Recombinant biglycan promotes bone morphogenetic protein-induced osteogenesis.

The aim of this study was to determine the effects of glutathione-S-transferase-fused recombinant biglycan (GST-BGN) on craniofacial bone regeneration. We recently demonstrated a positive effect of tissue-derived BGN on bone morphogenetic protein 2 (BMP-2) function, which is exerted likely via the BGN core protein. Here, we investigated the effects of GST-BGN lacking any posttranslational modifications on BMP-2 function in vitro and in vivo. In the C2C12 cell culture system, BMP-2-induced Smad 1/5/8 phosphorylation and alkaline phosphatase activity were both enhanced by the addition of GST-BGN. For the in vivo effect, we employed a Sprague-Dawley rat mandible defect model utilizing 1 µg (optimal) or 0.1 µg (suboptimal) of BMP-2 combined with 0, 2, 4, or 8 µg of GST-BGN. At 2 weeks post-surgery, newly formed bone was evaluated by microcomputed tomography and histologic analyses. The results revealed that the greatest amounts of bone within the defect were formed in the groups of suboptimal BMP-2 combined with 4 or 8 µg of GST-BGN. Also, bone was well organized versus that formed by the optimal dose of BMP. These results indicate that recombinant BGN is an efficient substrate to promote low-dose BMP-induced osteogenesis.

Mitotic catastrophe and cell death induced by depletion of centrosomal proteins.

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Increased matrix metalloproteinase-2 and bone sialoprotein response to human coronal caries.

BACKGROUND: It has been suggested that host matrix metalloproteinase-2 (MMP-2) present in dentin may be involved in caries progression, however, its response to caries is not known. Bone sialoprotein (BSP) has been implicated in dentin mineralization and MMP-2 modulation. OBJECTIVE: To identify and compare the distribution of MMP-2 and BSP in healthy human coronal dentin and those with early caries. METHODS: Freshly extracted 3rd molars and premolars with and without early caries were fixed, demineralized and subjected to immunohistochemistry using a monoclonal anti-MMP-2 antibody and monoclonal anti-BSP antibody with an avidin-biotin complex method. Immunoreactivity was visualized with 3,3'-diaminobenzidine substrate and observed under light microscopy. RESULTS: Immunohistochemical analysis revealed that MMP-2 and BSP are not detected in the tubule lumens of healthy dentin. However, intense immunoreactivity for MMP-2 and BSP was detected in association with the full length of the caries-affected dentinal tubules. The MMP-2 and BSP at the dentino-enamel junction appeared unaltered. CONCLUSION: The results indicate that MMP-2 and BSP may be actively secreted by odontoblasts in response to carious insult. MMP-2 and BSP accumulation in the caries-affected dentinal tubules may indicate their potential involvement in the host defense mechanism which results in calcification of regions affected by the carious process.

Simulation of membrane fouling considering mixed liquor viscosity and variation of shear stress on membrane surface.

Simulation of membrane fouling in MBR was conducted considering accumulation, detachment and consolidation of extracellular polymeric substances accumulated on membrane surface. The fluctuation of shear stress working on membrane surface and the influence of the viscosity of mixed liquor were considered for the evaluation of shear stress. A flat-sheet-type membrane module was used and the change of trans-membrane pressure was measured in a laboratory-scale MBR reactor. Shear stress working on membrane surface caused by aeration was measured by a shear force sensor changing viscosity of bulk liquid. Effective shear stress on membrane surface was defined in the model as the sum of time-averaged shear stress and three times of standard deviation. The increase in the trans-membrane pressure was accurately simulated by the developed model suggesting validity of the developed fouling model and the idea of the effective shear stress on membrane surface.

Role of glycosaminoglycans of biglycan in BMP-2 signaling.

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, µCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function.

AID-induced T-lymphoma or B-leukemia/lymphoma in a mouse BMT model.

Activation-induced cytidine deaminase (AID) diversifies immunoglobulin through somatic hypermutation (SHM) and class-switch recombination (CSR). AID-transgenic mice develop T-lymphoma, indicating that constitutive expression of AID leads to tumorigenesis. Here, we transplanted mouse bone marrow cells transduced with AID. Twenty-four of the 32 recipient mice developed T-lymphoma 2-4 months after the transplantation. Surprisingly, unlike AID-transgenic mice, seven recipients developed B-leukemia/lymphoma with longer latencies. None of the mice suffered from myeloid leukemia. When we used nude mice as recipients, they developed only B-leukemia/lymphoma, presumably due to lack of thymus. Analysis of AID mutants suggested that an intact form with SHM activity is required for maximum ability of AID to induce lymphoma. Except for a K-ras active mutant in one case, specific mutations could not be identified in T-lymphoma; however, Notch1 was constitutively activated in most cases. Importantly, truncations of Ebf1 or Pax5 were observed in B-leukemia/lymphoma. In conclusion, this is the first report on the potential of AID overexpression to promote B-cell lymphomagenesis in a mouse model. Aberrant expression of AID in bone marrow cells induced leukemia/lymphoma in a cell-lineage-dependent manner, mainly through its function as a mutator.

[Mitral valve repair with concomitant coronary bypass for partial rupture of an anterior papillary muscle].

We report a case of severe mitral regurgitation due to partial rupture of an anterior papillary muscle. A 63-year-old man was admitted to a hospital with heart failure. He was treated with diuretic agents effectively. Echocardiography demonstrated severe mitral regurgitation with prolapse of posterior leaflet and small mass-like structure on the prolapsed segment that was diagnosed the thickened leaflet. Coronary angiography revealed total occlusion of left anterior descending artery (LAD) filled with good collateral from right posterior descending artery and severe diffuse stenosis of circumflex artery (Cx). The patient underwent surgery on the 33rd day after admission with heart failure. At surgery, we recognized rupture of one of the heads of anterior papillary muscle that was entangled in chordae of the prolapsed segment. Mitral valve repair and coronary revascularization to LAD and Cx was successfully performed. His postoperative course was uneventful, and he was discharged on the 28th postoperative day.

Effect of bubble flow velocity on drag-force and shear stress working on submerged hollow fibre membrane.

This study is aimed at elucidating the mechanism by which rising air bubbles induce shear stress on hollow fibre membrane surfaces. Shear stress on hollow fibre membrane surfaces (laterally-set and vertically-set) caused by aeration was measured directly using a two-direction load sensor. In the laterally-set hollow fibre module, time-averaged upward-direction shear stress on the membrane surface was compared to theoretical shear stress values considering the effect of water flow on membrane surface. Measured time-average shear stress values were almost 200 times larger than theoretical values implying strong interactions between bubbles and solid surface. In the vertically-set membrane module, velocity measurement of bubble flow using laser Doppler velocimeter revealed that drag force working on membrane surface was closely related to upward-direction water velocity. Also fluctuation of drag force and shear force on membrane surface was found to be related to velocity fluctuation (turbulence).

Influence of extracellular polymeric substances on nitrogen removal in an intermittently-aerated membrane bioreactor.

Influence of EPS on fouling of intermittent aeration MBR reactor (denitrification MBR) was investigated changing intermittent aeration cycle (10 minute-cycle and 120 minute-cycle) in laboratory-scale reactors using synthetic wastewater. EPS were extracted from bacterial cells using cation resin method and molecular weight fractioning of EPS was conducted using gel chromatography. In both of the reactors, nitrogen removal rate was almost 100% after 50th day although DO concentration was not very high during the aerated phase because of accumulation of nitrifying bacteria in the reactors. In the 120 minutes-cycle reactor, trans-membrane pressure increased more rapidly than in the 10 minutes-cycle reactor. The reason might be that EPS of more than 1000 kDa, which are the main fouling substances, are produced more rapidly in the 120 minute-cycle condition. It was also found that three peaks at around 100 kDa, 500 kDa and 2000 kDa are prominent in EPS in intermittent-aeration MBR irrespective of cycle and higher molecular weight EPS are decomposed to smaller molecular weight EPS on membrane surface.

Package plant of extended aeration membrane bioreactors: a study on aeration intensity and biofouling control.

Biofouling control is important for effective process of membrane bioreactor (MBR). In this study, phenomena of biofouling for immersed type extended aeration MBR with two different anti-fouling aeration intensities were studied through a laboratory set up. The objectives of this study were (a) to observe biofouling phenomena of MBR that operates under different anti-fouling bubbling intensity, and simultaneously monitors performance of the MBR in organic carbon and nutrients removal; (b) to compare effectiveness of detergent and detergent-enzyme cleaning solutions in recovering biofouled membranes that operated in the extended aeration MBR. For MBR, which operated under continuous anti-fouling aeration, deposition and accumulation of suspended biomass on membrane surface were prohibited. However, flux loss was inescapable that biofilm layer was the main problem. Membrane cleaning was successfully carried out with detergent-enzyme mixture solutions and its effectiveness was compared with result from cleaning with just detergent solution.

Effect of loading rate and intermittent aeration cycle on nitrogen removal in membrane separation activated sludge process.

The performance of the submerged membrane separation activated sludge process with intermittent aeration was investigated in a laboratory scale experiment by changing organic loading rate and intermittent aeration cycle. A rectangular PVC tank was used as an aeration tank, in which a flat-sheet type Micro-Filtration membrane made of poly-olefin with a pore size of 0.2 microm was submerged. Organic loading rate to the reactor was set at 0.3 and 0.8 g-TOC/L/day. C/N ratio in the feed was set at around 5.0 for every condition. Aeration cycle was changed from 10 min-10 min (aeration-stop) to 120 min-120 min in different organic loading conditions. Flux through the membrane was set at 0.25 m/day. Membrane fouling proceeded rapidly in 0.8 g-TOC/L/day conditions. However, when organic loading rate was 0.3 g-TOC/L/day, bacterial metabolic substances were degraded rapidly compared to the production, thereby decreasing viscosity in mixed liquor. Nitrogen removal rate was between 60% and 80% for 0.8 g-TOC/L/day loading, and between 50% and 65% for 0.3 g-TOC/L/day loading. And the nitrogen removal was highest in 40 min to 60 min aeration cycle conditions. Too short aeration cycle did not result in sufficiently long anoxic periods for denitrification while too long a cycle resulted in unnecessary anaerobic periods after depletion of nitrate. Intermittent aeration was effective also for decreasing viscosity in mixed liquor.

Comparative study on microbial removal in immersed membrane filtration (IMF) with and without powdered activated carbon (PAC).

This paper describes an investigation on the effect of microbial removal using IMF for high quality drinking water production. The comparison of IMF and IMF-PAC configuration was carried out in the study to highlight the importance of PAC in the system. The specific objective of this study was to study the effect of PAC adsorption in the IMF-PAC system particularly in removing microbial substances from contaminated raw water. A bench scale IMF-PAC configuration using a flat sheet microfiltration membrane was set up for experimental purposes. Experimentally, the result has shown high removal of microbial substances with the IMF-PAC system compared to IMF. The result of E. coli removal achieved was below the detectable level due to the microbial size, which is bigger than membrane pore size. The addition of PAC has shown a direct effect on total microbial removal. The adsorption of microbial onto PAC surfaces reduced the amount of smaller microbial present in permeate samples. As a conclusion, the configuration of IMF is a promising separation process in removing microbial substances, especially when the system is combined with PAC.

Performance of an extended aeration-microfiltration (EAM) reactor in treating high strength industrial wastewater.

A laboratory study was conducted on an Extended Aeration-Microfiltration (EAM) reactor in treating a food industry wastewater. The reactor contained horizontally laid hollow fibre microfiltration (MF) units that were fully submerged. The MF units were connected to a peristaltic pump that was used to extract permeate continuously under suction pressure. Continuous aeration from beneath the modules provided the crossflow effect to the MF units. Active activated sludge was used in the start-up where the sludge was mixed together with the feed water at a Food/Microorganisms (F/M) value of about 0.1. Primary effluent with Chemical Oxygen Demand (COD) values ranged between 1,500 and 3,000 mg/l was used as feed water. The EAM reactor was operated for nearly three months without initiating cleaning of the MF units. A suction pressure of 0.9 bar and Mixed Liquor Suspended Solids (MLSS) of over 5,500 mg/l were reached when nearing the end of the three month operation period. Permeate COD and turbidity reduction of over 97% and 99% respectively, were achieved. Prior to this, the MF module arrangements were studied; where vertically arranged modules were found to perform poorly as compared to the horizontally laid modules, in terms of clean water permeate flux.

[A case of extracardiac unruptured aneurysm of sinus of Valsalva with obstruction of the right coronary artery and aortic regurgitation].

A 67-year-old female was admitted to our hospital with heart failure. Echocardiography, chest CT and MRA revealed an unruptured aneurysm of sinus of Valsalva. Cardiac catheterization and angiography detected obstruction of the ostium of the right coronary artery and moderate aortic regurgitation. Operative findings showed an extracardiac unruptured aneurysm arising from the right sinus of Valsalva. The orifice of the aneurysm was closed with Gore-Tex patch, then aortic valve replacement combined with single CABG to RCA was simultaneously performed. The pathological findings strongly suggested its acquired origin.

[Serological cross-reaction among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody method].

We studied the serological cross-reactions among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody (IFA) method, using sera from 8 patients with cat scratch disease (CSD), 13 patients with C. pneumoniae infection and 12 patients with acute Q fever. B. henselae IgG antibody was negative in 13 patients with C. pneumoniae infection, and was positive in 3 (titers being 1:64) of 12 patients with Q fever, whereas B. henselae IgM antibody was negative in all the patients with C. pneumoniae infection or Q fever. C. burnetii IgG antibody was removed by absorption of these 3 sera with C. burnetii antigens, whereas B. henselae IgG antibody did not change. C. pneumoniae IgG antibody was positive in 3 (titers being 1:125 in two, 1:32 in one) of 8 patients with CSD. Both C. pneumoniae and B. henselae IgG antibody titers were significantly reduced by absorption of these 3 sera with B. henselae antigens. C. burnetii IgG or IgM antibodies were negative in all patients with CSD. In conclusion, no serological cross-reaction between B. henselae and C. burnetii was observed. On the other hand. B. henselae IgG antibody cross-reacted to C. pneumoniae antigens, whereas C. pneumoniae IgG antibody did not cross-react to B. henselae antigens. Our findings suggest that determination of B. henselae IgG or IgM antibodies were not influenced by C. pneumoniae and C. burnetii antigens.

Resolution and synthesis of optically active alcohols with immobilized ovalbumin and pea protein as new bio-catalysts.

It was found that ovalbumin stereoselectively oxidized one of the enantiomers of p-substituted racemic alcohols, thereby providing optically active alcohols with high optical purities. It was found out that, when used appropriately in combination with immobilized pea protein, immobilized ovalbumin made it possible to resolve and synthesize racemic 1-(2-naphthyl)ethanol, 1-phenylethanol, and 1-phenyl-1-propanol. Immobilized ovalbumin could be continuously recycled at least three times without lowering the yield and purity of the products. These results suggested that cereals, beans, and ovalbumin might have additional fourth function among conventional foods. Namely, there might contain nutritional, sensory, biologically regulatory and bio-catalytic functions in conventional foods.

Intractable Q fever treated with recombinant gamma interferon.

A 3-year-old boy with Q fever received several kinds of antibiotics including minocycline, but spiking fever and positive PCR of Coxiella burnetii continued for several months. He became asymptomatic and his abnormal laboratory data normalized after the administration of gamma interferon three times a week.

B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

Asymmetric synthesis of the core structure of (-)-CP-263,114.

The enantioselective construction of a fully functionalized core structure of (-)-CP-263,114 (1), containing most of the required functionality for total synthesis, was conducted through sequential radical fragmentation-reductive olefination.