头风病证候诊断标准的研究

本研究通过复习古今中外有关文献,从宏观与微观、主观与客观等方面对头风病还候进行了规范的、定量的全面概括;对302例进行临床调研;运用现代数学理论和科学统计方法进一步补充及完善,确定了"头风病证候诊断标准"。该标准可反应头风病的病因、病机和不同的证候组合,具有可计量性、可重复性及简便易行的特点,为头风病临床辨证施治、多中心大协作研究及新药开发与利用打下了基础,为中医疾病的病与证的规范化和计量化的研究提供了依据。     

大鼠胚胎视网膜节细胞神经诱向因子及其受体的免疫组织化学定位及原位定量研究

用视网膜节细胞神经诱向因子(RGNTF)单克隆抗体及RGNTF抗独特型单克隆抗体对大鼠胚胎16～18d眼球切片进行了免疫组织化学定位及定量研究。结果表明,鼠胚视网膜节细胞的RGNTF含量从胚16～20d非常显著地减少(P<0.01),RGNTF受体的数量从胚16d到18d显著增加(P<0.05),从胚18d到20d又非常显著减少(P<0.01)。以上提示,16～18d的鼠胚视网膜节细胞既含大量RGNTF,又有大量RGNTF的受体,RGNTF对胚胎视网膜节细胞的存活及分化可能有自调节功能。20d的鼠胚视网膜节细胞的RGNTF及其受体都非常显著减少,提示节细胞在形态上分化完成后,其对RGNTF的自分泌立即显著降低,这可能是节细胞自然死亡的主要原因。     

Alkaloid profiling of crude and processed Veratrum nigrum L. through simultaneous determination of ten steroidal alkaloids by HPLC-ELSD

Veratrum nigrum L., a traditional Chinese herb, has been used for treatment of hypertension, blood-stroke, excessive phlegm, epilepsy, etc. Steroidal alkaloids were well-known as both bioactive and toxic constituents of Veratrum species, the toxicity of which the traditional processing procedure can reduce. To reveal the mechanism of processing V. nigrum L., a high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) method was developed for the simultaneous determination of ten steroidal alkaloids in crude and processed V. nigrum L., comparison with ultrasound extract of crude V. nigrum L. With a Venusil XBP-C(18) analytical column, the analytes were separated efficiently using the mobile phase consisted of (A) 0.03% aqueous triethylamine (TEA) and (B) acetonitrile in a gradient program. The parameters for ELSD were set: S.C. (Spray Chamber)=35 degrees C, D.T. (Drift Tube)=70 degrees C, GAS=50 psi. All calibration curves showed good linear regression (gamma>or=0.9990) within the tested range. Additionally, reproducibility for the quantification of ten alkaloids in V. nigrum L. with intra- and inter-day variations of less than 5.0% was observed. The obtained alkaloid profiles performed by this newly established method, provided valuable information for the differentiation of crude and processed V. nigrum L. and for the explanation of the different toxicity.     

Quantitative aspects of the analysis of the monoclonal antibody trastuzumab using high-performance liquid chromatography coupled with electrospray mass spectrometry

The analytical possibilities of quantification of the intact monoclonal antibody trastuzumab by high-performance liquid chromatography coupled with electrospray mass spectrometry (HPLC-ESI-MS) were investigated. To clarify the results obtained by LC-MS, complementary experiments were performed using direct UV-spectrophotometry and high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV). A polystyrene-divinylbenzene (POROS) column was applied with gradient elution using formic acid 0.08% (v/v) in water and formic acid 0.08% (v/v) in acetonitrile as mobile phase for chromatographic analysis. Quantification on LC-MS was performed by using the peak area of the total ion current (TIC) chromatograms of one charge state. Non-linearity and sensitivity loss were the major limitations observed with the LC-MS method, of which the non-linearity is most likely caused by detector saturation. The sensitivity loss during analysis could be reduced by lowering the MS source temperature. This parameter is critical in creating a robust LC-MS system for the quantitative analysis of trastuzumab.     

Validation of a quantitative NMR method for suspected counterfeit products exemplified on determination of benzethonium chloride in grapefruit seed extracts

A 1H-nuclear magnetic resonance (NMR) spectroscopy method for quantitative determination of benzethonium chloride (BTC) as a constituent of grapefruit seed extract was developed. The method was validated, assessing its specificity, linearity, range, and precision, as well as accuracy, limit of quantification and robustness. The method includes quantification using an internal reference standard, 1,3,5-trimethoxybenzene, and regarded as simple, rapid, and easy to implement. A commercial grapefruit seed extract was studied and the experiments were performed on spectrometers operating at two different fields, 300 and 600 MHz for proton frequencies, the former with a broad band (BB) probe and the latter equipped with both a BB probe and a CryoProbe. The concentration average for the product sample was 78.0, 77.8 and 78.4 mg/ml using the 300 BB probe, the 600MHz BB probe and CryoProbe, respectively. The standard deviation and relative standard deviation (R.S.D., in parenthesis) for the average concentrations was 0.2 (0.3%), 0.3 (0.4%) and 0.3mg/ml (0.4%), respectively.     

Rapid and reliable quantification of reovirus type 3 by high performance liquid chromatography during manufacturing of Reolysin

Reolysin, a human reovirus type 3, is being evaluated in the clinic as an oncolytic therapy for various types of cancer. To facilitate the optimization and scale-up of the current process, a high performance liquid chromatography (HPLC) method has been developed that is rapid, specific and reliable for the quantification of reovirus type 3 particles. Using an anion-exchange column, the intact virus eluted from the contaminants in 9.78 min at 350 mM NaCl in 50mM HEPES, pH 7.10 in a total analysis time of 25 min. The virus demonstrated a homogenous peak with no co-elution of other compounds as analyzed by photodiode array analysis. The HPLC method facilitated the optimization of the purification process which resulted in the improvement of both total and infectious particle recovery and contributed to the successful scale-up of the process at the 20 L, 40 L and 100 L production scale. The method is suitable for the analysis of crude virus supernatants, crude lysates, semi-purified and purified preparations and therefore is an ideal monitoring tool during process development and scale-up.     

Quantitative determination of underivatized polyamines by using isotope dilution RP-LC-ESI-MS/MS

A rapid, sensitive and selective method using LC-MS/MS was developed and validated for the simultaneous quantitative determination of five polyamines N(1),N(12)-diethylspermine (DESpm), N-ethylspermine (EtSpm), N(1)-ethylspermidine (EtSpd), spermidine (Spd) and N(1)-ethyl-1,3-diaminopropane (EtDAP) without any derivatization steps. The LC-MS/MS system was operated using the positive electrospray ionization (ESI) mode. The chromatographic separation only took 10min and was performed on a reversed phase C18 column with 0.1% heptafluorobutyric acid as the ion-pairing agent and acetonitrile gradient. Stable, deuterium labelled internal reference compounds of the five analytes were included in the quantification. The lower limit of quantification for all of the five analytes was 0.03muM and the method was linear for DESpm, EtSpd, Spd and EtDAP over the range of 0.03-60muM and for EtSpm over the range of 0.03-30muM. Correlation coefficients (R(2)) were always >0.995 for all the analytes. The precision of the overall method ranged from 0.2 to 9.7% as intra-day variability and from 0.9 to 6.8% as inter-day variability. The intra-day and inter-day accuracy of the assay ranged between 87.6-109.8% and 89.6-106.6%, respectively. The method has been applied successfully to quantify metabolites of DESpm as a substrate for recombinant human polyamine oxidase.     

Motor unit number estimation (MUNE): Where are we now?

Estimation of the number of motor units (MUNE) in specific muscles is important to monitor outcome in progressive neurogenic disorders, with potential application in clinical trials. However, in spite of recent developments to identify the most convenient technique for MUNE, all current methods have individual shortcomings. It is essential to understand the scientific concepts that support MUNE and the many methods already proposed. In particular, the core role of the compound muscle action potential (CMAP) size in the estimation process is undervalued. Operator-dependent variation in CMAP amplitude or area is the main factor underlying MUNE stability. At present, MUNIX, as standardized in many centers, is probably the best accepted method. Future developments should be based on full understanding of the neurophysiological concepts underlying the MUNE calculation, in order to find a quick, well-tolerated, operator-friendly and reliable method to apply more universally in clinical practice.     

Neurobiological Mechanisms for Semantic Feature Extraction and Conceptual Flexibility

Signs and symbols relate to concepts and can be used to speak about objects, actions, and their features. Theories of semantic grounding address the question how the latter two, concepts and real-world entities, come into play and interlink in symbol learning. Here, a neurobiological model is used to spell out concrete mechanisms of symbol grounding, which implicate the “association” of information about sign and referents and, at the same time, the extraction of semantic features and the formation of abstract representations best described as conjoined and disjoined feature sets that may or may not have a real-life equivalent. The mechanistic semantic circuits carrying these feature sets are not static conceptual entries, but exhibit rich activation dynamics related to memory, prediction, and contextual modulation. Four key issues in specifying these activation dynamics will be highlighted: (a) the inner structure of semantic circuits, (b) mechanisms of semantic priming, (c) task specificity in semantic activation, and (d) context-dependent semantic circuit activation in the processing of referential, existential, and universal statements. These linguistic-semantic examples show that specific mechanisms are required to account for context-dependent semantic function or conceptual “flexibility.” Static context-independent concepts as such are insufficient to account for these different semantic functions. Whereas abstract amodal models of concepts did so far not spell out concrete mechanisms for context-dependent semantic function, neuronal assembly mechanisms offer a workable perspective.