850 nm波长微激光对肥大细胞脱颗粒及释放组胺的影响

目的:研究850 nm波长微激光对肥大细胞(RBL-2H3 (Rat basophilic leukemia))照射后组胺释放的影响.方法:肥大细胞铺于96板上,密度为5×104/孔,12 h后细胞经D-Hank's缓冲液清洗3次用于实验.肥大细胞经微激光照射15 min后,轻轻吸取细胞上清液,加入等量0.1 mmol/L的邻苯二甲醛,充分混匀,室温下反应20 min后,于荧光酶标仪上测量混合物的荧光光谱.结果:微激光照射之后的肥大细胞上清液,能发出440 nm左右的荧光,说明850 nm微激光能使肥大细胞脱颗粒,从而判断850 nm微激光能作为仿灸仪器的光源.     

低强度激光血管内照射治疗的机理初探

用532nm的激光作为激光光源分别测量正常血液、血浆及HBsAg阳性血液、血浆的荧光光谱。血液光谱的测量结果显示,两种血液樯本都在630nm及710nm附近出现有荧光峰值。从光谱学的角度分析,可得出激光照射血液治疗的内在机制在激光照射可增强血液的携氧能力,从而促进了机体的新陈代谢。血浆图谱测量结果显示,正常血浆及HBsAg阳性血浆在738nm处的荧光强度有显著差异,可作为临床光谱诊断中判断HBsAg阳性的标准和依据。对HBsAg阳性血液的生化检测结果显示,He-Ne激光照射可抑制或杀伤乙肝病毒。     

微激光照射肥大细胞对胞内钙离子浓度的影响

目的:研究850 nm波长微激光对肥大细胞照射后胞内钙离子浓度的影响。方法:实验前12 h,将肥大细胞接种于激光共聚焦专用培养皿中,然后用D-Hank’s液洗涤3次,加入2 m L D-Hank’s液,按照照射时间不同共分六组(Control,1 min,2 min,2.5 min,3 min,4 min),其中,空白对照组不进行激光照射处理;激光照射后,弃去D-Hank’s液,加入含有Fluo-3/AM的D-Hank’s液1 m L,放入培养箱中孵育30 min后,用D-Hank’s液洗涤3次去除胞外多余的Fluo-3/AM,再加入含有10%FBS的D-Hank’s液2 m L,置激光扫描共聚焦显微镜检测。结果:空白对照组中细胞内未见荧光,说明此时胞内无钙离子,但从1 min开始在细胞中发现荧光,而且发现随着照射时间的加长,其荧光强度越来越强,这可能与微激光照射时间越长从而刺激胞内出现更多的钙离子有关。从上面的实验说明850 nm微激光能作为仿灸仪器的光源。     

He-Ne激光照射对血液及其组分荧光光谱影响的实验研究

为研究弱激光照射对人血液携氧能力的影响及机制,我们用荧光仪分别测量了He-Ne激光照射前后正常血液及其组分（血浆、红细胞）的荧光光谱,研究了激光照射导致的光谱变化,并分析了光谱变化与血液携氧能力改变的关系。实验结果显示：全血液标本在490nm及614nm附近有荧光峰值;血浆的荧光则主要分布在420－500nm之间;红细胞在500nm及614nm附近有荧光。He-Ne激光照射后,全血液及红细胞在614nm处的荧光谱都有较明显的变化,且较相似。由此可得出结论,He-Ne激光照射可影响血液的携氧能力。     

一种酵母细胞生长现象的实时单细胞拉曼光谱观察

用拉曼镊子观察单个即发活性干酵母(Saccharomy cescerevisiae)细胞在2.0%葡萄糖溶液中的活化过程,收集其拉曼光谱。结果发现,在某一批次的产品中,酵母细胞的1364cm-1峰强度随着细胞的活化而显著增加,531cm-1、652cm-1、1053cm-1等源自葡萄糖或葡萄糖基的信号峰也会随细胞的生长而增强,随后增强的还有1432cm-1、1448cm-1、1561cm-1等源自脂类物质的峰,而源自蛋白质及脂类的1000cm-1、1445cm-1、1655cm-1等峰的信号强度基本不变,酵母细胞代谢活跃的标志峰1603cm-1也基本不变。该批次产品中,10次实验有7次观察到上述现象,而在别的批次产品中并没有观察到该现象。用单细胞拉曼光谱实时记录了这一特殊的生长现象。     

正常和癌变胃粘膜组织的共焦拉曼光谱初探

目的:分析研究胃正常和癌变粘膜组织的拉曼光谱特征,为拉曼光谱应用于胃癌的临床检测诊断奠定基础。方法:收集胃镜检查中活检的19例正常和12例癌变胃粘膜组织标本,采用785 nm激发光拉曼光谱仪进行拉曼光谱采集。比较分析胃正常和癌变粘膜组织的拉曼光谱特征差异并研究其区分正常和癌变组织的价值。结果:1)特征峰1 098 cm-1、1 444 cm-1、1 555 cm-1、1 660 cm-1等在胃癌组织中发生了移位,平均位移(2.57±1.28)cm-1,以红移为主;2)癌变组织中相对峰强比I1087 cm-1/I1207 cm-1≥1.87,其区别胃癌和正常胃粘膜组织的准确率、灵敏度和特异度分别为87.1%、83.3%、89.5%;3)癌组织中增加了表征蛋白质的特征峰1 262 cm-1、1 586 cm-1,但同时减少了表征蛋白质和脂质特征峰1 172 cm-1。结论:拉曼光谱不仅可以准确区分正常和癌变,而且可以探索癌变相关的分子生化改变。拉曼光谱在胃癌的跟踪发现和检测诊断中具有良好前景。     

动脉粥样硬化斑块的微区拉曼光谱检测

为探讨动脉粥样硬化斑块的微区拉曼光谱特征,以球囊损伤日本长耳白兔右侧颈总动脉后予以高脂饮食喂养,在实验过程中监测体重和血脂变化情况。3个月后,以中国斑点蝰蛇毒和组胺加以触发使斑块破裂,将动物处死并查找动脉硬化斑块,动脉组织经大体病理分类后,进行微区拉曼光谱及病理检测。结果显示:动脉粥样硬化斑块的拉曼光谱图在1450及1660cm-1处均有明显的胆固醇等脂质特征峰。特征峰曲线下相对面积统计结果表明:明显动脉粥样硬化斑块的谱峰下相对面积(5.80×10-3±3.51×10-3)显著高于轻度动脉粥样硬化组织(2.01×10-3±1.49×10-3)及正常动脉组织(1.01×10-3±0.94×10-3),P<0.05。正常动脉组织拉曼光谱曲线较光滑,无明显特征峰。血栓形成处拉曼光谱图荧光背底较强,未见特征谱峰。该研究结果证明微区拉曼光谱可以对动脉粥样硬化斑块的胆固醇等脂质含量进行特异性定量检测,表明微区拉曼光谱是评估动脉粥样硬化程度及斑块稳定性的可行方法。     

美白祛斑药物对强脉冲光作用下的鼠皮黑色素的影响

观察美白祛斑药物对强脉冲光(intensive pulse light, IPL)作用下的鼠皮黑色素的影响,探讨IPL作用下,皮肤的黑色素的增生和美白药物之间的作用关系,以期探寻美白药物在减少光子嫩肤术并发症的作用机制.以小白鼠作为研究对象,用IPL在一定的波长及能量密度照射活体小鼠皮肤, 在照射后15 min涂抹低浓度的维A酸霜,分别在照射后的4 h、5 d,用显微镜观察涂霜剂、没涂霜剂的皮肤黑色素及其周边组织的变化情况;在照射后的1 d用多光子显微镜观察涂霜剂、没涂霜剂皮肤组织的变化.讨论了美白祛斑药物在光子嫩肤过程中抑制黑色素生成的过程,为如何减少光子嫩肤术并发症提供参考和借鉴.     

微藻细胞油脂含量的快速检测方法

以斜生栅藻(Scenedesmus obliquus JNU20)为实验材料,采用尼罗红(NR)荧光光谱法和傅里叶变换红外光谱法(FTIR)测定该藻细胞中的油脂含量。研究结果表明NR的最佳染色条件为:染色前微波处理40 s,二甲基亚砜体积分数1%, NR最终质量浓度1.5 μg/ml,染色时间5 min,染色温度40℃。比较了NR荧光光谱法、FTIR与传统重量法测定的该藻在不同时相的油脂积累情况,利用激光共聚焦显微镜观察了细胞中油体形成的动态过程。实验结果表明NR荧光光谱法、FTIR与传统重量法测定的结果显著相关(R²=0.9258, R²=0.9844),但NR荧光光谱法和FTIR更简便快速,研究结果为规模化筛选高含油量藻株及跟踪产油微藻油脂积累过程奠定了基础。     

Q开关激光爆破技术对豚鼠皮肤色素的影响

目的研究Q开关激光爆破术对豚鼠黑素细胞的影响及照射周边组织的变化,为临床治疗皮肤色素病变提供实验依据。方法用Q开关-YAG激光分别照射豚鼠黑色毛区及棕色毛区(波长分别用1064nm和530nm,光斑直径2mm),实验动物20只,随机分四组,分别于照射后间隔7d、10d、14d取材,照射前取材作对照,10%甲醛固定,冰冻切片,分别用HE和DOPA反应显示黑素细胞。结果照射后皮肤黑色素颗粒逐渐减少至消失,照射后30d黑毛区与棕毛区肉眼见照射区皮肤变白,毛也变白,HE染色皮肤、毛囊及毛未见黑色素颗粒,DOPA反应表皮黑色素细胞、毛囊和毛均呈阴性反应,部分豚鼠棕毛区毛及毛囊见黄色色素颗粒。结论波长1064nm和532nm Q-YAG激光对豚鼠皮肤黑色素细胞和黑色素颗粒的破坏效果显著;但对棕色色素清除效果较差。波长1064nm Q-YAG激光对豚鼠皮肤黑色素消减与照射次数有关,与照射间隔时间长短无关。     

Recent applications of fluorescence correlation spectroscopy in live systems

Fluorescence correlation spectroscopy (FCS) is a widely used technique in biophysics and has helped address many questions in the life sciences. It provides important advantages compared to other fluorescence and biophysical methods. Its single molecule sensitivity allows measuring proteins within biological samples at physiological concentrations without the need of overexpression. It provides quantitative data on concentrations, diffusion coefficients, molecular transport and interactions even in live organisms. And its reliance on simple fluorescence intensity and its fluctuations makes it widely applicable. In this review we focus on applications of FCS in live samples, with an emphasis on work in the last 5 years, in the hope to provide an overview of the present capabilities of FCS to address biologically relevant questions.     

Interaction between hydroxyethyl starch and propofol: computational and laboratorial study

Background: Hydroxyethyl starch (HES) is one of the most used colloids for intravascular volume replacement during anesthesia. Aim: To investigate the existence of a chemical interaction between HES and the anesthetic propofol by in vitro propofol dosing, computational docking, and examination of a complex between propofol and HES by infrared (IR), ultraviolet (UV), and ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Methods: Ten samples with human plasma mixed with HES or lactated Ringers (n?=?5 for each fluid) were prepared, and the propofol free fraction was quantified until 50?min, using gas chromatography-mass spectrometry. The docking study was performed between HES and propofol and compared with controls. The binding affinities between HES and the small molecules were evaluated by binding free energy approximation (ΔGb, kJ?mol^?1). The IR, UV, and NMR spectra were measured for propofol, HES, and a mixture of both obtained by the kneading method. Results: Propofol concentrations were significantly lower in the HES samples than in the LR samples (p?=?.021). The spectroscopic characterization of propofol combined with HES revealed differences in spectra and docking studies reinforced a potential interaction between propofol and HES. Conclusions: Propofol and HES form a complex with different physical-bio-chemical behavior than the single drugs, which may be an important drug interaction. Further studies should evaluate its clinical effects.     

Synthesis and conformational studies of peptides fromE. coli pilus proteins recognized by the chaperone PapD

Summary Adhesive pili in uropathogenicE. coli are composed of a few different types of proteins which are assembled into the pilus by the chaperone PapD. Peptides from the C-terminus of these pilus proteins have been prepared by Fmoc solid-phase synthesis. Use of a polyethyleneglycol polystyrene resin was found to be essential for successful synthesis. The conformational propensities of the peptides were analyzed by CD and¹H NMR spectroscopy, with special focus on PapG296-314 from the pilus adhesin which has previously showed the tightest binding to PapD. PapG296-314 was found to be flexible in different solutions, but with a significant propensity to adopt a -conformation. Interestingly the peptide is bound as a -strand in a crystalline complex with PapD. The peptides from three other pilus proteins could only be investigated in trifluoroethanol wher they displayed considerable -helicity in contrast to PapG296-314. These results suggest that conformational factors provide part of the explanation for the differential binding of pilus-related peptides to PapD.Abbreviations CD circular dichroism - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate - COSY two-dimensional correlated spectroscopy - DMF dimethylformamide - DMSO dimethylsulfoxide - FAB-MS fast-atom-bombardment mass spectroscopy - Fmoc 9-fluorenylmethoxycarbonyl - Gal galactose - HOBt 1-hydroxybenzotriazole - MeCN acetonitrile - MSNT 1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - PEG-PS resin polyethyleneglycol polystyrene resin - ROESY two-dimensional rotating frame nuclear Overhauser enhancement spectroscopy - SDS sodium dodecylsulfate - TFA trifluoroacetic acid - TFE 2,2,2-trifluoroethanol - TOCSY two-dimensional total correlation spectroscopy These authors have made equal contributions to the work presented.correspondence should be addressed.     

Light-induced Difference Terahertz Spectroscopy

Visible/near-infraredlaser-induced difference spectroscopy basedon a time-domain terahertz system has beendeveloped, and used to study copperpathancyonine. We find that the absorptionpeak of this molecule at 1.08 THz changessignificantly under 790 nm laserexcitation, suggesting that we haveobserved the first evidence of vibrationalmode changes in the THz range induced byvisible/near-infrared light.     

Biophysical characterization of double-stranded oligonucleotides using ETBR and isothermal fluorescence spectroscopy: implication for SNP genotyping

The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. − dF/dT (or dA/dT) vs T, lnK (or RlnK) vs T_M, ΔG vs T_M, ΔS vs T_M and ΔH vs T_M are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. AY151039) of chromosome 13 having either A or G mutation at position − 26.     

Predicting Mab product yields from cultivation media components, using near-infrared and 2D-fluorescence spectroscopies

The yield of monoclonal antibody (Mab) production processes depends on media formulation, inocula quality, and process conditions. As in industrial processes tight cultivation conditions are used, and inocula quality and viable cell densities are controlled to reasonable levels, media formulation and raw materials lot-to-lot variability in quality will have, in those circumstances, the highest impact on process performance. In the particular Mab process studied, two different raw materials were used: a complex carbon and nitrogen source made of specific peptones and defined chemical media containing multiple components. Using different spectroscopy techniques for each of the raw material types, it was concluded that for the complex peptone-based ingredient, near-infrared (NIR) spectroscopy was more capable of capturing lot-to-lot variability. For the chemically defined media containing fluorophores, two-dimensional (2D)-fluorescence spectroscopy was more capable of capturing lot-to-lot variability. Because in Mab cultivation processes both types of raw materials are used, combining the NIR and 2D-fluorescence spectra for each of the media components enabled predictive models for yield to be developed that out-performed any other model involving either one raw material alone, or only one type of spectroscopic tool for both raw materials. For each particular raw material, the capability of each spectroscopy to detect lot-to-lot differences was demonstrated after spectra preprocessing and specific wavelength regions selection. The work described and the findings reported here open up several possibilities that could be used to feed-forward control the process. These include, for example, enabling specific actions to be taken regarding media formulation with particular lots, and all types of predictive control actions aimed at increasing batch-to-batch yield and product quality consistency at harvest.     

An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A2 and amphiphilic molecules

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant.     

Redox and specific effects of vanadium ions on respiratory enzymes

The effects of vanadium ions on the activities of enzymes of aerobic and anaerobic respiratory chains were investigated in vitro and in situ employing ¹H-, ¹⁴N-, ³¹P- and ⁵¹V- nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and spectrophotometry. Vanadate and vanadyl ions produced either non-specific redox or specific activation or inhibition of respiratory enzymes. The oxidants molybdate and chromate and the reductant dithiothreitol were used to distinguish between non-specific and specific effects of vanadium ions on enzyme activities. The results suggested that components of anaerobic respiratory chains were more susceptible to vanadium ions than those of the aerobic respiratory chain