15 research outputs found
Thermal inactivation and conformational lock studies on glucose oxidase
In this study, the dissociative thermal inactivation
and conformational lock theories are applied for the
homodimeric enzyme glucose oxidase (GOD) in order to
analyze its structure. For this purpose, the rate of activity
reduction of glucose oxidase is studied at various temperatures
using b-D-glucose as the substrate by incubation of
enzyme at various temperatures in the wide range between
40 and 70 �C using UV–Vis spectrophotometry. It was
observed that in the two ranges of temperatures, the
enzyme has two different forms. In relatively low temperatures,
the enzyme is in its dimeric state and has normal
activity. In high temperatures, the activity almost disappears
and it aggregates. The above achievements are confirmed
by dynamic light scattering. The experimental
parameter ‘‘n’’ as the obvious number of conformational
locks at the dimer interface of glucose oxidase is obtained
by kinetic data, and the value is near to two. To confirm the
above results, the X-ray crystallography structure of the
enzyme, GOD (pdb, 1gal), was also studied. The secondary
and tertiary structures of the enzyme to track the thermal
inactivation were studied by circular dichroism and
fluorescence spectroscopy, respectively. We proposed a
mechanism model for thermal inactivation of GOD based
on the absence of the monomeric form of the enzyme by
circular dichroism and fluorescence spectroscopy
Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia
Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor
The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses
Abstract Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are dynamic modules enriched in subset of lipids and specialized proteins that determine their structure and functions. The MERCs regulate lipid transfer, autophagosome formation, mitochondrial fission, Ca2+ homeostasis and apoptosis. Since these functions are essential for cell biology, it is therefore not surprising that MERCs also play a critical role in organ physiology among which the immune system stands by its critical host defense function. This defense system must discriminate and tolerate host cells and beneficial commensal microorganisms while eliminating pathogenic ones in order to preserve normal homeostasis. To meet this goal, the immune system has two lines of defense. First, the fast acting but unspecific innate immune system relies on anatomical physical barriers and subsets of hematopoietically derived cells expressing germline-encoded receptors called pattern recognition receptors (PRR) recognizing conserved motifs on the pathogens. Second, the slower but very specific adaptive immune response is added to complement innate immunity. Adaptive immunity relies on another set of specialized cells, the lymphocytes, harboring receptors requiring somatic recombination to be expressed. Both innate and adaptive immune cells must be activated to phagocytose and process pathogens, migrate, proliferate, release soluble factors and destroy infected cells. Some of these functions are strongly dependent on lipid transfer, autophagosome formation, mitochondrial fission, and Ca2+ flux; this indicates that MERCs could regulate immunity