Secretion and assembly of type IV and VI collagens depend on glycosylation of hydroxylysines

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. These hydroxylysine-linked carbohydrates were shown recently to be indispensable for the formation of basement membranes (Ruotsalainen, H., Sipil?, L., Vapola, M., Sormunen, R., Salo, A. M., Uitto, L., Mercer, D. K., Robins, S. P., Risteli, M., Aszodi, A., F?ssler, R., and Myllyl?, R. (2006) J. Cell Sci. 119, 625-635). Analysis of LH3 knock-out embryos and cells in this work indicated that loss of glycosylated hydroxylysines prevents the intracellular tetramerization of type VI collagen and leads to impaired secretion of type IV and VI collagens. Mice lacking the LH activity of LH3 produced slightly underglycosylated type IV and VI collagens with abnormal distribution. The altered distribution and aggregation of type VI collagen led to similar ultrastructural alterations in muscle to those detected in collagen VI knockout and some Ullrich congenital muscular dystrophy patients. Our results provide new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion, assembly, and distribution of highly glycosylated collagen types.     

Cloning and characterization of saponin hydrolases from Aspergillus oryzae and Eupenicillium brefeldianum

We purified saponin hydrolases from Aspergillus oryzae PF1224 and Eupenicillium brefeldianum PF1226. It was confirmed that the enzymes from A. oryzae PF1224 (Sda1) and E. brefeldianum PF1226 (Sde1) are glycoproteins with molecular masses of 82 and 90 kDa respectively. The deduced amino acid sequences of each enzyme from the cloned genes (sda1 or sde1) showed approximately 50% homology with that of the saponin hydrolase Sdn1 from Neocosmospora vasinfecta var. vasinfecta PF1225 (DDBJ accession no. AB110615). When sda1 and sde1 were expressed in the host Trichoderma viride under the control of the cellobiohydrolase I gene promoter, recombinant proteins were secreted with molecular masses of 77 and 67 kDa respectively. These recombinant enzymes hydrolyzed soyasaponin I to soyasapogenol B and triose, and its substrate specificities for glycosides were similar to that of Sdn1, but the specific activities of these enzymes were lower than that of Sdn1.     

Genetic control of trichome branch number in Arabidopsis: the roles of the FURCA loci

We are using trichome (hair) morphogenesis as a model to study how plant cell shape is controlled. During a screen for new mutations that affect trichome branch initiation in Arabidopsis, we identified seven new mutants that show a reduction in trichome branch number from three branches to two. These mutations were named furca, after the Latin word for two-pronged fork. These seven recessive mutations were placed into four complementation groups that define four new genes: FURCA1, FURCA2, FURCA3 and FURCA4. The trichome branch number phenotype indicates that the FURCA genes encode positive regulators of trichome branch initiation. Analysis of double mutants suggests that primary and secondary branch initiation events are not genetically distinct, but rely on the levels of partially redundant groups of regulators of trichome branch initiation. Based on the analysis of both epistatic and additive genetic interactions between the FURCA genes and other genes that control trichome branch number, we propose a model that explains how these genes interact to control trichome branch initiation. This model successfully predicts the phenotypes of all the single and double mutants examined and suggests points of control of the trichome branch pathway.     

A simple and efficient method for isolating trichomes for downstream analyses

Arabidopsis trichomes are an excellent cell type to address many questions in plant biology including the control of cell shape, endoreplication, and cell expansion. Because trichomes comprise such a small percentage of the cells of a leaf, biochemical analyses of trichomes are limited. To overcome this limitation, we developed a method for removing trichomes from the leaf surface. Our method allows the isolation of intact trichomes for use in downstream applications such as cell wall analysis, immunolocalization of trichome proteins, analysis of DNA content, and proteomics. Also, this method will facilitate the isolation of trichomes from practically any plant species.     

Measurements of mouse pulmonary artery biomechanics

BACKGROUND: Robust techniques for characterizing the biomechanical properties of mouse pulmonary arteries will permit exciting gene-level hypotheses regarding pulmonary vascular disease to be tested in genetically engineered animals. In this paper, we present the first measurements of the biomechanical properties of mouse pulmonary arteries. METHOD OF APPROACH: In an isolated vessel perfusion system, transmural pressure, internal diameter and wall thickness were measured during inflation and deflation of mouse pulmonary arteries over low (5-40 mmHg) and high (10-120 mmHg) pressure ranges representing physiological pressures in the pulmonary and systemic circulations, respectively. RESULTS: During inflation, circumferential stress versus strain showed the nonlinear "J"-shape typical of arteries. Hudetz's incremental elastic modulus ranged from 27 +/- 13 kPa (n = 7) during low-pressure inflation to 2,700 +/- 1,700 kPa (n = 9) during high-pressure inflation. The low and high-pressure testing protocols yielded quantitatively indistinguishable stress-strain and modulus-strain results. Histology performed to assess the state of the tissue after mechanical testing showed intact medial and adventitial architecture with some loss of endothelium, suggesting that smooth muscle cell contractile strength could also be measured with these techniques. CONCLUSIONS: The measurement techniques described demonstrate the feasibility of quantifying mouse pulmonary artery biomechanical properties. Stress-strain behavior and incremental modulus values are presented for normal, healthy arteries over a wide pressure range. These techniques will be useful for investigations into biomechanical abnormalities in pulmonary vascular disease.     

Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide

Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.     

Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives: high-affinity ligands to the alpha 2 delta subunit of voltage gated calcium channels

A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the alpha2delta subunit of voltage-gated calcium channels has been described. Substitutions in the aryl ring of the molecule were generally not tolerated, and resulted in diminished binding to the alpha2delta subunit. Modifications to the pyridazine ring revealed numerous permissive substitutions, and detailed SAR studies were carried out in this portion of the molecule. Replacement of the pyridazine ring methyl group with an aminomethyl functionality provided greatly improved potency over the initial lead. The initial lead compound displayed good rat pharmacokinetic properties, and was shown to be efficacious in the Chung model for neuropathic pain in rats.     

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae)

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.