Xanthine oxidase mediates cytokine-induced,but not hormone-induced bone resorption

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have been implicated as mediators of osteoclastic bone resorption. Xanthine oxidase (XO) a ubiquitous enzyme is widely known for its production of these ROS. We therefore evaluated the potential of XO as a source of ROS in cytokine-and hormone-induced bone resorption. XO activity in rat calvarial osteoblasts was found to be significantly elevated upon stimulation by the cytokines, TNFalpha and IL-1beta. These cytokines also caused a dose related increase in bone resorption of mouse calvariae, which was significantly inhibited by catalase (10 IU/ml). Allopurinol, the competitive inhibitor of XO, also caused a dose related (1-50 microM) inhibition of TNFalpha (20 ng/ml) and (0.01-10 microM) IL-1beta (50 IU/ml)-induced bone resorption, respectively. PTH- and 1,25-(OH)2 Vitamin D3-induced bone resorption could also be inhibited by catalase (100 IU/ml) but was unaffected by allopurinol, indicating that another mediator, other than XO, is required for hormone-induced bone resorption. These results demonstrate, that modulation of the redox balance in the bone microenvironment, which contains XO, can affect the bone resorbing process. Therefore, XO may play a pivotal role in cytokine-induced bone resorption and, if manipulated appropriately, could show a therapeutic benefit in inflammatory bone disorders such as RA.     

Rapid monitoring of diabetes-induced lipid peroxidation by Fourier transform infrared spectroscopy: evidence from rat liver microsomal membranes

Increased oxidative stress is the consequence of either enhanced reactive oxygen species (ROS) production or attenuated ROS scavenging capacity, resulting in tissue damage that in most instances is assessed by the measurement of lipid peroxides. In the current study, diabetes-induced lipid peroxidation in rat liver microsomal membranes was investigated by Fourier transform infrared (FT-IR) spectroscopy at different temperatures. The olefinic (CH) band at 3012 cm-1 was used to probe diabetes-induced lipid peroxidation. The intensity and area values of this band of diabetic samples were found to be increased significantly (P<0.05) compared with nondiabetic samples. The increase in olefinic band intensity is attributed mainly to the lipid peroxidation end products. The results of the FT-IR study were found to be in agreement with biochemical studies that revealed a significant increase in malondialdehyde levels of diabetic samples compared with control samples (P<0.05) using the thiobarbituric acid test.     

Levels of iron, zinc, and copper in aqueous humor, lens, and serum in nondiabetic and diabetic patients

The purpose of this study was to investigate iron (Fe), zinc (Zn), and copper (Cu) levels of aqueous humor, lens, and serum in nondiabetics and diabetics and to determine the effects of diabetes on Fe, Zn, and Cu contents in the lens. Fe, Zn, and Cu contents of aqueous humor, lens, and serum samples of 19 patients (9 nondiabetic patients with a mean age of 62.3±5.4 yr, and 10 diabetic patients with a mean age of 59.5±5.9 yr) were analyzed by atomic absorption spectrometry using a prospective study design. The lens levels of Cu in diabetic patients were significantly higher compared with nondiabetic patients (p=0.02); however; there was no difference in the other elements (Zn, Fe; p=0.28, p=0.74, respectively). The levels of Fe, Zn, and Cu in the aqueous humor and serum of diabetic patients were not found to be statistically significant when compared to nondiabetics (p=0.46, p=0.11, p=0.18, and p=0.22, p=0.43, p=0.72, respectively). These results demonstrate that increased Cu content of the lens presumably has a greater association with the development of lens opacification in diabetics than Zn and Fe content.     

Effective extracellular production of Bacillus stearothermophilus esterase by pH-stat modal fed-batch culture of recombinant Bacillus brevis

An automated two-component substrate feeding strategy with a pH-stat modal fed-batch culture using a high pH limit was developed to effectively porduce esterase from a hyperprotein exreting Bacillus brevis HPD31 harboring a plasmid pHSC131 which carries a Bacillus stearothermo philus esterase gene. First, the effect of single- and multi-substrate feedings on the growth and activity of the excreted esterase was investigated. Then a two-component (polypepton + glucose) feeding using different feed rates was studied. Highest activity of the excreted esterase (34 U/mL) was obtained when the concentrations of poly-pepton and glucose in the nutrient feed solution were 250 and 41.60 g/L respectively. The absence and excessive amount of glucose in the nutrient feed solution was ineffective for the exracellular esterase formation because without glucose the increase in cell concentration was minimum while excessive amount of glucose favored cell growth at the expense of the esterase production. It is believed that the mechanism of enzyme excretion is growth dependent and that a higher cell growth of the host is in effect unfavorable for the enzyme production. The feed rate, automatically controlled by the direct signal of the pH change, at 0.30 mL/pulse was found optimum for the esterase production while lower (0.15 mL/pulse) and higher (0.67 mL/pulse) feed rates did not produce good results. The activity of the excreted esterase was increased more than eight times from 4 U/mL obtained in the conventional batch culture to 34 U/mL obtained in this study. The esterase productivity was likewise increased more than threefold. (c) 1992 John Wiley & Sons, Inc.     

Identification and characterisation of a Theileria annulata proline‐rich microtubule and SH3 domain‐interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria‐induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co‐immunoprecipitation, we identified a CLASP1/CD2AP/EB1‐containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14‐3‐3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta‐p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain‐interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1‐binding SxIP motif, as well as functional SH3 domain‐binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non‐infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.     

The impact of OGG1, MTH1 and MnSOD gene polymorphisms on 8-hydroxy-2′-deoxyguanosine and cellular superoxide dismutase activity in myocardial ischemia–reperfusion

Ischemia–reperfusion (I/R) injury, by inducing oxidative DNA damage, is one of the leading causes of increased patient morbidity and mortality in coronary artery by-pass grafting (CABG) surgery. 8-Hydroxyguanine (8-OHG) is an important oxidative base lesion. The 8-oxoguanine glycosylase (hOGG1) and hMTH1, which have several polymorphisms, remove 8-OHdG from the nucleotide pool. We investigated whether there are any correlations the biomarkers of oxidative stress (superoxide dismutase; SOD and 8-OHdG in serum) with genotype for two DNA repair genes (OGG1 and MTH1) and an antioxidant enzyme gene (manganese superoxide dismutase; MnSOD). Therefore, we measured DNA damage (8-hydroxy-2-deoxyguanosine; 8-OHdG) and endogenous antioxidant activity (SOD) at five different time points (T1, before anesthesia; T2, after anesthesia; T3, after ischemia; T4, after reperfusion and T5, after surgery). and also, MnSOD and MutT homolog 1 (MTH1) genes polymorphisms were genotyped by polymerase chain reaction–restricted fragment length polymorphism (PCR–RFLP) in patients undergoing coronary artery by-pass grafting (CABG) surgery. No statistically significant differences were detected in the levels of 8-OHdG and SOD in serum in terms of OGG1 Ser326Cys, MTH1 Val83Met and MnSOD Ala16Val genetic polymorphisms. Our results suggest that OGG1, MTH1 and MnSOD gene polymorphisms are not genetic risk factors for I/R injury.