Cytoplasmic gamma‐tubulin complex from brain contains nonerythroid spectrin

The newer member of the tubulin superfamily, γ‐tubulin, is known to mediate microtubule nucleation from the centrosome of eukaryotic cells with the aid of some other proteins. The major amount of γ‐tubulin is believed to be located in the centrosome before the onset of mitotic division. However, a considerable amount has been found in the cytoplasm in the form of a complex whose function is not well known. Microtubules are most abundant in brain tissues and brain microtubules have been extensively used in many in vitro studies. Thus, it is relevant to use brain tissue to characterize cytoplasmic γ‐tubulin complex. Here we show that cytoplasmic γ‐tubulin in brain tissues exists as a ring complex as in other tissues. Interestingly, along with the common members of the γ‐TuRC reported from several tissues and species, the purified brain cytoplasmic complex contains some high molecular weight proteins including α and β nonerythroid spectrin which are not found in other tissues. Immunohistochemical studies of brain tissue sections also show the co‐localization of γ‐tubulin and spectrin. The possible implications have been discussed. J. Cell. Biochem. 110: 1334–1341, 2010. © 2010 Wiley‐Liss, Inc.     

Attempts to characterize the mechanisms involved in the growth inhibition of Mycobacterium microti in interferon-γ or tumor necrosis factor-α activated J774A.1 cells

Abstract The growth of Mycobacterium microti was inhibited within J774A. 1 macrophage cells activated with either interferon-γ or tumor necrosis factor-α. Activation with interferon-γ or tumor necrosis factor-α alone did not stimulate the production of nitrite in J774A. 1 cells. Interferon-γ but not tumor necrosis factor-a increased the production of hydrogen peroxide in a concentration dependent manner but scavengers of reactive oxygen species did not influence the growth inhibiting effect of interferon-γ within J774A.1 cells. Both interferon-γ and tumor necrosis factor-α enhanced the fusion of M. microti containing phagosomes with lysosomes and the ultimate degradation of bacteria. Our results showed that growth inhibition of M. microti within interferon-γ or tumor necrosis factor-a stimulated J774A. 1 cells was independent of reactive oxygen intermediate and reactive nitrogen intermediate production.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

Studies of Glu-416 Variants of β-Galactosidase (<Emphasis Type="Italic">E. coli</Emphasis>) Show that the Active Site Mg<Superscript>2+</Superscript> is Not Important for Structure and Indicate that the Main Role of Mg<Superscript>2+</Superscript> is to Mediate Optimization of Active Site Chemistry

Variants of β-galactosidase with Valine and with Glutamine replacing Glutamate-416 did not have a Mg²⁺ bound at the active site even at high Mg²⁺ concentrations (200 mM). They had low catalytic activity and the pH profiles were very different from those of the native enzyme. In addition, substrates, substrate analogs, transition state analogs and galactose bound very poorly. However, the orientation and conformation of the Mg²⁺ ligands (residues 416, 418, and 461) as well as the B-factors of these three side chains did not change significantly. The structures, conformations and B-factors of other active site residues were also essentially unchanged. These studies show that the active site Mg²⁺ is not necessary for structure and is, therefore, mainly important for modulating the chemistry and mediating the interactions between the active site components.