Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

A New Enzymatic Cycling Technique for Glutamate Determination in Brain Microdialysates

A quantitative analysis of glutamate in brain dialysate was made by using an enzymatic cycling technique. This method made it possible to measure the concentration of glutamate in dialysate collected at 30-s intervals. Dialysates were collected from Mongolian gerbil hippocampus before, during, and after two 90-s ischemic insults at an interval of 5 min. An extracellular increase in levels of glutamate was already observed in samples collected during a 30-60 s period after the onset of each ischemia, and the levels of glutamate were maximal at the end of each period of ischemia (approximately a fourfold increase). The increased levels of glutamate rapidly returned almost to preischemic levels by 30 s of recirculation. This method will provide more precise information about temporal changes in the extracellular glutamate concentration in the brain during ischemia.     

Biosynthesis of cat hemoglobins A and B: Amino terminal acetylation of the β chain of HbB

Acetylation of the amino terminal serine of the β chains of cat HbB occurs during synthesis of hemoglobin in a mRNA dependent rabbit reticulocyte lysate protein synthesizing system in the presence of acetyl-CoA and cat reticulocyte mRNA. Both of the major cat hemoglobins, the nonacetylated HbA and acetylated HbB, are synthesized efficiently in the rabbit lysate system. The acetylation of HbB-β chains occurs during the biosynthesis of these proteins. Radioautography of tryptic peptide maps reveals that acetylation occurs specifically at the amino terminal serine of HbB-β globin, and not on HbA-β globin or on the α chain common to both hemoglobins A and B. Because of the similarity of the structures of HbA-β and HbB-β globin, it is suggested that the amino terminal residue determines whether the peptide chain is recognized for acetylation by a ribosomal acetyltransferase.     

Structural studies of urinary oliogosaccharides from patients with mannosidosis

Eight neutral oligosaccharide fractions were obtained from the pooled urine of two patients with mannosidosis by Bio-Gel P2 and Bio-Gel P4 column chromatography. The structures of seventeen oligosaccharides were determined by monosaccharide composition analysis, methylation studies, acetolysis, Smith degradation, and ¹³C NMR analysis. Three of the proposed structures, Manα1-3Manβ1-4GlcNAc, Manα1-2Manα1-3Manβ1-4GlcNAc, and Manα1-2Manα1-2Manα1-3Manβ1-4GlcNAc are identical to those first published by Norden et al. (N. E. Norden, A. Lundblad, S. Svennson, P. A. Ockerman, and S. Autio, 1973. J. Biol. Chem.248, 6210–6215; N. E. Norden, A. Lundblad, S. Svennson, and S. Autio, 1974. Biochemistry13, 871–874). Thirteen of them, Manα1-3Manα1-6(Manα1-3)-Manβ1-4GlcNAc, Manα1-3Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAc, and 11 isomers of (Manα1-2)_0–4[Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc], are the same as those first published by Yamashita et al. (K. Yamashita, Y. Tachibana, K. Mihara, S. Okada, H. Yabuuchi, and A. Kobata, 1980, J. Biol. Chem.255, 5126–5133); a tetrasac-charide, Manα1-6(Manα1-3)Manβ1-4GlcNAc, is newly reported and several other structural possibilities are proposed.     

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal (M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.     

Neural induction: Historical views and application to pluripotent stem cells

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state‐of‐the‐art stem cell research.     

Synthesis of anilino-monoindolylmaleimides as potent and selective PKCbeta inhibitors

We report herein synthesis of PKCbeta-selective inhibitors possessing the novel pharmacophore of anilino-monoindolylmaleimide. Several compounds of this series exhibited IC50's as low as 50 nM against human PKCbeta2. One of the most potent compounds, 6l, inhibited PKCbeta1 and PKCbeta2 with IC50 of 21 and 5 nM, respectively, and exhibited selectivity of more than 60-fold in favor of PKCbeta2 relative to other PKC isozymes (PKCalpha, PKCgamma, and PKCepsilon).     

A selective inducible NOS dimerization inhibitor prevents systemic,cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.