共查询到20条相似文献,搜索用时 31 毫秒
1.
These studies focused on a new radiolabeling technique with copper (64Cu) and zirconium (89Zr) for positron emission tomography (PET) imaging using a CD45 antibody. Synthesis of 64Cu-CD45 and 89Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC) was assessed in vitro (viability, population doubling times, colony forming units). hPBSC viability was maintained as the dose of 64Cu-TETA-CD45 increased from 0 (92%) to 160 µCi/mL (76%, p>0.05). Radiolabeling efficiency was not significantly increased with concentrations of 64Cu-TETA-CD45 >20 µCi/mL (p>0.50). Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p<0.05). For 89Zr, there were no significant differences in viability (p>0.05), and a trend towards increased radiolabeling efficiency was noted as the dose of 89Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0–40 µCi/mL (p<0.05). A greater than 2,000 fold-increase in the level of 89Zr-Df-CD45 labeling efficiency was observed when compared to 64Cu-TETA-CD45. Similar to 64Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05) (growth, colony formation). Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×106 hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI), then 0.3 mCi of 89Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that 89Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high. 相似文献
2.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1991,18(7):695-703
Direct labeling involves 99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind 99mTc. The direct 99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with 99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for 99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the 99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good 99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use. 相似文献
3.
4.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1992,19(6):669-677
The in vitro labeling and stability of 99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the 99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The 99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the 99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between 99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N2S2) compound. The Fab′ with IgG2a isotype displayed tighter binding to 99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N2S2 challenge and incubation with human plasma. The in vivo biodistribution of five 99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable 99mTc labeling of antibody fragments from three of the major murine isotypes. 相似文献
5.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1986,13(3):235-243
Polyvinyl alcohol sponge (PVA) is a widely used angiographic embolic agent. The radiolabeling of PVA can accurately identify particle localization and may decrease the possibility of patient morbidity from embolization to distal sites. We incorporated 99mTc sulfur colloid (SC) into PVA by heating. Animal experiments demonstrated the in vivo stability of the 99mTc SC-PVA complex and the efficacy of external imaging. 99mTc SC-PVA biodistribution data and external NaI(Tl) scintillation probe counts were performed, to assess anatomic localization. Embolization with this complex was performed in a patient. 相似文献
6.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1989,16(4):365-369
Radiolabeled hemoglobin may be a useful tool in the study of the body distribution of hemoglobin solutions developed as plasma expanders with oxygen-transporting capacity. The present investigation compares the suitability of two radiolabeling techniques for hemoglobin. 125I labeling of hemoglobin with Iodogen® as iodinating agent caused major changes in the chromatographic behaviour and an accelerated plasma clearance of the labeled hemoglobin in rats. A recently developed two-step procedure for 99mTc labeling gave better results. The label had only minimal influence on the chromatographic behaviour of hemoglobin. In vivo, no free label occurred in the circulation and no transfer of the label to other plasma proteins took place. The plasma clearance of 99mTc-labeled hemoglobin in rats was slowed. However, this could be explained entirely by diminishing glomerular filtration, probably by inhibition of the dissociation of the hemoglobin molecule into dimers. The plasma clearance of hemoglobin modified by intramolecular cross-linking, which prevents dissociation of the molecule into dimers and thus excretion by the kidney, was not influenced by the label.We conclude that the 99mTc labeling procedure is suitable for in vivo distribution studies of hemoglobin when it is taken into account that the urinary excretion is underestimated. For cross-linked hemoglobin, which is more promising as plasma expander, no such restriction exists. 相似文献
7.
Lei Yin Qian Zhao Ling Li Su Lei Zhang Xue Qi Chen Chao Ma Lei Kang Meng Liu Chun Li Zhang Ping Yan Rong Fu Wang 《PloS one》2013,8(7)
Objective
The α7 nicotinic acetylcholine receptors (nAChRs) play a vital role in the pathophysiology of neuropsychiatric diseases such as Alzheimer’s disease and depression. However, there is currently no suitable positron emission tomography (PET) or Single-Photon Emission Computed Tomography (SPECT) radioligands for imaging α7 nAChRs in brain. Here our aim is to radiosynthesize a novel SPECT radioligand 131I-CHIBA-1001 for whole body biodistribution study and in vivo imaging of α7 nAChRs in brain.Method
131I-CHIBA-1001 was radiosynthesized by chloramine-T method. Different conditions of reaction time and temperature were tested to get a better radiolabeling yield. Radiolabeling yield and radiochemical purities of 131I-CHIBA-1001 were analyzed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) system. Whole body biodistribution study was performed at different time points post injection of 131I-CHIBA-1001 in KM mice. Monkey subject was used for in vivo SPECT imaging in brain.Result
The radiolabeling yield of 131I-CHIBA-1001 reached 96% within 1.5∼2.0 h at 90∼95°C. The radiochemical purity reached more than 99% after HPLC purification. 131I-CHIBA-1001 was highly stable in saline and fresh human serum in room temperature and 37°C separately. The biodistribution data of brain at 15, 30, and 60 min were 11.05±1.04%ID/g, 8.8±0.04%ID/g and 6.28±1.13%ID/g, respectively. In experimental SPECT imaging, the distribution of radioactivity in the brain regions was paralleled with the distribution of α7 nAChRs in the monkey brain. Moreover, in the blocking SPECT imaging study, the selective α7 nAChR agonist SSR180711 blocked the radioactive uptake in the brain successfully.Conclusion
The CHIBA-1001 can be successfully radiolabeled with 131I using the chloramine-T method. 131I-CHIBA-1001 can successfully accumulate in the monkey brain and image the α7 acetylcholine receptors. 131I-CHIBA-1001 can be a candidate for imagingα7 acetylcholine receptors, which will be of great value for the diagnosis and treatment of mental diseases. 相似文献8.
Introduction
Visualization of tumor angiogenesis using radionuclide targeting provides important diagnostic information. In previous study, we proved that an arginine-arginine-leucine (RRL) peptide should be a tumor endothelial cell specific binding sequence. The overall aim of this study was to evaluate whether 99mTc-radiolabeled RRL could be noninvasively used for imaging of malignant tumors in vivo, and act as a new molecular probe targeting tumor angiogenesis.Methods
The RRL peptide was designed and radiosynthesized with 99mTc by a one-step method. The radiolabeling efficiency and radiochemical purity were then characterized in vitro. 99mTc-RRL was injected intravenously in HepG2 xenograft-bearing BALB/c nude mice. Biodistribution and in vivo imaging were performed periodically. The relationship between tumor size and %ID uptake of 99mTc-RRL was also explored.Results
The labeling efficiencies of 99mTc-RRL reached 76.9%±4.5% (n = 6) within 30–60 min at room temperature, and the radiochemical purity exceeded 96% after purification. In vitro stability experiment revealed the radiolabeled peptide was stable. Biodistribution data showed that 99mTc-RRL rapidly cleared from the blood and predominantly accumulated in the kidneys and tumor. The specific uptake of 99mTc-RRL in tumor was significantly higher than that of unlabeled RRL blocking and free pertechnetate control test after injection (p<0.05). The ratio of the tumor-to-muscle exceeded 6.5, tumor-to-liver reached 1.98 and tumor-to-blood reached 1.95. In planar gamma imaging study, the tumors were imaged clearly at 2–6 h after injection of 99mTc-RRL, whereas the tumor was not imaged clearly in blocking group. The tumor-to-muscle ratio of images with 99mTc-RRL was comparable with that of 18F-FDG PET images. Immunohistochemical analysis verified the excessive vasculature of tumor. There was a linear relationship between the tumor size and uptake of 99mTc-RRL with R2 = 0.821.Conclusion
99mTc-RRL can be used as a potential candidate for visualization of tumor angiogenesis in malignant carcinomas. 相似文献9.
《Bioorganic & medicinal chemistry》2016,24(10):2251-2256
Prostate-specific membrane antigen (PSMA) is expressed strongly in prostate cancers and is, therefore, an attractive diagnostic and radioimmunotherapeutic target. In contrast to previous reports of PMSA-targeting 99mTc-tricarbonyl complexes that are cationic or lack a charge, no anionic 99mTc-tricarbonyl complexes have been reported. Notably, the hydrophilicity conferred by both cationic and anionic charges leads to rapid hepatobiliary clearance, whereas an anionic charge might better enhance renal clearance relative to a cationic charge. Therefore, an improvement in rapid clearance would be expected with either cationic or anionic charges, particularly anionic charges. In this study, we designed and synthesized a novel anionic 99mTc-tricarbonyl complex ([99mTc]TMCE) and evaluated its use as a single-photon emission computed tomography (SPECT) imaging probe for PSMA detection. Direct synthesis of [99mTc]TMCE from dimethyl iminodiacetate, which contains both the asymmetric urea and succinimidyl moiety important for PSMA binding, was performed using our microwave-assisted one-pot procedure. The chelate formation was successfully achieved even though the precursor included a complicated bioactive moiety. The radiochemical yield of [99mTc]TMCE was 12–17%, with a radiochemical purity greater than 98% after HPLC purification. [99mTc]TMCE showed high affinity in vitro, with high accumulation in LNCaP tumors and low hepatic retention in biodistribution and SPECT/CT studies. These findings warrant further evaluation of [99mTc]TMCE as an imaging agent and support the benefit of this strategy for the design of other PSMA imaging probes. 相似文献
10.
A study of lipid bilayer membrane stability using precise measurements of specific capacitance 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
S H White 《Biophysical journal》1970,10(12):1127-1148
A method is described for measuring the specific capacitance (Cm) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10-2 M KCl show that Cm depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), Cm increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm2 with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. Cm of final-state membranes depends upon applied voltage (Va) and obeys the equation Cm = C0 + βVa2 where Va VDC + VrmsAC. C0 and β depend upon temperature; C0 decreases linearly with temperature while β increases linearly. At 20°C, C0 = 0.559 ±0.01 (SD) μF/cm2 and β = 0.0123 ±0.0036 (SD) (μF/cm2)/(mv2) and at 34°C, C0 = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in Cm are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves. 相似文献
11.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1990,17(2):229-232
An improved method for utilization and purification of 99Mo from spent 99Mo/99mTc generators has been described. After washing the generator with saline to remove the generated 99mTc, followed by 2 mL 5 M NaOH containing a few drops of H2O2, the 99Mo was quantitatively eluted from the generator with 5 mL 5 M NaOH. The alkaline eluate containing 99Mo was contaminated with partially dissolved alumina. In the present method, an anion-exchange resin Dowex 1 × 8 column was used for purification of 99Mo from the contaminating alumina. The resultant 99Mo was of high purity and contained < 1 μg Al3+/mL 99Mo solution, as estimated by atomic absorption. The eluted 99Mo was used for radiolabeling of monoclonal antibodies (Mab) to verify the theoretical modeling of an in vivo generator for radioimmunotherapy, and the results of this study will be published elsewhere. 相似文献
12.
Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m−2 h−1 for cyanobacterial and lichen crust, respectively. Complete denitrification to N2 was further confirmed by an 15NO3− tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m−2 h−1 for cyanobacterial and lichen crust, respectively. Strikingly, N2O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m−2 h−1 from the cyanobacterial and lichen crust, respectively, with N2O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N2O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N2O gas emission and potentially reduces desert soil fertility. 相似文献
13.
Ju?ara Ribeiro Franca Giselle Foureaux Leonardo Lima Fuscaldi Tatiana Gomes Ribeiro Lívia Bomfim Rodrigues Renata Bravo Rachel Oliveira Castilho Maria Irene Yoshida Valbert Nascimento Cardoso Simone Odília Fernandes Sebasti?o Cronemberger Anderson José Ferreira André Augusto Gomes Faraco 《PloS one》2014,9(4)
The purpose of the present study was to develop and assess a novel sustained-release drug delivery system of Bimatoprost (BIM). Chitosan polymeric inserts were prepared using the solvent casting method and characterized by swelling studies, infrared spectroscopy, differential scanning calorimetry, drug content, scanning electron microscopy and in vitro drug release. Biodistribution of 99mTc-BIM eye drops and 99mTc-BIM-loaded inserts, after ocular administration in Wistar rats, was accessed by ex vivo radiation counting. The inserts were evaluated for their therapeutic efficacy in glaucomatous Wistar rats. Glaucoma was induced by weekly intracameral injection of hyaluronic acid. BIM-loaded inserts (equivalent to 9.0 µg BIM) were administered once into conjunctival sac, after ocular hypertension confirmation. BIM eye drop was topically instilled in a second group of glaucomatous rats for 15 days days, while placebo inserts were administered once in a third group. An untreated glaucomatous group was used as control. Intraocular pressure (IOP) was monitored for four consecutive weeks after treatment began. At the end of the experiment, retinal ganglion cells and optic nerve head cupping were evaluated in the histological eye sections. Characterization results revealed that the drug physically interacted, but did not chemically react with the polymeric matrix. Inserts sustainedly released BIM in vitro during 8 hours. Biodistribution studies showed that the amount of 99mTc-BIM that remained in the eye was significantly lower after eye drop instillation than after chitosan insert implantation. BIM-loaded inserts lowered IOP for 4 weeks, after one application, while IOP values remained significantly high for the placebo and untreated groups. Eye drops were only effective during the daily treatment period. IOP results were reflected in RGC counting and optic nerve head cupping damage. BIM-loaded inserts provided sustained release of BIM and seem to be a promising system for glaucoma management. 相似文献
14.
STEREOLOGICAL ANALYSIS OF MAMMALIAN SKELETAL MUSCLE : I. Soleus Muscle of the Adult Guinea Pig 总被引:14,自引:7,他引:7
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 ± 0.06% (mean ± standard error, n = 5 animals) and the nuclei volume was 0.86 ± 0.20% of the fiber volume. The total mitochondrial volume was 4.85 ± 0.66% of the fiber volume with about one-third being found in an annulus within 1 µm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 ± 0.4%. The T system has a volume of 0.14 ± 0.01% and a surface area of 0.064 ± 0.005 µm2/µm3 of the fiber volume. The surface area of the sarcolemma is 0.116 ± 0.013 µm2/µm3 which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 ± 0.33% and the surface area is 0.97 ± 0.09 µm2/µm3. The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 ± 0.19% and surface area is 0.24 ± 0.05 µm2/µm3. The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 ± 0.24% and 0.51 ± 0.08%, and the surface areas of the I and A band reticulum are 0.56 ± 0.07 µm2/µm3 and 0.16 ± 0.04 µm2/µm3, respectively. The Z line width, myofibril and fiber diameters were measured. 相似文献
15.
Purpose
To examine whether 99mTc(V)-DMSA could be used as a non-invasive measure of cancer cell proliferation.Methods
Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with 99mTc(V)-DMSA in media for 60 min at 37°C. DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-3H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups.Results
In all cell lines rates of 99mTc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-3H]thymidine incorporation and expression of PCNA and Ki-67.Conclusion
Rates of 99mTc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo. 相似文献16.
myo-Inositol homeostasis in foetal rabbit lung 总被引:2,自引:2,他引:0
In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h−1·g of tissue−1) was higher than that found in adult lung tissue (23.2±1.0nmol·h−1·g of tissue−1). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na+-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h−1·g of tissue−1) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue. 相似文献
17.
C. S. R. Gooden D. E. Snook Agamemnon A. Epenetos 《Cell biochemistry and biophysics》1993,22(1-3):49-59
It is essential in any method for radiolabeling antibody with99mTc that the labeling procedure is rapid and reliable, producing a highly stable99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (99mTc and125I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (99mTc or125I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of125I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the99mTc equivalent corresponds to free iodide. 相似文献
18.
1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-14C]adenine, [8-14C]guanine and [8-14C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 4·7 μm) and hypoxanthine phosphoribosyltransferase (Ki 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of Ki for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug. 相似文献
19.
Validation of a Green Fluorescent Protein-Labeled Strain of Vibrio vulnificus for Use in the Evaluation of Postharvest Strategies for Handling of Raw Oysters
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
S. L. Drake D. Elhanafi W. Bang M. A. Drake D. P. Green L. A. Jaykus 《Applied microbiology》2006,72(11):7205-7211
In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45°C), freeze-thaw tolerance (−20o and −80°C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5°C), cold adaptation (15°C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log10 after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (102 CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 103 and >104 CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish. 相似文献
20.
The thyroid uptake at 20 minutes of intravenously administered Technetium-99m (99mTc) was measured in 117 patients with a standard scintillation counter. Patients were divided into three groups on the basis of clinical assessment, four-hour 131I uptake, triiodothyronine (T-3) resin uptake, and protein-bound iodine measurements.In 31 patients with no evidence of thyroid disease the mean 99m Tc uptake was 1·8% ±S.D. 1·1%. In 32 patients with thyroid enlargement who were euthyroid the mean uptake was 2·5% ±S.D. 2·2%. In 54 thyrotoxic patients the mean uptake was 17·7% with a range of 4·1 to 44%, all cases having an uptake above the upper limit of normal (4·0%). These results agree closely with reported uptake studies using scanning techniques. In seven patients the extrathyroidal neck activity was measured by using a scanner, and the mean was 6·3% of the extrathyroidal total body radioactivity comparing favourably with an assumed 6% used in our calculations.We have shown that the measurement of the thyroid uptake of 99mTc with a scintillation counter is of value, and that it is not necessary to use scanning techniques in the diagnosis of thyrotoxicosis. Advantages of 99m Tc are minimal radiation, reduction in patient and laboratory time, and low cost. 相似文献