Incubation Time Points Research Articles

Effects of dehydration on cardiovascular development in the embryonic American alligator (Alligator mississipiensis)

Effects of dehydration on reptilian embryonic cardiovascular function are unknown. Here, we present the first morphological and physiological data quantifying the cumulative effects of four acute dehydration events on the embryonic American alligator, Alligator mississipiensis. We hypothesized that dehydration would alter embryonic morphology, reduce blood volume and augment the response to angiotensin II (Ang II), a key osmotic and blood volume regulatory response element in adult vertebrates. Drying events at 30%, 40%, 50%, and 60% of embryonic incubation reduced total egg water content by 14.43±0.37g, a 3.4 fold increase relative to controls. However, embyronic blood volume was greater in the dehydration group at 70% of embryonic incubation compared to controls (0.39±0.044mLg−1 and 0.22±0.03mLg−1, respectively), however, both groups were similar at 90% of incubation (0.18±0.02mLg−1 in the controls and 0.23±0.03mLg−1 in the dehydrated group). Dehydration altered the morphological phenotype and resulted in an overall reduction in embryonic mass at both incubation time points measured. Dehydration also altered the physiological phenotype, resulting in embryonic alligators that were relatively bradycardic at 90% of incubation. Arterial Ang II injections resulted in a dose dependent hypertension, which increased in intensity over the span of incubation studied. While progressive incubation altered the Ang II response, dehydration had no impact on the cardiovascular responses to the peptide. Quantification of Ang II type-1 receptor protein using western blot analysis illustrated that dehydration condition and incubation time point did not alter protein quantity. Collectively, our results show that dehydration during embryonic development of the American alligator alters embryonic morphology and baseline heart rate without altering arterial pressure and response to Ang II.

Heat impact upon the infectivity of hepatitis B virus in serum

This article was to explore the impact of temperature on hepatitis B virus infectivity. HBV positive serum with a HBV DNA titer of 1.33 × 10(8) copies/ml was aliquots into 23 Ep tubes with 1.5 ml, 100 µl in one tube.15 tubes were incubated at 37, 56 and 65°C for 0, 30, 60, 120 and 600 minutes, respectively. The other 8 tubes were incubated at 98°C for 0, 5, 10 and 30 minutes, respectively. Post-treated serum at all time points were selected to infect HepG-2 cell. When 18 hours after infection, these cells were extensively washed with phosphate buffered saline. Cells were harvested after the addition of fresh culture medium to culture cells for 48 hours. HBV DNA was detected by FQ-PCR. HBV DNA was detected in cells that were infected by serum at 37°C and 56°C for 30, 60, 120 and 600 minutes, respectively. The titers for the cells incubated at 37°C were (4.85 ± 1.71) × 10(5), (3.85 ± 1.76) × 10(5), (1.67 ± 0.67) × 10(5), (7.86 ± 1.03) × 10(4) copies/ml, and those for the cells incubated at 56°C were (4.01 ± 0.16) × 10(5), (9.77 ± 0.97) × 10(4), (6.36 ± 0.65) × 10(4), (5.05 ± 0.24) × 10(3) copies/ml at different incubation time points. For the cells incubated at 65°C for 60 and 120 minutes, HBV DNAs were (5.15 ± 7.28) × 10(3) and (7.56 ± 10.60) × 10(2) copies/ml, respectively, which were much lower than those in the controls cells ((6.79 ± 1.48) × 10(5) copies/ml). The results of HBV DNA were different (F = 104.4, P < 0.001) in groups treated with different temperature, and results of HBV DNA were also different (F = 144.0, P < 0.001) in groups processed for different period of time. Temperature and processing time had interaction (F = 23.6, P < 0.001). After heating at 98°C for 10 minutes and boiling for 5 minutes, the HBV DNA copy number ((3.02 ± 4.26) × 10(2), (4.31 ± 6.09) × 10(2) copies/ml) in infected cells decreased by about 10 folds than that in the control group ((6.79 ± 1.48) × 10(5) copies/ml). HBV DNAs were not detected in cells that were infected by serum which was heated at 98°C for 30 minutes and boiled for 10 minutes. The infectivity of HBV serum in vitro was relatively stable at low temperature, and it would lose its infectivity in short period of time at high temperature.

In vitro and in vivo neo-cartilage formation by heterotopic chondrocytes seeded on PGA scaffolds

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12weeks in vivo construct size reductions were higher compared with 6weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.

Surface structures and osteoblast response of hydrothermally produced CaTiO 3 thin film on Ti–13Nb–13Zr alloy

This study investigated the surface characteristics and in vitro biocompatibility of a titanium (Ti) oxide layer incorporating calcium ions (Ca) obtained by hydrothermal treatment with or without post heat-treatment in the Ti–13Nb–13Zr alloy. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, atomic force microscopy and contact angle measurements. In vitro biocompatibility of the Ca-containing surfaces was assessed in comparison with untreated surfaces using a pre-osteoblast cell line. Hydrothermal treatment produced a crystalline CaTiO 3 layer. Post heat-treatment at 400 °C for 2 h in air significantly decreased water contact angles in the CaTiO 3 layer ( p < 0.001). The Ca-incorporated alloy surfaces displayed markedly increased cell viability and ALP activity compared with untreated surfaces ( p < 0.001), and also an upregulated expression of various integrin genes (α1, α2, α5, αv, β1 and β3) at an early incubation time-point. Post heat-treatment further increased attachment and ALP activity in cells grown on Ca-incorporated Ti–13Nb–13Zr alloy surfaces. The results indicate that the Ca-incorporated oxide layer produced by hydrothermal treatment and a simple post heat-treatment may be effective in improving bone healing in Ti–13Nb–13Zr alloy implants by enhancing the viability and differentiation of osteoblastic cells.

Large-scale 2-D DIGE studies - guidelines to overcome pitfalls and challenges along the experimental procedure

Journal of Integrated Omics *Corresponding author: PD Dr. Martin von Bergen. UFZ, Helmholtz-Centre for Environmental Research, Department of Proteomics, Permoserstr. 15, 04318 Leipzig, Germany. Fax: +49-341-2351786. Email Address: Martin.vonbergen@ufz.de. | DOI: 10.5584/jiomics.v1i1.50 Franziska Dautel et al., 2010 | Journal of Integrated Omics 170-179:171 dealing with gel-to-gel discrepancies, labeling inefficiencies, and dyeand batch effects. Gel-to-gel discrepancies arise from run-time differences, variances in the loaded protein amounts or dye-front deformations [4]. Accounting for these differences is important for both 2-DE and 2-D DIGE. However, the dye-effect is specific for DIGE-projects, as the application of three different fluorophores can cause preferential dye-protein binding, variances in the fluorescent signal and background and differences in gel migration of the labeled proteins. As a result, protein abundances are not directly comparable when the proteins are labeled differently in various samples [5-7]. In addition, the experimental execution for a large number of samples is often divided into several batches of 6 or 12 gels. As a consequence, results of protein expression often cluster with the performed batches rather than with the individual samples and replicates. The goal is to identify spots that are truly differentially expressed, while accounting for statistical issues such as the multiple testing problem. This multiple testing problem states the accumulation of false positives as a general property of confidence-based statistical tests. These tests are applied across multiple features such as individual spots in DIGE to detect significantly altered changes in protein abundance [8]. This study reports an experimental design for a 2-factor analysis (time and concentration): murine hepatoma cells (Hepa1c1c7) were treated with the procarcinogen benzo-apyrene (B[a]P) and protein concentrations were quantified using 2-D DIGE (Fig. 1). Differential protein expression induced by B[a]P (or active B[a]P-metabolites) has previously been studied in different cellular models using one incubation time point and several B[a]Por B[a]P-metabolite concentrations [9-13]. In contrast, this B[a]P-protein expression analysis sampled four incubation time points at one toxic (5 μM) and one sub-acute B[a]P-concentration (50 nM), which required the processing of 36 samples in total [14]. In order to process the data originating from these experiments, a statistical analysis pipeline was developed to account for dyeand batch effects and to extract concentrationand time-dependent protein profiles. 2. Material and Methods 2.1 Cell culture and BaP exposure Murine hepatoma cells (Hepa1c1c7, ATCC No. CRL-2026; LGC Promochem, Wesel, Germany) were cultured as described elsewhere [15]. The cells were exposed to 50 nM B[a]P (Sigma-Aldrich, Steinheim, Germany), 5 μM B[a]P or DMSO for 2, 4, 12 and 24 h. Three independent biological replicates of all treatments were prepared. 2.2 DIGE and Data Analysis 2.2.1 Difference gel electrophoresis Cells were washed and lysed according to the procedure previously described [16]. Protein extracts were prepared and labeled according to manufacturer’s recommendations (GE Healthcare, Uppsala, Sweden). A Cy2-labeled common internal standard for all gels was prepared from a mixture of all samples IPG strips (24 cm, pH range 3-10 NL; GE Healthcare, Freiburg, Germany), which were rehydrated overnight and focused for 100,000 Vhrs using an Ettan IPGphor 3 isoelectric focusing unit (GE Healthcare, Freiburg, Germany) as described earlier [17]. Second dimension separation was performed using an Ettan DALTtwelve electrophoresis system (GE Healthcare, Uppsala, Sweden) on 12 % SDS-PAGE gels. The gels were scanned using the Ettan DIGE Imager Scanner (GE Healthcare, Uppsala, Sweden).

Improved pre‐osteoblast response and mechanical compatibility of ultrafine‐grained Ti–13Nb–13Zr alloy

Metallic implantation materials having high yield strength, low elastic modulus, and non-cytotoxic alloying elements would be advantageous for the long-term stability of implants. This study assessed the surface and mechanical properties, and also in vitro osteoconductivity of ultrafine-grained (UFG) Ti-13Nb-13Zr alloy produced by dynamic globularization without any severe deformation for future biomedical applications as an endosseous implant material. The surface characteristics and mechanical properties were investigated by orientation image microscopy, contact angle measurements, optical profilometry, and uniaxial tension tests. Mouse calvaria-derived pre-osteoblastic cell (MC3T3-E1) attachment, spreading, viability, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on UFG Ti-13Nb-13Zr alloy were compared with coarse-grained (CG) Ti-13Nb-13Zr and CG Ti-6Al-4V alloys. Dynamic globularized Ti-13Nb-13Zr alloy has an ultrafine grain size (0.3 μm) and an excellent combination of yield strength and elastic modulus compared with CG alloys, which displayed significantly lower water contact angles compared with CG alloys (P<0.05). The UFG and CG Ti-13Nb-13Zr alloys displayed significantly increased cellular attachment compared with CG Ti-6Al-4V alloy (P<0.05). The UFG Ti-13Nb-13Zr supported better cell spreading and more numerous focal adhesions. ALP activity (P<0.05) and mRNA expressions of the osteoblast transcription factor genes (osterix, Runx2) and marker gene for osteoblast differentiation (osteocalcin) were markedly increased in cells grown on the UFG substrate compared with CG substrates at early incubation timepoints. Enhanced pre-osteoblast response to UFG Ti-13Nb-13Zr substrate is attributable to the non-cytotoxic alloying elements and the submicron scale grain size contributes to the superior surface hydrophilicity and abundant grain boundaries favorable for cell behavior. These findings indicate that dynamic globularized UFG Ti-13Nb-13Zr alloy is promising for load-bearing endosseous implant material because of excellent mechanical and biological compatibilites.

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm(2) at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF(164), play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.

The antioxidative effect of Iranian Mentha pulegium extracts and essential oil in sunflower oil

The aim of the present study was to evaluate antioxidative activities of the essential oil, methanol and water extracts of Iranian pennyroyal in vegetable oil during storage. Different concentrations (0, 200, 400, 600, 800 and 1000ppm) of essential oil, water and methanol extracts and beta-hydroxy toluene (BHT; 200ppm) were added to sunflower oil emulsion in the presence of cupric ions and incubated for 7days at 60°C. Peroxide values (PVs) and thiobarbituric acid reacting substances (TBARS) levels were measured in each day up to day of seven. Furthermore, antioxidant capacity of the essential oil and extracts were determined using DPPH and β-carotene–linoleic acid methods. Values were compared among groups in each incubation time points using ANOVA. Results showed that DPPH and β-carotene–linoleic acid assay findings on the Mentha pulegium extracts were comparable to those found on BHT. Furthermore, in all incubation time points, M. pulegium extracts lowered PVs and TBARS levels when compared to the control (p<0.001). In this respect, water extract was more potent than the methanol extract. Essential oil did not show considerable antioxidative effect. It seems that water extract of M. pulegium is a potent antioxidant which makes it as a potential antioxidant for oil and oily products during storage.

Lentivirus Immobilization to Nanoparticles for Enhanced and Localized Delivery From Hydrogels

Hydrogels can provide a controllable cell microenvironment for numerous applications in regenerative medicine, and delivery of gene therapy vectors can be employed to enhance their bioactivity. We investigated the delivery of lentiviral vectors from hydrogels, and employed the immobilization of lentivirus to hydroxylapatite (HA) nanoparticles as a means to retain and stabilize vectors within hydrogels, and thereby increase delivery efficiency. Entrapment of the vector alone within the hydrogel maintained the activity of the virus more effectively compared to the absence of a hydrogel, and release was slowed with an increasing solid content of the hydrogel. Association of the lentivirus with HA increased the activity of the complexes, with HA increasing the virus activity for 72 hours. Cells seeded onto lentivirus-HA-loaded hydrogels had a decreased number of infected cells outside of the hydrogel relative to the absence of HA. In vivo studies with collagen hydrogels loaded with lentivirus and HA-lentivirus demonstrated sustained and localized transgene expression for at least 4 weeks, with increased expression using the lentivirus-HA complex. This strategy of nanoparticle immobilization to stabilize and retain vectors is broadly applicable to hydrogels, and may provide a versatile tool to combine gene therapy and biomaterials for applications in regenerative medicine.

Heparin and commercial bothropic antivenom against the paralyzing effect of Bothrops jararacussu snake venom

The crude venom of Bothrops jararacussu (Bjssu) is known to induce muscular paralysis in vitro. Many studies have shown that various substances, including heparin, neutralize the damage caused by snake venom. In the present study, the ability of heparin (Hep) and commercial bothropic antivenom (CBA) to neutralize neuromuscular effects of Bjssu venom, at different time-points, was analyzed. Mouse phrenic nerve-diaphragm preparation was used through a conventional myographic technique, following five different protocols: Group 1 was incubated with Bjssu (40 µg/mL) without any other treatment; Groups 2 and 3 were pretreated with heparin (1 µL/mL) and CBA (120 µL/mL), respectively, for 15 minutes before venom addition; Group 4 after 50% neuromuscular blockade induced by Bjssu crude venom received 1 µL/mL of heparin while Group 5 received a mixture of Hep:CBA:Bjssu. Control preparations (Tyrode) were treated with Hep and CBA (mean ± SEM; n = 3-6). After 120 minutes of venom incubation, Group 1 preparations presented twitch-tension of 12 ± 2%. However, in Groups 2 and 3, the neutralizations were 92 ± 1.9% and 81 ± 6%, respectively. The heparin addition, after 50% neuromuscular blockade by Bjssu, produced 40 ± 6% muscular response after 120 minutes of incubation. Hep:CBA:Bjssu mixture displayed a protective effect of 84 ± 10% against venom action. In conclusion, heparin and commercial bothropic antivenom efficiently neutralized the neurotoxic effects caused by B. jararacussu crude venom, even at different incubation time-points.

LC-ESI MS/MS quantification of atropine and six other antimuscarinic tropane alkaloids in plasma

We have developed and validated a quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) procedure for the simultaneous determination of seven natural and semisynthetic tropane alkaloids in plasma: atropine (d-hyoscyamine/l-hyoscyamine), cocaine, homatropine, ipratropium, littorine, N-butylscopolamine, and scopolamine. Plasma and serum samples were precipitated for deproteinization (recovery 88-94%), followed by reversed-phase-based liquid chromatography prior to positive electrospray ionization for detection by multiple reaction monitoring using a linear ion trap quadrupole mass spectrometer. All analytes were quantified using cocaine-d3 as an internal standard suitable and reliable for robust, precise (coefficient of variation 2-13%), and accurate (87-122%) measurement within a linear range of 3 orders of magnitude (0.05-50 ng/ml plasma). The method was exemplarily applied to stability studies in phosphate-buffered saline, human serum, and rabbit serum. Each alkaloid was incubated separately and samples were taken at distinct incubation time points. Supernatants of diverse alkaloids at corresponding time points were pooled and subjected to simultaneous LC-ESI MS/MS quantification. This combinatorial analysis design allowed us to analyze the stability of samples with a drastically reduced number of chromatographic runs. In the presence of rabbit serum, all tropane alkaloids tested were degraded significantly within minutes to hours, with the exception of the stable semisynthetic compounds ipratropium and N-butylscopolamine. In contrast, in the presence of equal concentrations of human serum, no degradation was observed for any of the compounds, with the exception of cocaine. Relevant enzymes involved in enzymatic degradation are discussed.

The role of receptor protein tyrosine kinase MERTK and intracellular Ca2+ playing in the phagocytosis of human retinal pigment epithelial cells

Objective To investigate the role of intraeellular Ca2+ and MERTK in the phagocytosis of human retinal pigment epithelial (RPE) cells, and reveal the relationship between MERTK and intraeellular Ca2+. Methods The euhured RPE cells were incubated with rod outer segments (ROS) at 37℃, the phagoeytosis was terminated at different incubation time points. The concentration of intraeellular Ca2+ was assayed by Fluro-3/AM loading methods combined with fluorescence microscope and CCD system, and the mRNA level of MERTK gene was measured by reverse transcription polymerase chain reaction (RT-PCR). Treating the RPE cells with stimulator (A23187)or inhibitor(verapamile)of intraeellular Ca2+ to observe the changes of MERTK gene expression. Results ROS adhered to hRPE cells at the 15th minute, and the ingestion saturated at the 24th hour. The concentration of intracellular Ca2+ increased at the 15th minute, and kept the high level in 24 hours. The level of MERTK mRNA increased at the 5th minute, and kept the high level duration the whole incubation. When RPE cells were treated by A23187, the expression of MERTK increased in a dose-dependent manner. After RPE cells was pretreated by A23187, the expression level of MERTK was higher in the proceeding incubation groups than which in the control group except at the 3rd hour. When RPE cells were treated by verapamil, the expression level of MERTK decreased in a dose-dependent manner. After RPE cells were pretreated by verapamil , the expression level of MERTK was lower in all the proceeding incubation groups than which in the control group (P<0.05). Conclusion MERTK gene and Ca2+ play an important role in sustaining RPE cells phagoeytizing ROS. As an up-stream regulator, the receptor tyrosine kinase MERTK keeps RPE cells phagocytizing ROS by starting the intracellular Ca2+. Key words: Pigment epithelium of eye/pathophysiology; Phagoeytosis/physiology; Ca2+MERTK gene

High throughput, non-invasive and dynamic toxicity screening on adherent cells using respiratory measurements

A dynamic respiration assay based on luminescence decay time detection of oxygen for high throughput toxicological assessment is presented. The method uses 24-well plates (OxoDishes) read with the help of a sensor dish reader placed in a humidified CO 2-incubator. Adherent primary rat hepatocytes and the human hepatic cell line Hep G2 were exposed to known toxic compounds. Dissolved oxygen concentration, a measure of respiration, was measured with an oxygen sensor optode immobilized in the centre of each well. The cells were maintained in the dishes during the assay period and can afterwards be processed for further analyses. This dynamic, non-invasive measurement allowed calculation of 50% lethal concentrations (LC 50) for any incubation time point giving concentration–time-dependent responses without further manipulation or removal of the cells from the incubator. Toxicokinetic profiles are compared with Sulforhodamine B assay, a common cytotoxicity assay. The novel assay is robust and flexible, very easy to carry out and provides continuous online respiration data reflecting dynamic toxicity responses. It can be adapted to any cell-based system and the calculated kinetics contributes to understanding of cell death mechanisms.

DNA delivery in vitro via surface release from multilayer assemblies with poly(glycoamidoamine)s

Localized controlled release of nucleic acid therapeutics could be an effective way to reduce the extracellular barriers associated with systemic delivery. Herein, we have used the layer-by-layer film deposition approach to construct ultrathin multilayer assemblies for in vitro controlled release of plasmid DNA (pDNA). Layer-by-layer assemblies containing alternate layers of cationic poly( l-tartaramidopentaethylenetetramine) ( T4), and anionic pDNA were fabricated. The film thickness and the absorbance at 260 nm for different T4/pDNA multilayer assemblies were characterized by ellipsometry and UV–vis spectrophotometry, respectively. The results indicated an increased loading capacity of pDNA with respect to an increase in the number of T4/pDNA bilayers deposited. For the controlled-release studies we incubated the bilayers coated on quartz slides in phosphate-buffered saline (PBS) at 37 °C and collected the media at different incubation time points. The collected PBS samples were characterized for pDNA release by complexing solutions containing the released pDNA with Lipofectamine 2000 and following cellular pDNA uptake via flow cytometry and GFP gene expression assays with HeLa cells. The study showed that the multilayer films started to release pDNA after 1 day of incubation and increased after 7 days of incubation. Assays monitoring green fluorescent protein (GFP) expression in HeLa cells indicated that about 20% of the cells were positive for GFP expression at all sample time points up to 11 days. Although an increase in cells positive for Cy5-pDNA was found as the incubation time increased, the number of cells positive for GFP expression remained constant over the same time frame.

Simplified estimation of forage degradability in the rumen assuming zero-order degradation kinetics

SUMMARYIn the present paper, a simplified procedure using few in situ data points is derived and then evaluated (using a large database) against reference values estimated with the standard nylon bag first-order kinetics model. The procedure proposed involved a two-stage mathematical process, with a statistical prediction of some degradation parameters (such as lag time) and then a kinetic model derived by assuming degradation follows zero-order kinetics to determine effective degradability in the rumen (E). In addition to the estimation of washout fraction and discrete lag, which is common to both procedures, the simplified procedure requires measurement of dry matter losses at one incubation time point only. Thus, interference of the animal rumen will be much reduced, which will lead to increased capacity for feed evaluation. Calibration of the zero-order model against the first-order model showed that suitable estimates of E can be obtained with disappearance at 24, 48 or 72 h as the single incubation end time point. The strength of the calibration is such that an end incubation time point as low as 24 h may be sufficient, which may reduce substantially the total incubation time required and thus the impact on the experimental animal. Relevant regression equations to predict reference values of parameters such as lag time or E are also developed and validated.

Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin's endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs.

In vitro assessment of the pharmacodynamic properties of DB75, piperaquine, OZ277 and OZ401 in cultures of Plasmodium falciparum

Using synchronized cultures of Plasmodium falciparum, the time- and concentration-dependent growth changes of erythrocytic parasite stages to DB75, piperaquine, OZ277 and OZ401 were investigated in vitro over a concentration range of approximately 1-100x the IC(50) of piperaquine, OZ277 and OZ401 and approximately 10-1000x the IC(50) of DB75. The effects of timed in vitro exposure (1, 6, 12 or 24 h) were monitored by the incorporation of [(3)H]hypoxanthine into the parasite nucleic acids. After 1 h of exposure to the highest concentration of the compound followed by removal of the compound, the growth of all stages of P. falciparum was reduced to < 34% for DB75 and 15% for piperaquine, OZ277 and OZ401 compared with untreated control parasites. At this time point, no stage-specific effects were observed at any of the concentrations. Strong inhibition (< or = 10% growth) of all parasite stages was observed when the parasites were exposed to 10x or 100x the IC(50) of OZ277 and OZ401 for > or = 6 h. At the 6 h incubation time point, DB75 was more active against mature parasite stages, with the IC(50)s of young ring forms elevated up to 7-fold. This trend was observed up to 12 h, but was only statistically significant at the lowest concentration. Interestingly, the stage-specific effect of DB75 on ring forms was not detectable when washing procedures were omitted. This indicates a cytostatic action of DB75 on P. falciparum ring forms. The current study suggests that P. falciparum ring stages are less susceptible to DB75. A milder and often statistically insignificant stage-specific trend was observed for piperaquine, whereas OZ277 and OZ401 were equally active against the erythrocytic parasite stages.

Evaluation of a rapid agglutination method for detection of equine red cell surface antigens (Ca and Aa) as part of pretransfusion testing

Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. Serum was obtained from a horse with anti-Ca antibodies and from another horse with anti-Aa antibodies. The presence of agglutinating antibodies was confirmed with antibody screening. Venous blood samples, collected in citrate-phosphate-dextrose, were obtained from 21 horses of various breeds. Samples were blood typed in the Veterinary Medical Teaching Hospital Hematology Laboratory using standard methodology. Washed RBCs from each of the 21 horses were incubated individually with anti-Ca and anti-Aa sera at dilutions of 1:4, 1:8, and 1:16 for 15 and 30 minutes at room temperature and 37 degrees C. Of the 21 horses, 13 were identified as Aa+/Ca+, four were Aa+/Ca-, two were Aa-/Ca+, and two were Aa-/Ca-. All 17 Aa-positive horses had a positive agglutination reaction at all dilutions of anti-Aa serum, incubation times, and temperatures, while all Aa-negative horses were negative. Each Ca-positive horse had a positive agglutination reaction at all incubation time points and temperatures up to the 1:16 dilution of the anti-Ca serum. All Ca-negative horses were negative at all times, temperatures, and dilutions of anti-Ca serum. Use of the modified protocol on 26 hospitalized horses resulted in accurate typing, based on complete antibody screens. These results support the hypothesis that equine RBCs can be blood typed using a rapid (15 minute) protocol, at room temperature, for the presence of Ca and Aa antigens using equine-derived antisera. This technique may be beneficial for pretransfusion testing of equine patients in an emergency setting.

Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin

The mechanism of action of gabapentin is still not well understood. It binds to the α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels but has little acute effect on calcium currents in several systems. However, our recent results conclusively demonstrated that gabapentin inhibited calcium currents when applied chronically but not acutely, both in heterologous expression systems and in dorsal root ganglion neurons 1. In that study we only examined a 40 hour time point of incubation with gabapentin, and here we have extended these results to include the effect of up to 6 and 20 hours incubation with gabapentin on calcium channel currents formed from CaV2.1/β4/α2δ-2 subunits. Gabapentin was significantly effective to inhibit the currents if included for 17-20 hours prior to recording, but it did not produce a significant inhibition if included for 3-6 hours. We previously concluded that gabapentin acts primarily at an intracellular location, requiring uptake into cells. However, this effect is mediated by α2δ subunits, being prevented by mutations in either α2δ-1 or α2δ-2 that abolish gabapentin binding 1. Furthermore, we also showed that the trafficking of α2δ-2 and CaV2 channels was disrupted by gabapentin. Here we have also extended that study, to show that the cell-surface expression of CaV2.1 is not reduced by chronic gabapentin if it is co-expressed with α2δ-2 containing a point mutation (R282A) that prevents gabapentin binding

Signaling Pathways of All-Trans Retinoic Acid (ATRA)-Induced Apoptosis in Mantle Cell Lymphoma (MCL).

MCL, characterized by the t(11;14), results in the overexpression of cyclin D1, and typically has an aggressive clinical course. The disease is not curable by conventional therapy, and new modalities are needed. A MCL cell line, Granta, has been studied and has a complex karyotype and genetic alterations of cell cycle regulatory genes. ATRA is a metabolic derivative of retinoic acid (RA). Upon binding with ATRA, its receptors become transcriptionally activated and transactivate their target genes leading to cell growth arrest or apoptosis. We hypothesized that ATRA would lead to apoptosis or cell growth arrest in Granta MCL cells. We used ATRA nanodisks for increased solubilization and in vivo delivery. Reactive oxygen species (ROS) were measured with fluorescence-activated cell sorting (FACS) following incubation at 6 hours (h) and 24 h with ATRA (12.5–50nM). At 6 h, ATRA induced dose-dependent generation of ROS, which returned to baseline at 24 h. The levels of ROS in untreated controls were unchanged over all the incubation time points. To determine the effects of ATRA on apoptosis, Granta cells were treated with various concentrations for 6 h, 24 h, and 48 h; apoptosis was measured with annexinV/propidium iodide (annexin/PI) binding and FACS analysis. We found increasing apoptosis in Granta at 24 h, reaching significant levels at 48 h. At 48 h, the lowest doses of ATRA (12.5nM) resulted in >70% increase (as a percentage of control) in annexin/PI. To determine the biologic consequences of ATRA exposure in Granta, RARα protein levels were measured in control and ATRA treated cells at 2 h, 6 h, and 24 h. RARα protein was marginally increased at 6 h and significantly downregulated following 24 h ATRA exposure (50nM). Furthermore, ATRA significantly increased p21 protein expression at 24 h as assessed by immunoblotting. RT-PCR data showed that SESN1(T2) (an oxidant gene) transcript was virtually absent in Granta, and that SESN2 and CDKN1A transcripts were not affected by ATRA exposure. In summary, these data demonstrate that ATRA, complexed with nanodisk delivery particles, stimulates ROS generation as early as 6 h in Granta cells. In addition, ATRA induced significant apoptosis at 48 h, downregulated RARα protein, and increased p21 protein. Further studies investigating the role of ATRA in MCL are warranted.