Minutes Incubation Time Research Articles

Organotypic soft-tissue co-cultures: Morphological changes in microvascular endothelial tubes after incubation withiodinated contrast media.

Clinical complications like thrombosis or anaphylaxis have been described to go along with the intra-venous or intra-arterial injection of iodinated contrast media (CM). It has been suggested that the administration of CM affects rheological parameters and thereby causes reduced blood velocity in microvessels. In vitro studies revealed significant buckling of endothelial cells after exposure to CM reducing the lumen of vessels. The aim of this study was to test the influence of CM on three-dimensional microvascular tubules with open lumina within an organotypic soft-tissue co-culture assay in vitro. This model, which is based on the co-culture of endothelial cells and fibroblasts, allows the analysis and quantitation of different parameters of microvascular endothelial capillary structures. Human dermal fibroblasts and human dermal microvascular endothelial cells were co-cultured for 10 days. Fibroblasts were adapted to the endothelial cell medium before co-culture and allowed to proliferate as well as produce extracellular matrix. The co-cultures were exposed to three different CM, i.e., Iomeprol (Imeron 400MCT), Iodixanol (Visipaque 320) or Iohexol (Accupaque 350) for 1.5 minutes or 5.0 minutes, respectively. For this, a mixture of CM and cell culture medium in a ratio of 30% CM by volume was prepared. After fixation in methanol/acetone, the endothelial cells were immunolabeled with the endothelial marker anti-CD31 and the tubular structures were assessed morphometrically. In the organotypic soft-tissue co-cultures with fibroblasts, the endothelial cells developed three-dimensional capillary-like structures which expanded via sprouting branches. After incubation with the different CM, the numbers of endothelial tubes (p = 0.001) and their lengths (p = 0.003) were significantly lower after the 5 minutes incubation time, when compared to the 1.5 minutes incubation time. The tubular diameters were significantly reduced after 5 minutes (p < 0.001), when compared to the 1.5 minutes incubation duration. Interestingly, Iomeprol and Iodixanol induced an elongation of the tubular branches during incubation duration of 1.5 minutes (p = 0.015). However, after 5 minutes incubation, the tubular branches were drastically shorter in the presence of Iomeprol and Iodixanol than the tubular branches of the control (p = 0.007). All CM exerted a negative effect on the parameters of in vitro blood vessel development.

Enzymatic added extraction and clarification of fruit juices–A review

ABSTRACTEnzymatic treatment for juice extraction is most commonly used now a days. The enzymatic process is claimed to offer a number of advantages over mechanical-thermal comminution of several fruit pulps. Enzymes are an integral component of modern fruit juice manufacturing and are highly suitable for optimizing processes. Their main purposes are: increase extraction of juice from raw material, increase processing efficiency (pressing, solid settling or removal), and generate a final product that is clear and visually attractive. Juice extraction can be done by using various mechanical processes, which may be achieved through diffusion extraction, decanter centrifuge, screw type juice extractor, fruit pulper and by different types of presses. Enzymatic treatment prior to mechanical extraction significantly improves juice recovery compared to any other extraction process. Enzymatic hydrolysis of the cell walls increases the extraction yield, reducing sugars, soluble dry matter content and galacturonic acid content and titrable acidity of the products. Enzymatic degradation of the biomaterial depends upon the type of enzyme, incubation time, incubation temperature, enzyme concentration, agitation, pH and use of different enzyme combinations. We can conclude from the technical literature that use of the enzymes i.e. cellulases, pectinases, amylases and combination of these enzymes can give better juice yield with superior quality of the fruit juice. Pectinase enzyme can give maximum juice yield i.e. 92.4% at 360 minutes incubation time, 37°C incubation temperature and 5 mg/100 g of enzyme concentration. Whereas the combination of two enzymes i.e. pectin methyl esterase (PME) and polygalacturonase (PG) at 120 minutes of incubation time, 50°C of incubation temperature and 0.05 mg/100 gm of enzymatic concentration can give the maximum yield of 96.8% for plum fruits. This paper discusses the use of enzymes in fruit juice production focusing on the juice recovery, clarity and effect of the particular enzyme on the biochemical properties of the fruit juices.

Characterization of Crude Xylanase Produced by Edible Mushroom Pleurotus eryngii

Xylanase has been increasingly forthcoming in recent years because of their possible involvement in numerous industrial processes including bioconversion of lignocellulose derived sugars in to fuels, processing food and the paper and fiber industries. Edible mushrooms are emerging as important source of xylanolytic enzymes and this study has concentrated to produce and characterize xylanases by Pleurotus eryngii. The crude enzyme was characterized on the basis of various parameters such as incubation time, substrate specificity, substrate concentration, enzyme volume, buffer, pH, pH stability, temperature, temperature stability, and effect of various metal ions or compounds. The xylanase activity was noted maximum at 15 minutes of incubation time, 2.0% xylan and 0.5 ml enzyme volume. The highest enzyme activity was found at pH 4.5, whereas xylanase exhibited maximum stability in the range of pH 4.0 to 10.0. The maximum xylanase activity was noted at 60°C, while enzyme was most active and retains more than 40% activity at 90°C within 10 minutes of incubation. ZnCl2 (10mM) stimulated the xylanase activity as compare to other metal ions or compounds. It is concluded that Pleurotus eryngii is capable to produce pH stable and thermostable xylanase for industrial purposes.

Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

Recombinant Factor VIIa (rFVIIa) Mediated Activation of Prothrombin Complex Concentrates (PCCs). Studies on the Comparison of Novoseven with a Biosimilar Product

IntroductionRecombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for same indication (AryoSeven™, AryoGen, Tehran-Iran). The two products exhibit comparable compositional and biochemical profile. To compare the activation profile of NovoSeven® and AryoSeven™, two commercially available protein complex concentrates (PCCs) were used. The activation profiles were studied utilizing SDS- gel electrophoresis profile and immunoblotting using anti-rabbit recombinant thrombin antibody.Materials & MethodsNovoSeven®, AryoSeven™, Profilnine® SD (Grifols Biologicals Inc. USA), and Beriplex® (CSL Behring Canada, Inc.) were obtained commercially. Recombinant human thrombin antibodies were generated in rabbits. Recothrom® was commercially obtained (Zymogenetic, Seatle,Wa- USA and Medicine Company ,Parsippany, NJ –USA). Tissue factor RecombiPlasTin 2G was obtained from Instrumentation Laboratory Company (Bedford- USA).NovoSeven® and AryoSeven™ were diluted with saline to make working solution of 1mg/ml. Further dilutions were made with saline to obtain 0.5 µg/ml, 1.25µg/ml, and 2.5µg/ml rFVIIa preparations. To make working solution of PCCs, PCCs were diluted to 10 U/ml in saline. The sample mixtures contain 2U/ml PCCs, 0.1, 0.25, 0.5 µg/ml rFVIIa, and with RecombiPlasTin 2G at 1:5 ratio. The samples mixtures incubated in 5, 15, and 30 minutes at 37°c. SDS-PAGE (Pierce Biotechnology) and immunoblotting carried against Recothrom®.ResultsProfilnine® SD activated by RecombiPlasTin 2G resulted in conversions of prothrombin to prethrombin and thrombin at 5 to 30 minutes incubation time as evident by the generation of distinct bands at 50kDa and 36 kDa. However, addition of rFVIIa at final concentration range of 0.25 and 0.5µg/ml to the same mixture resulted in total conversion of prothrombin to thrombin at 30 minutes with a doublet bands at 36 kDa. Recombinant FVIIa alone failed to generate thrombin in native Profilnine® SD at 5 to 30 minutes as evident by the absence of 36 kDa band in the SDS-PAGE and Immunoblotting studies. Further studies comparing NovoSeven® and AryoSeven™ (FC: 0.1µg/ml) in the activated Profilnine® SD resulted in a comparable and progressive generation of thrombin at 5 and 15 minutes incubation time. At 5 and 15 minutes residual prethrombin bands were noted which was less intense at 15 minute incubation time in both rFVIIa preparations. Although rFVIIa alone unable to generate thrombin in native Beriplex®, the presence of rFVIIa in activated Beriplex did not change the activation profile. Activated Beriplex® by RecombiPlasTin 2G resulted in complete conversion of prothrombin to thrombin at 30 minutes incubation time. A complete generation of thrombin was noted in both activated PCCs in the presence of rFVIIa at final concentrations of 0.25µg/ml and 0.5µg/ml. At 5 minutes residual prothrombin and prethrombin bands were noted in the presence of rFVIIa at final concentration of 0.1µg/ml. However, at 15 minutes prothrombin bands were completely disappeared and prethrombin bands were less intense at same concentration of 0.1µg/ml of rFVIIa. The results obtained by Western blot analysis confirmed the differential activation of Profilnine® SD and Beriplex®. However, both the NovoSeven® and AryoSeven™ exhibited similar activation profiles.ConclusionsThese results indicate that the activation of PCCs by both AryoSeven™ and NovoSeven® result in comparable generation of thrombin. Furthermore, while both rFVIIa produced similar activation profile, PCCs based differences in the activation profile were noted. These studies provide an in vitro profiling of the effects of activated FVIIa on the generation of thrombin and related enzymes to demonstrate biosimilarity of the branded and generic versions of these haemostatic products. DisclosuresNo relevant conflicts of interest to declare.

Immobilization of &lt;i&gt;Lactobacillus plantarum&lt;/i&gt; B134 Cells using Sodium Alginate for Lactose Hydrolysis in UHT Milk

Hydrolysis of lactose in milk by β-galactosidase from immobilized bacterial cells has the potential to alleviate the problem of lactose intolerance. The present study was aimed to immobilize cells of L. plantarum strain B134 and evaluate their efficiency in hydrolyzing lactose in ultra high temperature (UHT) milk. Immobilized cells were generated by mixing cell suspensions with solutions of sodium alginate and calcium chloride. The β-galactosidase activity of the immobilized cells was tested by determining their ability in hydrolyzing lactose in UHT milk (whole milk and skimmed milk). Results showed that cells of L. plantarum strain B134 were entrapped optimally using a combination of 1 % sodium alginate, 100 mM calcium chloride and 12 % w/v cell suspension. The highest β-galactosidase activity was achieved at pH 6.5 and a temperature of 45 ºC for 5 minutes incubation time. The immobilization efficiency achieved was 28.95 %. The immobilized cells could reduce lactose by up to 85.45 % in UHT whole milk and 91.26 % in UHT skimmed milk. The times required for that reduction of lactose in UHT whole milk and UHT skimmed milk were 12 hours and 9 hours respectively. The immobilized cells could be re-used up to 4 times for efficient lactose hydrolysis for both types of milk. Therefore, immobilized cells of L. plantarum B134 have the potential to be used for lactose hydrolysis in UHT milk.

Techniques to Quantify the Size of Protein Colloids in Amyloid Fiber Formation

A new method for the analysis of protein colloidal diameter has been developed using three existing protein concentration quantification techniques, absorption at 280 nm, colloidal gold assay, and DC protein assay. Protein colloids are formed in the process of aggregation and are thought to be intermediates in protein self-assembly and formation of amyloid fiber. Deposition of the protein fibers in tissues leads to numerous human diseases including Alzheimer’s. Lysozyme was incubated at pH 2.0, 55°C, an environment conducive to amyloid fiber formation. The protein colloids present in the supernatant of the samples after centrifugation were studied over a time course of 30 days. The OD 280 assay detects total protein concentration based on absorption of radiation in the near UV. The colloidal gold assay and DC protein assay only measure colloidal sphere surface protein concentration. Due to the surface plasmon resonance, the light absorption spectrum changes when proteins bind to colloidal gold particles. Using the measured protein concentration on the surface of protein colloids along with the total measured protein concentration in the entire protein colloidal spheres, an interior protein concentration for all colloids is obtained. The protein colloidal sphere size can be calculated by using the ratio between the interior protein concentration and total protein concentration. Results indicate that the colloidal gold assay, DC protein assay, and OD 280 assay can be used to quantify the size of the protein colloids. The colloidal gold assay and DC protein assay are both independently effective in analysis of surface protein concentration in protein colloids. The DC protein assay was found to be much quicker in data production as it only requires 15 minutes of incubation time. The DC protein assay was also more reliable than the colloidal gold assay in accuracy and precision of results.

In situ study of the impact of acidic and neutral deposition pH on alkane phosphate film formation and stability on TiO2

TiO2 has been used as a model system for the surface of medical implant devices based upon titanium alloys. Self Assembled Monolayers (SAM) can be applied via a specific phosphate interaction to modify the surface properties of the TiO2 substrate. Here it is demonstrated via in situ Quartz Crystal Microbalance and Neutron Reflectometry experiments that the deposition of dodecyl phosphate onto TiO2 proceeds more rapidly at pH 4.5 than at pH 7.0. Conversely, the film stability was enhanced for films deposited at pH 7.0, demonstrating that while the initial association with the surface is driven by electrostatics it does not determine the SAM density. While the adsorbed amount appeared to be relatively constant after a few minutes incubation time it was found that washing with buffer removed about 50% of the adsorbed material after these short incubation times. With incubation time of the order of hours the proportion of the film washed off the surface decreased demonstrating that the specific phosphate–TiO2 interaction was a slow process. The slower initial surface interaction at pH 7.0 therefore allowed greater re-arrangement of the dodecyl phosphate resulting in more complete and robust monolayers than at pH 4.5. This was demonstrated by washing the film with buffer of increasing pH of up to 9.5. For SAM longevity on titanium alloys it is clear that slow deposition at pH 7 produces more robust films than rapid deposition at lower pH values.

Fractional laser‐assisted delivery of methyl aminolevulinate: Impact of laser channel depth and incubation time

Pretreatment of skin with ablative fractional lasers (AFXL) enhances the uptake of topical photosensitizers used in photodynamic therapy (PDT). Distribution of photosensitizer into skin layers may depend on depth of laser channels and incubation time. This study evaluates whether depth of intradermal laser channels and incubation time may affect AFXL-assisted delivery of methyl aminolevulinate (MAL). Yorkshire swine were treated with CO2 AFXL at energy levels of 37, 190, and 380 mJ/laser channel and subsequent application of MAL cream (Metvix) for 30, 60, 120, and 180 minutes incubation time. Fluorescence photography and fluorescence microscopy quantified MAL-induced porphyrin fluorescence (PpIX) at the skin surface and at five specific skin depths (120, 500, 1,000, 1,500, and 1,800 µm). Laser channels penetrated into superficial (∼300 µm), mid (∼1,400 µm), and deep dermis/upper subcutaneous fat layer (∼2,100 µm). Similar fluorescence intensities were induced at the skin surface and throughout skin layers independent of laser channel depth (180 minutes; P < 0.19). AFXL accelerated PpIX fluorescence from skin surface to deep dermis. After laser exposure and 60 minutes MAL incubation, surface fluorescence was significantly higher compared to intact, not laser-exposed skin at 180 minutes (AFXL-MAL 60 minutes vs. MAL 180 minutes, 69.16 a.u. vs. 23.49 a.u.; P < 0.01). Through all skin layers (120-1,800 µm), laser exposure and 120 minutes MAL incubation induced significantly higher fluorescence intensities in HF and dermis than non-laser exposed sites at 180 minutes (1,800 µm, AFXL-MAL 120 minutes vs. MAL 180 minutes, HF 14.76 a.u. vs. 6.69 a.u. and dermis 6.98 a.u. vs. 5.87 a.u.; P < 0.01). AFXL pretreatment accelerates PpIX accumulation, but intradermal depth of laser channels does not affect porphyrin accumulation. Further studies are required to examine these findings in clinical trials.

Essential Oil Composition and DPPH Scavenging Activity of Endemic Tanacetum mucroniferum Hub. –Mor. & Grierson from Turkey

Water-distilled essential oil from flowers of Tanacetum mucroniferum from Turkey were analyzed by GC and GC-MS. The flower oil of T. mucroniferum was characterized with oxygenated monoterpenes and oxygenated sesquiterpenes. Main components of the oil were 1,8-cineole 21.9 % and camphor 6.4 %. Antioxidant activity of the oil was determined at various concentrations and incubation time by DPPH radical protocol. Highest activity was observed at 20 mg/mL concentration (22.31 %) with 60 minutes incubation time. When compared with positive control α-tocopherol (96.59 %) at same conditions oil showed low DPPH scavenging activity. T. mucroniferum was reported to be an intermediate species between T. sipikorense and T. aucheranum. Essential oil composition of T. mucroniferum was investigated for the first time and similarities with previously reported T. aucheranum essential oil was compared.

Impaired sperm aggregation in Adam2 and Adam3 null mice

Previous studies have shown that sperm from Adam2 and Adam3 knockout mice have defective migration in the female reproductive tracts and cannot bind to the egg's zona pellucida (ZP), which leads to infertility. Here, we report that Adam2 and Adam3 knockout sperm have severely impaired sperm aggregation and that this defect is not restored over time during in vitro cultivation, suggesting the requirement of ADAM2 and ADAM3 in normal sperm association.

Improvement of enzyme stability via non-covalent complex formation with dextran against temperature and storage lifetime

Improvement of enzyme stability via non-covalent complex formation with dextran against temperature and storage lifetime The optimal methodology to prepare the novel modified enzyme, polymer-enzyme complex, was developed to give a high catalytic activity in aqueous solution. The non-covalent complexes of two different enzymes (horseradish peroxidase and glucose oxidase) were prepared with various molar ratios (nD/nE 0,05; 0,1; 1; 5; 10; 15; 20) by using 75kDa dextran. The thermal stabilities of the obtained complexes were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time for pH 7. The complexes with the molar ratio nD/nHRP: 10 and nD/nGOD: 5 showed the highest thermal stability. Its activity was very high (ca. 1,5-fold higher activity than pure enzyme for HRP-dextran complexes) and almost the same between applying one hour incubation time and without incubation, and could also be measured at high temperatures (70, 80 °C). We finally succeeded in preparing dextran-enzyme complexes which showed higher activity than pure enzyme in aqueos solution at all temperatures for pH 7. In addition, the mentioned complexes at pH 7 had very long storage lifetime compared to purified enzyme at +4 °C; which is considered as a good feature for the usage in practice.

Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-beta1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.

Stabilization of horseradish peroxidase by covalent conjugation with dextran aldehyde against temperature and pH changes

Abstract Stabilization of Horseradish Peroxidase (HRP; EC 1.11.1.7) against temperature and pH via the formation of the conjugates obtained by multipoint covalent bonding of dextran aldehyde (DA) to the enzyme were studied. Hence, three different molar weighted dextrans (17.5 kD, 75 kD, 188 kD) were covalently bonded to purified enzyme with different molar ratios (nHRP/nDA 20/1, 10/1, 1/1, 1/5, 1/10, 1/15, 1/20). The thermal stabilities of the obtained conjugates were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time. Conjugates formed were characterized by gel-permeation chromatography (GPC) and fluorescence techniques. The conjugate synthesized using dextran 75 kDa with nHRP/nDA 1/10 molar ratio showed better thermal stability than other conjugates and purified enzyme at pH 7. This conjugate also has wider activity pH range than purified enzyme. In addition, mentioned conjugate at pH 7 had very long storage lifetime compared to purified enzyme at +4°C and room temperature; which is considered a favorable feature for usage in practice.

Évaluation analytique de la trousse ST AIA-PACK 27.29 (équivalent CA 15-3) de Tosoh bioscience sur l'automate AIA 21 et comparaison avec la trousse AIA-PACK 27.29 VIDAS CA 15-3 bioMérieux, AXSYM CA-15-3 Abbott

We report here evaluation results from analytical features related to ST-AIA PACK 27.29 (10 minutes incubation time format) which measures MUC-1 protein, a breast tumour marker; and results' comparison versus a pool of 113 patients with, in one hand, 2 others methods used (VIDAS CA15.3 and AXSYM CA 15.3) and on the other hand, a third method (AIA PACK 27.29, a 40 minutes incubation time format, used in our daily routine lab. Systems used are AIA 21 and VIDAS, both installed within our Hospital lab and AXSYM, installed in the city commercial lab. The “SFBC VALTEC protocol – version 1997 – 2000” has been followed with all those tests. The Results of the evaluation tests, repeatability, reproducibility, dilution, correlation for ST-AIA PACK 27.29, are fully compliant with VALTEC criteria. Assay range for this kit is from 1 to 400 U/ml. Results from different correlations between ST AIA-PACK 27.29 and AIA PACK 27.28 are Y = 0.84X – 0.18 (r = 0.98), and Axsym are Y = 0.76X + 0.34 (r = 0.97) and Vidas are Y = 0.92 X + 0.24 (r = 0.92). As conclusion, the performed method gives results with acceptable analytical criteria, compliant with the current state of the art, and correlated with the 2 others methods used. The analytical system allowing performing this kit highlights the main advantages of the AIA machines line as far as quality insurance is concerned: Pipetting thanks to individual tips directly onto the primary tube, use of unitary tests cups which guarantee excellent reagents stability and flexibility are the main points of interest and to be taken into account.

LABELING OF MIBI (METOXY ISOBUTYL ISONITRYL) WITH TECHNETIUM-99m AS A PERFUSION MYOCARDIAC IMAGING RADIOPHARMACEUTICAL

Coronary artery disease is one the major causes of death in Indonesia. For early diagnosis of the disease, it is deemed necessary to visualize the myocardium blood flow perfusion. 99mTc-MIBI labeled compound is one of the agent that could be used for such purpose. The labeling of MIBI with technetium-99m under some variations of several parameters has been carried out. The labeling efficiency was determined by counting the radiochemical purity using ITLC-SG 60 thin layer chromatography using two kinds of solvents, i.e. methanol and 0.9% NaCl solutions. The optimum labeling condition was achieved at the pH = 5.5 – 6.0, 1 mg of [Cu(MIBI)4BF4, 0.060 mg SnCl2.2H2O and 10 minutes incubation time in a boiling water bath, gave ± 99% of labeling efficiency. Under the formulation stated above, MIBI dry kit produces could give the radiochemical purity of more than 95%. Key words : metoxy isobutyl isonitril (MIBI), technetium-99m, labeling, myocardiac perfusion imaging.

Near-real-time determination of hydrogen peroxide generated from cigarette smoke

The ability to monitor hydrogen peroxide (H2O2) in aqueous smoke extracts will advance our understanding of the relationship between cigarette smoke-induced oxidative stress, inflammation, and disease and help elucidate the pathways by which the various smoke constituents exert their pathogenic effects. We have demonstrated, for the first time, the measurement of H2O2 production from cigarette smoke without prior separation of the sample. Cigarettes were tested on a commercial smoking machine, such that the whole smoke or gas vapor phase was bubbled through phosphate buffered saline solution at pH 7.4. Aliquots of these solutions were analyzed using an Amplex Red/horseradish peroxidase fluorimetric assay that required only a 2 minute incubation time, facilitating the rapid, facile collection of data. Catalase was used to demonstrate the selectivity and specificity of the assay for H2O2 in the complex smoke matrix. We measured approximately 7-8 microM H2O2 from two reference cigarettes (i.e., 1R4F and 2R4F). We also observed 9x more H2O2 from whole smoke bubbled samples compared to the gas vapor phase, indicating that the major constituent(s) responsible for H2O2 formation reside in the particulate phase of cigarette smoke. Aqueous solutions of hydroquinone and catechol, both of which are particulate phase constituents of cigarette smoke, generated no H2O2 even though they are free radical precursors involved in the production of reactive oxygen species in the smoke matrix.

IFN-beta decreases adhesion and transmigration capacities of lymphocytes in Guillain-Barré syndrome.

The adhesion capacities, transmigration capacities, and integrin expression of lymphocytes from patients with Guillain-Barré syndrome incubated with interferon-beta were studied. Interferon-beta induced a dose-dependent inhibition of lymphocyte adhesion to recombinant vascular adhesion molecule-1 (p < 0.0001) and recombinant intercellular adhesion molecule-1 (rICAM-1) (p < 0.01) without modulation of very late activation molecule-4 and lymphocyte function-associated antigen-1 expressions and a dose-dependent decrease of lymphocyte transmigration across fibronectin (p < 0.0001). Inhibition of adhesion to rICAM-1 was similar after long (18 hours) or short (5 minutes) incubation time. These results support the potential therapeutic benefit of interferon-beta in Guillain-Barré syndrome.

A new rapid acetylcholinesterase histochemical method for the intraoperative diagnosis of Hirschsprung's disease and intestinal neuronal dysplasia.

The most commonly used acetylcholinesterase (AChE) method for the diagnosis of Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND) was first introduced in 1964 by Morris Karnovsky and Logan Roots. This technique requires about 80 - 120 minutes incubation time and cannot be used for the intraoperative diagnosis of HD and IND. To avoid these limitations, in 1994 Kobayashi et al first proposed an accelerated modified method in two different versions, the first using diaminobenzydine (DAB) reagent, the second using 4-chloro-1-naphthol as final reagent. In the present study, we propose a new rapid variation of AChE staining which avoids the use of DAB and naphthol, notably toxic reagents, but follows the same acceleration principle of Kobayashi's technique. Our modified rapid AChE requires a total incubation time of only 8 minutes, which is compatible with intraoperative histochemical examination purposes. Intraoperative seromuscular or full-thickness intestinal biopsies were obtained from 92 children affected by intestinal dysganglionoses. The biopsies were frozen and cut in 15 microm cryostatic sections. Rapid AChE was performed with a special incubation medium using 3-amino-9 ethylcarbazole (AEC) as chromogenic substance. The two complementary histochemical techniques alpha-naphthylesterase (ANE) and lactate-dehydrogenase (LDH) were also used intraoperatively for the staining of ganglion cells. The diagnosis was confirmed postoperatively with conventional AChE Karnovsky technique, comparing the extensions of hyperganglionic, hypoganglionic and aganglionic segments in each studied case. The new rapid AChE modified method can identify ganglion cells and fibers using a dark brown precipitate. In all the cases studied, the intestinal innervation pattern identified with this modified technique was similar to that obtained with Karnovsky AchE. Seventy-eight HD, 8 isolated IND and 6 HD associated with an evident IND segment were diagnosed. This new rapid AChE histochemical technique avoids the use of DAB and naphthol, and can thus be considered safe for operators. Rapid AChE is a valid tool for both the evaluation of aganglionosis extension and for the identification of IND pattern during surgery. We recommend this very reliable method for the intraoperative diagnosis of HD and IND, in association with other enzymatic markers of ganglion cells (ANE or LDH). We propose the following diagnostic protocols: a) for preoperative histochemical study: conventional AChE plus LDH and NADPH-diaphorase; b) for intraoperative study: rapid AChE plus ANE.

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells

The effects of microgravity on Jurkat cells — a T-lymphoid cell line — was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.