Intracellular distribution of actin in cells of the orgen of Corti: A structural basis for cell shape and motility

Immunofluorescence staining and phalloidin labeling have provided localization of actin in the sensory and supporting cells of the inner ear at the light microscopic level. However, with electron microscopy, neither actin nor actin filaments have been found in the outer hair cell body. This paper describes various techniques utilized to preserve and identify cytoplasmic actin at the ultrastructural level. Post-embedding staining of Lowicryl K4M sections, pre-embedding staining of permeabilized cells of the organ of Corti, pre-embedding staining of vibratome sections, and pre-embedding staining of permeabilized dissociated cells documented the presence of actin, but each of these techniques was best suited to localize actin in specific parts of the cell. Cytoplasmic actin was labeled when isolated cells were lightly fixed and membranes were permeabilized with detergent—conditions under which the cell ultrastructure was compromised. Under conditions of optimal fixation, cytoplasmic filaments embedded in the dense granular matrix of the hair cell cytoplasm were observed.     

Freeze-fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/μm². In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/μm². A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.     

日立835与8800氨基酸分析仪的应用与比较

L 8 8 0 0的Thr -Ser、Gly -Ala、Ile -Leu分离度较 835有很大的提高 ,最低检测浓度为 3pmolAsp ,而 835则为 30pmolAsp ,分析时间也较 835缩短 ,成本大大降低而效率大为提高。比较日立 835 - 5 0型与L - 880 0型氨基酸分析仪在分析中的实际应用情况 ,对这两种机型的应用做个总结 ,使二者得到更合理、充分的利用。     

高效液相色谱法测定复方苯甲酸软膏中苯甲酸及水杨酸的含量

研究用高效液相色谱法测定复方苯甲酸软膏的含量。用甲醇做溶剂,70℃水浴溶解软膏,冰浴析出基质,使基质与水杨酸、苯甲酸分离,采用Zorbax-SB C_(18)柱,0.1Mol/L磷酸氢二钠:甲醇(60:40)作为流动相,流速0.8mL/min。在240nm波长处,用外标法测定含量。苯甲酸、水杨酸浓度分别在0.01～0.2mg/mL范围内线性关系良好,R=0.9996。最低检测限为200ng/mL。日内精密度为0.01%～0.04%,0.019%～0.11%,日间精密度为0.045%～0.74%,0.039%～0.19%。方法回收率苯甲酸为:98.31% (n=4),RSD0.79%。水杨酸为:98.54% (n=4),RSD为1.62%。基质凡士林对苯甲酸、水杨酸的测定无干扰。试验证明本方法操作简便、快速,结果准确,可用于该制剂的含量测定。     

Differentiation of isomeric amino acid residues in proteins and peptides using mass spectrometry

Characterization and differentiation of isomers in biological macromolecules using mass spectrometry is one of the most significant challenges facing scientists in the field. The capability of high‐resolution MS instruments along with the development of new fragmentation methods now provides the ability to indirectly differentiate between some isomers. This ability has enabled mass spectrometry to evolve into a multidisciplinary technique incorporating areas such as pharmaceutical research, proteomics, polymer science, medicine, environmental chemistry, and recently archeology. This article aims to review recent developments in mass spectrometry methodologies in the identification of structural and spatial isomers in biological macromolecules, such as aspartic acid and isoaspartic acid (Asp/IsoAsp), leucine and isoleucine (Leu/Ile), glutamic acid and γ‐glutamic acid, and D/L enantiomers. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:609–625, 2012     

Combined molecular docking,homology modeling and DFT method for the modification of bovine serum albumin (BSA) to improve fluorescence spectroscopy for phthalate acid esters chelated with BSA

While phthalate acid esters (PAEs) cannot fluoresce alone, they can be detected by fluorescence spectroscopyafter chelation with bovine serum albumin (BSA). In this study, the types of amino acid residues at the active site of PAEschelated with BSA were determined using molecular docking technology. A modification scheme of BSA with higherdetection sensitivity fluorescence spectroscopy for PAEs was proposed based on the docking results and constructedfor a novel BSA structure with a higher detection sensitivity of fluorescence spectroscopy using a homologousmodeling method. Density functional theory (DFT) was employed to explore the influence before and after BSAmodification on PAEs’ detection through fluorescence spectroscopy. The results showed that the docking scoresbetween BSAs and dimethyl phthalate (DMP), dibutyl phthalate (DBP) and di-n-octyl phthalate (DNOP) wereincreased up to 26.45%, 16.82% and 16.30%, respectively, indicating that the active site modification of BSA couldenhance the binding affinity between BSA and PAEs. The fluorescence intensity of PAEs chelated with modified BSAswere calculated. The fluorescence intensity of fluorescence spectroscopy for DMP, DBP and DNOP chelated withBSAs after modification was increased up to 2.8-, 104.51- and 62.43-fold, respectively, which achieved the purpose oftheoretically modifying BSA to improve the detection sensitivity of fluorescence spectroscopy for PAEs.     

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: Calibration curves and electron beam sensitivity

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K Kα P/B or Y = (P/B) · Z²/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10–25 kV for Ca standards. K standards were also independent when P/B was corrected for Z²/A. Background counts in the otoconia (B_o) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. B_o was not dependent on the spectra acquisition time, with the exception of B_o under Kα K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z²/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.     

微柱液相色谱法同时检测食品中苯甲酸和山梨酸

通过自构建的微柱液相色谱系统上色谱条件的优化,建立了一种同时检测食品中苯甲酸和山梨酸的新方法。食品中苯甲酸和山梨酸经提取后,6分钟内达到基线分离,与常规高效液相色谱法相比,分析时间缩短,流动相消耗减少。在1～25 mg/L浓度范围内线性关系良好,相关系数分别为0.9999和0.9997。苯甲酸和山梨酸的回收率均在94.7%～103.1%之间,三次平行加标回收率测试,相对标准偏差分别为1.12%和1.29%。该方法稳定性好,回收率高,能快速、准确的检测食品中苯甲酸和山梨酸。     

On the method of revealing enamel structure by acid etching. Aspects of optimization and interpretation

The study aimed at finding an optimal combination of acid concentration and etching time when nitric acid is used as etchant for the study of the finer details of human dental enamel structure. Four hundred 2–3‐mm‐thick segments of facio‐lingually sectioned human third molar crowns were assigned to 20 groups with 20 specimens in each group, each group differing with respect to acid concentration (0.1, 1, 2.5, and 5%) and etching time (15, 30, 45, 90, and 180 s). After etching and preparation, specimens were observed in the scanning electron microscope (SEM). Surface roughness/topography increased with increasing acid concentration and increasing etching time, but not in a linear fashion; generally, prisms tended to go from flat‐surfaced to cone‐shaped and prism sheaths from fissure‐like to wedge‐shaped. Intragroup variations and intergroup similarities were considerable. The two major enamel factors determining the etch effect are crystal orientation and prism sheath properties. Other factors, such as distribution of porosities and crystal quality, also contribute probably. Slight to moderate topography is best for observing the finer enamel structure, for example, etching with concentrations in the range 0.1–1% and with etching times in the range 15–90 s, the stronger the acid, the shorter the time. The depth effect of nitric acid is judged to be relatively small. Considerable variations in expression of prism cross‐striations were observed. SEM observations of acid‐etched enamel in carefully selected planes are a powerful method for the study of enamel structure, bearing in mind the artifactual aspects of the observed surface.     

Measurement of absolute tracer concentrations in tissue sections by using digital imaging fluorescence microscopy. Application to the study of plasma protein uptake by the arterial wall

Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to other studies employing DIFM. Nonlinearities were found to arise from offsets in the video digitizers, from background fluorescence and stray light within the microscope and from the transfer characteristics of the intensified CCD camera. Camera gain controls showed complex behaviour. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in response were caused by the geometry of the microscope optics and by the image intensifier. However, the ratios between areas were not affected by light intensity or camera gain settings. Measured intensities were independent of the depthwise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not concentration dependent. Methods used to correct these effects and obtain a uniform, linear and constant relationship between concentration and grey level are described. Using the system and appropriate corrections, in vivo uptake of sulphorhodamine-B-labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean uptake of tracer and its local variation were quantitatively similar to those previously obtained with nonmicroscopic methods.     

用HPLC测定当归中阿魏酸含量方法的研究

建立HPLC法测定当归中阿魏酸的含量方法。色谱柱采用Thermo ODS HY-PERSIL(250mm × 4.6 mm,5 μ m),流动相为乙腈-0.3%乙酸溶液(体积比为30 : 70),流速:1.0mL/min,检测波长:322nm,进样量为10μL,柱温:35℃。阿魏酸在0.1～10 μ g/mL范围内,线性关系良好(r= 0.9998)。平均回收率为101.58%,RSD为1.57%。该方法灵敏、准确、简便易行、重复性好,可用于中药当归药材的质量控制。     

毛细管气相色谱法检测面粉中的过氧化苯甲酰和山梨酸

过氧化苯甲酰和山梨酸是在面粉中常用的添加剂。本文采用毛细管气相色谱法检测面粉中的过氧化本甲酰,方法准确、快速。     

直接进样高效液相色谱-柱后衍生法检测水中草甘膦和氨甲基膦酸

本文介绍了直接进样高效液相色谱-柱后衍生法对水中草甘膦和氨甲基膦酸的检测方法。水样直接进样,经高效液相色谱分离后草甘膦和氨甲基膦酸被衍生成强荧光物质,以荧光检测器进行检测;本文还对该方法的准确性和精密度进行了试验,结果表明该法对饮用水中草甘膦和氨甲基膦酸的最低检测限分别为0.015mg/L、0.011mg/L,相对标准偏差（RSDs,n=5）分别为1.63%、0.75%,加标回收率分别为107.52%、109.62%。该方法快速、灵敏度高、精密度好,能够满足生活饮用水对草甘膦及氨甲基膦酸的检测要求。     

Determination of carnosic acid and carnosol in Rosmarinus officinalis L. by high-performance capillary electrophoresis

Carnosol and carnosic acid in Rosmarinus officinalis L. were separated by high-performance capillary electrophoresis using a 50?cm capillary, a 12?mmol/L 80:20 pH 9.9 borax:methanol buffer using 25?kV of applied voltage at 30°C with detection at 280?nm. Rosmarinus officinalis L. was extracted with methanol with ultrasound at 170?W and 50?kHz for 20?min. The precision, repeatability, and recovery of the method were evaluated. The calibration relationships for carnosol and carnosic acid were y?=?2.331x?+?5.92 from 0.01 to 0.160?mg/mL and y?=?7.980x?+?4.32 from 0.005 to 0.080?mg/mL, respectively. The established method allows the separation and quantification of carnosic acid and carnosol in R. officinalis from various locations in China.     

On the friction and wear performance of boric acid lubricant combinations in extended duration operations

Lubrication is critical for minimizing wear in mechanical systems that operate for extended time periods. Developing lubricants that can be used in engineering systems without replenishment – particularly those that are environmentally friendly – is very important for increasing the functional lifetime of mechanical components. In the present investigation, extended duration pin-on-disk experiments were carried out to determine the relative performance of a wide range of lubricant combinations in a commercial brake valve assembly. In the experiments, the lubricants were initially applied to the disk surface but were not replenished over a sliding distance of more than 6000 m. The experimental results revealed that the environmentally friendly lubricant boric acid was highly ineffective for reducing the wear in the surfaces tested. When combined with a commercial transmission fluid, however, the boric acid mixture proved to be highly effective in terms of both friction and wear performance. Based on the success of the combined lubricant experiments, the boric acid was then mixed with canola oil to form a completely natural lubricant combination. Based on further pin-on-disk experiments, this lubricant combination yielded the best wear performance of all the lubricants tested. The importance of these results, as related to the use of the natural lubricant combination in other engineering systems such as sheet metal stamping, was subsequently ascertained and discussed.     

Hypothalamic regulatory peptides and their receptors: Cytochemical studies of their role in regulation at the adenohypophyseal level

Hypothalamic regulatory peptides bind to specific receptors on target cells in the pituitary and control secretion. They in turn can be regulated at the pituitary level by steroid and peptide modulators. Affinity cytochemical techniques are important tools for the identification of specific target binding sites for these regulatory peptides. This presentation reviews the work in which potent, biotinylated ligands of gonadotropin releasing hormone (bio-GnRH), corticotropin releasing hormone (bio-CRH), and arginine vasopressin (bio-AVP) were applied to study the target cell responses. Bio-GnRH, bio-CRH, and bio-AVP bind to membrane receptors on specific anterior pituitary cells. Dual labeling for either gonadotropin or adrenocorticotropin (ACTH) antigens further identified the target cells. After 1–3 minutes, the label was in patches or capped on the surface. After 3 minutes, it was internalized in small vesicles and sent to receptosomes and vacuoles in the Golgi complex. Eventually the biotinylated peptides, or a metabolite, was found in the lysosomes (multivesicular bodies) and a subpopulation of secretory granules. The route and rate of uptake was similar to that described for the classical receptor-mediated endocytosis process. In contrast, intermediate lobe corticotropes internalized the bio-CRH in less than 1 minute. The route through the Golgi complex appeared to be bypassed. Instead the labeled peptide was in vesicles, on the membranes of scattered vacuoles, and in multivesicular bodies. Modulation of ligand binding by steroids showed that changes in receptor numbers correlated with changes in the number of cells that bound the ligand. In male rats, dihydrotestosterone reduced the percentage of GnRH-bound cells by 50%. Most of the reduction appeared in cells that stored luteinizing hormone (LH) antigens. In diestrous female rats, estradiol increased the percentage of bio-GnRH-bound cells. However, the steroid decreased the percentage of GnRH-bound cells in cells from proestrous rats. Glucocorticoids decreased the percentage of CRH-bound corticotropes in as little as 10 minutes. Potentiation of secretion by these ligands was correlated with increases in the percentage of ligand-bound cells. AVP pretreatment of corticotropes increased the percentage of cells that bound bio-CRH. It also increased the rate of receptor-mediated endocytosis of CRH and changed the route so that the Golgi complex was bypassed. This effect could be mimicked by activation of its second messengers (calcium and protein kinase C). Similarly, CRH pretreatment increased the percentage of corticotropes that bound AVP. Thyrotropin releasing hormone (TRH) pretreatment also increased the percentage of thyrotropes that bound AVP. Finally, calcium or sodium channel blockers altered CRH binding so that fewer cells were labeled. This binding by CRH was not dependent on extracellular calcium and tests with a calcium channel agonist showed that it was related to activation of calcium channels. To summarize, these affinity cytochemical studies have identified specific target cells in the pituitary for GnRH, CRH, and AVP. They have also identified heterogeneity in the population. They have demonstrated new information about the direct modulatory effects of steroids, ion channels, and neuropeptides on neuropeptide binding by subpopulations of these target cells.     

Localization of Ca2+ -stores and tissue compartments with a Ca2+ -binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy

The addition of 10 mM CaCl₂ to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca²⁺ L_2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO₄) revealed a small but significant calcium signal.     

Hyaluronic acid inhibited the upregulation of heat shock protein 70 in human chondrocytes from osteoarthritis and Kashin-Beck disease

This study aimed to investigate the effect of hyaluronic acid (HA) on the expression of heat shock protein70 (HSP70) in chondrocytes isolated from patients with osteoarthritis (OA) and Kashin-Beck disease (KBD). Thechondrocytes were collected from OA and KBD patients, and chondrocytes isolated from patients of accident injurieswere used as the control. The chondrocytes were treated with HA at different doses. HSP70 expression in chondrocytesat both mRNA and protein levels was tested by PCR and Western blot analysis. Compared with control, both mRNAand protein levels of HSP70 were higher in chondrocytes from KBD and OA. However, HA at the dose of 500 μg/mL significantly inhibited HSP70 expression levels in both KBD and OA groups (P < 0.05). In conclusion, HSP70 ishighly expressed in chondrocytes of patients of OA and KBD. HA intervention inhibits the upregulation of HSP70 inchondrocytes of OA and KBD patients and could be a promising agent for treatment of OA and KBD.