Sensitive detection of fluorescence in western blotting by merging images.

The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

Severe gangrene accompanied by varicella zoster virus-related vasculitis mimicking rheumatoid vasculitis.

Herpes zoster infection occurs more frequently and severely in immunosuppressed populations. However, the condition sometimes presents with atypical clinical manifestations of the skin, which makes it difficult to reach a correct diagnosis. We experienced a case of acral gangrene caused by varicella zoster virus (VZV)-related vasculitis in a rheumatoid arthritis (RA) patient. Histologically, necrotic vasculitis was observed; however, there were initially no findings in the epidermis suggestive of a viral infection. We thought that the skin ulcer was related to rheumatoid vasculitis. However, an immunohistochemical analysis for VZV confirmed VZV infection in the vascular endothelium of the dermis, leading to effective treatment with intravenous acyclovir. Since various pathogenic skin phenotypes are observed in RA patients, modified according to the status of immunosuppression, clinicians must recognize the variation in typical and atypical manifestations in order to manage these patients.

Enantioselective construction of a polyhydroxylated pyrrolidine skeleton from 3-vinylaziridine-2-carboxylates: synthesis of (+)-DMDP and a potential common intermediate for (+)-hyacinthacine A1 and (+)-1-epi-australine.

We report an enantioselective synthesis of the polyhydroxylated pyrrolidine alkaloid (+)-DMDP. The key steps in the synthesis were guanidinium ylide mediated asymmetric aziridination, stereospecific ring opening of trans-3-vinylaziridine-2-carboxylate with an oxygen nucleophile, iodine-mediated 5-endo-trig amino cyclization, and Prévost displacement. In addition, a potential common intermediate for the polyhydroxylated pyrrolizidine alkaloids (+)-hyacinthacine A(1) and (+)-1-epi-australine was synthesized from a diastereoisomeric cis-aziridine coformed in the asymmetric aziridination using the same strategy. A rationale for the diastereoselectivity observed for the iodine-mediated amino cyclization reactions is proposed on the basis of the heats of formation of the products.

An experimental study of bone grafting using rat milled tooth.

PURPOSE: The aim of this study was to develop a novel bone graft material that used extracted teeth. MATERIALS AND METHODS: Thirty-six 10-week-old male Wistar rats were used. The incisors were extracted, immediately frozen and milled, mixed with hydroxypropyl cellulose (HPC), and injected into the socket. The remaining rats received HPC alone, or the socket was left to heal untreated. Socket healing and bone formation in all three groups were evaluated by three-dimensional image analysis from microcomputed tomography examination and histologic observation. RESULTS: Quantitative morphologic measurements demonstrated that bone formation was significantly stimulated in the group that received milled tooth and HPC at 2 and 4 weeks after extraction compared to that of the control (untreated) group, which showed normal healing without any intervention. Histologic observation revealed that the compound of milled tooth and HPC promoted early healing of the socket and initiation of bone formation in the surrounding area. Interestingly, HPC injection alone decreased bone formation and bone mineral content at 2 weeks and then increased bone formation at 4 weeks. CONCLUSION: A bone graft material composed of milled tooth promotes early healing and bone formation, while HPC, which is chemically stable in vivo, affects bone formation in the extraction socket.

Crystallization and preliminary X-ray crystallographic studies of XynX, a family 10 xylanase from Aeromonas punctata ME-1.

Xylanases catalyze the hydrolysis of beta-1,4-glycosidic linkages within the xylan backbone. XynX is a xylanase from Aeromonas punctata ME-1 and belongs to glycoside hydrolase family 10. While most xylanases show endo-type catalytic activities, XynX shows exo-like catalytic activities, selectively producing xylobiose from birchwood xylan. In this study, XynX was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 79.0, b = 88.6, c = 93.2 A, and diffracted to beyond 1.8 A resolution.

[A patient care experience of the psychologically impaired cancer patient against chemotherapy--the successful outpatient case report using a sedative agent to reduce vomiting].

We would like to present a case report on how we managed to reduce an adverse effect of chemotherapy, specifically vomiting and nausea with a sedative agent diazepam, when a patient was intravenously injected with chemotherapeutic agents at the outpatient department. The patient was a 39-year-old female who experienced total hysterectomy and bilateral oophorectomy due to ovarian ca. (stage III) on May 29, 2002. She used to have complained of severe vomiting whenever undergoing 4-day inpatient chemotherapies every month from June 2002 to April 2003. In the last chemotherapy, she suffered vomiting prior to a day before the infusion and after 5 days of the infusion. Because of her experience, the patient refused to undergo the following chemotherapy. Based on discussions with her, use of sedatives was chosen to relieve vomiting. Due to relief of stress, the patient slept and relaxed by means of a sedative effect during and after administration of chemotherapy. Since the reduction of vomiting, she has been accepting further chemotherapies.

Osteoclast differentiation in ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2).

Osteoclast differentiation in the process of ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2) was examined to clarify the relationship between osteoclast development and rhBMP-2-induced bone formation. A combination of rhBMP-2 with a porous microsphere (PMS) and blood clot was implanted subcutaneously on the bilateral chest muscles of rats. Tartrate-resistant acid phosphatase (TRAPase) activity, cathepsin K (cath K), and calcitonin receptor (CTR), as markers of osteoclasts and their precursors, were examined using enzyme and immunohistochemical analysis up to 7 days after implantation. Mononuclear cells positive for TRAPase, cath K, and CTR first appeared on day 3 in connective tissue surrounding the PMS after implantation of rhBMP-2. Simultaneously, alkaline phosphatase activity became detectable in mesenchymal cells in the connective tissue. Electron microscopy demonstrated some mononuclear cells with abundant mitochondria and poorly developed rough endoplasmic reticulum in the proximity of mesenchymal cells. However, there was no evidence of cartilage or bone matrix formation on day 3. Osteoclasts in various stages of development, classified by the pattern of immunoreactivity for cath K, were observed by day 7. The polarized intracellular distribution of cath K was found only in osteoclasts attached to bone matrix. In conclusion, we have demonstrated for the first time the appearance of osteoclast precursors before bone matrix formation induced by rhBMP-2, suggesting that bone matrix is not a prerequisite for osteoclast precursor recruitment. Furthermore, we suggest that differentiation into polarized functional osteoclasts is accomplished when the osteoclasts attach to the bone matrix.