Phospholipid transfer protein is present in human tear fluid

The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 +/- 2.4 microg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 +/- 1.8 micromol mL(-)(1) h(-)(1), was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 degrees C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer.     

Nested double PCR法对献血者血浆中HCV-RNA的检出——兼评作为献血筛选试验的Anti-HCV抗体检查

本文报道了在位于5’端非编码区的引物的诱导下用Nested double PCR技术检测献血者血浆中HCV-RNA的方法。我们发现Nested double PCR敏感性及特异性均优于单次PCR。anti-HCV(C100-3)阳性血浆样品中只有35.2％(19/54)能同时被证实为PCR阳性。由于anti-HCV(C100-3)假阳性率太高,作为献血筛选试验检测方法不能令人满意,而本文报导的Nestel double PCR方法可以弥补anti-HCV试验的不足。     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

Analysis of human tear fluid by Fourier transform infrared spectroscopy

The purpose of this research is to find some useful spectroscopic factors in human tear fluid contents to monitor diurnal changes of the physicochemical ocular conditions noninvasively. All tear fluid samples were collected with glass microcapillary tubes from both eyes of three donors and analyzed by Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). We measured the peak intensities at 2852, 1735, 1546, and 1242 cm(-1), and the peak intensity ratios among those peaks in the second derivative spectra. We found significant diurnal and individual variations in those peak intensities for tear fluid obtained from right and left eyes. Among these variations, we observed significant changes in tear samples between right and left eyes. In this case the peak intensity ratio between 1242 (phosphate ester) and 2852 cm(-1) (fatty acid methylene) of right eye tear fluid was increased in the afternoon (1600 to 1900 h), while that of left eye tear fluid did not change significantly. In the ratio between 1242 (phosphate ester) and 1546 cm(-1) (amide II), the difference was not observed between both eyes. We conclude that the difference in diurnal variations of biochemical constituents between right and left eye tear fluids could be monitored noninvasively and nondestructively by FTIR technique and this method could be useful in the future for tear diagnoses.     

DNA persistence after treatment of Lyme borreliosis

One hundred twenty-four patients—53 with neuroborreliosis, 48 with erythema migrans, and 23 with Lyme arthritis—were tested in a prospective study for the presence of the DNA of Borrelia burgdorferi sensu lato in plasma, cerebrospinal fluid (CSF), urine, and synovial fluid by nested polymerase chain reaction (PCR). Specific DNA was detected using five amplification systems simultaneously: three targeted chromosomal genes encoding 16S rDNA, flagellin, and p66; and two plasmid sequences of OspA and OspC. Patients were examined clinically and by PCR before and after treatment and again after 3 and 6 months. Before treatment, the specific DNA was detected in 78 patients (62.9 %). Forty-one neuroborreliosis patients were DNA-positive (77.4 %), with CSF positivity in 26 patients, urine in 25, and plasma in 16. Twenty-six erythema migrans patients were DNA-positive (54.2 %), with plasma positivity in 18 cases and urine in 14. Eleven Lyme arthritis cases (47.8 %) were DNA positive (six in urine, five in plasma, and four in synovial fluid). The frequency of PCR positives was comparable in CSF and urine, and it was lower by approximately 50 % in plasma. Specific DNA was also found in a significant number of patients in later testing periods: 48 patients after treatment, 29 patients after 3 months, and 6 patients after 6 months. The prolonged PCR positivity was not explainable by persistent infection according to the clinical manifestations of the disease. Possible explanations of the problem are discussed.     

盘羊肺炎支原体感染PCR 检测方法的建立与初步应用

羊支原体性肺炎,是由支原体引起的一类高度接触性传染病,又称羊传染性胸膜肺炎(Infections pleuropneumonia of sheep and goats),临床症状主要表现为高热、咳嗽、消瘦、     

Interaction of phospholipid transfer protein with human tear fluid mucins

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.     

Species differences in tears; Comparative investigation in the chimpanzee (Pan troglodytes)

This paper describes evolutionary divergence in composition of tear fluid among some mammals, and discusses the implications of these differences with regard to the choice of appropriate animals for use as models in ophthalmic research. For the first time a comprehensive investigation of tear fluid in the chimpanzee (Pan troglodytes) is presented in which tear fluid was collected during narcosis of eight chimpanzees. Total protein in chimpanzee tear fluid (8.8±0.3 g/l) is not significantly different from total protein of human tear fluid (10.0±0.6 g/l). The values in tear fluid for lysozyme (6.2±1.5 mg Hen Egg Lysozyme, HEL/ml), peroxidase (115±18 U/ml), and amylase (3.5±0.4 U/ml) in chimpanzees were significantly different from those of human lysozyme (11.8±1.6 mg HEL/ml), peroxidase (70±5 U/ml), and amylase (1.0±0.2 U/ml). Polyacrylamide gelelectrophoresis of tear fluid of the chimpanzees shows in comparison with human tear fluid an additional low-molecular protein (<14 kiloDalton).     

Erythropoietin levels in human tear fluid

Erythropoietin level was evaluated in blood plasma and tear fluid of humans with normal functions of eye and normal blood characteristics. We examined 21 patients. Principle ability of erythropoietin level detection in patient's tear fluid ascertained.     

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

The T-cell receptor Vβ repertoire in naive and human immunodeficiency virus-1 (HIV-1) infected chimpanzees

Using a panel of human T-cell receptor (TCR) variable region β chain (Vβ) polymerase chain reaction (PCR) primers, we performed cross-sectional and longitudinal analyses of the TCR Vβ repertoire in naive and HIV-1 infected chimpanzees. We demonstrate that our TCR PCR primer panel will support amplification of chimpanzee cDNA from most of the TCR Vβ families. However, no differences in TCR Vβ expression were found between the naive and HIV-1 infected chimpanzees, unlike the TCR Vβ repertoire perturbation found in HIV-1 infected human subjects. This finding suggests that a complete TCR repertoire in HIV-1 infected chimpanzees is associated with the maintenance CD4 + T-cell numbers and lack of progression to AIDS.     

Microelements in seminal plasma of infertile men infected with Ureaplasma urealyticum

The purpose of this study was to assess the association among male infertility, infection of Ureaplasma urealyticum (Uu), and microelements in seminal fluid. Semen analysis and cultivation of Uu were carried out on 160 samples of seminal fluid. The concentrations of microelements, such as arsenic (As), molybdenum (Mo), magnesium (Mg), lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn) in the samples were measured by an inductively coupled plasma quantometer (ICP). The ratios Cu/Zn and Cd/Zn in the poor spermatic quality group were obviously higher than those in seminal plasma of the group with normal spermatic quality (p<0.01 and p<0.05, respectively), whereas the concentrations of As, Mg, Mo, and Pb showed no difference in the two groups. The ratios Cu/Zn and Cd/Zn and the concentrations of As and Mg in seminal plasma infected with Uu were markedly higher than those not infected with Uu (p<0.05, p<0.01, p<0.05, and p<0.05, respectively), whereas the concentrations of Mo and Pb showed no statistical difference. The ratios Cu/Zn and Cd/Zn and the concentrations of As and Mg in seminal plasma of the semen with poor spermatic quality and Uu infection were obviously higher than those not infected with Uu (p<0.05), whereas the concentrations of Mo and Pb showed no statistical difference. Abnormally high ratios Cu/Zn and Cd/Zn as well as an overdose of As were found to be predisposed to Uu infection. Uu infection resulted in an increase of the ratios Cu/Zn and Cd/Zn and the concentrations of As and Mg in seminal fluid, which therefore caused spermatic quality decline.     

Polymorphic microsatellite DNA markers from the Oriental Fruit Fly Bactrocera dorsalis (Hendel)

Six polymorphic microsatellite loci are isolated from the Oriental fruit fly Bactrocera dorsalis (Hendel), an agricultural pest in Asia, including Taiwan. To assess their potential utility as high‐resolution genetic markers, polymerase chain reaction (PCR) primers, amplification conditions, and an automated fluorescence detection protocol were developed. In analyses of 71 individual flies from six different areas of Taiwan, allele numbers ranged from five to 25 were detected for each locus. The observed heterozygosity ranged between 0.268 and 0.737 among these loci. No linkage disequilibrium was found. These microsatellite markers have potential utility to population structure and gene flow studies of B. dorsalis (Hendel).     

In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

纳米金对PCR扩增效率影响的机制探讨

近年来,纳米金粒子对聚合酶链式反应（polymerasechainreaction,PCR）的增效作用倍受关注,但是其具体的机制仍未明确提出。研究发现在PCR反应中,纳米粒子的增效作用是存在最佳浓度的,增加DNA聚合酶或者小牛血清蛋白（BSA）可以消除纳米金粒子导致的抑制。我们认为,纳米金粒子可能起到了类似于聚合酶B亚基的作用,提高了DNA聚合酶的延伸能力;而过量纳米金对PCR的抑制作用可能与纳米金结合单链DNA产生的位阻效应有关。     

Development of a PCR/Ligase Detection Reaction/Nanogold-Based Universal Array Approach for the Detection of Low-Abundant DNA Point Mutations

The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5′-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.     

聚合酶链反应技术检测禽网状内皮组织增殖病病毒

目的建立聚合酶链反应(PCR)技术检测禽网状内皮组织增殖病病毒(REV)的方法。方法提取感染REV-T和脾坏死病毒(SNV)的SPF鸡胚成纤维细胞DNA为模板,利用前病毒长末端重复序列(LTR)区引物进行扩增。采集肿瘤病鸡,以及人工感染REV 28 d后鸡肝脏、脾脏、肾脏、心脏、胸腺、法氏囊等器官,进行扩增。同时将采集的脏器组织,进行HE染色和免疫组化试验(IHC)。结果REV-T感染的组织未检测出电泳条带,而SNV感染的细胞中检测到了一条300bp特异而清晰的电泳条带,而且SNV感染的鸡组织中,PCR方法检测到了特异的条带。通过HE染色和免疫组化技术观察到了肿瘤组织,肿瘤细胞的形态、分布。结论PCR检测REV更快捷,特异更好。     

Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance During the Purification Of Mammalian Cell Substrate Derived Biotherapeutics

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.