Osteoclastic activity induces osteomodulin expression in osteoblasts

Bone resorption by osteoclasts stimulates bone formation by osteoblasts. To isolate osteoblastic factors coupled with osteoclast activity, we performed microarray and cluster analysis of 8 tissues including bone, and found that among 10,490 genes, osteomodulin (OMD), an extracellular matrix keratan sulfate proteoglycan, was simultaneously induced with osteoclast-specific markers such as MMP9 and Acp5. OMD expression was detected in osteoblasts and upregulated during osteoblast maturation. OMD expression in osteoblasts was also detected immunohistochemically using a specific antibody against OMD. The immunoreactivity against OMD decreased in op/op mice, which lack functional macrophage colony stimulating factor (M-CSF) and are therefore defective in osteoclast formation, when compared to wild-type littermates. OMD expression in op/op mice was upregulated by M-CSF treatment. Since the M-CSF receptor c-Fms was not expressed in osteoblasts, it is likely that OMD is an osteoblast maturation marker that is induced by osteoclast activity.     

Observation of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis

One of the most well known characteristics for Parkinson's disease (PD) is a polymerization of wild-type or mutant alpha-synuclein into aggregates and fibrils, commonly observed as Lewy bodies and Lewy neuritis in PD patients. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation are not well understood yet. In the present study, structural properties and propensities to form fibrils of wild-type, A30P, E46K, and A53T alpha-synucleins were investigated using fluorescence and circular dichroism (CD) methods. The results from these studies were analyzed using singular value decomposition (SVD) method which estimates a number of conformationally independent species for a given process. The time-dependent CD spectra of the wild-type alpha-synuclein indicated a multi-step process in the fibril formation, and SVD analysis using the time-dependent CD spectra revealed that five or nine intermediates were formed at the early stage of fibrillation.     

Spontaneous adenocarcinoma immunoreactive to cyclooxygenase-2 and transforming growth factor-beta1 in the buccal salivary gland of a Richardson's ground squirrel (Spermophilus richardsonii).

The ground squirrel is used as an experimental animal because of its unique biological nature. A 3-year-old female Richardson's ground squirrel developed a mass, 1.5 cm in diameter, in the buccal mucosa. The mass consisted of neoplastic epithelial cells showing acinar, ductular, intraductal papillary, solid, and lobular growth patterns; the cells were immunoreactive to cytokeratin, cyclooxygenase-2 (a marker of malignancy) and TGF-beta1. After resection, the tumor recurred with increased area having a solid or lobular pattern with little differentiation. This tumor was diagnosed as an adenocarcinoma arising from the buccal gland, the first case reported in the ground squirrel. A prominent desmoplastic reaction was present. The interstitial cells reacted to alpha-smooth muscle actin and vimentin, indicating a myofibroblastic nature, presumably induced by epithelial TGF-beta1.     

Net primary productivity and spatial distribution of vegetation in an alpine wetland,Qinghai-Tibetan Plateau

To initially describe vegetation structure and spatial variation in plant biomass in a typical alpine wetland of the Qinghai-Tibetan Plateau, net primary productivity and vegetation in relationship to environmental factors were investigated. In 2002, the wetland remained flooded to an average water depth of 25 cm during the growing season, from July to mid-September. We mapped the floodline and vegetation distribution using GPS (global positioning system). Coverage of vegetation in the wetland was 100%, and the vegetation was zonally distributed along a water depth gradient, with three emergent plant zones (Hippuris vulgaris-dominated zone, Scirpus distigmaticus-dominated zone, and Carex allivescers-dominated zone) and one submerged plant zone (Potamogeton pectinatus-dominated zone). Both aboveground and belowground biomass varied temporally within and among the vegetation zones. Further, net primary productivity (NPP) as estimated by peak biomass also differed among the vegetation zones; aboveground NPP was highest in the Carex-dominated zone with shallowest water and lowest in the Potamogeton zone with deepest water. The area occupied by each zone was 73.5% for P. pectinatus, 2.6% for H. vulgaris, 20.5% for S. distigmaticus, and 3.4% for C. allivescers. Morphological features in relationship to gas-transport efficiency of the aerial part differed among the emergent plants. Of the three emergent plants, H. vulgaris, which dominated in the deeper water, showed greater morphological adaptability to deep water than the other two emergent plants.     

Concise synthesis of dideoxy-epigallocatechin gallate (DO-EGCG) and evaluation of its anti-influenza virus activity

Dideoxy-epigallocatechin gallate (DO-EGCG) (2), a simplified analog of naturally occurring EGCG (1), was efficiently prepared by directly introducing a ketone group at C3 and successive reduction to the sec-alcohol with 2,3-cis stereochemistry. Compound 2 showed potent anti-influenza virus activity, indicating that the hydroxyl substituents on the A-ring are not crucial for anti-influenza virus activity.     

Formation of hydroxyapatite provides a tunable protein reservoir within porous polyester membranes by an improved soaking process

Biomineralization on porous polyester membranes was examined using an improved alternate soaking process (ASP). The effect of ion migration for the formation of hydroxyapatite (HAp) was shown to be crucial. Ion migration was improved by reducing the surface tension by mixing ethanol into an aqueous solution. The resulting hybrid materials were evaluated in terms of calcium content; structure using scanning electron microscopy (SEM), X-ray diffraction (XRD), and infrared spectroscopy (IR); and protein adsorption. The amount of formed HAp was controlled by the number of ASP cycles and also through the ethanol content of the mixed solvent. As the formation of HAp increased, the formed structure could be verified using SEM, IR, and XRD. Protein adsorption was investigated using albumin, gamma-globulin, and fibrinogen, and the amount of adsorbed protein was well-correlated with that of the formed HAp. This result shows that the total amount of the adsorbed proteins can be regulated by the HAp content. In summary, a tunable protein reservoir was formed on a porous polyester membrane.     

Analysis of bile acid glutathione thioesters by liquid chromatography/electrospray ionization-tandem mass spectrometry

The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 degrees C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI-tandem mass spectrometry, where CA-GSH was detected on the product ion mass chromatograms monitored with stable and abundant dehydrated positive-ion [M+HH(2)O](+) at m/z 680.3 and fragmented negative-ion [GSHH](-) at m/z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro.     

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.     

New xanthine oxidase inhibitors from the fruiting bodies of Tyromyces fissilis

Excessive uric acid production, which causes gout and hyperuricemia, can be blocked by inhibiting xanthine oxidase (XO). However, some agents to block on XO often cause side effects, thereby necessitating the identification of new inhibitors. During the screening of XO inhibitors from various mushroom extracts, we found that a methanolic extract of the fruiting bodies of Tyromyces fissilis, an inedible and non-toxic fungus, showed inhibitory activity. Both n-hexane and ethyl acetate layers, obtained by partitioning this extract exhibited XO inhibitory activity. Subsequently, using an activity-guided separation method, eight active compounds (1–8) were isolated. The structures of five of the new compounds, 2–4, 6, and 7, were elucidated by spectral analysis and chemical derivatization. All compounds had a salicylic acid moiety with an aliphatic group at the C-6 position. Notably, 2-hydroxy-6-pentadecylbenzoic acid (1) showed the highest level of XO noncompetitive inhibition (58.9 ± 2.2% at 25 µM).