Permeability barrier disruption alters the localization and expression of TNFα/protein in the epidermis

Previous studies have shown that (1) epidermal TNF mRNA levels are increased following acute disruption of the cutaneous permeability barrier; (2) this increase is maximal at 1 h and decreases to control levels by 8 h; and (3) in essential fatty acid-deficient (EFAD) mice, a chronic model of barrier perturbation, TNF mRNA levels are also elevated several-fold over controls. In the present study we determined, using immunocytochemical procedures, epidermal TNF protein levels following either acute of chronic barrier disruption and the localization of any increase. Frozen, paraffin and Antibed sections of skin were incubated with polyclonal anti-mouse TNF antisera and detection was accomplished by either immunoperoxidase or fluorescence procedures. We found that (1) TNF-immunoreactive protein was present in normal mouse epidermis, and was primarily localized to the upper nucleated layers where it displayed a diffuse cytosolic pattern; (2) acute disruption of the barrier with acetone or tape-stripping resulted in TNF staining that was more intense throughout all of the nucleated epidermal cell layers in comparison with normal epidermis; (3) the increase in TNF staining occurred as early as 2 h after barrier disruption; and (4) increased TNF staining was also observed in the stratum corneum of EFAD mice. These results indicate that epidermal TNF protein levels increase after both acute and chronic barrier disruption, and are consistent with the hypothesis that TNF may signal and/or coordinate portions of the cutaneous response to barrier disruption.     

Validation of an in vivo extraction method for human stratum corneum ceramides

A topical acetone/diethylether (A/E) lipid extraction method was evaluated for its suitability for use in the study of stratum corneum lipids in various skin disorders. Its efficiency was compared in vitro with topical chloroform/methanol (C/M) extraction and with the classical integral C/M extraction (submerged tissue) of stratum corneum or whole epidermis. To estimate the depth of lipid removal by A/E extraction, light microscopic and freeze-fracture electron microscopic studies were carried out on A/E and C/M topically treated skin samples. The in vivo experiments consisted of topical A/E extraction and of classical C/M extraction of scrapings of the stratum corneum. Transepidermal water loss (TEWL) was measured before and after topical A/E extraction and after every scraping procedure, and correlated with TEWL values found after stripping of the stratum corneum. The total amount of lipid found with both topical extraction procedures was lower than that found with the integral extraction of the stratum corneum. Light microscopy showed that topical C/M extraction induced cell damage in the living epidermal cell layers. Great interindividual variation in overall lipid composition was shown in the in vitro experiments irrespective of the extraction protocol used. However, the ceramide (CER) profiles in a single skin sample from the same subject were similar irrespective of the protocol used, and a uniformity in the CER profiles was found in skin samples from different subjects. Similar results were obtained with in vivo topical A/E extractions: marked interindividual variation was seen in overall lipid composition, but not in the CER profile. Furthermore, the CER profiles found using the A/E extraction procedure both in vivo and in vitro were similar. The CER profiles were also found to be identical throughout the stratum corneum, as revealed by scraping experiments. Since the CER profiles are though to play a major role in the stratum corneum barrier function, the non-invasive A/E extraction of epidermal lipids seems to be suitable for clinical application.     

The anticonvulsant activity of acetone, the major ketone body in the ketogenic diet, is not dependent on its metabolites acetol, 1,2-propanediol, methylglyoxal, or pyruvic acid

BACKGROUND: Acetone, one of the principal ketone bodies elevated during treatment with the ketogenic diet, exhibits anticonvulsant properties that may contribute to the seizure protection conferred by the diet. The anticonvulsant mechanism of acetone is unknown, but it is metabolized to several bioactive substances that could play a role. METHODS: Acetone and its major metabolites-acetol, 1,2-propanediol, methylglyoxal, and pyruvic acid-were assessed for anticonvulsant activity in two mouse seizure models. Various doses of the substances administered intraperitoneally were characterized for their ability to elevate the threshold for clonic seizures induced by intravenous infusion of pentylenetetrazol (PTZ) and for protection against tonic seizures induced by subcutaneous bolus administration of 4-aminopyridine (4-AP). The inverted-screen test was used to assess acute neurological toxicity. RESULTS: Acetone (1-32 mmol/kg, i.p.), in a dose-dependent fashion, elevated the PTZ threshold and conferred protection against 4-AP seizures (ED(50), 26.3 mmol/kg). Effective doses of acetone (10-32 mmol/kg) did not cause motor impairment in the inverted-screen test (TD(50), 45.7 mmol/kg). In doses 10-fold greater than the minimally effective dose of acetone (3.2 mmol/kg), the metabolites acetol, 1,2-propanediol, and pyruvic acid were inactive in the PTZ model. At higher doses that produced motor impairment, acetol and 1,2-propanediol (but not pyruvic acid) did elevate the PTZ threshold. Methylglyoxal had both proconvulsant and anticonvulsant actions, and had substantial toxicity, producing respiratory distress, motor impairment, and death. None of the acetone metabolites protected against 4-AP seizures. CONCLUSIONS: This study confirms the broad-spectrum anticonvulsant properties of acetone and indicates that the seizure protection conferred is unlikely to result from its major metabolic products.     

A novel preparation method for PLGA microspheres using non-halogenated solvents

We investigated a new preparation method for microspheres based on an Oil/Water type emulsion solvent evaporation method using non-halogenated solvents. This method is based on phase separation between acetone and aqueous glycerol. For the preparation of microspheres by this method, a solution of poly(dl-lactic-co-glycolic acid) (PLGA) in acetone and aqueous glycerol containing poly(vinyl alcohol) were used as the dispersed and continuous phases in the emulsification process, respectively. Vitamin B₁₂ was used as the model drug. The formation of PLGA microspheres was observed above 60% glycerol in the continuous phase. The yield and encapsulation efficiency of the PLGA microspheres was about 80%, which was the maximum yield obtained with 70% glycerol. The release of vitamin B₁₂ lasted for three weeks.     

雾化吹干技术在头孢哌酮钠生产中的研究与应用#br#

目的 雾化吹干技术成功应用在头孢哌酮钠生产中。方法 通过雾化发生器，将无菌过滤的注射用水雾化，用无菌氮气将雾化的水汽通入产品湿粉中置换残留的有机溶媒。结果 应用雾化吹干技术得到的头孢哌酮钠产品，丙酮残留可降低至0.35%，远低于欧洲药典(EP) 9.0和中国药典(CP) 2015版质量标准≤2.0%，产品整体质量水平和稳定性得到较大提升。结论 该技术操作简单，工时短，可有效提高产能，适用于产业化生产。     

标本处理方法对尿液蛋白电泳的影响

目的探讨不同蛋白沉淀方法对尿液标本蛋白电泳效果的影响。方法采集尿液标本8份,分别采用丙酮沉淀法、TCA／丙酮沉淀法及ACN／TFA沉淀法对尿液标本进行蛋白沉淀,然后进行双向电泳,比较3种标本处理方法后蛋白电泳蛋白点数及图像清晰程度。结果TCA／丙酮沉淀后电泳图谱清晰,丙酮沉淀法及ACN/TFA沉淀后单边电泳图谱具有较多横纹干扰条带,尿液标本TCA／丙酮沉淀后电泳蛋白点数高于丙酮沉淀法及ACN/TFA沉淀法,差异具有统计学意义。结论在丙酮沉淀前应用TCA可以消除干扰等电聚焦的盐类物质,进一步降低标本的导电能力,并且能够最大程度地提取尿液中的蛋白质。     

GSTM1和CYP2E1遗传多态及饮酒因素对苯酚和丙酮作业工人DNA损伤的影响

目的　研究GSTM 1和CYP2E1基因型与苯酚、丙酮生产工人外周血淋巴细胞DNA损伤的关系。方法　应用PCR RFLP方法对苯酚、丙酮作业工人和非接触对照人员的GSTM1和CYP2E1基因型进行检测 ,采用“彗星”电泳技术检测外周血淋巴细胞DNA的损伤。结果　苯酚、丙酮作业工人中GSTM1基因缺陷型个体的人均彗星细胞数为 (1 34± 1 38) ,彗星细胞尾长与总长的比值为 (0 17± 0 15 ) ,与基因正常型个体比较 ,差异有显著性 (P <0 0 5 ) ,CYP2E1突变型酒精消耗个体人均彗星细胞数为 (1 88± 0 83) ,彗星细胞尾长与总长的比值为 (0 2 2± 0 11) ,均明显高于野生型非酒精消耗个体 ,差异有显著性 (P <0 0 5 ,P <0 0 1)。结论　苯酚、丙酮作业工人的DNA损伤因GSTM 1和CYP2E1基因型的不同存在差异 ,饮酒可加重损害作用     

A microscopic survey on the efficiency of well-known routine chemical fixatives on cryosections

This study was designed to analyze and compare tissue preservation efficiency of acetone (AC), formaldehyde (FA) and paraformaldehyde (PFA) on cryosections. Brain, kidney, heart and liver tissue of adult Balb/c mice were fixed with either FA or PFA prior to cryosectioning, or fixed with AC alone immediately after cryosectioning. Hematoxylin and eosin staining showed that AC is a poor fixative in preserving the general tissue and cellular organization. PFA, and to a lesser extent FA, produced significantly better results. Another set of cryosections were further analyzed to test the properties of those fixatives to preserve proteins from specific cell structures. Cytokeratin filaments, F-actin filaments and nuclei were immunolabeled and examined using confocal microscopy. Results demonstrated that, overall, PFA is the best fixative tested. However, FA fixation gave poor results in preserving neuronal tissues. Immunofluorescence confirmed the inefficiency of AC fixation, after which no specific labelling of cytokeratin filaments was detectable. Nevertheless, actin filaments were detectable on AC-fixed samples, a finding that was supported by the quantification of fluorescein-phalloidin binding to F-actin. Overall, the data suggest that AC fixation is unacceptable for preservation of most samples, whereas FA and PFA fixation should be chosen according to the tissues and proteins to be studied.     

顶空气相色谱法测定盐酸普鲁卡因中的残留溶剂

目的：建立盐酸普鲁卡因中乙醇、丙酮残留量的测定方法。方法：采用顶空气相色谱法,以水为溶解介质,色谱柱为DB一1301石英毛细管柱,柱温80℃,检测器为FID,检测器温度为250℃,进样口温度为150℃,结果：乙醇、丙酮的线性范围分别为0．025～2．0nag·ml-1（r=0．9999）和0．025—2．0mg·ml-1（r=0．9999）;平均回收率分别为99．6％（RSD=0．6％）和99．5％（RSO：1．1％）;定量限分别为4．79μg·ml-1和3．01μg·ml-1。结论：方法简单、准确,可用于盐酸普鲁卡因中乙醇和丙酮残留量的测定。     

Lymph node preparation in resected colorectal carcinoma specimens employing the acetone clearing method

For the histopathological evaluation of resected colorectal carcinoma specimens, the currently required minimum number of lymph nodes that need to be examined to ensure accurate staging is 12. In some cases, however, this number of lymph nodes cannot be obtained by the conventional preparation method, based on formalin-fixed fatty tissue. Since prognostic accuracy correlates with the number of lymph nodes examined, a repeat work-up of the fatty tissue after soaking in acetone for 24h is a means of achieving the required minimum of 12 lymph nodes in many cases. Our department, which deals with an average of 307 colorectal cancer resection specimens per year, received between May 2004 and November 2006 80 cases (10.4%) in which the number of detected lymph nodes was less than 12. Owing to the use of conventional preparation methods, the required number of 12 lymph nodes could not be found, and subsequent acetone clearance of fatty tissue for a repeat examination was carried out. On average, 4.4 additional lymph nodes having an average size of 2mm were discovered. Accessory lymph node metastases were found in 9 cases. In 2 of these, the UICC classification had to be revised. In 1 case, a lymph node metastasis was detected after acetone clearance in an initially nodal negative carcinoma. Acetone clearance is an appropriate option to identify accessory lymph nodes in colorectal carcinoma specimens for daily routine use, and also constitutes an excellent instrument for internal quality control.