Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

Characterization of Actinobacillus actinomycetemcomitans Hemolysin

Twenty out of 33 Actinobacillus actinomycetemcomitans strains formed hemolytic colonies on horse blood agar plates under anaerobic conditions. The hemolytic activity found in A. actinomycetemcomitans strain 137HE was examined. This activity was detected in the late exponential to early stationary phases of growth. Human erythrocytes were the most susceptible, followed by rabbit, sheep, horse and swine red blood cells. The majority of activity was detected in the cell-associated vesicle fraction. Zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extract from whole cells was semipurified by ammonium sulfate precipitation, preparative isoelectric focusing (IEF) and gel-filtration chromatography to yield a major band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 12 kDa. Heating at 80 C for 30 min and treatment with proteinase K or trypsin resulted in complete disappearance of the hemolytic activity. Sulphydryl reagents enhanced activity and small amounts of cholesterol inhibited it. In summary, we demonstrated the presence of hemolysin in A. actinomycetemcomitans, and examined and characterized it.     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors.

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor gamma) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes.     

Vegetation patterns in relation to micro-scale landforms in hilly land regions

Spatial structures of landforms as constants affecting vegetation patterns are discussed based on analyses of hilly land regions, mostly in the vicinity of Sendai, northeast Japan. The lower part of a hillslope is characterized by relatively active processes of soil erosion, landslides and slope failure. It supports a plant community different from that on the upper part of the hillslope. These two parts are termed the lower hillslope and the upper hillslope, respectively. The upper hillslope consists of valley heads with no stream water nor stream channels. Since a valley head is comprised of several micro-scale landform units, an upper hillslope can be subdivided into these landform units. Plant communities vary in their species compositions and structures as well as in some other ecological characteristics paralleling changes in their respective micro-scale landform units. However, the variations in species composition within upper hillslopes are not as extreme as those between the upper and lower hillslopes. xx]Papers presented at the Vth INTECOL Congress at YOKO-hama 1990.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

On-line state recognition in a yeast fed-batch culture using error vectors

The physiological states with respect to cell growth and ethanol production in a yeast fed-batch culture expressed in linguistic form could be recognized on-line by fuzzy inferencing based on error vectors. The error vector was newly defined here in a macroscopic elemental balance equation. The physiological states for cell growth and ethanol production were characterized by error vectors using many experimental data from fed-batch cultures. Fuzzy membership functions were constructed from the frequency distributions of the error vectors and state recognition was performed by fuzzy inferencing. In particular, an unusual physiological state for a yeast cultivation, in which aerobic ethanol production was accompanied by very low cell growth, could be recognized accurately. According to the results of the state recognition, an energy parameter, the P/O ratio in the metabolic reaction model was adaptively estimated, and the cell growth was successfully evaluated with the estimated P/O. (c) 1995 John Wiley & Sons, Inc.