Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

The influence of severe long-term exercise on the mouse hippocampus

To determine whether severe long-term exercise affects on the brain, we investigated the mice brain after 12-week treadmill exercise. The mice (ddN, male, 25-35 g in body weight) were divided into severe, mild, and non-exercise group. Mice in severe groups ran on a treadmill at a speed of 25 m/min for 12 weeks and mice in mild group ran on a treadmill at a speed of 10 m/min for 12 weeks. The mice were killed by transcardial perfusion with 0.1 M phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde in 0.1 M PB. The another sets of mice were fixed by 2.5% glutaraldehyde-2% osmium tetroxide for electromicroscope (EM). The brains were serially sectioned in the coronal plane at a thickness of 20-microns with a vibratome and then processed for histology, by means of hematoxylin-eosin (HE) staining and immunohistochemistry. Fifty % of mice in severe exercise showed hyperchromatic and shrunken nerve cells with nuclear pyknosis (dark neuron) in the hippocampus, but not in mild exercise and non-exercise groups. The immunoreactivity of microtuble associated protein-2 (MAP-2) decreased, while the heat-shock protein/cognate 70 (HSP/C 70) increased in the hippocampus of severe exercise group. Many destroyed mitochondria were observed in dark neurons by Electron micrograph. These findings suggested that severe long-term exercise might damage hippocampal neurons.     

36-kDa microfibril-associated glycoprotein (MAGP-36) is an elastin-binding protein increased in chick aortae during development and growth

MAGP-36 was discovered in porcine aorta in 1989 and is thought to be one of the microfibril-associated proteins. MAGP-36 has been localized on the surface of elastic fibers or laminae in immunohistochemical studies. However, its functional role in the aorta is obscure. Herein, we report on the binding activity of MAGP-36 to components of the aortic wall and its accumulation pattern in the aorta during development and growth. In vitro, MAGP-36 bound to elastin and collagen in a Ca(2+)-dependent manner, and mediated the adhesion of human aortic smooth muscle cells. This cell adhesion mostly depended on the RGD-containing domain of MAGP-36. We examined the accumulation of MAGP-36 with quantitative Western blot analysis and immunoelectron microscopy in chick aortae during development and growth. The amount of MAGP-36 increased on the surface of elastic fibers or laminae between days 14 and 34 after the start of incubation, and reached a plateau at about 53 days. This accumulation of MAGP-36 roughly correlated with an increase in blood pressure for this period. Thus, MAGP-36 might be a bridging protein that connects elastin to other components of the aortic wall and might play a role in maintaining the integrity of the aortic structure under arterial pressure.     

Identification of two calcineurin alpha isoforms in bovine brain by two different monoclonal antibodies.

Calcineurin (calcium- and calmodulin-stimulated phosphatase) alpha subunit purified from bovine brain was found to be composed of two polypeptides, 61 KDa (alpha 1) and 59 KDa (alpha 2). The two peptides were separated and extracted from polyacrylamide gel. The immuno-peptide mapping of the purified peptides by partial proteolysis showed that the 59-KDa polypeptide was not a degradative product of the 61-KDa polypeptide. The interaction of the enzyme with two monoclonal antibodies, Vj6 and Vd3, raised against bovine brain calcineurin revealed that the 61-KDa polypeptide was recognized by both Vj6 and Vd3, whereas the 59-KDa one was recognized only by Vj6. These results indicate that there are at least two isoforms of calcineurin alpha subunits in bovine brain.     

Identification of tranilast-binding protein as 36-kDa microfibril-associated glycoprotein by drug affinity chromatography, and its localization in human skin

To elucidate the molecular mechanism involved in the suppression of keloids and hypertrophic scars by tranilast, we investigated the target protein of tranilast in bovine skin and aorta. A specific tranilast-binding protein was isolated from both tissues by drug affinity chromatography and was identified as 36-kDa microfibril-associated glycoprotein (36-kDa MAGP). Binding of 36-kDa MAGP to tranilast seemed to be specific since 36-kDa MAGP could be eluted from the drug affinity column by tranilast itself and also binding of 36-kDa MAGP to other anti-allergy drugs (amlexanox and cromolyn) is significantly weaker than that to tranilast. Light and electron microscopic immunohistochemistry detected the protein at the periphery of elastic fibers in normal human skin. In hypertrophic scar tissue, however, 36-kDa MAGP was located on small bundles of microfibrils. These findings provide support for the concept that elastogenesis occurs in scar tissue and 36-kDa MAGP might be one of the targets for tranilast.     

Metabolic activities of partially degenerated hypertrophic chondrocytes: gene expression of hyaluronan synthases

Ultrastructural aspects of hypertrophic chondrocytes in hamster and mouse epiphysial cartilage were examined in relation to their metabolic activities. With the hypertrophic change, cytoplasmic vacuolization proceeded leaving the partially intact endoplasmic reticulum (ER). In the hypertrophic cells, cytoplasmic hyaluronan was stained with the biotinylated hyaluronan-binding region (b-HABR) of aggrecan, and mRNAs of hyaluronan synthase (Has 1, Has 2 and Has 3) were detected by in situ hybridization. When the epiphysial cartilage was cultured in the presence of 35S, 3H-GlcNAc, 3H-proline or 14C-palmitic acid, vacuolated late hypertrophic chondrocytes were labeled with these radioactive precursors. The evidence indicates that late hypertrophic chondrocytes are metabolically active, which appears to be essential for the enlargement of chondrocytes.     

F-actin bundling protein from Physarum polycephalum: purification and its capacity for co-bundling of actin filaments and microtubules

An F-actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F-actin bundling protein in Physarum extract was passed through a DEAE-cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S-300 HR followed by chromatography on CM-Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS-PAGE, and the molecular weight was estimated to be 52,000. This F-actin bundling protein is referred to as the 52 kDa protein. Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F-actin and microtubule bundles was Ca(2+)-insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross-reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F-actin bundling protein from HeLa cells) (Yamashiro-Matsumura and Matsumura: J. Biol. Chem. 260:5087-5097, 1985). When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co-bundles composed of both filaments formed. This is the first reported example in which an F-actin bundling protein induced co-bundling of actin filaments and microtubules.     

Cell differentiation of alveolar epithelium in the developing rat lung: ultrahistochemical studies of glycoconjugates on the epithelial cell surface

Glycoconjugates on the surface of pulmonary epithelial cells were ultrahistochemically examined in the fetal, neonatal and adult rat lung. Lectin and colloidal iron staining procedures were performed in combination with digestion using carbohydrate-degrading enzymes or methylation. The glycoconjugate composition of columnar cells at 16 days gestation was similar to that of cuboidal cells at 19 days gestation. Glycoconjugate differentiation on the cell surface occurred at 20 days gestation, and especially the loss of soybean agglutinin (SBA) binding sites could be detected on type II cells. The contents of Ricinus communis agglutinin-I (RCA-I) and Concanavalin A (Con A) binding sites on type II cells also began to decrease. On the contrary, the content of sulfated saccharides decreased on the surface of type I cells during development. Glycoconjugate differentiation on both type I and II cells was completed with the disappearance of hyaluronic acid and peanut agglutinin (PNA) binding sites; type I and II cells acquired a similar histochemical composition to that on adult type I and II cells at 5 days after birth. Both type I and II cells share a common early precursor cell, that is, the cuboidal epithelial cell at the canalicular stage.