Human brain hypoxanthine guanine phosphoribosyltransferase: structural and functional comparison with erythrocyte hypoxanthine guanine phosphoribosyltransferase

A rapid and simple method, based on GMP Sepharose affinity chromatography, was used for the purification of human brain hypoxanthine guanine phosphoribosyltransferase. A single protein band was detected by polyacrylamide gel electrophoresis of the native purified enzyme. A subunit molecular weight of 25,000 was estimated by SDS gel electrophoresis. The Km values for hypoxanthine and phosphoribosyl pyrophosphate were 50 and 111 microM, respectively. The Ki values for GMP and IMP with phosphoribosyl pyrophosphate were 21 and 37 microM, respectively. The purified enzyme from human brain did not differ significantly from the human erythrocyte one in amino acid composition. The brain and erythrocyte hypoxanthine guanine phosphoribosyltransferases showed complete immunochemical identity on Ouchterlony double diffusion.     

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE(2) biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE(2) generation. Arg(110), a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A(2) allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE(2) biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.     

Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. The keloid matrix contains excess collagen and glycosaminoglycans (GAGs), but lacks elastic fiber. However, the roles of these matrix components in the pathogenesis of keloid are largely unknown. Here, we show that elastin and DANCE (also known as fibulin-5), a protein required for elastic fiber formation, are not deposited in the extracellular matrix of keloids, due to excess accumulation of chondoitin sulfate (CS), although the expression of elastin and DANCE is not affected. Amount of CS accumulated in the keloid legion was 6.9-fold higher than in normal skin. Fibrillin-1, a scaffold protein for elastic fiber assembly, was abnormally distributed in the keloid matrix. Addition of purified CS to keloid fibroblast culture resulted in abnormal deposition of fibrillin-1, concomitant with significantly decreased accumulation of elastin and DANCE in the extracellular matrix. We propose that CS plays a crucial role in the development of keloid lesions through inhibition of elastic fiber assembly.     

Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress.

The present study was designed to clarify the role of radical oxygen species in testicular germ cell apoptosis induced by heat stress. Testicular cells isolated from immature rats were cultured with or without elevated temperature, and occurrence of apoptosis in these cells was defined by the appearance of DNA fragmentation following agarose gel electrophoresis and by flow cytometric quantification of apoptotic cells. At 32.5 degrees C, < 1% of cells showed signs of apoptosis throughout the culture period, whereas under heat stress, the proportion of apoptotic cells increased to 5% at 37 degrees C after 24 h of culture, or to 14% after 1-h exposure at 43 degrees C followed by 23-h culture at 32.5 degrees C. Similar to the effect of heat stress, exogenously supplied oxygen free radicals also induced apoptosis. In contrast, treatment with catalase significantly attenuated heat stress-induced apoptosis. Furthermore, heat stress of testicular cells was associated with an increased intracellular peroxide level as measured by a fluorescent probe, 2', 7'-dichlorofluorescin diacetate. In conclusion, our data indicate the involvement of radical oxygen species during testicular germ cell apoptosis induced by heat stress. This study provides a useful in vitro model for the study of testicular germ cell apoptosis.     

Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation

Microdomains in the plasma membrane (PM) have been proposedto be involved in many important cellular events in plant cells.To understand the role of PM microdomains in plant cold acclimation,we isolated the microdomains as detergent-resistant plasma membranefractions (DRMs) from Arabidopsis seedlings and compared lipidand protein compositions before and after cold acclimation.The DRM was enriched in sterols and glucocerebrosides, and theproportion of free sterols in the DRM increased after cold acclimation.The protein-to-lipid ratio in the DRM was greater than thatin the total PM fraction. The protein amount recovered in DRMsdecreased gradually during cold acclimation. Cold acclimationfurther resulted in quantitative changes in DRM protein profiles.Subsequent mass spectrometry and Western blot analyses revealedthat P-type H⁺-ATPases, aquaporins and endocytosis-related proteinsincreased and, conversely, tubulins, actins and V-type H⁺-ATPasesubunits decreased in DRMs during cold acclimation. Functionalcategorization of cold-responsive proteins in DRMs suggeststhat plant PM microdomains function as platforms of membranetransport, membrane trafficking and cytoskeleton interaction.These comprehensive changes in microdomains may be associatedwith cold acclimation of Arabidopsis.     

Host range factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) is an essential viral factor required for productive infection of NPVs in IPLB-Ld652Y cells derived from L. dispar

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.     

Applicability of food composition tables as a tool to estimate mineral and trace element intake of pre-school children in Japan: A validation study

Because dietary intakes of some minerals (including trace elements), especially iron (Fe), are insufficient for the needs of the general Japanese population, accurate estimation of mineral intake is important. This capability is especially necessary to preserve the health of Japanese children. Therefore, the current version of food composition tables (FCT) in Japan was evaluated for validity as tools to estimate dietary intake of minerals for children. For this purpose, 24 h food duplicate samples were collected from 292 pre-school children in Miyagi prefecture, Japan. From the weights of items and food codes, intakes of nine minerals were estimated taking advantage of the FCT. In parallel, amounts of minerals in each duplicate samples were instrumentally measured by ICP-AES for Ca, Cu, Fe, Mg, Mn, P and Zn, and by flame AAS for K and Na, both after wet-ashing. The distributions of the mineral amounts were essentially normal. The comparison of the FCT-based estimates (E) and instrumental measures (M) showed that the E/M ratio was close to 1 for Ca, K, Mn, P and Zn, suggesting that E may be a surrogate of M for Ca, K, Mn, P and Zn on a group basis. The ratio being larger than 1.2 for Cu, Fe, Mg and Na indicates that a risk of over-estimation exists when E is relied upon in place of M. On an individual basis, significant differences were detected for all 9 minerals suggesting that the use of E as a surrogate for M should be practiced with care for the estimation of mineral intake.