Construction of multi-functional extracellular matrix proteins that inhibits migration and tube formation of endothelial cells

Artificial extracellular matrix (ECM) proteins have been designed that have strong cell-adhesive activity and active functional units that inhibit network formation among vascular endothelial cells. A laminin-derived sequence (YIGSR) that blocks migration of vascular endothelial cells was designed to incorporate into an elastin-derived structural unit. The designed ECM fusion protein also had a cell-adhesive RGDN sequence that conferred an intense migration-inhibitory effect. The resultant ECM showed cell-adhesive activity superior to that of the synthetic YIGSR peptide and it blocked the migration of vascular endothelial cells. Furthermore, the designed ECM inhibited the angiogenic activity of a collagen gel. The engineering strategy of designing multi-functional ECM proteins could be applied to support novel tissue engineering techniques.     

Gold ions bio-released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury

Traumatic brain injury results in loss of neurons caused as much by the resulting neuroinflammation as by the injury. Gold salts are known to be immunosuppressive, but their use are limited by nephrotoxicity. However, as we have proven that implants of pure metallic gold release gold ions which do not spread in the body, but are taken up by cells near the implant, we hypothesize that metallic gold could reduce local neuroinflammation in a safe way. Bio-liberation, or dissolucytosis, of gold ions from metallic gold surfaces requires the presence of disolycytes i.e. macrophages and the process is limited by their number and activity. We injected 20-45 mum gold particles into the neocortex of mice before generating a cryo-injury. Comparing gold-treated and untreated cryolesions, the release of gold reduced microgliosis and neuronal apoptosis accompanied by a transient astrogliosis and an increased neural stem cell response. We conclude that bio-liberated gold ions possess pronounced anti-inflammatory and neuron-protective capacities in the brain and suggest that metallic gold has clinical potentials. Intra-cerebral application of metallic gold as a pharmaceutical source of gold ions represents a completely new medical concept that bypasses the blood-brain-barrier and allows direct drug delivery to inflamed brain tissue.     

Induction of endothelial apoptosis by 4-hydroxyhexenal.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.     

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

The 5′-untranslated region (5′-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5′-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5′-UTRs with high translation efficiency using a ribosome display technique. A 5′-UTR random library, comprised of 5′-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5′-UTR with high translation efficiency was obtained from random 5′-UTR library.     

Selection of a carbohydrate-binding domain with a helix-loop-helix structure

We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.     

Genetic studies on reference strains of mutans streptococci

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.     

Effects of Chloroplast DNA Content on the Cell Proliferation and Aging in Chlamydomonas reinhardtii

Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4–7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Received 7 September 2000/ Accepted in revised form 14 February 2001     

Morphological study on the 'seams' in the multiroot formation of rat molar teeth.

'Seams' of the root furcation with multiroots in rat molar teeth, termed by Lester and Boyde, were investigated using transmitted light and scanning electron microscopy in the formation process. The seam was formed in the junctional line of Hertwig's epithelial root sheaths. The seam formation will be classified into three types in the initial stage based on the position of the epithelial root sheaths approaching each other: (1) the close junction or the very narrow slit or gap formed between them, (2) the clear gap containing mesenchymal cells and (3) the gap containing one blood vessel. When the roots were formed, the seam was formed as follows: (1) the slight ridge composed of the cellular cementum, (2) the proliferation or the depression in the dentine formation and (3) the accessory or lateral canal of the root. These structures were variously combined with each other into one seam, although the slight ridge was very common.     

Identification of human ST2 protein in the sera of patients with autoimmune diseases.

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS-PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS-PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.     

Accuracy evaluation of sleep-wake stage analysis with SleepSign Ver2.0

Sleep and Biological Rhythms -