Recent advances in fluorescent labeling techniques for fluorescence microscopy

Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.     

Rutaecarpine attenuates osteoclastogenesis by impairing macrophage colony stimulating factor and receptor activator of nuclear factor κ‐B ligand‐stimulated signalling pathways

Rutaecarpine is a major alkaloid isolated from Evodia rutaecarpa. Here, we investigated the effects of rutaecarpine on osteoclast differentiation induced by macrophage colony stimulating factor (M‐CSF) and receptor activator of nuclear factor κ‐B ligand (RANKL) in bone marrow‐derived macrophages (BMMs). Treatment with rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of BMM‐derived osteoclasts. Mechanistically, rutaecarpine decreased the protein level of nuclear factor of activated T cells cytoplasmic‐1 (NFATc1) and the phosphorylation of other signalling pathways during the osteoclast differentiation. Thus, rutaecarpine may be useful as a therapeutic agent for the treatment of bone diseases.     

Traumatic unilateral temporomandibular joint dislocation overlooked for more than two decades

Forward dislocation of the temporomandibular joint commonly can be easily diagnosed and successfully reduced by manual repositioning. In this report, we discuss a rare case of prolonged temporomandibular dislocation that had persisted for more than 20 years because the otolaryngologist and dentist had missed the dislocation. This patient underwent open reduction and mandibular joint plasty with preoperative orthodontic therapy. It is possible that strong pain and mouth-closing disability may gradually remit and only deviated mandibular prognathism like malocclusion may persist. Therefore, abnormal occlusion warrants careful attention to temporomandibular joint dislocation.     

A case of lung infection due to Mycobacterium abscessus (M. abscessus) complicated with primary macroamylasemia]

We report a case of lung infection due to Mycobacterium abscessus (M. abscessus), complicated with primary macroamylasemia. A 76-year-old man was admitted to our hospital in August 2002 because of bloody sputum and an abnormal shadow found on chest radiography. The patient had had pulmonary tuberculosis from 1998 to 2000. He was found to be antacid bacillus-positive (Gaffky 5) on examination of the sputum in August 2002, but after hospitalization was negative for tuberculosis bacillus on sputum examination by the PCR method. We had suspected the presence of non-tuberculous mycobacterial disease since the patient's admission, and had started a regime of three drugs: clarithromycin, rifampicin, and ethambutol. The bacteria were identified as M. abscessus in a later sputum culture examination. It was noticed that the blood amylase level was high, and the disease was diagnosed as primary macroamylasemia. Such a case of lung infection due to M. abscessus complicated with macroamylasemia has rarely been reported in Japan.     

A case of MPO-ANCA-related vasculitis with chronic alveolar hemorrhage]

It has been reported that alveolar hemorrhage caused by ANCA-associated vasculitis occurs in the seriously acute stage. However, we report a rare case of chronic alveolar hemorrhage in a 26-year-old woman who had been on maintenance hemodialysis because of rapidly progressive glomerulonephritis (RPGN) since the age of 17. On a regular checkup at her clinic, chest radiographs revealed diffuse micronodular opacities in both lung fields when she was 22 years of age, and anemia was identified at 24. Before the patient was referred to our hospital, the shadows on the radiographs remained almost unchanged, whereas the anemia, for reasons unknown, slowly deteriorated. She was referred to our hospital because of micronodular opacities detected on a chest radiogram in an annual health check at the age of 26. She was asymptomatic, but her laboratory data showed a normochromic anemia (hemoglobin 6.3 g/dl), and the serum level of MPO-ANCA was 195 EU. Bronchoalveolar lavage at bronchoscopy macroscopically revealed bloody fluids containing hemosiderin-laden macrophages. Histological examination of the biopsy specimen by video-assisted thoracoscopic lung biopsy revealed pauci-immune pulmonary capillaritis and alveolar hemorrhage. She was diagnosed as having MPO-ANCA-related vasculitis, especially microscopic polyangiitis preceding RPGN, and the clinical course suggesting alveolar hemorrhage was progressing slowly. The diagnosis was therefore "chronic alveolar hemorrhage". We emphasize that it is necessary to consider alveolar hemorrhage when a patient who has been on maintenance hemodialysis for RPGN has a combination of anemia and diffuse micronodular opacities, even if the condition is not accompanied with any respiratory symptoms.     

Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice

Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.     

Appetite loss may be induced by lower serum ghrelin and higher serum leptin concentrations in subarachnoid hemorrhage patients

Background

Patients with aneurysmal subarachnoid hemorrhage (SAH) typically develop appetite loss. However, the mechanisms regulating appetite are not understood. Ghrelin and leptin, both of which signal nutritional status and energy storage levels to the hypothalamus, are essential elements of the appetite system. Thus, the goal of this study was to investigate the relationship between appetite and ghrelin and leptin concentrations in patients with SAH.

Methods

Blood plasma or serum profiles and appetite status were measured in 19 patients with SAH who underwent aneurysmal clipping within 48 hours of SAH onset. Appetite status was measured using dietary oral calorie intake. All outcome variables were measured at an early (day 3) and late (day 8) time point after SAH onset (day 0).

Results

Of the 19 patients studied, 6 (31.6%) showed lower dietary oral calorie intake at the late time point than at the early time point. In these patients with appetite loss, plasma hemoglobin (P < 0.02), albumin (P < 0.01), glucose (P < 0.01), plasma insulin (P < 0.04), and serum ghrelin (P < 0.03) concentrations were lower at the late time point than at the early time point. Serum leptin was higher at the late time point than at the early time point (P < 0.02).

Conclusion

In SAH patients, appetite loss may be induced by lower serum ghrelin and higher serum leptin concentrations resulting from high plasma glucose and insulin levels due to a catecholamine surge following SAH.