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Fluorescence Microscope (fluorescence + microscope)
Selected Abstracts5-Aminolevulinic Acid-Based Photodynamic Therapy in Leukemia Cell HL60,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2004Su-Juan Zhang ABSTRACT A study to explore the optimal experimental parameters and the photosensitization of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) in promyelocytic leukemia cell HL60 has been conducted, in which HL60 cells and their control groups, peripheral blood mononuclear cell (PBMC), first are incubated with different concentrations of ALA in dark for different periods of time and then followed by irradiating with different wavebands for different fluences. Fluorescence microscope and spectrofluorometer have been used to detect the fluorescence of protoporphyrin IX (PpIX) endogenously produced by ALA. The response of the cells to ALA-PDT was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay (interval between irradiation and the MTT assay is 24 h) and by flow cytometry (the length of time between irradiation and the flow assay is 30 min). MTT results will reflect the relative number of metabolically active mitochondria in the population. Propidium iodide uptake in flow cytometry will test for membrane damage. The results of parameter experiments were obtained: 1 × 105/mL HL60 cell was first incubated with 1 mmol/L ALA in dark for 4 h and the maximum fluorescence of PpIX level appeared; then irradiated with 410 nm (4 mW/cm2) for 14.4 J/cm2 and maximum photodamage to membrane and mitochondrial function of HL60 cell resulted. With the normal granulocytes, such response was not detected. Therefore a hypothetical idea can be brought forward that ALA-based PDT can be used for inactivation of leukemia cell HL60 and these optimal parameters may be useful for clinical application. [source] Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survivalDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2009Laura Lossi Abstract Apoptosis can be modulated by K+ and Ca2+ inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca2+ signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K+/Ca2+ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K+]e and [Ca2+]i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K+]e under conditions of immaturity, is independent of extracellular Ca2+ and operates via IP3 channels. The second leads to influx of extracellular Ca2+ following activation of ryanodine channels in the presence of physiological [K+]e, when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Development of microreactor array chip-based measurement system for massively parallel analysis of enzymatic activityELECTRONICS & COMMUNICATIONS IN JAPAN, Issue 4 2009Yosuke Hosoi Abstract Microarray chip technology such as DNA chips, peptide chips, and protein chips is one of the promising approaches for achieving high-throughput screening (HTS) of biomolecule function since it has great advantages in feasibility of automated information processing due to one-to-one indexing between array position and molecular function as well as massively parallel sample analysis as a benefit of downsizing and large-scale integration. Mostly, however, the function that can be evaluated by such microarray chips is limited to affinity of target molecules. In this paper, we propose a new HTS system and enzymatic activity based on microreactor array chip technology. A prototype of the automated and massively parallel measurement system for fluorometric assay of enzymatic reactions was developed by the combination of microreactor array chips and a highly sensitive fluorescence microscope. Design strategy of microreactor array chips and an optical measurement platform for the high-throughput enzyme assay are discussed. © 2009 Wiley Periodicals, Inc. Electron Comm Jpn, 92(4): 35,41, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10056 [source] Investigating DNA migration in pulsed fields using a miniaturized FIGE systemELECTROPHORESIS, Issue 23 2008Xiaojia Chen Abstract PFGE is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here, we present a simple miniaturized FIGE apparatus capable of separating DNA fragments up to 32.5,kb in length within 3,h using a modest applied potential of 20,V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N,0.5 over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively. [source] Fast quantitative determination of diuretic drugs in tablets and human urine by microchip electrophoresis with native fluorescence detectionELECTROPHORESIS, Issue 16 2007Kamal Tolba Abstract Microchip electrophoresis (MCE) with native fluorescence detection has been applied for the fast quantitative analysis of pharmaceutical formulations. For this purpose, methods for fast separation and sensitive detection of the unlabeled diuretic drugs, amiloride, triamterene, bendroflumethiazide (BFMTZ), and bumetanide were developed. An epifluorescence setup was used enabling the coupling of different lasers into a commercial fluorescence microscope. The detection sensitivity of different excitation light sources was compared utilizing either a HeCd laser (,exc,=,325,nm), a frequency quadrupled Nd:YAG laser (,exc,=,266,nm), or a mercury lamp (,exc,=,330,380,nm). At optimal conditions using the HeCd laser, the drugs were separated within 15,s with LODs less than 1,,g/mL for the four compounds. A linear relationship between concentration and peak area was obtained in the concentration range of 0.05,20,,g/mL with a mean correlation coefficient of around 0.996 for all analytes. The method was successfully applied to the analysis of the respective drugs in commercial formulations and in human urine without interference from other constituents. These data show that MCE has a great potential for reliable drug analysis. [source] Impact of Kerogen Heterogeneity on Sorption of Organic Pollutants.ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2009Abstract The overall goal of the present study was to establish correlations between organic pollutant sorption and physicochemical properties of kerogen materials. Three coal samples, each representing a typical kerogen type, were used as the starting materials. A thermal technique was employed to treat the kerogen materials under seven different temperatures ranging from 200 to 500C to simulate different diagenetic history. These samples were systematically characterized for their chemical compositions, functionalities, physical rigidity, and optical properties. The results showed that the chemical, spectroscopic, and optical microscopic properties of each kerogen series changed consistently as a function of treatment temperature or kerogen maturation. The oxygen-to-carbon atomic ratio decreased from 0.29, 0.12, and 0.07 for the original lignite (XF0), fusinite (HZ0), and lopinite (LP0) samples, respectively, to 0.07, 0.06, and 0.04 for XF7, HZ7, and LP7, respectively, that underwent the highest temperature treatment. The hydrogen-to-carbon atomic ratio exhibited similar reducing trend, which is consistent with the aromaticity increasing from 45 to 58% of the original samples to 76 to 81% of highly mature samples. Under the fluorescence microscope, the organic matrix changed from yellow (original lignite sample) and red-brown (original lopinite sample) to colorless for the samples of higher maturation. The measured reflecting index increased from the original samples to the highly mature samples. Moreover, the original and the slightly matured samples exhibited very different chemical compositions and structural units among the three types due to the difference in their source materials. As the kerogen maturation increased, such differences decreased, indicating highly mature kerogen became homogenized regardless of the source material. [source] Effects of tumour necrosis factor alpha and interleukin-6 on progesterone and calcium ionophore-induced acrosome reactionINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2009F. Lampiao Summary For human spermatozoa to successfully fertilize the oocyte, they need to undergo a timely acrosome reaction (AR). Factors which disturb the AR may lead to fertilization failure. The objective of this study was to investigate the effects of two cytokines namely tumour necrosis factor alpha (TNF-,) and interleukin-6 (IL-6) on the spontaneous, calcium ionophore-induced and progesterone-induced human sperm AR. Twenty-two normal semen samples were treated with increasing concentrations of TNF-, and IL-6 after spermatozoa were isolated by a double wash swim-up method. The AR was induced by calcium ionophore A23187 and progesterone. The AR was determined by using fluorescein isothiacyanate Pisum sativum agglutinin and observed under fluorescence microscope. Both TNF-, and IL-6 could decrease the spontaneous, ionophore and progesterone-induced AR (p < 0.05) in a dose-dependent manner. TNF-, showed a more potent inhibiting effect than IL-6 by inhibiting the AR at lower concentrations. This study has demonstrated that TNF-, and IL-6 play a role in inhibiting both the non-physiological as well as physiologically elicited AR by calcium ionophore and progesterone respectively. [source] Effect of an enamel matrix protein derivative (Emdogain®) on ex vivo dental plaque vitalityJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 11 2001Anton Sculean Abstract Background: A common clinical observation following surgical periodontal therapy with an enamel matrix derivative (Emdogain®) is the improved healing of the soft tissues and the limited inflammation of the operated areas. These clinical observations are empirical and difficult to explain. One of the factors influencing the early wound healing might be a potential antimicrobial effect of Emdogain®. Aim: To investigate the effect of Emdogain® on the vitality of ex vivo supragingival dental plaque and to compare this effect to that of a standard 0.2% chlorhexidine solution. Materials and Methods: 24 patients suffering from adult periodontitis were included in the study. At the beginning of the experiment, all participants were given a professional tooth cleaning. For the following 4 days, they had to refrain from any kind of oral hygiene measures. At day 5, from each of the volunteers, a voluminous plaque biofilm sample was taken with a sterile curette from the vestibular surfaces of the 1st lower molars and divided into 5 equal parts. Each part was mounted with 5 ,l of the following solutions: (1) NaCl, (2) enamel matrix derivative dissolved in water (EMD), (3) enamel matrix derivative dissolved in the vehicle (Emdogain®), (4) vehicle (propylene glycol alginate, PGA), (5) 0.2% chlorhexidine digluconate (CHX). After a reaction time of 2 min the test solutions were sucked off, and subsequently the biofilm was stained with a fluorescence dye. The vitality of the plaque flora after the treatments was evaluated under the fluorescence microscope (VF%). Results: Plaque samples treated with NaCl showed a mean vitality of 76.8±8%. The EMD, Emdogain®, PGA and CHX showed VF values of 54.4±9.2, 21.4±10.6%, 19.6±11.6% and 32.3±11.8%, respectively. Emdogain®, PGA and CHX showed statistically highly significant reductions (p<0.0001) in terms of bacteria vitality when compared to water (negative control) and EMD. Both Emdogain® and PGA were found to be statistically significantly different compared to CHX (p<0.001) (positive control). Conclusion: The results of this study indicate that Emdogain® might have an antibacterial effect on the vitality of the ex vivo supragingival dental plaque flora. Zusammenfassung Hintergrund: Eine allgemeine klinische Beobachtung nach parodontalchirurgischer Therapie mit einem Schmelzmatrixderivat (Emdogain®) ist die verbesserte Heilung des Weichgewebes und die begrenzte Entzündung des operierten Gebietes: Diese klinischen Beobachtungen sind empirisch und schwierig zu erklären. Ein Faktor, der die frühe Wundheilung beeinflusst, könnte ein potentieller antimikrobieller Effekt von Emdogain® sein. Ziel: Untersuchung des Effektes von Emdogain® auf die Vitalität von ex vivo supragingivaler dentaler Plaque und Vergleich dieses Effektes zu demjenigen einer Standard 0.2%igen Chlorhexidinlösung. Material und Methoden: 24 Patienten, die an einer Erwachsenen-Parodontitis litten, wurden in diese Studie aufgenommen. Zu Beginn der Studie wurde bei allen Teilnehmern eine professionelle Zahnreinigung durchgeführt. An den folgenden 4 Tagen wurden keine oralen Hygienemaßnahmen erlaubt. Am Tag 5 wurde von jedem Teilnehmer eine voluminöse Plaquebiofilmprobe mit einer sterilen Kürette von der vestibulären Oberfläche des ersten unteren Molaren genommen und in 5 gleiche Teile aufgeteilt. Jeder Teil wurde mit 5 ,l der folgenden Lösungen gemischt: (1) NaCl, (2) Schmelzmatrixderivat in Wasser gelöst (EMD), (3) Schmelzmatrixderivat in einem Vehikel gelöst (Emdogain®), (4) Vehikel (Propylenglycolalginat, PGA), (5) 0.2%iges Chlorhexidindiglukonat (CHX). Nach einer Reaktionszeit von 2 Minuten wurden die Testlösungen aufgesaugt und folgend der Biofilm mit Fluoreszenzfarbstoff gefärbt. Die Vitalität der Plaqueflora nach den Behandlungen wurde unter dem Vitalfluoreszenzmikroskop (VF%) evaluiert. Ergebnisse: Die Plaqueproben, die mit NaCl behandelt wurden, zeigten eine mittlere Vitalität von 76.8±8%. Das EMD, Emdogain®, PGA und CHX zeigten VF Werte von 54.4±9.2%, 21.4±10.6%, 19.6±11.6% und 32.3±11.8%. Emdogain®, PGA und CHX zeigten statistisch signifikant höhere Reduktionen (p<0.0001) in Beziehung zur bakteriellen Vitalität, wenn zu Wasser (negative Kontrolle) und EMD verglichen wurde. Sowohl Emdogain® und PGA waren statistisch signifikant unterschiedlich zu CHX (p<0.0001) (positive Kontrolle). Schlussfolgerung: Die Ergebnisse dieser Studie zeigten, dass Emdogain® einen antibakteriellen Effekt auf die Vitalität von supragingivaler dentaler ex vivo Plaqueflora haben könnte. Résumé Origine: Une observation clinique courante durant un traitement parodontal chirurgical à l'aide de protéines de la matrice améllaire (Emdogain®) est une meilleure guérison des tissus mous et une inflammation moindre. Ces observations cliniques sont empiriques et difficiles à expliquer. Un des facteurs influençant la guérison précoce peut être un effet antimicrobien de l'EMD. But: Le but de cette étude a été d'évaluer l'effet de l'Emdogain® sur la vitalité de la plaque dentaire sus-gingivale ex vivo et de comparer cet effet avec une solution de chlorhexidine 2%. Matériaux et Méthodes: 24 patients souffrant de parodontite de l'adulte ont été inclus dans cette étude. Au début de l'expérience, tous les participants ont recu un nettoyage dentaire professionnel. Pendant les 4 journées suivantes, ils ont dû arrêté toute hygiène buccale. Au jour 5, une quantité de plaque dentaire volumineuse a étééchantillonné des surfaces vestibulaires des premières molaires inférieures de chaque volontaire à l'aide d'une curette stérile et divisée en 5 parts égales. Chaque partie a été montée avec 5 ,l des solutions suivantes: (1) NaCl, (2) EMD: dérivé de la matrice améllaire dissout dans l'eau (3) Emdogain®: dérivé de la matrice améllaire dissout dans son véhicule, (4) PGA: le véhicule propylène glycol alginate, (5) CHX: chlorhexidine 0.2%. Après un temps de réaction de 2 min, les solutions tests ont été aspirées et le biofilm dentaire a été imprégné d'un colorant de fluorescence. La vitalité de la flore de la plaque dentaire après ces traitements a étéévaluée sous microscopie à fluorescence (VF%). Résultats: Les échantillons de plaque traités avec NaCl possèdaient une vitalité moyenne de 76.8±8%. L'EMD, Emdogain®, PGA, et CHX avaient des valeurs VF respectives de 54.4±9.2%, 21.4±10.6%, 19.6±11.6% et 32.3±11.8%. Emdogain®, PGA, et CHX réduisaient la vitalité bactérienne de manière très hautement significative (p<0.0001) lorsque ces solutions étaient comparées aux contrôle négatif NaCl et à EMD. Tant Emdogain® que PGa étaient différents comparés au contrôle positif CHX (p<0.001). Conclusions: Les résultats de cette étude indiquent que Emdogain® pourrait avoir un effet antibactérien sur la vitalité de la flore se trouvant dant la plaque dentaire sus-gingivale ex vivo. [source] Pathology of lumbar nerve root compression Part 1: Intraradicular inflammatory changes induced by mechanical compressionJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2004Shigeru Kobayashi Study design: This study is to investigate the intraradicular inflammation induced by mechanical compression using in vivo model. Objectives: The relationship between the intraradicular edema and nerve fiber degeneration induced by mechanical compression was determined in the nerve root. Summary of background data: Recently some studies reported that mechanical compression increased microvascular permeability of the endoneurial capillaries and resulted in an intraradicular inflammation. These changes may be an important factor of the pathogenesis of radiculopathy. However, the natural courses of the intraradicular inflammation after mechanical compression are still poorly understood. Methods: In dogs, laminectomy was performed at L7 and the seventh nerve root was exposed to compression at 7.5 gram force (gf) clipping power. The animals were evaluated at 1 and 3 weeks after clipping. After the appropriate period of nerve root compression, Evans blue albumin (EBA) was injected intravenously. The nerve root sections were divided into two groups. The sections were used to investigate the status of the blood,nerve barrier function under the fluorescence microscope. The other sections were used for light and transmission electron microscopic study. Results: After 1 and 3 weeks, intraradicular edema was observed not only at the site of compression but also in the peripheral zone of a compressed anterior root and in the central zone of a compressed posterior root. The evidence of active Wallerian degeneration was also seen in the area of intraradicular edema. In addition, the nerve roots showing Wallerian degeneration were infiltrated by inflammatory cells, such as macrophages and mast cells. Conclusions: Inflammatory reaction, such as Wallerian degeneration, breakdown of blood,nerve barrier and appearance of macrophage, may be deeply involved in radiculitis arising from mechanical compression, and these factors seem to be important in the manifestation of radiculopathy. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] A capillary holder for scanning detection of capillary isoelectric focusing with laser-induced fluorescenceJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2009Katsuyoshi Takahashi Abstract A holder for a 12 cm long capillary was designed for scanning LIF detection of CIEF. The polyimide coat of a fused-silica capillary has been removed, and 1.5 mm diameter flanges have been attached near both ends. The holder is fixed on the stage of a fluorescence microscope via a translational stage, and a capillary guide is directly fixed on the microscope stage. The guide has a groove and a pressure plate for the capillary to slide in. The holder has two pulling plates with slits of 1 mm to accept the capillary just inside the flanges. The slits and the groove of the guide have been aligned. The motion of the translational stage brings the pulling plate into contact with the flange at the pulled side, and slides the capillary through the guide. The other end of the capillary is free and produces no strain on the capillary. When the motion of the stage is reversed, an unstrained contact is achieved at the other end. The baseline noise from scanning was only 50% larger than that without scanning. The fluorescence-signal variation during scanning was about 4% of the total signal, which was about twice that without scanning. [source] Application of a microfluidic device for counting of bacteriaLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2006K.-I. Inatomi Abstract Aims:, To develop a miniaturized analytical system for counting of bacteria. Methods and Results:,Escherichia coli cells were used throughout the experiments. The system consists of a microfluidic chamber, a fluorescence microscope with a charge-coupled device (CCD) camera and syringe pumps. The chamber was made of a silicone rubber (30 × 30 mm and 4 mm high). The E. coli cells were flowed from a micro-nozzle fabricated in the chamber and detected with the CCD camera. The individual cells were indicated as signal peaks on a computer. The cell counts showed a good correlation compared with that of a conventional plate counting method, and results of the simultaneous detection of live and dead cells were also presented. Conclusions, Significance and Impact of the Study:, The system having a small disposable nozzle has the advantages for low cost and safe medical or environmental analysis, when compared with a conventional flow cytometer. This is the first step of the development of a one-chip microbe analyzer. [source] Anticaries effect of compounds extracted from Galla Chinensis in a multispecies biofilm modelMOLECULAR ORAL MICROBIOLOGY, Issue 6 2008Q. Xie Introduction:,Galla Chinensis is a leaf gall known to have some antibacterial effects. Using an in vitro biofilm model of dental plaque, the present study aimed to evaluate the anticaries effects of Galla Chinensis and its chemical fractions. Methods:, A four-organism bacterial consortium (Streptococcus sanguis, Streptococcus mutans, Actinomyces naeslundii, Lactobacillus rhamnosus) was grown on hydroxyapatite (HA) discs, bovine enamel blocks, and glass surfaces in a continuous culture system and exposed to repeated solution pulses. Galla Chinensis extracts, sucrose solutions, and sodium fluoride solutions were pulsed into different flow cells. The pH value of the planktonic phase in each flow cell was recorded and the bacteria colonizing the biofilm on the HA discs were counted. Enamel blocks were observed using a polarized microscope and lesion depth was evaluated. The biofilm morphology was examined with a fluorescence microscope and the images captured were analyzed on an image analysis system. Results:, When Galla Chinensis extract, its chemical fraction, or fluoride was added to the sucrose solution, the planktonic phase pH remained higher than that in the sucrose alone. A lower level of colonization on the HA surface was also observed in the groups to which Galla Chinensis and fluoride were added compared with the control sucrose group, and this was reflected in both the total viable count and the biofilm imaging, which showed fewer cariogenic bacteria and a less compact biofilm, respectively. Enamel demineralization in both the fluoride group and the Galla Chinensis group was significantly less than that in the sucrose group. Conclusions:,Galla Chinensis and fluoride may inhibit the cariogenicity of the oral biofilm. Galla Chinensis appears to be a promising source of new agents that may prevent dental caries. [source] Apoptosis of circulating lymphocyte in rats with unilateral ureteral obstruction: Role of angiotensin IINEPHROLOGY, Issue 5 2005SOMCHIT EIAM-ONG SUMMARY: Background: Unilateral ureteral obstruction (UUO) could induce increased renal angiotensin II (ANG II), which enhances apoptosis of renal tubular cells and renal tissue loss. Systemic ANG II is also increased in UUO. There are no data available about whether UUO can induce apoptosis of circulating lymphocytes or not. Methods: UUO or sham-operated male Wistar rats (n = 8 in each group) were fed a drinking solution containing water, angiotensin II receptor type 1 antagonist (ARA; losartan, 500 mg/L) or angiotensin-converting enzyme inhibitor (ACEI; enalapril: 200 mg/L) for 1 day or 7 days. Blood samples were collected and circulating lymphocyte cells were separated. The apoptotic cells were detected by in situ terminal deoxynucleotidyl transferase (TdT assay)-mediated digoxigenin/antidigoxigenin conjugated fluorescein method and counted under a fluorescence microscope. The apoptotic index was calculated. Results: UUO caused marked increases in the apoptotic index of circulating lymphocytes in UUO rats at both 1 day and 7 days when compared with the respective sham groups (P < 0.001). Neither ARA nor ACEI treatment had an effect on the apoptotic index values in the UUO rats at 1 day. In the UUO rats at 7 days, the apoptosis of circulating lymphocytes was markedly decreased from 29.2 ± 2.7% to 11.9 ± 2.7% (P < 0.01) in the ARA-treated rats and to 7.6 ± 2.7% (P < 0.001) in the ACEI-treated rats. Conclusion: UUO, via stimulation of ANG II, could promptly enhance apoptosis of circulating lymphocytes. The apoptosis persisted throughout the 7 days of the study. Prolonged UUO would impair lymphocyte cell immunity and the host defense mechanism. Continuous treatment with either ARA or ACEI could abrogate ANG II-stimulated circulating lymphocyte apoptosis. [source] An efficient gene transfer method mediated by ultrasound and microbubbles into the kidneyTHE JOURNAL OF GENE MEDICINE, Issue 1 2005Hiromi Koike Abstract Background Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. Methods In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. Results Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70,80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 × 105 relative light units (RLU)/g tissue; 50%: 31.3 × 105 RLU/g tissue; 100%: 57.9 × 105 RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. Conclusions Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease. Copyright © 2004 John Wiley & Sons, Ltd. [source] ORIGINAL RESEARCH,BASIC SCIENCE: Immunohistochemical Description of Cyclic Nucleotide Phosphodiesterase (PDE) Isoenzymes in the Human Labia MinoraTHE JOURNAL OF SEXUAL MEDICINE, Issue 3 2007Stefan Ückert PhD ABSTRACT Introduction., Up until now, only minimal research has been carried out on those female genital organs known to contribute to the normal cycle of sexual arousal and orgasm. Some findings indicated that there might be a significance of cyclic nucleotide-mediated pathways in the control of the normal function of female genital tissues. Aim., To elucidate, by means of immunohistochemistry, the distribution of the phosphodiesterase (PDE) isoenzymes 1, 3, 4, 5, 10, and 11 in the human labia minora. Main Outcome Measures., The amount of immunohistochemical staining specific for cyclic adenosine monophosphate (cAMP)- and/or cyclic guanosine monophosphate (cGMP)-degrading PDE isoenzymes was detected. Methods., Human labial tissue was obtained from four female cadavers (age at death: 18,42 years). Vibratome sections prepared from formaldehyde-fixated tissue specimens were incubated with primary antibodies directed against the respective PDE isoenzymes. Sections were then incubated with fluorochrome (fluorescein isothiocyanate, Texas Red)-labeled secondary antibodies. Visualization was commenced by means of a laser fluorescence microscope. Results., Immunostaining indicating the expression of PDE4 and PDE5 was abundantly observed in the smooth musculature of vessels interspersing the tissue. Immunoreactions specific for PDE3 were recognized in epithelial and subepithelial layers, sebaceous glands, and interstitial or neuroendocrine-like single cells located in the epithelium. Signals related to PDE10 and PDE11 were limited to the epithelium or glandular-like structures, respectively. Conclusions., Our results, for the first time, demonstrate the presence of cAMP- and cGMP-PDE isoenzymes in the human labia minora and give a hint to a significance of PDE4 and PDE5 in the control of labial vascular tissue function. Ückert S, Oelke M, Albrecht K, Stief C, Jonas U, and Hedlund P. Immunohistochemical description of cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the human labia minora. J Sex Med 2007;4:602,608. [source] Stroma remodelling during healing of corneal surface irregularities induced by PTKACTA OPHTHALMOLOGICA, Issue 4 2007Marios Panagiotopoulos Abstract. Purpose:, To study the histopathology of the remodelling process in the stroma after excimer-laser-induced corneal irregular injuries. Methods:, Seven New Zealand white rabbits received in one eye a transepithelial plano photoablation (60 µm) and an additional plano ablation (25 µm). On the denuded stroma, an electron microscopy specimen grid was placed and another 25 µm ablation was applied to produce surface irregularities. Dichlorotriazinyl aminofluorescein (DTAF) was then applied for 45 seconds. Another seven right eyes of seven rabbits were ablated the same way but without using the grid, resulting in a plano ablation. All the rabbits were killed at weekly intervals after treatment. The harvested corneas from both eyes were further processed for haematoxylin-eosin staining and were also stained with monoclonal antibodies directed against Ki-67 antigen and ,-smooth muscle actin (,-SMA). All specimens were examined under light and fluorescence microscope. Results:, The corneal wounds were covered by epithelium during the first week. The 25 µm × 25 µm × 25 µm stromal irregularities were clearly discernible up to 3 weeks after treatment, during which time they melted and disappeared. A homogeneous zone was formed in which stroma cells laid down an initially disorganized stroma. This was sharply visible under a fluorescence microscope as a dark area between the dichlorotriazinyl aminofluorescein (DTAF) fluorescent stroma and autofluorescent epithelium. Very little response was seen in the plano-ablated wound microscopically and in terms of positive stained cells. Conclusion:, As the irregularities are flattened and the homogenous zone becomes repopulated with keratocytes forming extracellular matrix material (ECM), the cornea regains its previous architecture in both groups. The irregular wound surface promotes wound-healing reactivity, a process that allows the cornea to compensate for the irregularities and heal to a functional state. [source] PROTECTIVE ROLE OF A NOVEL ERYTHROCYTE-DERIVED DEPRESSING FACTOR ON BLOOD VESSELS OF RENOVASCULAR HYPERTENSIVE RATSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2007Huan Pang SUMMARY 1We have isolated a novel human erythrocyte-derived depressing factor (EDDF) that has a significant antihypertensive effect in various rat models of hypertension. The aim of the present study was to examine the mechanisms of action of EDDF on vascular function in two-kidney, one-clip (2K1C) renovascular hypertensive rats. 2The EDDF was prepared from human erythrocytes. Experiments were performed in 18 male Wistar rats. The vascular ring perfusion assay and a two-photon laser scanning fluorescence microscope (TMP) were used to evaluate the vascular contractile response. The effects of EDDF on phenylephrine (PE)- and noradrenaline (NA)-induced vascular contraction were evaluated in 2K1C hypertensive rats. The proliferation and DNA synthesis in vascular smooth muscle cells (VSMC) were determined using the [3H]-TdR (thymidine) incorporation and 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Flow cytometry, reverse transcription,polymerase chain reaction and western blots were used to measure cell cycle and apoptotic profiles, platelet-derived growth factor (PDGF)-A expression and the activity of extracelluar signal-regulated kinase (ERK)-1/2, as well as the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4. 3At 10,5 g/mL, EDDF significantly decreased the PE- and NA-induced hypertensive vascular contraction. In addition, EDDF inhibited DNA synthesis in primary VSMC from 2K1C rats. The mRNA expression of PDGF-A in VSMC was twofold higher in 2K1C rats compared with control rats, whereas EDDF significantly inhibited the increment in PDGF-A mRNA expression. In addition, EDDF inhibited the phosphorylation of ERK1/2 and decreased the expression of cyclin D1 and CDK4; p21 (Cip1) levels were increased after treatment with EDDF. 4In conclusion, EDDF inhibits VSMC proliferation in 2K1C rats through G0/G1 cell cycle arrest. The effects may be mediated, in part, by enhanced expression of p21 (Cip1) and the inhibition of ERK1/2 phosphorylation and the expression of cyclin D1/CDK4 and PDGF-A. [source] | |||||||||||||