姜黄薄壁细胞原位拉曼光谱研究

用拉曼光谱原位分析姜黄橙黄色及黄色薄壁细胞中的物质。德宏、广西及西双版纳姜黄三种样品橙黄色细胞的拉曼光谱非常相似,较强峰出现在1 632/1 633/1 637、1 599/1 601/1 605、1 184/1 186/1 190 cm-1,中等强度的峰出现在1 529/1 528/1 534、1 425/1 426/1 430、1 306/1 308/1 311、1 235/1 235/1 239、1 167/1 168/1 173、1 122/1 125/1 130、967/969/975 cm-1。三种姜黄薄壁细胞中出现的强峰、次强峰位置、峰型都一致,说明三种姜黄橙色薄壁细胞中物质的主要成分相同。与姜黄素(curcumin)拉曼光谱的主要的19条谱线比较,在姜黄橙色细胞拉曼谱的17条谱峰中有16条与之对应。而黄色薄壁细胞中大部分谱线与支链淀粉的谱峰有较好的对应关系。用密度泛函理论计算了姜黄素的拉曼光谱,并对谱线进行了初步的归属。     

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。     

体外联合使用指纹区及高波数区拉曼光谱诊断胃癌

目的:研究正常胃粘膜和胃癌组织在拉曼光谱指纹区(800-1 800 cm-1)和高波数区(2 800-3 000 cm-1)的光谱特征,并将其联合使用建立胃癌诊断模型。方法:收集38例正常胃粘膜和37例胃癌组织活检标本,采用785 nm激发光拉曼光谱仪进行拉曼光谱采集。比较正常胃粘膜和胃癌组织在指纹区和高波数区的拉曼光谱异同,使用偏最小二乘判别分析(PLS-DA)结合留一法交叉验证建立诊断模型。结果:1)胃癌组织在853 cm~(-1),879cm~(-1),1 003 cm~(-1),1 047 cm~(-1),1 173 cm-1,1 304 cm~(-1),1 319 cm~(-1),1 338 cm~(-1),1 374 cm-1、2 932 cm-1谱峰处与正常胃粘膜的拉曼峰强度差异有统计学意义(P0.05)2)将拉曼光谱指纹区和高波数区联合,利用PLS-DA建立胃癌诊断模型的敏感性为94.59%(35/37),特异性为86.84(33/38),正确率为90.6%(68/75)。结论:正常胃粘膜和胃癌组织在拉曼光谱指纹区和高波数区均有显著差异,将上述两区联合使用建立模型诊断胃癌能取得良好的诊断效果。     

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。     

二甲基亚砜对离体猪皮肤组织的拉曼光谱的影响

拉曼光谱技术作为鉴定生物分子种类最有力的分析工具之一,具有快速、简单、无损、准确等优点.目前,拉曼光谱已被国内外学者广泛开展了在人体组织的应用研究,但由于生物组织具有高散射性,因此限制了拉曼光谱对其的检测深度.本文主要采用光透明剂--二甲基亚砜对组织拉曼光谱的影响进行研究,对离体猪皮组织的不同深度（100 μm,200 μm,300 μm,400 μm）拉曼光谱强度随处理前、后不同时间（0 min,10 min,20 min,30 min,60 min）的变化进行对比.结果发现,不管是经二甲基亚砜处理前还是后,都出现随着距猪皮组织表面的深度加深,其拉曼光谱强度都不断减少;同时发现各层猪皮组织随着处理后时间的加长,其特征峰的强度不断加强,信噪比逐渐提高,在处理后的60 min效果最好;而且出现了在经二甲亚砜处理前看不见的峰（1 126 cm-1和1 426 cm-1）.结果表明：5%DMSO对组织的处理能增强其拉曼光谱的强度,同时也能使拉曼光谱仪的信噪比提高,并且使组织谱图中特征峰也得到相应的增加.     

皮肤组织显微共聚焦拉曼光谱成像研究

显微共聚焦拉曼光谱成像技术(Confocal Raman Microspectroscopy Imaging,CRMI)能够对样品微区进行精确无损的拉曼光谱分析和光谱图像扫描,提供生物样品的无损高分辨光学信息。本项研究工作,利用CRMI技术实验获取了正常人体离体皮肤组织的拉曼光谱特征,并结合典型特征峰的扫描图像,探讨了脂类、蛋白质等成分在皮肤真皮层的分布特点。实验发现皮肤组织真皮层内胶原蛋白的拉曼特征峰1 248 cm-1强度及其空间分布尤为突出,这一实验结果与组织学中胶原纤维占真皮结缔组织95%的事实相符。实验结果显示,CRMI技术能够全面诠释生物组织内部生化组成与分布信息,在实验描述皮肤组织病理变化的分子生物学机制方面具有广阔的应用前景。     

利用PCA-LDA和参数-CART方法对直肠癌血清拉曼光谱的分析

本文利用拉曼光谱技术检测由直肠癌的发生导致的血清成分的变化。比较了直肠癌患者和对照组之间血清拉曼光谱的差异,并对术后直肠癌患者血清拉曼光谱的变化也进行了比较,以监测术后治疗效果。结果表明在某些波数位置不同组的拉曼峰有统计学意义的变化,这些变化反应了血清中相应的生物物质的改变。之后,主成分分析(PCA)及峰强比参数这两种方法被用于原始拉曼光谱的特征变量的提取。将线性判别分析(LDA)和分类回归树(CART)两种判别分析法用于特征变量的判别分析。PCA-LDA和参数-CART方法的诊断准确率分别为87%和90%。     

近红外光谱测定小麦粉常规营养成分的模型优化

目的：应用近红外光谱（NIR）结合偏最小二乘法（PLS）建立小麦粉常规营养成分蛋白质、水分和脂肪的含量预测模型,并选择最佳模型。方法：收集117份小麦粉样品的近红外光谱,化学法测定蛋白质、水分和脂肪的含量,利用主成分分析（PCA）随机分组,81份样品用于构建模型、36份样品用作验证模型的预测能力。探讨波长范围和光谱预处理方法对所建模型预测能力的影响。结果：3个营养成分预测能力最好的模型分别是：对于蛋白质,预处理采用矢量归一化（SNV）,波长选取7 505.9~5 446.2 cm-1和4 605.4~4 242.8 cm-1,预测模型的RPD值是7.02;对于水分,无预处理,波长选择全谱12 800~3 960 cm-1,模型的RPD值是6.83;对于脂肪,无预处理,波长在9 000~4 000 cm-1,模型的RPD值是5.06。结论：近红外光谱法可以实现对小麦粉常规营养成分的快速预测,通过选择波长范围和光谱预处理方法可以显著提高模型的预测能力。     

菹草红色类胡萝卜素的拉曼光谱特性研究

用MgO 硅藻土 (1∶2 ,w/w)柱层析和硅胶G薄层层析 (TLC)从菹草中分离纯化了三种红色素r1、r2和r3,其极性大小排序依次为 :虾青素 >r1>r2 >r3>角黄素 >β 胡萝卜素。以虾青素和 β 胡萝卜素为对照 ,运用显微拉曼技术对它们作了测试分析研究。在拉曼光谱的“指纹区”(<180 0cm-1) ,r1、r2和r3都在 15 2 7cm-1处有最强的特征谱线 ,对应的是c=c伸缩振动 (υc=c) ,r1在 116 2cm-1、r2和r3在 115 7cm-1有较强的特征谱线 ,对应的是c c伸缩振动(υc c) ,r1、r2和r3在 10 0 7cm-1有较强的特征谱线 ,对应的主要是甲基与碳链之间的摇摆振动 ;r1、r2和r3与虾青素及 β 胡萝卜素在 2 0 0 0— 4 0 0 0cm-1区也有很好的对应关系。实验结果充分证明菹草红色素r1、r2和r3属于类胡萝卜素化合物 ,r1、r2和r3之间及与虾青素和 β 胡萝卜素的拉曼光谱都存在差异 ,说明它们的细微结构不同 ,其具体结构有待进一步研究。     

生物组织拉曼光谱数据的统计分析

拉曼光谱技术在生命科学领域已取得较为广泛的研究与应用,如何从低信噪比、质量差的拉曼光谱信号提取并充分利用谱图中所含信息,对光谱后续分析、样品的归类等至关重要.本文首先明确了生物组织拉曼光谱统计分析中几个重要的概念,进而对拉曼光谱数据的预处理,光谱分析研究中较为常用的几种多元统计方法进行了归纳、对比与分析.     

毛姜花油细胞原位拉曼光谱研究

为避免复杂的样品的制备及提取过程,最大限度避免精油活性成分变化,常温下,用拉曼光谱原位分析毛姜花油细胞中精油。样品切片后置于共聚焦显微拉曼光谱仪下,用10倍物镜可观察到油细胞。油细胞精油的拉曼光谱与1,8-桉油精拉曼光谱非常相似。以毛姜花油细胞/1,8-桉油精的拉曼峰为序,较强峰出现在2928/2 921、647/652 cm~(-1),次强峰出现在540/545、808/813、915/920、926/930、1 012/1 016、1 075/1 080、1 270/1273、1 427/1 432 cm~(-1)。在油细胞中出现的强峰、次强峰与1,8-桉油精的拉曼峰一致,说明毛姜花油细胞中油的主要成分为1,8-桉油精。毛姜花油细胞的25条拉曼峰都与1,8-桉油精的拉曼峰有很好的对应关系。     

The Resonance Raman Spectra of β-Carotene Molecules in the Photosystem Ⅱ Reaction Center D1/D2/Cyt b-559 Complex

The resonance Raman spectrum of β-carotene in photosystem Ⅱ (PS Ⅱ )reaction center complex was characterized by four main bands, peaking at 1532 (νl), 1156 (ν2), 1010 (ν3) and 970 (ν4) cm -1, respectively, with several additional small Raman bands in the region between 1100 cm-1 and 1500 cm-1 It was suggested that β-carotene molecules of the reaction center complex were in all-trans configuration. The resonance Raman spectrum of an acetone extract from the reaction center complex also showed four main bands. The peak position of νl, ν3 and ν4 band shifted 5 cm-1 to the shorter wave number. The most dramatic changes were the reduction of the intensity of ν4. From the above results it was demon- strated that the conformation of β-carotene molecules in the PS Ⅰ reaction center was not the same as that of free β-carotene molecules in solution, but similar to that of carotenoid molecules in the photosynthetic bacterial reaction center, in other words, they are likely to be in a twisted conformation.     

Effect of the G.T mismatch on backbone and sugar conformations of Z-DNA and B-DNA: analysis by Raman spectroscopy of crystal and solution structures of d(CGCGTG) and d(CGCGCG)

The Z-DNA crystal structures of d(CGCGTG) and d(CGCGCG) are compared by laser Raman spectroscopy. Raman bands originating from vibrations of the phosphodiester groups and sensitive to the DNA backbone conformation are similar for the two structures, indicating no significant perturbation to the Z-DNA backbone as a result of the incorporation of G.T mismatches. Both Z structures also exhibit Raman markers at 625 and 670 cm-1, assigned respectively to C3'-endo/syn-dG (internal) and C2'-endo/syn-dG conformers (3' terminus). Additional Raman intensity near 620 and 670 cm-1 in the spectrum of the d(CGCGTG) crystal is assigned to C4'-exo/syn-dG conformers at the mismatch sites (penultimate from the 5' terminus). A Raman band at 1680 cm-1, detected only in the d(CGCGTG) crystal, is assigned to the hydrogen-bonded dT residues and is proposed as a definitive marker of the Z-DNA wobble G.T pair. For aqueous solutions, the Raman spectra of d(CGCGTG) and d(CGCGCG) are those of B-DNA, but with significant differences between them. For example, the usual B-form marker band at 832 cm-1 in the spectrum of d(CGCGTG) is about 40% less intense than the corresponding band in the spectrum of d(CGCGCG), and the former structure exhibits a companion band at 864 cm-1 not observed for d(CGCGCG). The simplest interpretation of these results is that the conventional B-form OPO geometry occurs for only 6 of the 10 OPO groups of d(CGCGTG). The remaining four OPO groups, believed to be those at or near the mismatch site, are in an "unusual B" conformation which generates the 864 cm-1 band.(ABSTRACT TRUNCATED AT 250 WORDS)     

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm-1 shift from 1306 cm-1 in aqueous solution to 1314 cm-1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm-1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm-1; in water, a strong hydrogen-bonding solvent, 1306 cm-1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm-1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

Raman spectroscopy of interferon-induced 2',5'-linked oligoadenylates

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.     

A Raman study of low frequency intrahelical modes in A-, B-, and C-DNA.

We have obtained low frequency (less than 200 cm-1) Raman spectra of calf-thymus DNA and poly(rI).poly(rC) as a function of water content and counterion species and of d(GGTATACC)2 and d(CGCGAATTCGCG)2 crystals. We have found that the Raman scattering from water in the first and second hydration shells does not contribute directly to the Raman spectra of DNA. We have determined the number of strong Raman active modes by comparing spectra for different sample orientations and polarizations and by obtaining fits to the spectra. We have found at least five Raman active modes in the spectra of A- and B-DNA. The frequencies of the modes above 40 cm-1 do not vary with counterion species, and there are only relatively small changes upon hydration. These modes are, therefore, almost completely internal. The mode near 34 cm-1 in A-DNA is mostly internal, whereas the mode near 25 cm-1 is dominated by interhelical interactions. The observed intensity changes upon dehydration were found to be due to the decrease in interhelical distance. Polymer length appears to play a role in the lowest frequency modes.     

Protein influences on porphyrin structure in cytochrome c: evidence from Raman difference spectroscopy

To probe the details of protein heme interactions, we have developed a Raman difference spectroscopic technique, which allows reliable detection of very small, approximately equal to 0.01 cm-1, frequency differences. When this technique is applied to heme proteins, structural differences in the protein which perturb the porphyrin macrocycle may be examined by obtaining Raman difference data on the porphyrin vibrational modes which are strongly enhanced in the Raman spectrum produced with visible laser excitation. We report here Raman difference spectroscopic data on cytochromes c from 24 species. The differences in the Raman spectrum of the porphyrin between the cytochromes c of any two species are small, confirming that all of the cytochromes we have examined have the same "cytochrome fold". However, many small (0.02-2 cm-1) but systematic differences were detected which indicate structural differences among these proteins. These differences could be classified into three different groups and interpreted in terms of different types of structural variations resulting from specific differences in the amino acid sequences. First, direct interactions between near-heme residues and the porphyrin influence the electron density in the pi orbitals of the porphyrin macrocycle. Second, variation in the residue at position 92, far removed from the heme, affects the frequency of the core-size marker line at 1584 cm-1. Third, the conformation near cysteine 14 affects the shape of the Raman mode which is sensitive to the pyrrole ring substituents (approximately 1313 cm-1). From these data we conclude that there are several ways in which the protein amino acid sequence may regulate the oxidation-reduction potential and several ways in which the sequence can modify the binding site between cytochrome c and its redox partners.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.