Regulation of I-branched poly-N-acetyllactosamine synthesis. Concerted actions by I-extension enzyme, I-branching enzyme, and beta1,4-galactosyltransferase I

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching beta1, 6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta1, 6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta1, 4-galactosyltransferases (beta4Gal-Ts), beta4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta4Gal-TI and i-extension beta1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta1-->4GlcNAcbeta1-->3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta4Gal-TI adds a galactose poorly to beta1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta1, 3-N-acetylglucosaminyltransferase and beta4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.     

Ca2+ mobilization and activation of extracellular acidification by carbachol in acutely dispersed cells from guinea pig detrusor: Fura 2 fluorometry and microphysiometry using the cytosensor

The study aim was to develop a simple in vitro model for pharmacophysiological investigation of urinary bladder smooth muscles. Smooth muscle cells from guinea pig detrusor were dissociated, and the suspended cells were stimulated with carbachol (CCh), an acetylcholine receptor agonist. Cytosolic Ca2+ levels were determined using Fura 2 fluorescence and extracellular acidification rates were monitored by the Cytosensor microphysiometer. CCh dose-dependently increased cytosolic Ca2+ levels and extracellular acidification rates, with EC50 values of approximately 1 microM. Both the acetylcholine muscarinic receptor antagonist atropine and the M3 muscarinic receptor-preferring antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the effects of CCh, three orders of magnitude more potently than the selective M2 muscarinic receptor antagonist, methoctramine. These data indicate the dominant role of M3 receptors in guinea-pig bladder but fail to show clear evidence of any functional role for M2 receptors. Since this finding agrees with a number of other studies using in vivo and in vitro models (1), cell suspensions such as these may prove to be simple tools for the pharmacological study of urinary bladder smooth muscle tissue.     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.     

Mammosomatotrophs develop within mammotroph clusters in bovine adenohypophysis

The existence and distribution of mammosomatotrophs (MS cells) containing growth hormone (GH) and prolactin (PRL) in bovine adenohypophysis were detailed by a combined method of mirror sections and immunohistochemical staining. MS cells always occurred in bovine adenohypophysis but their number was low. In the midsagittal plane, the cells were observed in the hind dorsal, hind ventral and fore ventral region abundant in GH and PRL cells. Whereas, in the zona tuberalis where GH and PRL cells were less frequent, MS cells were not detected. MS cells were invariably solitarily distributed within mammotroph (PRL cell) clusters but not within somatotroph (GH cell) clusters. The proportion of MS cells declined as the ages proceeded and the appearance was spatially related to the arrangement of PRL cells. These findings indicated that, in bovine adenohypophysis, MS cells were differentially distributed and occurred in PRL cell clusters. The results strongly suggest that MS cells originate in GH cells pre-existed within PRL cell clusters with special reference to the functional activation of PRL cells.     

Threshold-dependent DNA synthesis by pure pressure in human aortic smooth muscle cells: Gialpha-dependent and -independent pathways

Mechanical forces related to pressure and flow are important for the etiology of atherosclerosis and hypertension. We hypothesized the presence of mechanosensors that were solely sensitive to pure atmospheric pressure in the absence of shear and tensile stresses. A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells (HASMC). Pressure application of 140 to 180 mmHg produced DNA synthesis in a pressure-dependent manner. In contrast, pressure of 120 mmHg or less produced no significant change. Both extracellular signal-regulated kinase and c-Jun N-terminal kinase activities, but not p38 activity, were stimulated by pressures of more than 160 mmHg. Pertussis toxin (PTx) completely inhibited the pressure-induced increase of DNA synthesis under the high pressure of 200 mmHg. These data suggest that HASMC have a mechanosensing cellular switch for DNA synthesis which is sensitive to pure atmospheric pressure, and that the molecular switch is activated by pressure of more than 140 mmHg. The activation mechanism consists of PTx-sensitive and -insensitive pathways, and the former is activated by high pure pressure.     

Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.     

Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose

Lacto-N-tetraose (Galbeta1 -3GlcNAcbeta1-3Galbeta1-4Glc, LNT) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, LNnT) were enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to lactose. Lacto-N-triose II (GlcNAcbeta1-3Galbeta1-4Glc) was prepared first by the transfer of GlcNAc from UDP-GlcNAc to lactose by beta-1,3-N-acetylglucosaminyltransferase from bovine serum. The resulting lacto-N-triose II was converted into LNT and LNnT utilizing two kinds of beta-D-galactosidase-mediated transglycosylations. Thus, beta-D-galactosidase from Bacillus circulans ATCC31382 induced regioselective galactosyl transfer from o-nitrophenyl beta-D-galactoside to the OH-3" position of lacto-N-triose II, and commercially available beta-D-galactosidase from B. circulans to the OH-4" position of lacto-N-triose II. These convenient processes are suitable for large-scale preparations of LNT and LNnT. As another method, LNT was directly synthesized from lactose as an initial substance, utilizing lacto-N-biosidase (Aureobacterium sp. L-101)-mediated transglycosylation with Galbeta1-3GlcNAcbeta-pNP donor.     

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.