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Rapid and precise quantification of the infectivity of HIV is important for molecular virologic studies, as well as for measuring the activities of antiviral drugs and neutralizing antibodies. An indicator cell line, a CCD camera, and image-analysis software were used to quantify HIV infectivity. The cells of the P4R5 line, which express the receptors for HIV infection as well as beta-galactosidase under the control of the HIV-1 long terminal repeat, were infected with HIV and then stained 2 days later with X-gal to turn the infected cells blue. Digital images of monolayers of the infected cells were captured using a high resolution CCD video camera and a macro video zoom lens. A software program was developed to process the images and to count the blue-stained foci of infection. The described method allows for the rapid quantification of the infected cells over a wide range of viral inocula with reproducibility, accuracy and at relatively low cost.
Methods Mol Biol 2009
PMID:CCD camera detection of HIV infection. 1915 43

The performance of a homemade, simple, fluorescence-induced capillary electrophoresis (CE) detector is described here. It is based on LED as excitation source, a bifurcated optical fibre as a waveguide and a CCD as a photodetector. The connection of all the components is fairly easy, even for non-experts. This detector provides a low cost and rapid system for the determination of high-quantum-yield native fluorescence compounds and fluorescence derivatised compounds by CE with direct fluorescence determination. R-phycoerythrin and B-phycoerythrin were used as models for native fluorescence compounds and amine labelled with FITC were set as models for the fluorescence derivatised ones. Detection limits of 0.50 and 0.64 microg/mL for R-phycoerythrin and B-phycoerythrin and 1.6 x 10-(7) M for FITC-labelled 1,6-diaminohexane were achieved. The homemade LED-IF detector is not expected to displace the LIF-IF one, but offers another possibility and a cheaper way to solve simple analytical problems for determining biomolecules.
Methods Mol Biol 2009
PMID:Simple luminescence detector for capillary electrophoresis. 1915 44

Filters are a critical element in fluorescence detection used by many biosensors. One of the main limitations of the conventional optical filters used in biosensors is that they are limited to a single wavelength operation while numerous wavelengths are used in a typical fluorescence detection used for biosensing. Acousto-optic tunable filters (AOTFs) have the potential to overcome this limitation and provide both spectral and polarization information because they are wavelength agile and polarization sensitive. Such filters can be used to develop compact hyperspectral/polarization imagers. Such an imager can be readily used for real-time two-dimensional spectral imaging applications. These imagers are small, vibration-insensitive, robust, remotely controlled, and programmable and can be used in the spectral region from the ultraviolet (UV) to the near infrared (NIR). A minimal amount of data processing is required for AOTF imagers because they can acquire images at only select wavelengths of interest, and the selected wavelengths can be changed based on the sensing requirements. We use AOTFs made of KDP, MgF2, and TeO2, with a Si-based CCD camera to cover different spectral regions from the UV to the NIR. A liquid crystal variable retarder (LCVR) is used to obtain two orthogonally polarized images at each wavelength The user can write software to control the operation and image acquisition for an AOTF imager for a fully computer controlled operation.
Methods Mol Biol 2009
PMID:Biosensors technologies: acousto-optic tunable filter-based hyperspectral and polarization imagers for fluorescence and spectroscopic imaging. 1915 48

Electrochemiluminescent (ECL) arrays containing polymer ([Ru(bpy)(2)(PVP)(10)](2+), PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H(2)O(2)-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 approximately chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.
Mol Biosyst 2009 Feb
PMID:Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays. 1915 62

An increasing number of patients are suffering from allergic diseases such as rhinoconjunctivitis, atopic eczema, uticaria, anaphylaxis, and food and drug allergies. Although it is possible to measure a multitude of allergen-specific IgE antibodies by radio or enzyme immunoassays in the patients' blood, these tests are expensive, time-consuming, and usually need a rather high volume of reagent solutions (allergens and blood). Protein microarrays offer the possibility to circumvent these limitations. The described in vitro allergy testing system is based on microscopic glass slides activated with glycidyloxypropyl-trimethoxysilane. Allergen solutions (allergen extracts and/or purified allergens; approximately 10 nL) are printed on the activated glass surface with a piezoelectric spotting machine. The protein components of the allergen solutions are immobilized on the modified glass surface via hydrophobic interaction and/ or covalent binding. After a blocking step, the slides are incubated with the respective diluted serum sample (approximately 25 microL serum required) and bound IgE antibodies are detected with a secondary horseradish peroxidase (HRP) labelled anti-human-IgE antibody via chemiluminescence. The measurement can be performed automatically with the so called PASA system. Test results are directly visualized with a CCD-camera. Analytical and clinical data have shown that the microarray-based test format offers significant advantages in time and costs compared with traditional test formats. The described allergen microarray demonstrated a sufficient qualitative reproducibility and enabled the distinction between allergic and non-allergic patients. Detection limits of 0.35 kU/L (r Bet v1), 0.16 kU/L (PLA2), 1.9 kU/L (Der p1), and 41 kU/L (total IgE) were achieved.
Methods Mol Biol 2009
PMID:Detection of known allergen-specific IgE antibodies by immunological methods. 1921 17

This chapter introduces the demonstration of specific antibody detection by using a microbead-based assay with quantum dot (QD) fluorescence on a polydimethylsiloxane (PDMS) microfluidic chip. The microfluidic chip is designed to isolate a single microbead where the binding reaction of antibodies occurs on the surface. The microfluidic chip is fabricated on a glass substrate using a transparent silicone elastomer, PDMS, for easy access of monitoring and flexible gate operations to capture the single microbead. For antibody detection, a sequence of functionalized assays has been performed in the fabricated chip, including the capturing of microbeads, antibody injection into a microchamber, quantum dot injection, and fluorescence detection. Various concentrations of human IgG antibodies have been introduced to bind to a single microbead captured and isolated inside a designated microchamber in a small volume of 75 pL. Fluorescence detection is monitored using a CCD camera after the second binding with the QDs conjugated with anti-human IgG. In this experiment, a human IgG antibody concentration below 0.1 microg/mL has been successfully detected.
Methods Mol Biol 2009
PMID:Microfluidic chips designed for measuring biomolecules through a microbead-based quantum dot fluorescence assay. 1948 93

The polyphenols in fruits and vegetables may be partly responsible for the health-promoting effects attributed to fruit and vegetable intake. Although their properties have been relatively well studied, the activity of their metabolites, produced after ingestion, has been poorly investigated. Thus, the aim of this work was to study the potential anti-inflammatory effect of 18 polyphenol metabolites, derived from colon microbiota. They were screened by measuring prostaglandin E(2) (PGE(2)) production by CCD-18 colon fibroblast cells stimulated with IL-1beta. Metabolites that inhibited more than 50% PGE(2) production were hydrocaffeic (HCAF), dihydroxyphenyl acetic (dOHPA), and hydroferulic acid (HFER), that subsequently were tested with the writhing and paw pressure test in rodents where all three compounds showed an anti-inflammatory effect. The effect of HCAF administered orally (50 mg/kg) was also tested in the dextran sodium sulfate (DSS)-induced colitis model. Weight loss and fecal water content were more pronounced in DSS rats than in DSS-HCAF treated rats. HCAF treatment diminished the expression of the cytokines IL-1beta, IL-8, and TNF-alpha, reduced malonyldialdehyde (MDA) levels and oxidative DNA damage (measured as 8-oxo-2'-deoxyguanosine levels) in distal colon mucosa. These results indicate that HCAF, dOHPA, and HFER have anti-inflammatory activity in vitro and in vivo.
Mol Nutr Food Res 2009 Aug
PMID:Polyphenol metabolites from colonic microbiota exert anti-inflammatory activity on different inflammation models. 1955 20

Pyrosequencing is a real-time DNA sequencing method. It is based on the transformation of pyrophosphates, released during DNA elongation by DNA polymerase, into measurable light. During DNA elongation, a single pyrophosphate molecule is released following incorporation of a single nucleotide. In the pyrosequencing reaction, released pyrophosphates are then rapidly converted by sulfurylase to adenosine triphosphate, which in turn is utilized by luciferase to produce light. Within standardized conditions, this reaction is accomplished in a few milliseconds and the light produced can be registered with a CCD camera. Therefore, it becomes possible to quantitatively measure the nucleotides incorporated. This approach has been automated in different platforms and can be used for a wide variety of applications, such as single-nucleotide polymorphism (SNP) genotyping, DNA sequencing, loss of heterozygosity analysis, and CpG methylation studies. Here we describe the entire process, focusing our attention on SNP genotyping, and giving examples of some other applications.
Methods Mol Biol 2009
PMID:Pyrosequencing for SNP genotyping. 1976 90

Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1/2-dependent. Aldosterone induced the rapid activation of ERK1/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1/2 was inhibited in cells suppressed in the expression of PKD1.
J Steroid Biochem Mol Biol 2010 Jan
PMID:Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation. 1980 26

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.
Mol Nutr Food Res 2010 Mar
PMID:The cytotoxic effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases. 1988 48


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