Computerized topographical analysis of functionally homogeneous neuronal sets.

A computer-assisted analysis of the spatial distribution of neurons having homogeneous characteristics is described in this paper. The camera lucida drawings of sections of a brain nucleus and the points representing the neurons labeled on the basis of a specific behavior of discharge rates were digitized on a personal computer Amiga 2000 or IBM compatible. Our software provided: a) the computerized, stereotaxically oriented reconstruction of the stored sections and of the plotted neurons; b) the identification within each section of the mass center (MC) of the units sharing a given behavior and of the area where the density of such neurons was maximal (MDA). The routine was tested on the spatial distribution of neuronal responses to serotonin in the lateral vestibular nucleus.     

How to discriminate between vibration syndrome and local fatigue of motorcyclists

Motorcyclists who work in some offices sometimes complained of coldness, pain and numbness of upper limbs. We studied how to discriminate between vibration syndrome and local fatigue of the motorcyclists. Subjects are 42 motorcyclists of an office in Aichi prefecture. 25 of them held several letters in their left hand when they delivered the letters. They complained of coldness, pain and numbness in the left upper limbs more than in the right limbs (p less than 0.01). We think that it is the local fatigue rather than the disorder of vibration syndrome that causes such symptoms. So it is very important to recognize the existence of local fatigue in order to know how to discriminate between vibration syndrome and local fatigue of the motorcyclists.     

Studies on the Structure of Gluco-oligosaccharides Obtained from the Partial Acid Hydrolyzate of Sclerotia of Sclerotinia libertiana

The defatted sclerotia powder was partially hydrolyzed with dilute acid, and the material obtained was fractionated by carbon column chromatography, separated into two disaccharides, three trisaccharides and three tetrasaccharides, respectively. In these hydrolyzates α, α-trehalose, Iaminaribiose, and gentiobiose were identified.     

Global stability of single-species diffusion volterra models with continuous time delays

In this paper we consider the stability property of single-species patches connected by diffusion with a within-patch dynamics of Volterra type and with continuous time delays. We prove that this system can only have two kinds of equilibria: the positive and the trivial one. By the assumption that the delay kernels are convex combinations of suitable non-negative and normalized functions, the linear chain trick gives an expanded system of O.D.E. with the same stability properties as the original integro-differential system. Homotopy function techniques provide sufficient conditions for the existence of the positive equilibrium and for its global stability. We also prove the local stability of any positive equilibrium and the local instability both of positive and trivial equilibria. The biological meanings of the results obtained are compared with known results from the literature. This work was performed under the auspices of G.N.F.M., C.N.R. (Italy) and within the activity of the Evolution Equations and Applications group, M.P.I. (Italy). I thank the Department of Applied Mathematics, Shizuoka University, Japan, which enabled me to visit Urbino.     

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.     

Changes in chromatin structure during aging of human skin fibroblasts

Human skin fibroblasts from embryo, 16-, 30- and 60-year-old adults were cultivated and passaged in vitro. Their chromatin structures were examined by the sensitivity to micrococcal nuclease and by electron microscopy. When the mode of DNA degradation by the nuclease was analysed during in vitro aging of the embryo skin fibroblasts, the discrete ladder of nucleosomal DNA became obscure in old cells. Analogous change of chromatin structure was also observed even in young cells as their donor ages increased. From the observation with electron microscopy, it became clear that chromatin of fibroblasts from 30-year-old adults does not have regularly spaced nucleosomes, compared with chromatin from embryo. These results suggest that the length of the linker DNA which connects core particles becomes to be heterogeneous by aging, both in vivo and in vitro in human skin fibroblasts.     

Molecular relationship between two types of phospholipase B from Penicillium notatum and reconstitution of active enzyme from its peptide fragments

Two types of phospholipase B of Penicillium notatum, the native type and the modified type that is thought to be generated by the introduction of some nicks into the native type of enzyme by the endogenous protease(s), were distinguished on a slab sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under a nonreducing condition. The native form migrated with a rate corresponding to 95K Da, whereas the modified form migrated more slowly, corresponding to 106K Da, presumably because of its more extended conformation. That the "106K" protein was indeed a nicked product of the "95K" protein was confirmed by amino acid analysis, peptide mapping, N- and C-terminal sequence analyses, and immunoblotting. The peptide fragments (70K and 37K + 32K) comprising the modified protein were isolated by gel filtration in the presence of SDS and 2-mercaptoethanol (the 32K peptide was suggested to be a partial proteolytic product of the 37K peptide). When the "95K" protein was subjected to the same treatment under denaturing condition, it retained a low, but significant, enzymatic activity; in contrast, the separated peptide fragments did not show any significant activity. By a coincubation of these fragments, however, a restoration of enzymatic activity was observed through a reformation of the active complex, corresponding to the original modified protein. The enzymatic activity of this complex was further increased by a treatment with guanidine X HCl, followed by dialysis. The association of peptide fragments appears to occur through the formation of interpeptidal disulfide bonds.     

Sulfhydryl modification and activation of phenylalanine hydroxylase by dinitrophenyl alkyl disulfide

A new family of asymmetric thiol-disulfide exchange reagents, the dinitrophenyl alkyl disulfides (DNPSSR), was used to modify rat liver phenylalanine hydroxylase. The results indicate that the enzyme has two different types of reactive sulfhydryl (SH) residues per subunit. One SH residue was modified selectively by a DNPSSR having a neutral and hydrophilic alkyl group, and this modification was accompanied by appreciable activation of enzyme; the other SH residue was modified only by an anionic DNPSSR, and this modification did not result in activation. The catalytic properties of phenylalanine hydroxylase activated by DNPSSR were similar to those of the N-ethylmaleimide- (NEM-) modified enzyme, but the process of activation by DNPSSR was quite different from modification with NEM. An analysis of the reaction kinetics of the modification and of catalysis by the modified enzyme suggests that DNPSSR modification causes a change in the subunit interaction leading to a loss of the negative cooperativity normally seen with phenylalanine hydroxylase.     

Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization

Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3-6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.