微量标本Western blot在诊断肢带型肌营养不良2A型中的应用

目的 探讨微量标本Western blot在诊断肢带型肌营养不良2A型中的应用.方法 对73例以肢体近端肌肉受累为首要临床表现的进行性肌营养不良患者行开放式骨骼肌活体组织检查,标本行组织化学染色以及抗dystrophin-N、C、R,α、β、γ、δ-sarcoglycan,dysferlin,caveolin-3单克隆抗体免疫组织化学染色.对其中已经除外dystrophin、sarcoglycans、dysferlin、caveolin-3蛋白异常的29例患者再行骨骼肌Western blot抗calpain-3、caveolin-3单克隆抗体免疫反应.结果 共有10例患者被确诊为肢带型肌营养不良2A型,其临床特点均为肢体近端肌无力起病,血清肌酸激酶不同程度升高,肌电图呈肌源性改变.组织化学染色见肌纤维大小不一,可见不同程度肌纤维变性、坏死和再生,结缔组织增生,6例见分叶状肌纤维;免疫组织化学染色见肌纤维中dystrophin、sarcoglycan、dysferlin、caveolin-3蛋白均正常表达.Western blot发现该10例患者相对分子质量94 000条带(calpain-3)与Duchenne/Becker型肌营养不良对照相比,呈完全(8例)或部分(2例)缺失;30 000区域附加条带均呈弱表达,22 000区域条带(caveolin-3)均正常表达.结论 骨骼肌微量标本Western blot是诊断LGMD2A的有效方法,适用于临床LGMD的分型诊断.     

Involvement of puromycin‐sensitive aminopeptidase in proteolysis of tau protein in cultured cells,and attenuated proteolysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) mutant tau

In tauopathies, tau protein is hyperphosphorylated, ubiquitinated, and accumulated in the brain; however, the mechanisms underlying this accumulation remain unclear. To gain an understanding of the role of proteases in the metabolism of tau protein, in the present study we evaluated the effects of protease inhibitors in SH‐SY5Y human neuroblastoma cells and COS‐7 cells transfected with the tau gene. When cells were treated with 0.1–10 µmol/L of lactacystin and 1.0–20 µmol/L of MG‐132 (inhibitors of proteasome), 0.1–10 µmol/L of CA‐074Me (a cathepsin inhibitor), and 0.1–2 µmol/L of puromycin (a puromycin‐sensitive aminopeptidase (PSA) inhibitor) for up to 24 h, there were no significant changes in tau protein levels. However, pulse‐chase experiments demonstrated that the proteolysis of tau protein in SH‐SY5Y cells was attenuated following treatment of cells with 200 nmol/L puromycin. Increased tau protein levels were also observed in SH‐SY5Y cells treated with short interference (si) RNA to PSA to inhibit the expression of PSA. These data suggest that PSA is a protease that catalyses tau protein predominantly in SH‐SY5Y cells. The protein metabolism of tau‐containing mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) was also investigated using pulse‐chase experiments. The results indicate attenuated proteolysis of tau in cells transfected with mutant tau genes after 48 h. Further immunocytochemical analysis and subcellular fractionation experiments revealed that the mutations did not alter the intracellular distribution of tau and suggested that impaired accessibility of tau to PSA is unlikely to account for the attenuated proteolysis of tau protein. Western blotting with phosphorylation‐dependent antibodies revealed that phosphorylation levels of tau at Thr²³¹, Ser³⁹⁶, and Ser⁴⁰⁹ were increased in cells transfected with V337M, R406W, and R406W mutant tau genes, respectively. Together, the data suggest that attenuated proteolysis of FTDP‐17 mutant tau may be explained by increased phosphorylation levels, resulting in resistance to proteolysis.     

Isomer pattern and elimination of dioxins in workers exposed at a municipal waste incineration plant

The aim of this study was to clarify patterns of serum concentrations of dioxins in the employees of a waste incineration plant and to estimate elimination rates and half-lives of serum dioxin isomers, and the maximum serum concentrations of dioxin isomers at the time of plant shutdown. Sixteen subjects participating 3 times or more in annual health examinations during an 8-yr period from 2000 to 2007 were recruited for this study. Serum concentrations of dioxins expressed as TEQ/g lipid decreased gradually after plant shutdown with the highest decrease observed in polychlorinated dibenzofurans (PCDFs) followed by polychlorinated deibenzo-p-dioxins (PCDDs) and then coplanar PCBs. The serum toxic equivalency (TEQ) concentrations of PCDF and PCDD congeners in the employees were higher than those in the general population survey by the Ministry of the Environment, Japan, whereas the serum concentrations of coplanar PCBs were similar to those in the general population. The estimated half-lives and elimination rates of PCDDs and PCDFs in the highly exposed workers increased compared with the moderately exposed workers. The estimated geometric mean serum concentrations of PCDDs, PCDFs and total dioxins at the time of plant shutdown were 35, 53 and 107 pg TEQ/g lipid, respectively.     

Posterior Auricular Artery-Middle Cerebral Artery Bypass: A Rare Superficial Temporal Artery Variant with Well-developed Posterior Auricular Artery—Case Report

The posterior auricular artery (PAA) is one of the branches of the external carotid artery, but is usually too small for use as a donor artery for middle cerebral artery (MCA) territory revascularization. An extremely unusual case of PAA-MCA anastomosis was performed in a patient requiring MCA territory revascularization because the superficial temporal artery (STA) parietal branch was absent and the PAA was large enough. A 65-year-old man developed mild motor weakness in the right extremities caused by multiple small infarctions. Single photon emission computed tomography (CT) revealed deterioration of the vascular reserve capacity in the left MCA area. Cerebral angiography showed severe stenosis in the C2 portion of the left internal carotid artery, absence of the parietal branch of the left STA, and a well-developed PAA extending to the parietal area. The patient underwent STA (frontal branch)-MCA and PAA-MCA double anastomosis, and has suffered no stroke or transient ischemic attack. The STA with no bifurcation is known as a rare variation. The PAA also occurs with size variations but well-developed PAA is thought to be extremely rare. PAA can be used as a donor artery for MCA territory revascularization if the vessel size is suitable. Preoperative evaluation of the anatomy is mandatory for harvesting the arteries.     

Increased serum levels of squamous cell carcinoma-related antigen in pemphigus

Serum levels of squamous cell carcinoma-related antigen (SCC-RAG) were measured in five cases of pemphigus, five cases of bullous pemphigoid and 18 cases of benign and malignant dermatoses other than SCC. The SCC-RAG titres were significantly raised in four of five patients with pemphigus, while they remained within the normal range in the other dermatoses except in one case. In three pemphigus cases in whom serial measurements were made, SCC-RAG levels seemed to be related to disease activity. The SCC-RAG levels in blister fluids were much higher than those in serum, suggesting that the skin is a major source of serum SCC-RAG. These results show that SCC-RAG is increased not only in SCC, but also in some cases of pemphigus, and suggest that pemphigus antibodies may cause the production or release of SCC-RAG.     

Delayed adrenal insufficiency long after unilateral adrenalectomy: Prolonged glucocorticoid therapy reduced reserved secretory capacity of cortisol

A 51-year-old woman with Cushing's syndrome underwent unilateral adrenalectomy for left adrenal adenoma. After 7 years of prednisolone treatment (with some interruptions), followed by 4 years of total withdrawal from prednisolone treatment, she presented with hypotension, weight loss, general fatigue, nausea, hyponatremia and hypoglycemia. These clinical features together with a low response in the rapid adrenocorticotropic hormone test led to the diagnosis of acute adrenal insufficiency. Relatively low serum adrenocorticotropic hormone levels in the face of increased demand for cortisol during adrenal crisis suggested a disordered hypothalamic-pituitary function, indicating secondary adrenal insufficiency. This patient demonstrated the etiology of acute adrenal insufficiency long after unilateral adrenalectomy in association with subsequent glucocorticoid therapy. A reduction in the reserved secretory capacity of cortisol after prolonged prednisolone treatment was considered to have induced secondary adrenal insufficiency, even after 4 years of total withdrawal from prednisolone.     

C677T polymorphism of methylenetetrahydrofolate reductase gene affects plasma homocysteine level and is a genetic factor of late-onset Alzheimer's disease

Background: Elevated plasma homocysteine levels are known as a risk for atherosclerotic vascular disease and venous thrombosis and have been shown as a risk for late‐onset Alzheimer's disease (LOAD). Method: To examine the effect of genetic factors predisposing to elevated plasma homocysteine levels on the occurrence of LOAD, we determined the genotype of a C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene and a variable number tandem repeat (VNTR) spanning exon 13–intron 13 boundary of cystathionine β‐synthase (CBS) gene in patients with LOAD and community‐based control subjects. Results: Logistic regression indicated that the MTHFR‐T allele was a risk for LOAD (P < 0.05), independently from apolipoprotein E‐?4 (APOE‐?4) allele. Kaplan–Meier tests showed that in APOE‐?4 non‐carriers, individuals with the MTHFR‐TT genotype have occurences of LOAD earlier than those with the MTHFR‐CC genotype (P < 0.05). Multiple regression analysis indicates that MTHFR‐T allele increases plasma homocysteine levels (P = 0.0002), while the number of X chromosomes decreases (P = 0.01). Plasma homocysteine level was not correlated with age, plasma albumin reflecting nutritional condition, and the dose of APOE‐?4 allele. The CBS‐20 VNTR allele showed the same trend to increase plasma homocysteine level as the MTHFR‐T allele, but a risk effect for LOAD was not evident. Conclusion: A genetic propensity for elevated plasma homocysteine levels, explained by the MTHFR‐T allele encoding defective enzymatic function, is involved in the development of LOAD, particularly in APOE‐?4 non‐carriers, and that homocysteine metabolism could be a preventive target to LOAD in the elderly.     

Case Report: Mucin-producing cystic neoplasm of the pancreas with onset in childhood

Mucin-producing tumours of the pancreas have been recently reported with increasing frequency and most cases have occurred in middle-aged and elderly people. In the present report, a case of a 21-year-old man with mucinous cystadenoma of the pancreas is reported. He had a long history of recurrent pancreatitis from the age of 8. When he was aged 10, the first branch of the main pancreatic duct was shown to be enlarged on endoscopic retrograde pancreaticography (ERP). A series of ERP studies and computed tomography scans performed over a period of 11 years demonstrated continuing growth of this enlargement of the pancreatic duct. Pancreaticoduodenectomy was performed and the patient has been well without further episodes of acute pancreatitis and has been free of recurrent tumour for 1 year.     

Epidemiological and clinical features of idiopathic pulmonary alveolar proteinosis in Japan

Abstract:   Idiopathic pulmonary alveolar proteinosis (IPAP) is a rare disease characterized by excessive amounts of lipoproteinaceous material in the alveolus. This report presents an interim analysis of nationwide epidemiological data from Japanese patients with pulmonary alveolar proteinosis, and the roles of serum markers for IPAP. (i) The nationwide demographic data from 166 Japanese patients with IPAP are shown. The female to male ratio was 1:2, and the average age was 51 ± 14 years old (age range: 15–79 years) at registration or diagnosis. A total of 30% of patients with IPAP have a poor clinical course. In total, 30% of patients were treated with whole lung lavage therapy (WLL). Under WLL, the patients significantly improved in the short term, but 40% of the patients who underwent WLL worsened again. A new strategy such as granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy for intractable PAP is required. (ii) The correlation of serum KL-6, carcinoembryonic antigen, surfactant proteins D and A, and LDH with disease severity suggests their potential as disease markers. In contrast, serum anti-GM-CSF antibody did not correlate with disease severity, but is a specific marker for the diagnosis of IPAP. The combined measurements of the serum markers may well prove very useful for both the diagnosis and the management of IPAP patients.