenolic antioxidants generally undergo certain chemical reactions on the occasion of their oxidation. As a result, some oxidizable, phenolic -OH moieties are reproduced in the polymeric products. The unusually large n-values of polyphenols and thus their higher radical scavenging activities may be ascribed to such reproduction of -OH moieties by oxidative polymerization.

An inhibitor of squalene epoxidase, NB-598, suppresses the secretion of cholesterol and triacylglycerol and simultaneously reduces apolipoprotein B in HepG2 cells.

NB-598, a specific inhibitor of squalene epoxidase, suppressed the secretion of cholesterol and triacylglycerol from HepG2 cells into the medium. L-654,969, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibited the secretion of cholesterol as potently as NB-598, but did not suppress the secretion of triacylglycerol. Both compounds decreased the intracellular cholesterol content almost equally, and neither of the compounds reduced the intracellular triacylglycerol content. The suppression of lipid secretion by NB-598 was associated with a significant reduction in apolipoprotein (apo) B secretion into the medium. Therefore, the suppression of lipid secretion by NB-598 may be caused by a reduction in the number of triacylglycerol-rich lipoprotein particles. In contrast, the suppression of cholesterol secretion by L-654,969 may be due to a modulation of lipoprotein lipid composition, since this agent did not reduce the secretion of apo B or triacylglycerol. The secretion of apo A-I was unaffected by either NB-598 or L-654,969. Pulse chase studies using [35S]methionine showed that the suppression of apo B secretion by NB-598 depended on an enhancement of intracellular degradation of apo B. These results indicate that the secretion of apo B from HepG2 cells is not regulated by the lipid synthesis alone, and suggest that the mechanism of the hypolipidemic effect of NB-598 involves the suppression of triacylglycerol-rich lipoprotein secretion from the liver as well as an inhibition of cholesterol synthesis in the liver.

Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1.

Chlorzoxazone, a muscle-relaxing drug, is metabolized by carbon-hydroxylation at position 6. Chlorzoxazone has been suggested as an in vivo probe for CYP2E1. We studied the specificity of such a substrate using vaccinia virus expressed human P450 forms and the effect of inhibitors for chlorzoxazone metabolism by human liver microsomes. The 6-hydroxylation of chlorzoxazone was mediated by CYP1A2 as well as by CYP2E1. The Km value of CYP1A2 and CYP2E1 for the reaction was 5.69 microM and 232 microM, respectively. However, the Vmax value of CYP2E1 for the reaction was approximately 8.5-fold higher than that of CYP1A2. The CYP1A inhibitor, alpha-naphthoflavone, as well as the CYP2E1 inhibitor, diethyldithiocarbamate, decreased chlorzoxazone 6-hydroxylation at a low substrate concentration by human liver microsomes. Our results raise questions about the suitability of chlorzoxazone as an in vivo probe for hepatic CYP2E1 activity. In human liver microsomal samples, the Km = 40 microM was different from either the Km of CYP1A2 or CYP2E1. We think that this discrepancy is due to the co-expression of similar levels of CYP1A2 and CYP2E1 in human liver. Furthermore, it is suggested that the role of CYP2E1 in 6-hydroxychlorzoxazone formation at the physiological chlorzoxazone concentration of 30-60 microM is almost the same when compared to that of CYP1A2.

Molecular analysis of a novel protein kinase in maturing rice seed.

Protein kinases play important roles in controlling biological functions. We employed PCR-based cloning technique to isolate a protein kinase gene from rice endosperm and obtained a novel protein kinase (REK) cDNA clone from a cDNA library constructed from maturing rice seed. The deduced amino acid sequence from the cDNA exhibited a high similarity to the wheat abscisic acid inducible protein kinase (PKABA1), including 11 conserved regions of the catalytic domain. REK belongs to the SNF1-related family that possesses abundant acidic amino acid resides in the C-terminal region. RT-PCR analysis showed that the REK gene is expressed in leaf and maturing seed, but not in stem and root. Bacterial recombinant REK showed autophosphorylation activity depending upon Ca2+. In addition, we isolated a REK genomic clone and determined its gene structure.

Cloning of the rice seed alpha-globulin-encoding gene: sequence similarity of the 5'-flanking region to those of the genes encoding wheat high-molecular-weight glutenin and barley D hordein.

A genomic clone encoding the rice endosperm major globulin (alpha-globulin) with an apparent molecular mass of 26 kDa was isolated, and its nucleotide (nt) sequence and transcription start point (tsp) were determined. The tsp was identical to that of the gene encoding the wheat high-molecular-weight (HMW) glutenin subunit. The consensus '-300 element' and an A + T-rich sequence exist upstream from the TATA box in the 5'-flanking region. A nt sequence of about 130 bp in the 5'-flanking region was found to be markedly homologous to those of the genes encoding the wheat HMW glutenin subunit and barley D hordein.

Complex formation of the nonstructural protein 3 of hepatitis C virus with the p53 tumor suppressor.

By co-immunoprecipitation analysis, we demonstrated that wt-p53 formed a complex with non-structural protein (NS) 3 of hepatitis C virus, both in the absence and the presence of NS4A, a viral cofactor that strongly associates with NS3. Deletional analysis revealed that a portion near the N-terminus of NS3 (amino acids (aa) 1055 and 1200), which is different from the NS4A binding site, was necessary for the complex formation with wt-p53. On the other hand, a portion near the C-terminus of wt-p53 (aa 301-360), which has been reported to contain the oligomerization domain, was important for the complex formation with NS3.

Nuclear localization and intramolecular cleavage of N-terminally deleted NS5A protein of hepatitis C virus.

The full-size NS5A (NS5A-F) of hepatitis C virus is localized in the cytoplasm despite the presence of a functional nuclear localization signal (NLS) in its C-terminal region (amino acids (aa) 354-362). In the present study, we demonstrated that a short stretch of sequence near the N-terminus of NS5A (aa 27-38) masked the functional NLS, preventing NS5A from being transported to the nucleus. This sequence, referred to as an NLS-masking sequence, was distinct from a nuclear export signal, as it did not actively target a protein to the cytoplasm. We also found that other sequences located at either an N- (aa 1-21) or a C-terminal region (aa 353-447) were responsible for targeting NS5A to the cytoplasm. Western blot analysis of the transfected cells revealed that NS5A mutants that had been N-terminally deleted by 66 aa or more were cleaved at a certain cleavage site, generating a common fragment of ca. 40 kDa. This result implies the possible presence of a cleavage site in the NS5A sequence around aa 150, which is exposed through conformational alteration upon the N-terminal deletions.

Stimulation of saphenous afferent nerve produces vasodilatation of the vasa nervorum via an axon reflex-like mechanism in the sciatic nerve of anesthetized rats.

The present study was intended to examine the physiological relevance of peptidergic afferent vasodilative fibers in the regulation of blood flow in the vasa nervorum, with special reference to the axon reflex. The response of nerve blood flow (NBF) in the sciatic nerve to electrical stimulation of saphenous nerve afferents was examined using laser Doppler flowmetry in anesthetized rats whose lumbosacral afferents and efferents had been disconnected from the spinal cord. Repetitive electrical stimulation of unmyelinated fibers in the central cut end of the saphenous nerve produced an increase in NBF in the sciatic nerve ipsilateral to the stimulation, independent of changes in mean arterial blood pressure. This increase was abolished by topical application of a calcitonin gene-related peptide (CGRP) receptor antagonist, hCGRP (8-37). In conclusion, NBF in the sciatic nerve is regulated via an axon reflex-like mechanism by unmyelinated afferent CGRP containing vasodilators with collaterals in the saphenous nerve.

Cilostazol, a selective cAMP phosphodiesterase inhibitor, dilates retinal arterioles and increases retinal and choroidal blood flow in rats.

The effects of cilostazol, a selective cyclic AMP (cAMP) phosphodiesterase inhibitor, on retinal and choroidal blood flow and retinal arteriole diameter were examined in anesthetized rats. The retinal and choroidal blood flow was measured using laser Doppler flowmetry, and the diameter of the retinal arterioles was measured using digital video microscopy. Cilostazol was administered by two routes; systemically via the intravenous route, and directly into the retinal vessels via the intra-arterial route. When administered intravenously, 1 mg/kg of cilostazol produced a biphasic blood flow response, composed of an initial decrease which was dependent on a depressor response of mean arterial pressure, and a subsequent slight but significant increase which was independent of changes in mean arterial pressure. When administered intra-arterially over a 2-min period, 40-55 and 400-440 microg of cilostazol both produced an increase in the blood flow in a dose-dependent manner, while a depressor effect was observed only at the dose of 400-440 microg. The diameter of the retinal arterioles was increased after the intra-arterial injection of cilostazol (400 microg). It is concluded that intra-arterially administered cilostazol induces vasodilation of the retinal arterioles of rats, which results in an increase in blood supply to the retina, independent of changes in mean arterial pressure.