Cell Culture Growth Medium Research Articles

Selective removal of copper from complex biological media with an agarose-immobilized high-affinity PSP ligand.

The elucidation of metal-dependent biological processes requires selective reagents for manipulating metal ion levels within biological solutions such as growth media or cell lysates. To this end, we immobilized a phosphine sulfide-stabilized phosphine (PSP) ligand on agarose to create a resin for the selective removal of copper from chemically complex biological media through simple filtration or centrifugation. Comprised of a conformationally preorganized phenylene-bridged backbone, the PSP-ligand binds Cu(I) with a 1:1 stoichiometry and exhibits a pH-independent Cu(I) dissociation constant in the low zeptomolar range. Neither Zn(II), Fe(II), nor Mn(II) interact with the ligand at millimolar concentrations, thus offering a much-improved selectivity towards copper over other commonly employed solid-supported chelators such as Chelex 100. As revealed by X-ray fluorescence elemental analysis, the immobilized chelator effectively removes copper from cell culture growth media and cell lysate isolated from mouse fibroblasts. In addition to preparing copper-depleted media or celllysates for biological studies, PSP-immobilized ligands might prove equally useful for applications in radiochemistry, materialsscience, and environmental science.

Towards the Standardization of Mesenchymal Stem Cell Secretome-Derived Product Manufacturing for Tissue Regeneration.

Mesenchymal stem cell secretome or conditioned medium (MSC-CM) is a combination of biomolecules and growth factors in cell culture growth medium, secreted by mesenchymal stem cells (MSCs), and the starting point of several derived products. MSC-CM and its derivatives could be applied after injuries and could mediate most of the beneficial regenerative effects of MSCs without the possible side effects of using MSCs themselves. However, before the clinical application of these promising biopharmaceuticals, several issues such as manufacturing protocols and quality control must be addressed. This review aims to underline the influence of the procedure for conditioned medium production on the quality of the secretome and its derivatives and highlights the questions considering cell sources and donors, cell expansion, cell passage number and confluency, conditioning period, cell culture medium, microenvironment cues, and secretome-derived product purification. A high degree of variability in MSC secretomes is revealed based on these parameters, confirming the need to standardize and optimize protocols. Understanding how bioprocessing and manufacturing conditions interact to determine the quantity, quality, and profile of MSC-CM is essential to the development of good manufacturing practice (GMP)-compliant procedures suitable for replacing mesenchymal stem cells in regenerative medicine.

Asymmetric depth-filtration: A versatile and scalable method for high-yield isolation of extracellular vesicles with low contamination.

We developed a novel asymmetric depth filtration (DF) approach to isolate extracellular vesicles (EVs) from biological fluids that outperforms ultracentrifugation and size‐exclusion chromatography in purity and yield of isolated EVs. By these metrics, a single‐step DF matches or exceeds the performance of multistep protocols with dedicated purification procedures in the isolation of plasma EVs. We demonstrate the selective transit and capture of biological nanoparticles in asymmetric pores by size and elasticity, low surface binding to the filtration medium, and the ability to cleanse EVs held by the filter before their recovery with the reversed flow all contribute to the achieved purity and yield of preparations. We further demonstrate the method's versatility by applying it to isolate EVs from different biofluids (plasma, urine, and cell culture growth medium). The DF workflow is simple, fast, and inexpensive. Only standard laboratory equipment is required for its implementation, making DF suitable for low‐resource and point‐of‐use locations. The method may be used for EV isolation from small biological samples in diagnostic and treatment guidance applications. It can also be scaled up to harvest therapeutic EVs from large volumes of cell culture medium.

Electrochemical sensor based on rGO/Au nanoparticles for monitoring H2O2 released by human macrophages

Increased oxidative burden contributes to the pathogenesis of most inflammatory diseases and is associated with aging and chronic inflammation. Macrophages contribute to the generation of reactive oxygen species (ROS) within inflamed tissues. Currently, ROS generation is measured using fluorescent probes and colorimetric/fluorimetric biochemical assays. Hydrogen peroxide (H2O2) diffuses through the cell membrane and can be monitored in the extracellular space. Herein, we present a sensor for H2O2 detection released by cells in culture supernatants. H2O2 sensing performance was evaluated using chronoamperometric detection. A sensitivity of 0.0641 μA μM−1 cm−2 with a limit of detection of 6.55 μM and excellent selectivity against many interferents was found. H2O2 release was also measured in conditioned medium from human THP-1 macrophages exposed to pro-oxidant and anti-oxidant treatments. The results were compared with those obtained by flow cytometry using the same cells stained with carboxy-H2DCFDA and MitoSOX Red, which detect intracellular ROS and mitochondrial superoxide, respectively. The addition of pro-oxidants lipopolysaccharide (LPS) and nigericin resulted in a significant increase in the cathodic current due to the H2O2 reduction, indicating an increased release of H2O2. The addition of 17-oxo-DHA, which inhibits LPS- and nigericin-dependent responses, decreased the LPS- and nigericin-induced release of H2O2. All the results obtained with the sensor were consistent with those obtained using flow cytometry. The operation of the sensor directly in the cell culture growth medium had no impact on cell viability. The sensor is highly sensitive, fast, and cost effective, and it can potentially be used for real time monitoring of oxidative stress.

A Novel Model of Long‐term Human Microvascular Endothelial Cell Serum Starvation

Microvascular endothelial cell injury and progressive loss of microvasculature in the dermis, kidney, retina and perineural beds is caused by hyper‐and hypoglycemic insults in individuals with Diabetes mellitus (DM). The consequence of microvascular injuries occurring over years to decades is the onset of DM‐associated co‐morbidities such as delayed wound healing, renal failure, vision loss and peripheral neuropathy. The Diabetes Control and Complications Trial noted a correlation between baseline endogenous C‐peptide serum levels and the delay in onset of DM‐associated co‐morbidities. In immortalized human microvascular endothelial cells (HMEC) and primary human dermal microvascular endothelial cells treatment with 1.5nM C‐peptide and 0.5nM insulin prevented hyperglycemia‐induced oxidative injury. In studies of these cells using immunofluorescence to track C‐peptide and the C‐peptide receptor, G‐protein coupled receptor 146 (GPR146), endothelial cells repeatedly demonstrated cytosolic stores of C‐peptide. Fetal bovine serum, a component of almost all cell culture growth media was determined to have high concentrations of C‐peptide. The aim of this study was to develop a model of long term endothelial serum starvation that would result in depleted internal stores of C‐peptide, but would prevent endothelial cells transformation to smooth muscle cells or fibroblasts, as endothelial progenitor cells are capable of differentiating into one of these three cell types. We hypothesized that cytosolic C‐peptide would reduce over time with serum starvation. To test this hypothesis, HMEC were incubated for up to two months in MCDB13 media supplemented with human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, basic fibroblast growth factor, insulin‐like growth factor‐1, ascorbic acid, and L‐glutamine. Serum starved HMEC were harvested at various time points and stained for C‐peptide (MAB80561 R&D Systems clone 915612) and DAPI or lysed with lysis buffer with 2‐mercaptoethanol followed by RNA isolation, cDNA preparation and RT‐PCR evaluation of targets. Cytosolic C‐peptide fluorescent signal diminished over time in a linear manner but was never completely abolished. HMEC serum starved for 30 days retained endothelial markers CD31, CD34, VEGFR2, had no change in smooth muscle markers Smoothelin, or Smooth muscle actin, and similarly, had no change in expression Vimentin a fibroblast marker relative to un‐serum starved HMEC controls as determined by RT‐PCR. The ability to serum starve HMEC long‐term and substantially reduce internal C‐peptide stores has the potential to enhance research to elucidate the pathways employed by C‐peptide in the prevention of hyperglycemia‐induced microvascular injury.Support or Funding InformationNIDDK

Bioengineered three-dimensional scaffolds to elucidate the effects of material biodegradability on cell behavior using POSS-PEG hybrid hydrogels

Tissue-engineered three-dimensional (3D) scaffolds are required to have a suitable degradation period and biocompatibility. Polyhedral oligomeric silsesquioxane−poly(ethylene glycol) (POSS−PEG) hybrid hydrogels with different degradation rates were prepared by varying the proportion of two cross-linkers, namely degradable PEG-diester-dithiol and non-degradable PEG-dithiol. These hydrogels were used as 3D scaffolds for the encapsulation, culture, and differentiation of cells to elucidate the effects of material biodegradability on cell behavior. Human umbilical vein endothelial cells (HUVECs) encapsulated in the 3D hybrid hydrogels were round-shaped with a cell survival rate of 98%. As the content of degradable cross-linker in the hybrid hydrogels was increased, the number of cells increased more rapidly, and the cell morphology gradually changed from slender spindle to round. Bone marrow mesenchymal stem cells (BMSCs) encapsulated in the 3D hybrid hydrogels grew and proliferated in both osteogenic differentiation media and cell culture growth media. As the content of the degradable cross-linker was increased, the osteogenic differentiation gradually increased, as indicated by the presence of calcium nodes. These results demonstrate that the degradation rates of the hybrid hydrogels had an effect on cell compatibility, morphology, and differentiation.

Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.

BackgroundThe majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as “methionine dependence” or “methionine stress sensitivity” in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.ResultsUsing a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.ConclusionThis study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.

Evaluation of PD 404,182 as an anti-HIV and anti-herpes simplex virus microbicide.

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672-681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD's virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC50) of PD, when diluted in seminal plasma, is ∼1 μM, similar to the IC50 determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC50], >300 μM), freshly activated human peripheral blood mononuclear cells (CC50, ∼200 μM), and primary CD4(+) T cells, macrophages, and dendritic cells (CC50, >300 μM). PD also exhibited high stability in pH-adjusted Dulbecco's phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD's microbicidal candidacy in animals and humans.

Magnesium degradation as determined by artificial neural networks

Magnesium degradation under physiological conditions is a highly complex process in which temperature, the use of cell culture growth medium and the presence of CO2, O2 and proteins can influence the corrosion rate and the composition of the resulting corrosion layer. Due to the complexity of this process it is almost impossible to predict the parameters that are most important and whether some parameters have a synergistic effect on the corrosion rate. Artificial neural networks are a mathematical tool that can be used to approximate and analyse non-linear problems with multiple inputs. In this work we present the first analysis of corrosion data obtained using this method, which reveals that CO2 and the composition of the buffer system play a crucial role in the corrosion of magnesium, whereas O2, proteins and temperature play a less prominent role.

Abstract 4046: Kinetic quantification of caspase-3/7 mediated apoptosis using live-cell imaging

Abstract Apoptosis, the biological process by which cells undergo programmed cell death, is required for normal tissue maintenance and development. However, aberrations in apoptotic signaling networks are implicated in numerous human diseases including neurodegeneration and cancer. Stimulation of either the extrinsic or intrinsic apoptotic pathways triggers a signaling cascade that typically results in the activation of executioner caspase-3. Numerous in vitro assays have been designed to measure the activation of caspase-3. The majority of these assays utilize reagent substrates that incorporate the DEVD motif which is recognized by both activated caspases 3 and 7. Such assays result in single, user-defined time point measurements of caspase-3/7 activity and typically require multiple wash steps or cell lifting prior to data collection; potentially resulting in the loss of cells or critical data in experiments where cells undergo apoptosis at different rates according to treatment conditions. In this study, we optimized an assay system incorporating a bi-functional reagent containing the DEVD motif linked to a DNA dye for use on the IncuCyte FLR imaging system. When added to cell culture growth medium, this relatively inert, non-fluorescent molecule crosses the cell membrane where it is available to be a substrate for activated caspase-3/7. The reaction product is a green fluorescent DNA dye that labels the nuclei of cells undergoing apoptosis. Our data show that this strategy can quantitatively measure the kinetic activation of caspase-3/7 through extrinsic stimulation of human tumor derived MDA-MB-231 breast adenocarcinoma cells, HeLa cervical adenocarcinoma cells, A549 lung epithelial carcinoma cells, and HT 1080 fibrosarcoma cells following treatment with TNFα and cycloheximide. Moreover, we exemplify the use of this strategy in 96-well plate screening protocols using both HT 1080 and MDA-MB-231 cells to measure the induction of caspase-3/7 activity in the presence of staurosporine, a well-known general protein kinase inhibitor and inducer of caspase-3/7 mediated apoptosis with Z’ values α0.6. We provide evidence that this stable, kinetic, mix-and-read assay can provide both quantitative and qualitative assessments of caspase-3/7 activity using fluorescent images and high-definition phase contrast images, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4046. doi:1538-7445.AM2012-4046

Abstract 3129: Regulation of expression and role of the pigment epithelium derived factor in Ras-mediated pancreatic oncogenesis

Abstract Mutated and constitutively activated Ras oncoproteins are found in ∼33% of all human cancers. In particular, Ras mutations are found in a majority of pancreatic cancers (90%), and aberrant Ras activation has been shown to be critical for promoting pancreatic adenocarcinomas. Ras-mediated transformation is associated with downregulation of Pigment Epithelium Derived Factor (PEDF), an anti-angiogenic and lipid regulatory factor shown to suppress oncogenic K-Ras-induced invasive disease in vivo. PEDF has also been shown to have anti-tumorigenic activity on tumor cells themselves. Specifically, mice with chronically active mutant KRAS(12D) background form precancerous lesions with high frequency, and knockout of PEDF expression specifically in pancreatic tissue resulted in a significant aggravation of the precancerous phenotype. The mechanism(s) of this biological phenotype is unclear and the specific role of PEDF as a potential tumor suppressor gene in Ras-mediated oncogenesis remains unresolved. Western analyses were used in this study to investigate the expression of PEDF in K-Ras-transformed and nontransformed human pancreatic duct-derived cells (HPNE) and their surrounding cell culture growth media. We observe that PEDF is upregulated both intracellularly under growth and serum-starved conditions and extracellularly under serum-starved conditions in K-Ras-transformed human pancreatic cells. We are also investigating the role of exogenous PEDF in cell proliferation induced by oncogenic variants of K-Ras. While no significant difference was observed in cell proliferation between the PEDF-treated and nontreated cells (whether they be K-Ras-transformed and nontransformed cells) in growth conditions, a decrease in cell proliferation was observed in the PEDF-treated K-Ras-transformed cells under serum-starved conditions. By better understanding the link between Ras and PEDF we will be able to assess the contribution and therapeutic potential of PEDF to formation and progression of pancreatic cancer while investigating novel targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3129.

Confounding experimental considerations in nanogenotoxicology

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Purification of the densonucleosis virus by tangential flow ultrafiltration and by ion exchange membranes

Virus capture is critical in a number of applications. In gene therapy and vaccine production, large-scale purification of virus vectors is often essential. In the manufacture of biopharmaceuticals, validation of virus clearance is critical. Tangential flow filtration is frequently used in the biotechnology industry for the purification of proteins. Also preparative chromatography is widely used in downstream purification procedures of biopharmaceutical products. Replacement of resins by membranes as chromatographic supports overcomes many of the limitations associated with resin-based chromatography. In particular, adsorptive membranes may be ideally suited for virus capture. Purification of the Aedes aegypti densonucleosis virus (AeDNV, a nonenveloped single-stranded mosquito-specific parvovirus, particle size about 26 nm) using tangential flow filtration and also using anion and cation exchange membranes was investigated. The results obtained here indicate that 30, 50, and 100 kDa membranes reject the virus particles, whereas 300 kDa membranes allow virus particles to pass into the permeate. The decrease in permeate flux for the 300 kDa ultrafiltration membrane is much greater than for the other membranes. This indicates a possible entrapment of virus particles in the membrane pores. The permeate flux and level of protein rejection are strongly affected by the cell culture growth medium. AeDNV particles may be adsorbed by both anion and cation exchange membranes by adjusting the pH of the feed stream. However, strongly basic anion exchange membranes were the most effective in adsorbing AeDNV particles.

Transformation of human urothelial cells (UROtsa) by as and cd induces the expression of keratin 6a.

BackgroundCadmium and arsenite can directly and malignantly transform the UROtsa cell line. The tumor heterotransplants produced from these transformed cells have histologic features consistent with human bladder cancer. Previous microarray analysis of total RNA from the parental and transformed cells suggested that keratin 6a was overexpressed as a result of cell transformation.ObjectivesOur goals were to verify overexpression of keratin 6a in Cd2+- and As3+-transformed UROtsa cells, the corresponding tumor heterotransplants, and human bladder cancer biopsy specimens and to assess what factors may be involved in keratin 6a overexpression.MethodsExpression was assessed with real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. We used the effect of addition and deletion of potential growth factors in the cell culture growth medium to assess possible pathways used in keratin 6a overexpression.ResultsCd2+- and As3+-transformed cells grown in serum-containing growth medium, as well as the derived tumor heterotransplants, overexpressed keratin 6a mRNA and protein compared with UROtsa cells grown in serum-containing growth medium. Immunostaining of keratin 6a in tumor heterotransplants showed focal staining of the tumor cells that was localized to the cytoplasm. Focal immunostaining of keratin 6a was also found in some but not all archival patient specimens of high-grade bladder cancer, confirming translation of the results to human bladder cancer. Studies on growth factor deletion and addition indicated that the level of keratin 6a expression was regulated by the presence of both insulin and epidermal growth factor (EGF). In contrast, growth factors had no effect on the elevated levels of keratin 6a expression found in transformed UROtsa cells.ConclusionsOur present studies suggest that keratin 6a expression may be a biomarker for malignant urothelial cells that possess an activated EGF and or insulin growth factor pathway.

Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: A preliminary study

Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 microL and photomicrographed. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10(2) promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Purification of Densonucleosis Virus by Tangential Flow Ultrafiltration

Purification at commercial scale of viruses and virus vectors for gene therapy applications and viral vaccines is a major separations challenge. Tangential flow ultrafiltration has been developed for protein purification. Here tangential flow ultrafiltration of parvoviruses has been investigated. Because these virus particles are small (18-26 nm), removal of host cell proteins will be challenging. The results obtained here indicate that 30, 50, and 100 kDa membranes reject the virus particles, whereas 300 kDa membranes allow some virus particles to pass into the permeate. The decrease in permeate flux for the 300 kDa ultrafiltration membrane is much greater than for the 30, 50, and 100 kDa membranes, indicating possible entrapment of virus particle in the membrane pores. The permeate flux and level of protein rejection is strongly affected by the cell culture growth medium. The results indicate that when developing a new process, it is essential that the cell culture and purification operations be developed in parallel.

Paired Cysteine Mutagenesis to Establish the Pattern of Disulfide Bonds in the Functional Intact Secretin Receptor

The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors.

Effects of Ca and H2O2 added to RPMI on the fretting corrosion of Ti6Al4V.

Titanium and its alloys have demonstrated considerable success in various surgical procedures including orthopedic, dental, and cardiovascular surgery. However, particulate debris from corrosion and wear is present in a considerable quantity in tissue local to the implant. This study evaluated the effect of Ca, since it is present in both serum and bone, and H2O2, since it is produced through local inflammation, on the amount of titanium release. Four sets of Ti6Al4V plates and Ti6Al4V screws were used. Each set was designated to one of four solutions: RPMI (cell culture growth media), RPMI with CaCl2, RPMI with CaCO3, and RPMI with H2O2. A fretter was used to cause corrosion by creating micromotion between two screws and a two-hole plate of Ti6Al4V. After fretting for 72 h, weight loss of the plate and screws and the amount of Ti and vanadium (V) in solution was used to assess the amount of fretting corrosion which had occurred. Results of weight loss and Ti in solution indicated that the presence of H2O2 increased the amount of particulate debris produced in RPMI as compared with RPMI alone. The addition of CaCl2 to RPMI also increased both weight loss and Ti in solution compared with RPMI alone. The addition of CaCO2, however, did not give values significantly different from RPMI alone. Comparison of weight loss and Ti in solution indicated that the increase in fretting corrosion was not different between RPMI with CaCl2 and RPMI with H2O2. The particulate wear debris from the four solutions was black in color and the size of the particulate produced was compared using a Coulter Multisizer. The results indicated that particles produced in the four solutions were not different, with mean values between 1.324 and 1.100 microns, and they were similar in size to the particulate found in tissues surrounding failed total hip replacements. In order to better understand the role of Ca in the fretting corrosion of Ti6Al4V, energy dispersive x-ray analysis (EDXA) using SEM was used to determine elemental composition of one countersink surface of a plate which had been run four times in RPMI with CaCl2. The presence of Ca in the bulk was not significant (% composition < 0.5%). However, Ca was present in two surface particles which were examined at a magnification of 55,000, with a Ca% composition of 63.2% and 19.2%. While results from this study indicate that both soluble Ca(CaCl2) and H2O2 increase the fretting corrosion of Ti6Al4V, the insoluble form of Ca, which would be found in bone and hydroxyapatite, has no effect. These data indicate that it is important to specify the media used in corrosion, dissolution, and elution experiments.

Comparison of the Gimenez Staining Method and Antigen Detection ELISA with Culture for Detecting Chlamydiae in Birds

Chlamydiosis, caused by Chlamydia psittaci, an obligate intracellular bacterium, is a serious health concern for various types of birds and their owners. Disease can occur as a chronic inapparent, subacute, acute, or peracute infection. Culture for chlamydiae is considered the “gold standard” for evaluating other methods, even though it probably does not have 100% sensitivity nor 100% specificity in live human subjects, and culture of multiple sample types from birds may increase the sensitivity. Multiple cultures may increase chances of recovering chlamydiae from live birds, but it probably is not advisable to delay treatment of birds critically ill with signs consistent with chlamydiosis. Monoclonal antibodies that detect chlamydial lipopolysaccharide have allowed development of an enzyme-linked immunosorbent assay (ELISA) for rapid detection. Two commercially available kits have been evaluated for use in birds. Microscopic examination of impressions of uncut surfaces of bird organ tissues and inoculated cell culture monolayers using the Gimenez staining method (GSM) also was reported as a rapid diagnostic procedure for examining dead birds. The purpose of this study was 2-fold: 1) to expand the comparison of 1 commercially available ELISAB with chlamydial isolation attempts from live birds and 2) to compare results of antigen-detection ELISA and the GSM with culture results on dead birds. Samples used in this study were submitted to the Texas Veterinary Medical Diagnostic Laboratory (TVMDL) for chlamydial testing. Cloacal swabs, choanal slit swabs, and fecal samples from live birds were submitted in chlamydial transport medium. Some fresh fecal samples also were submitted. Fresh fecal material was placed into chlamydial transport medium to make an approximate 20% (w:v) suspension, which was centrifuged at 1,000 x g for 15 minutes and then diluted 1:2 for isolation attempts. For the ELISA testing, 1.0 ml of fecal suspension was centrifuged at 3,000 x g for 20 minutes, and the supematant fluid was discarded. A swab was then used to collect the pellet. Visceral and other organs from dead birds necropsied by referring veterinarians or TVMDL personnel were processed for the GSM, ELISA, and isolation procedures. Impressions were most often made of liver and spleen uncut surfaces and occasionally of the pericardium, air sacs, or lungs. These impressions were stained by the GSM and examined microscopically. 4,5 Suspensions (20%, w:v) of pooled organ tissues for cell monolayer inoculation were made in cell culture growth medium and centrifuged for 15 minutes at 1,000 x g. For ELISA of tissues, swabs were inserted into spleen and liver and then air dried.