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Longer Incubation Time (longer + incubation_time)
Selected AbstractsDifferential mitogenic responses of human macrovascular and microvascular endothelial cells to cytokines underline their phenotypic heterogeneityCELL PROLIFERATION, Issue 3 2001I. Lang A variety of growth factors promote the complex multistep process of angiogenesis. The mitogenic activity of vascular endothelial growth factors (VEGFs) and placental growth factors (PlGFs), known as cytokines acting predominantly on endothelial cells, was tested on human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MIEC) and compared with the potency of the universally acting basic fibroblast growth factor (FGF-2). The cells were seeded at different cell numbers and incubated with various doses of growth factors for a period of 24,72 h in culture medium ± serum. Proliferation was determined by measuring the optical density after staining the cells with the tetrazolium salt WST-1. VEGF121 and VEGF165 increased the number of HUVEC and MIEC at low and high seeding densities various doses and incubation times. The efficiency of FGF-2 was less pronounced at high seeding densities of the cells under serum-free conditions. PlGF-1 and PlGF-2 stimulated mitogenesis on HUVEC only at low cell numbers and after a short incubation time by 125 ± 3% and 102 ± 5% (P < 0.001), respectively. Longer incubation times with the lower seeding density in the absence of FCS did not induce a significant stimulatory effect of the PlGFs. MIEC responded stronger to all growth factors. In particular under serum free conditions, PlGF-1 and PlGF-2 effectively stimulated cell proliferation by 247 ± 54% (P < 0.01) and 288 ± 40% (P < 0.05) at low cell numbers, and by 81 ± 13% (P < 0.05) and 49 ± 13% (P < 0.01), respectively, at high cell numbers. The addition of fetal calf serum caused a reduced proliferative response of all growth factors on both cell types related to the controls. In conclusion, MIEC and HUVEC differ in their proliferative response to VEGFs, PlGFs and FGF-2. [source] Morphological and biochemical changes associated with apoptosis induced by okadaic acid in human amniotic FL cellsENVIRONMENTAL TOXICOLOGY, Issue 5 2009Ming-luan Xing Abstract The marine toxin okadaic acid (OA) is an apoptosis inducer and a tumor promoter. During recent years, extensive studies have demonstrated that OA can induce apoptosis in a wide variety of cell types. In contrast to the relatively longer incubation time or higher treatment concentrations of OA in apoptosis shown previously, relatively lower concentrations (,100 nM) and shorter time (4 h) were designed in the current study to observe the toxic effects of OA in human amniotic cells (FL cells). The present study was undertaken to determine the morphological and biochemical changes of FL cells induced by OA. Results indicated that externalization of phosphatidylserine, cytoskeletal disruption, DNA strand breaks and decrease of Bcl-2 protein expression levels as well as increase of PP2A-A subunit protein were all involved in the apoptosis of FL cells induced by OA. This work not only provided further evidence of apoptosis induced by OA but also suggested that PP2A might play a pivotal role in apoptosis induced by protein phosphatases inhibitors. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009. [source] Reproductive strategies of Gammarus lacustris (Crustacea: Amphipoda) along an elevation gradientFUNCTIONAL ECOLOGY, Issue 4 2000Wilhelm F. M. Abstract 1.,The number of eggs, their size, mass and development time, and the starvation time of newly hatched young, was examined in four populations of Gammarus lacustris along an elevation gradient from prairie to alpine lakes (730 m to > 2300 m above sea level). Water temperature and ice-free season decreased with increasing altitude. 2.,Females in the alpine lake produced fewer but larger and heavier eggs than females in the prairie lake. Eggs produced by females in montane and subalpine lakes were intermediate in size, mass and number. Within populations, egg size was not related to the number of eggs or female size. 3.,The development time of eggs declined with an increase in incubation temperature. At all incubation temperatures, large eggs had a longer incubation time than small eggs. All eggs incubated at 4 °C failed to produce young. Young from large eggs were larger in size than young from small eggs. 4.,The starvation time of newly hatched young increased with decreasing temperature. However, slopes of regressions relating starvation time to temperature differed among populations. At 4 °C young from large eggs survived longer than young from small eggs. 5.,The high phenotypic plasticity in reproductive traits contributes to the success of G. lacustris in a wide range of aquatic habitats. It is predicted that in response to climate-induced warming, populations in currently cold montane and alpine lakes would shift their reproduction to produce more eggs of smaller size. However, the accurate prediction of the fate of populations between ecoregions will require knowledge of the extent to which these traits are under genetic control. [source] Rapid in vitro conversion of fosphenytoin into phenytoin in sera of patients with liver disease: Role of alkaline phosphatase ,JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2001Amitava Dasgupta Abstract Fosphenytoin, a phosphate ester pro drug of phenytoin, also cross-reacts with the fluorescence polarization immunoassay (FPIA) for phenytoin. We measured fosphenytoin concentrations using the FPIA kit and TDx analyzer. We prepared serum pools from normal volunteers and patients with liver disease. None of them received either fosphenytoin or phenytoin. Fosphenytoin standard solution (1 mg/ml) was prepared in water. We supplemented aliquots of normal and liver pools with known amounts of fosphenytoin and measured the concentrations at different time intervals. The conversion of fosphenytoin to phenytoin was slow in sera with normal alkaline phosphatase activities. The conversion was rapid in sera collected from patients with liver disease who also had high alkaline phosphatase activities. The observed concentrations were close to target concentrations within 0,2 min of supplementation with fosphenytoin. Surprisingly, the observed concentration then started to decline slightly but significantly with longer incubation time. In contrast, the observed concentration increased steadily in serum with normal alkaline phosphatase activity. For example, in the normal pool supplemented with 15.0 ,g/ml fosphenytoin (as the phenytoin equivalent), the observed concentrations were 6.9, 7.3, 7.7, 8.3, and 9.8 ,g/ml at 0,2, 10, 20, 30, and 60 min, respectively. However, in a serum pool prepared from patients with liver disease and supplemented with 15.0 ,g/ml of fosphenytoin (alkaline phosphatase: 2547 U/l), the observed phenytoin concentrations were 12.9, 12.1, 11.0, 10.7, and 10.7 ,g/ml at 0,2, 10, 20, 30, and 60 min, respectively. When we added alkaline phosphatase to the normal serum pool, we observed rapid conversion of fosphenytoin into phenytoin within 10 min, but the concentrations then declined with longer incubation time. However, when we repeated the experiment with protein-free ultrafiltrate, we observed rapid conversion of fosphenytoin to phenytoin, but the concentration did not decline with longer incubation time. J. Clin. Lab. Anal. 15:244,250, 2001. © 2001 Wiley-Liss, Inc. [source] Effects of Vegetable Juice Powder Concentration and Storage Time on Some Chemical and Sensory Quality Attributes of Uncured, Emulsified Cooked SausagesJOURNAL OF FOOD SCIENCE, Issue 5 2007J.J. Sindelar ABSTRACT:, Uncured, no-nitrate/nitrite-added meat products can be manufactured with vegetable juice powder (VJP) and a starter culture containing Staphylococcus carnosus, resulting in quality and sensory attributes similar to traditional cured products. The 1st objective of this study was to determine the effects of varying concentrations of VJP and incubation times (MIN-HOLD) on quality characteristics, including lipid oxidation, color, and cured meat pigment concentrations, of emulsified-frankfurter-style-cooked (EFSC) sausages over a 90-d storage period. The 2nd objective was to compare residual nitrate and nitrite content resulting from different processing treatments and the 3rd objective was to assess sensory properties of finished products. Four EFSC sausage treatments (TRT) (TRT 1: 0.20% VJP, 30 MIN-HOLD; TRT 2: 0.20% VJP, 120 MIN-HOLD; TRT 3: 0.40% VJP, 30 MIN-HOLD; TRT 4: 0.40% VJP, 120 MIN-HOLD) and a sodium nitrite-added control (C) were used for this study. No differences for lipid oxidation (TBARS) between any TRTs and C or over time were observed. No differences (P > 0.05) for CIE L* values were found between TRTs. CIE a* and reflectance ratio values revealed that TRTs 2, 4, and C were redder than TRTs 1 and 3 at day 0. Trained sensory intensity ratings for cured aroma, cured color, cured flavor, uniform color, and firmness determined that all but TRT 1 were similar to C. These results indicate a longer incubation time (120 compared with 30 min) was found more critical than VJP level (0.20% or 0.40%) to result in products comparable to a sodium nitrite-added control. [source] Differential apoptotic response of J774 macrophages to alumina and ultra-high-molecular-weight polyethylene particlesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2002Alain Petit We recently identified apoptosis in in vitro wear particle-stimulated macrophages. The recent explosion of interest in apoptosis lies in the fact that it is under positive and negative regulation through evolutionary conserved biochemical pathways. It may also be possible to modulate macrophage apoptosis in the treatment of periprosthetic osteolysis. The purpose of this study was to compare the macrophage response to identically sized particles of alumina ceramic (Al2O3) and ultra-high-molecular-weight polyethylene (UHMWPE) in terms of TNF-, release and induction of apoptosis. J774 mouse macrophages were incubated for 0,24 h in the presence of Al2O3 and UHMWPE particles. TNF-, release was measured by ELISA; Poly(ADP-ribose)polymerase (PARP) and caspase-3 expression was analyzed by Western blot; DNA fragmentation (DNA laddering) was visualized on agarose gel containing ethidium bromide. Al2O3 particles induced TNF-, release after 4 h incubation with concentrations reaching 483 and 800 pg/ml after 24 h with 125 and 250 particles/macrophage, respectively (control = 161 pg/ml) (P < 0.05 vs. control). The same concentrations of UHMWPE particles induced a much larger and significant TNF-, release after only 1 h incubation, increasing up to 6250 pg/ml after 24 h (P < 0.05 vs. control). Western blot analysis demonstrated that the active caspase-3 fragment (17 kDa) and the proteolytic PARP fragment (85 kDa) were expressed after 2 h incubation with 125 and 250 Al2O3 particles/macrophage. The active caspase-3 and the PARP fragment had lower expression and appeared after a longer incubation time (8 h) with 125 and 250 UHMWPE particles/macrophage. Finally, DNA fragmentation (DNA laddering) was observed after 16 h with 125 and 250 particles of Al2O3 per macrophage whereas no laddering was induced by UHMWPE particles even after 24 h incubation. This study shows that although both Al2O3 and UHMWPE particles induce TNF-, release, this stimulation was much greater (8,10 times higher) with UHMWPE than A12O3 (P < 0.05 vs. control). As well, the induction of apoptosis, as measured by activation of caspase-3, PARP cleavage and DNA laddering, is different for these two particles, being faster and more important with Al2O3 than UHMWPE. We hypothesize that the ability of Al2O3 to induce macrophage apoptosis may explain the lower TNF-, release observed with these particles and explain the differences seen in osteolysis patterns of ceramic,ceramic (CC) vs. metal,polyethylene (Mpe) articulations. In conclusion, apoptosis may be a major internal mechanism to decrease macrophage activity and may be a desired therapeutic endpoint. The identification of an apoptosis-related pathway in the macrophage response to ceramic particles provides crucial data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] High-performance liquid chromatographic determination of creatine kinase activity influenced by methylglyoxalBIOMEDICAL CHROMATOGRAPHY, Issue 2 2009Xiaofang Peng Abstract Protein glycation has been implicated in the development of diabetic complications and other health disorders, which mainly arise from accumulation of advanced glycation endproducts (AGEs) in vivo. Methylglyoxal (MGO), a typical reactive intermediate carbonyl formed in early glycation process, can react non-enzymatically with N -terminal amino groups on proteins, leading to their inactivation and generation of detrimental AGEs. Recently, it was reported that activity of creatine kinase (CK, EC 2.7.3.2) could be reduced or even eliminated completely after incubation with MGO in vitro. CK activity is usually determined by conventional colorimetric assays. However, these methods are not appropriate for monitoring the influence of MGO on CK activity since MGO can also directly react with creatine, a substrate of CK. In this study, an efficient and much more accurate HPLC approach was established to investigate the effect of MGO on CK activity. Aminoguanidine was utilized to eliminate interference from the undesirable reaction between residual MGO and creatine. It was found that higher concentrations of MGO and longer incubation time for CK and MGO caused more pronounced reduction in CK activity. This HPLC method greatly facilitates acquisition of kinetic data about CK reaction and through further improvement it may be adopted to rapidly screen potential inhibitors of MGO-induced glycation. Copyright © 2008 John Wiley & Sons, Ltd. [source] Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid , peptidesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001N. Demeester Abstract The Dutch (E22Q) and Flemish (A21G) mutations in the ,APP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three A,(12,42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 µm the aggregation of these peptides followed the order: A,(1,42) WT > A,(12,42) WT > A,(12,42) Flemish >,A,(12,42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their ,-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in ,-sheet and random coil content. Apoptosis was induced in neuronal cells by the A,(12,42) WT and Flemish peptides at concentrations as low as 1,5 µm, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length A,(1,42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of A, WT and Flemish peptides, which may play a role in the accelerated progression of dementia. [source] Dynamics of a Transgene Expression in Acute Rat Brain Slices Transfected with Adenoviral VectorsEXPERIMENTAL PHYSIOLOGY, Issue 4 2003C. E. L. Stokes We present a quantitative account of the expression dynamics of a transgene (enhanced green fluorescent protein, EGFP) in acute brain slices transfected with an adenoviral vector (AVV) under control of the human cytomegalovirus (HCMV) promoter. Micromolar concentrations of EGFP could be detected in brainstem and hippocampal slices as early as 7 h after in vitro transfection with a viral titre of 4.4 × 109 plaque-forming units (pfu) ml,1. Although initially EGFP appeared mainly in glia, it could be detected in neurones with longer incubation times of 10-12 h. However, fluorescence was never detected within some populations of neurones, such as hippocampal pyramidal cells, or within the hypoglossal motor nucleus. The density of cells expressing EGFP peaked at 10 h and then decreased, possibly suggesting that high concentrations of EGFP are toxic. The age of the animal significantly affected the speed of EGFP accumulation: after 10 h of incubation in 30-day-old rats only 4.88 ± 0.51 cells/10 000 ,m2 were fluorescent compared to 7.28 ± 0.39 cells/10 000 ,m2 in 12-day-old rats (P < 0.05). HCMV promoter-driven transgene expression depended on the activity of protein kinase A, and was depressed with a cAMP/protein kinase A antagonist (20 ,M Rp-cAMPS; P < 0.0005). This indicates that expression of HCMV-driven constructs is likely to be skewed towards cellular populations where cAMP-dependent signalling pathways are active. We conclude that acute transfection of brain slices with AVVs within hours causes EGFP expression in micromolar concentrations and that such transfected cells may remain viable for use in physiological experiments. [source] Predictive models of the combined effects of curvaticin 13, NaCl and pH on the behaviour of Listeria monocytogenes ATCC 15313 in brothJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2000A. Bouttefroy Thirty-three strains of Listeria monocytogenes belonging to different serotypes were tested for their sensitivity to curvaticin 13, an antilisterial bacteriocin produced by Lactobacillus curvatus SB13, using the well diffusion method in Institut Pasteur agar plates at 37 °C. No relationship between serotype and sensitivity was observed. The sensitivity of this species was strain-dependent and a large variation in tolerance to curvaticin 13 was observed. The combined effects of curvaticin 13 (0,160 AU ml,1), NaCl (0,6% w/v), pH values (5·0,8·2) and incubation time (0,24 h) were investigated on L. monocytogenes ATCC 15313 in trypcase soy,yeast extract broth at 22 °C. For this study, two Doehlert matrices were used in order to investigate the main effects of these factors and their different interactions. The results were analysed using the Response Surface Methodology. Curvaticin 13 had a major inhibitory effect and the response was NaCl concentration-, time- and pH-dependent. This inhibitory activity was the same at pH values between 6·6 and 8·2. Curvaticin 13 was bactericidic at acidic pH values, but the surviving cells resumed growth. For a short incubation time (12 h), the effectiveness of curvaticin 13 was maximal in the absence of NaCl. For longer incubation times (12,48 h), with high NaCl (6%) and curvaticin 13 concentrations (160 AU ml,1), the inhibition of L. monocytogenes was greater than that observed with NaCl or curvaticin 13 alone. [source] Molted feathers from clay licks in Peru provide DNA for three large macaws (Ara ararauna, A. chloropterus, and A. macao)JOURNAL OF FIELD ORNITHOLOGY, Issue 2 2009Kara J. Gebhardt ABSTRACT Conservation genetic analyses of wildlife have increased greatly in the past 10 yr, yet genetic studies of parrots are rare because of difficulties associated with capturing them and obtaining samples. Recent studies have demonstrated that molted feathers can provide a useful source of DNA, but success rates have varied considerably among studies. Our objective was to determine if molted macaw feathers from Blue-and-yellow Macaws (Ara ararauna), Scarlet Macaws (A. macao), and Red-and-green Macaws (A. chloropterus) collected from rainforest geophagy sites called clay licks could provide a good source of DNA for population genetic studies. Specific objectives were to determine (1) how nuclear DNA microsatellite amplification success and genotyping error rates for plucked macaw feathers compared to those for molted feathers collected from clay licks in the Amazon rainforest, and (2) if feather size, feather condition, species, or extraction method affected microsatellite amplification success or genotyping error rates from molted feathers. Amplification success and error rates were calculated using duplicate analyses of four microsatellite loci. We found that plucked feathers were an excellent source of DNA, with significantly higher success rates (P < 0.0001) and lower error rates (P= 0.0002) than for molted feathers. However, relatively high success rates (75.6%) were obtained for molted feathers, with a genotyping error rate of 11.7%. For molted feathers, we had higher success rates and lower error rates for large feathers than small feathers and for feathers in good condition than feathers that were moldy and broken when collected. We also found that longer incubation times and lower elution volumes yielded the highest quality DNA when extracting with the Qiagen DNeasy tissue kit. Our study demonstrates that molted feathers can be a valuable source of genetic material even in the challenging conditions of tropical rainforests, and our results provide valuable information for maximizing DNA amplification success rates when working with shed feathers of parrots. SINOPSIS Los análisis genéticos para la conservación de la vida silvestre han crecido en gran escala durante los últimos 10 años, pero el análisis genético de los loros son raros por las dificultades asociados con su captura y obtención de muestras. Estudios recientes han demostrado que plumas mudadas podrían proveer una fuente útil de ADN, pero las tasas de éxito varían considerablemente entre estudios. Nuestro objetivo fue determinar si las plumas mudadas de Ara ararauna, A. macao y A. chloropterus colectadas en sitios de bosque húmedo donde estas aves consumen el suelo, llamados colpas, podrían proveer una fuente útil de ADN para estudios de la genética de las poblaciones. Los objetivos específicos fueron determinar (1) como comparan las tasas de éxito de la amplificación de los microsatélites del ADN nuclear y las tasas de error en el análisis del genotipo de plumas, entre plumas colectadas directamente de los guacamayos y plumas colectadas en colpas en el bosque Amazónico, y (2) si el tamaño de la pluma, su condición, la especie o el método de extracción afecta el éxito de la amplificación de los microsatélites o las tasas de error en el análisis del genotipo de las plumas mudadas. Las tasas de éxito de amplificación y error fueron calculados usando análisis duplicados de cuatro loci de microsatélites. Encontramos que plumas colectadas directamente de las aves son una fuente excelente de ADN, con tasas de éxito significativamente más altas (P < 0.0001), y con menores tasas de error (P= 0.0002) que las plumas mudadas. Sin embargo, tasas de éxito relativamente altas (75.6%) fueron obtenidos de plumas mudadas, con una tasa de error en el análisis del genotipo de 11.7%. Para plumas mudadas, tuvimos tasas de éxito más altas y tasas de error menores para plumas grandes que para plumas pequeñas y para plumas en buena condición que para plumas que estaban cubiertos con hongos y quebradas cuando fueron colectadas. También encontramos que mayores periodos de incubación y menores volúmenes de elución proveían el ADN de mayor calidad cuando se extraía el ADN usando el kit de tejido Quiagen DNeasy. Nuestro estudio demuestra que las plumas mudadas pueden ser una fuente valiosa de materia genética, hasta en las condiciones de los bosques húmedos tropicales. Nuestros resultados proveen información valiosa para maximizar las tasas de éxito de la amplificación del ADN cuando se analizan las plumas mudadas de los loros. [source] Human prion strain selection in transgenic mice,ANNALS OF NEUROLOGY, Issue 2 2010Kurt Giles DPhil Objective: Transgenic (Tg) mice expressing chimeras of mouse and human prion proteins (PrPs) have shorter incubation periods for Creutzfeldt-Jakob disease (CJD) prions than mice expressing full-length human PrP. Increasing the sequence similarity of the chimeric PrP to mouse PrP, by reverting human residues to mouse, resulted in a Tg line, denoted Tg22372, which was susceptible to sporadic (s) CJD prions in ,110 days. Methods: Mice expressing chimeric mouse/human PrP transgenes were produced. The mice were inoculated intracerebrally with extracts prepared from the brains of patients who died of CJD. Onset of neurological dysfunction marked the end of the incubation time. After sacrifice of the Tg mice, their brains were analyzed for PrPSc and neuropathological changes. Results: Reversion of 1 additional residue (M111V) resulted in a new Tg line, termed Tg1014, susceptible to sCJD prions in ,75 days. Tg1014 mice also have shorter incubation periods for variant (v) CJD prions, providing a more tractable model for studying this prion strain. Transmission of vCJD prions to Tg1014 mice resulted in 2 different strains, determined by neuropathology and biochemical analysis, which correlated with the length of the incubation time. One strain had the biochemical, neuropathological, and transmission characteristics, including longer incubation times, of the inoculated vCJD strain; the second strain produced a phenotype resembling that of sCJD prions including relatively shorter incubation periods. Mice with intermediate incubation periods for vCJD prions had a mixture of the 2 strains. Both strains were serially transmitted in Tg1014 mice, which led to further reduction in incubation periods. Conversion of vCJD-like to sCJD-like strains was favored in Tg1014 mice more than in the Tg22372 line. The single amino acid difference therefore appears to offer selective pressure for propagation of the sCJD-like strain. Interpretation: These 2 Tg mouse lines provide relatively rapid models to study human prion diseases as well as the evolution of human prion strains. ANN NEUROL 2010;68:151,161 [source] | ||||||||||||||||