Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca2+ -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.

Effect of protein adsorption layers and solution treatments on hydroxyapatite deposition on polystyrene plate surfaces in simulated body fluids.

We have developed a method to functionalize polystyrene (PS) cell culture plates with hydroxyapatite (HAp) via protein adsorption layers such as human serum albumin (HSA) in simulated body fluids (SBFs). In order to investigate the versatility the method, in this study the effect of protein adsorption layers on HAp deposition on PS plate surfaces in SBF was evaluated. Pretreatments with alternate soaking process (ASP) using solutions containing calcium ions and phosphate ions followed by incubation with SBF for 24 h resulted in HAp deposition on PS plates with adsorption layers of HSA, type I collagen, hen egg white lysozyme, and poly L-glutamic acid, an acidic protein analogue: the deposition behaviors were correlated with adsorption ability and charge state of proteins. We also demonstrated that commercially available tissue culture-treated PS (TCPS) were directly coated with bone-like HAp using the same ASP and SBF processes. Human mesenchymal stem cells adhered and stretched on the HAp-coated TCPS plates in a similar manner to the case of the HAp-coated PS plates prepared via HSA adsorption layers. The results indicate that the present methods are useful for preparing bone-like HAp-coated cell culture plates that can be utilized function of adsorbed proteins and that are obtainable conveniently and at low-cost.

Necrotizing suppurative nephritis in a Japanese black feedlot steer due to Proteus mirabilis infection.

A Japanese black feedlot steer suddenly died after exhibiting astasia and cramping of the extremities. Necropsy of the animal revealed that the right kidney was enlarged and pale with severe nephrolithiasis. The urinary bladder displayed mucosal hemorrhage. Upon bacteriological investigation, Proteus mirabilis was isolated from the liver, spleen, right kidney, lungs and urine. Histopathological examination revealed necrotizing suppurative nephritis with the presence of numerous gram-negative bacilli and fibrinous suppurative cystitis with no bacilli. Immunohistochemical analysis revealed that the bacteria and cytoplasm of the macrophages stained positively with P. mirabilis antiserum. Electron microscopy revealed the presence of numerous bacteria in the renal tubules. To our knowledge, this is the first report describing the histopathological aspects of nephritis caused by P. mirabilis in cattle.

Surface functionalization of tissue culture polystyrene plates with hydroxyapatite under body fluid conditions and its effect on differentiation behaviors of mesenchymal stem cells.

The surfaces of polystyrene (PS) cell culture plates were functionalized with hydroxyapatite (HAp) under body fluid conditions utilizing protein adsorption layers and a pretreatment with an alternate soaking process (ASP) using solutions containing calcium and phosphate ions. Adsorption layers of human serum albumin (HSA) formed on the surface of each well of commercial 24-well PS plates by solution processes. CaCl2 and K2HPO4 solutions were alternately added to the wells, the plates were incubated to form the precursors, and this was followed by the addition of simulated body fluid (SBF) and a further incubation for 24h. These treatments resulted in the surfaces of the PS cell culture plates being completely covered with bone-like HAp. The coating of PS plates with HAp promoted the adhesion of mesenchymal stem cells (MSCs) and maintained cell growth that was as fast as that on tissue culture-treated PS (TCPS) plates. Osteogenic differentiation was greater, whereas adipogenic and chondrogenic differentiation was less in the culture on HAp-coated PS plates than in that on TCPS plates. The present method is useful for preparing HAp-coated PS plates at clean benches without the need for any expensive apparatus. HAp coated on PS plates by this method was a bone-like apatite with high bioactivity; therefore, the present HAp-coated PS plates are promising materials for assays of bone-related cells in the bone remodeling process.

A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF.

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses.

Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB.

Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca(2+) ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca(2+) and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca(2+)-dependent activation, while these regions are not required for phosphorylation-induced ROS production.

An outbreak of systemic granulomatous disease in cows with high milk yields.

Seven of 92 lactating Holstein cows on a dairy farm developed urticaria with alopecia and decreased milk production, and three of the seven died over the course of 7 to 18 days. Pathologic examination of the three cows, including the two dead and one euthanized cow, revealed that the skin, liver, spleen, kidneys, heart, salivary glands, pancreas, adrenal glands, mammary glands, lymph nodes, and trigeminal ganglia had lymphocytic to lymphogranulomatous inflammation. Inflammation predominated by lymphocytic infiltration was prominent in the heart, pancreas, mammary glands, adrenal gland, and trigeminal ganglia. Severe granulomatous inflammation with multinucleated giant cells was present in the spleen and kidneys. These lesions and their distributions were most similar to those seen in suspected cases of citrus pulp toxicosis and hairy vetch toxicosis. The outbreak of this disease resolved with the elimination of Citrus pulp from the feed. Immunohistochemical detection of lymphocytes and macrophage markers confirmed dramatic hyperplasia of CD3-positive T lymphocytes in these lesions. This strongly suggested that a type 4 hypersensitivity reaction played a role in the development of the lesions.