Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue.

Until now methods using tetramethylbenzidine (TMB) for electron microscopy (TMB-EM methods) are all unable to provide a maximum demonstration of transported horseradish peroxidase (HRP) while maintaining good ultrastructural tissue preservation. In order to solve this problem, we have attempted to adapt a newly developed, highly sensitive TMB method using sodium tungstate (ST) as the stabilizer (TMB-ST method) for HRP electron microscopic retrograde and anterograde fiber tracing. The present study shows that the TMB-ST method combined with diaminobenzidine-cobalt (DAB-Co) is more sensitive than existing TMB-EM methods and that ultrastructural details are well preserved with this combined method. The resultant reaction product complex after osmication is stable and is observed as characteristic crystal-like structures which are extremely electron dense and often aggregated into clumps. In contrast, the TMB-ST method without the DAB-Co step frequently produces a moderate electron-dense reaction product. Therefore, we recommend the TMB-ST method combined with DAB-Co for HRP electron microscopy.     

Hemangioma of the adrenal gland. Apropos of a case

A case of adrenal hemangioma is reported, displayed by computed tomography and confirmed by pathologic investigation. This rare tumor poses a difficult diagnostic challenge and rejoins the problem of incidentally discovered adrenal masses.     

Induction of FMLP-mediated calcium mobilization requires crosslinking of surface immunoglobulin in Daudi cells.

We have explored the requirements for the induction of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) response in Daudi cells after anti-immunoglobulin treatment. Our results indicate that (a) induction of responsiveness to FMLP was observed in Daudi only after crosslinking of surface immunoglobulin by anti-immunoglobulin; (b) this induced responsiveness was not observed in Ramos or Wil-2 cells; (c) the F(ab')2 fragment was sufficient for the induction of the FMLP response, but the Fab fragment and the Fc fragment were ineffective; (d) of the many agents active in B lymphocyte regulation which were tested, none were as effective as anti-immunoglobulin in the induction of the FMLP response; and (e) three inhibitors of calcium mobilization (W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide), PMA (phorbol 12-myristate 13-acetate), and colchicine), acting on distinct mechanisms, inhibited both the calcium mobilization due to anti-immunoglobulin and the induction of responsiveness to FMLP. Our results suggest important determinants in the induction of a calcium-mobilizing FMLP response in cells of B lymphocyte lineage include (a) the cell type, (b) a selective requirement for activation via surface immunoglobulin, and (c) crosslinking of the surface immunoglobulin.     

Mass spectrometric analysis of the structure of gamma II bovine lens crystallin.

The amino acid sequence of bovine gamma II-crystallin has been verified by a combination of electrospray and fast atom bombardment mass spectrometry. The molecular weight of gamma II, isolated by gel filtration and ion exchange chromatography, was determined to be 20,967 +/- 3 by electrospray mass spectrometry. Another aliquot of gamma II was completely digested by trypsin in a medium of 20% CH3CN and 0.1 M Tris, pH 8.2. The tryptic peptides were separated by reversed phase HPLC and identified by their molecular weights, as determined by fast atom bombardment mass spectrometry (FABMS). The identification of each peptide was confirmed by digesting the peptide further to give new peptides whose molecular weights were also determined by FABMS and related to the proposed amino acid sequences. The data from both types of mass spectrometric analyses were consistent with the sequence previously proposed by Hay et al. (J. Biol. Chem. 1987, 146, 332-338), including threonine at position 119. The FAB mass spectrum of one HPLC fraction suggested that disulfide bonding between Cys 18 and Cys 22 was present in at least half the protein preparation. Whether the Cys 18/Cys 22 disulfide bond was present in native gamma II or was produced during isolation or enzymic digestion could not be determined from these studies. Samples that had been stored for several weeks showed that several of the cysteines had become disulfide bonded. These studies illustrate the power of mass spectrometric techniques to accurately confirm the primary structure of proteins and to identify post-translational modifications.     

Althin/TIAN System1000 HDF血透机常见故障及检修

Althin／TIAN System1000 HDF血透机是美国Baxter公司较为先进的血液透析滤过装置。它采用的是平恒腔式的定容超滤控制系统．使得超滤量的控制更为精确。非在线式血液滤过的设计彻底保证了滤过液的安全性。人性化的液晶触摸屏给操作和维修带来及大的方便。由于血透机特殊的工作环境．年久的机器会出现一些问题。现将几例常见故障的检修提供给同行，供同行参考。     

兔动静脉短路环法诱导组织工程骨支架材料血管生成的初步研究

Objective To compare the effects of 2 vascular carriers, arteriovenous loop and arteri-ovenous bundle, on inducing angiogenesis in coral scaffold of vascularized tissue-engineered bone in animal models.Methods Thirty-six adult male New Zealand rabbits were randomized into 2 even groups.In group A, an arteriovenous loop (AVL) was formed by microsurgical anastomosis at the proximal ends between the femoral poptiteal artery and vein, and placed in the circular side groove of the coral block (6 mm × 8 mm × 10 mm) .In group B, flow-through vessels bundles of both femoral artery and vein were placed in the side grooves of the coral block.All the implants in 2 groups were wrapped by a micro-porous expand-ed-polytetrafluoroethylene (ePTFE) membrane, and fixed subcutaneously by suturing.Evaluation methods included gross morphological observations, histological examinations, India ink perfusion and vascular casting after 2, 4, 6 weeks.The density of blood vessels was analyzed by the statistical software SPSS 10.0.Results All the corals were encased by newly formed fibrovascular tissues in 2 groups.Ink-stained vessels distributed the surfaces and side grooves, and invaded the interspaces of corals.The degree of vascularization increased over the course of experiment.Blood vessel density demonstrated a significant continuous increase between 2 and 6 weeks after implantation in group A.The mean value of blood vessel density in group A (2 weeks 276.60±4.67, 4 weeks 517.20±10.66, 6 weeks 707.00 ±11.87) was significantly higher than in group B (2 weeks 153.60 ±7.16, 4 weeks 269.40±6.80, 6 weeks 279.20±6.53) (P <0.01).Vascular casting showed that in group A, significant blood vessels sprouted from all areas of the loop, espe-cially at the entrance of the arteriovenous pediele where the small tubes were densely interconnected.In group B, however, no blood vessels sprouted from the arteriovenous bundles and only some small vessels grew from the entrance and exit.Conclusions A vascularized coral model can be constructed by inserting an ar-teriovenous loop or an arteriovenous bundle, useful in vascular bone tissue engineering.The former, however, have stronger abilities to induce angiogenesis than the latter.     

乳腺囊性增生病癌变过程中部分因素变化的意义

检测乳腺囊性增生病（ＦＣＤ）经不典型增生到癌变部分因素的变化。结果提示：从因明显ＦＣＤ症状活检至癌变为２～１０年；从Ⅱ级以上不典型增生到临床癌变需２～７年；癌变率为３．１％。ＦＣＤ患者存在性激素分泌调控失常，血浆雌激素和催乳素含量增加，导致上皮细胞增生。乳腺一般性增生细胞的ＤＮＡ含量和超微结构与正常乳腺上皮细胞相似；无肿瘤相关抗原及异常基因产物表达。而发生在一般性增生基础上的不典型增生则呈现细胞基因物质ＤＮＡ含量增加，部分为超４Ｃ的多倍体细胞；同时出现细胞膜和细胞核超微结构异常；雌激素受体含量增加，对性激素的依赖性和敏感性增强；部分不典型增生细胞出现胚胎性肿瘤相关抗原和异常基因产物表达。随不典型增生程度加重至乳腺癌，上述诸因素的变化趋势具有明显规律性。提示ＦＣＤ上皮细胞从一般性增生经不典型增生至乳腺癌为细胞生物学连续逐渐变化的过程。部分不典型增生细胞中具有癌倾向的细胞生物学行为异常和表型变化与乳腺癌发生密切相关。细胞核ＤＮＡ含量等异常变化及程度可作为乳腺癌前病变发展程度的客观标志     

Evaluation of peripheral blood involvement of mantle cell lymphoma by fluorescence in situ hybridization in comparison with immunophenotypic and morphologic findings.

Mantle cell lymphoma is non-Hodgkin's B-cell lymphoma characterized by the t(11;14)(q13;q32) translocation. Peripheral blood involvement of mantle cell lymphoma is usually associated with a poor prognosis and therefore, its identification is clinically important. In this study, we performed cyclin D1/IgH-probe fusion fluorescence in situ hybridization analysis on 223 peripheral blood samples: 185 from 125 mantle cell lymphoma patients, and 38 normal controls. The cutoff values for the test were established using normal controls. Flow cytometry on peripheral blood and corresponding bone marrow samples was used to evaluate this test. In all, 26% of the 185 peripheral blood samples and 27% of the 161 corresponding bone marrow samples were flow cytometry positive for mantle cell lymphoma. The mean numbers of single and- double-fusion signals and the mean number of CD5/CD19-positive cells, absolute blood lymphocyte count, and white blood cell count were significantly higher in peripheral blood and corresponding bone marrow samples with mantle cell lymphoma-positive flow cytometry. Double-fusion signals were more specific than single-fusion ones. Fluorescence in situ hybridization was far more likely to be positive for mantle cell lymphoma when the peripheral blood and the corresponding bone marrow samples had positive flow cytometry results or morphology (P<0.01). Our study indicates that cyclin D1/IgH-fusion fluorescence in situ hybridization analysis could be used to determine the presence and character of circulating mantle cell lymphoma cells in peripheral blood, thus enhancing our ability to evaluate leukemic mantle cell lymphoma and minimum residual disease.