Filter Chambers Research Articles

Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro.

Impairing the motility of glioblastoma multiforme (GBM) cells is a compelling goal for new approaches to manage this highly invasive and rapidly lethal human brain cancer. Work here characterized an array of pharmacological inhibitors of membrane ion and water channels, alone and in combination, as tools for restraining glioblastoma spread in human GBM cell lines U87-MG and U251-MG. Aquaporins, AMPA glutamate receptors, and ion channel classes (shown to be upregulated in human GBM at the transcript level and linked to mechanisms of motility in other cell types) were selected as pharmacological targets for analyses. Effective compounds reduced the transwell invasiveness of U87-MG and U251-MG glioblastoma cells by 20-80% as compared with controls, without cytotoxicity. The compounds and doses used were: AqB013 (14 μM); nifedipine (25 µM); amiloride (10 µM); apamin (10 µM); 4-aminopyridine (250 µM); and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 µM). Invasiveness was quantified in vitro across transwell filter chambers layered with extracellular matrix. Co-application of each of the ion channel agents with the water channel inhibitor AqB013 augmented the inhibition of invasion (20 to 50% greater than either agent alone). The motility impairment achieved by co-application of pharmacological agents differed between the GBM proneural-like subtype U87-MG and classical-like subtype U251-MG, showing patterns consistent with relative levels of target channel expression (Human Protein Atlas database). In addition, two compounds, xanthurenic acid and caelestine C (from the Davis Open Access Natural Product-based Library, Griffith University QLD), were discovered to block invasion at micromolar doses in both GBM lines (IC50 values from 0.03 to 1 µM), without cytotoxicity, as measured by full mitochondrial activity under conditions matching those in transwell assays and by normal growth in spheroid assays. Mechanisms of action of these agents based on published work are likely to involve modulation of glutamatergic receptor signaling. Treating glioblastoma by the concurrent inhibition of multiple channel targets could be a powerful approach for slowing invasive cell spread without cytotoxic side effects, potentially enhancing the effectiveness of clinical interventions focused on eradicating primary tumors.

Pengaruh algoritma filter bubble dan echo chamber terhadap perilaku penggunaan internet

Introduction. This study aims to explore effects of bubble filter and echo chamber on information searching behaviour on the internet, how it affects the scope of the internet ecosystem and users, and what we can see from the behaviour.
 Data Collection Method. The paper used a mixed-methods approach with surveys, online discussions, and literature research. Twenty respondents between 19-21 years old participated in this exploratory study.
 Analysis Data. Data was obtained and calculations have automatically accumulated through Google Form. The user data analysis and discussion were conducted manually by considering aspects of rationality concerning the literature and previous research.
 Results and Discussions. The analysis results obtained were bubble filter and echo chambers were proven to affect internet users both in positive and negative ways.
 Conclusions. Bubble filter and echo chamber may have both positive and negative effects simultaneously, however, it depends on the user because the system has provided control features to reduce both features. Using internet wisely is also parts of the important aspect. 

Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display.

RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display. Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count. Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity. BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.

Effectiveness of Extra Oral Vacuum Aspirator During Dental Treatment in COVID -19 Pandemic

As COVID 19 pandemic has rapidly became a public health crisis of global concern, dental office is also affected. Due to the unique characteristics of dental office i.e., generating high volume of aerosol during the dental procedures, both dental health care professionals as well as the patients have increased risk of cross infection. Various devices like extra oral vacuum aspirator, high volume evacuator, and high efficiency particulate air filter and ultraviolent chambers in conjunction with personal protective equipment are used to reduce aerosols produced in dentistry. The objective of this review is to explore the effectiveness of extra oral vacuum aspirator to decrease number of airborne pathogens, such as the SARS- CoV-2 virus during the dental procedure.

Method for calculating a cylindrical ultrasonic cavitation filter chamber with a regeneration effect

Introduction. The creation of modern ultrasonic cavitation equipment for filtering working fluids in various technological processes is associated with the study of the interaction of a liquid load with ultrasonic emitters, taking into account the influence of the geometric shape of the cavitation chamber. The efficiency of the operation of ultrasonic cavitation devices for filtration is a rather urgent issue today and directly depends on the quality of this agreement. Problem statement. To build an ultrasonic filter with a regeneration effect and to increase its efficiency, it is necessary to create a calculation method that will take into account both the impedance of electro-acoustic emitters and the complexity of the geometric shape of the cavitation chamber. Development of calculation methods. The proposed method for calculating the geometric dimensions of the acoustic resonance system of the modular section of the tubular ultrasonic cavitator, which is excited by folded piezoelectric drive-emitters, which carry out longitudinal vibrations and are installed on the outer surface of the tubular vibrator. Simulation results. The results of modeling in the Abaqus Student Edition 2018 software environment are presented, confirming the possibility of creating ultrasonic flow cavitators, the vibrator, which are excited in a radial-flexion mode of vibration, for the technological process of ultrasonic cavitation filtration with the regeneration effect. Conclusions. The proposed calculation technique allows designing cylindrical ultrasonic cavitation filter chambers with a regeneration effect. Repeated practical verification of the presented method for calculating resonant acoustic systems has confirmed its sufficient accuracy under the applied assumptions.

Microfluidic Device for Screening for Target Cell-Specific Binding Molecules by Using Adherent Cells.

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells.

Fisetin Suppresses Human Osteosarcoma U-2 OS Cell Migration and Invasion via Affecting FAK, uPA and NF-ĸB Signaling Pathway In Vitro.

Evidence has indicated that fisetin induces cytotoxic effects in human cancer cell lines, including the inhibition of cell migration and invasion, however, the exact molecular mechanism of action of fisetin in human osteosarcoma cells remains unclear. The anti-metastatic mechanisms of fisetin in human osteosarcoma U-2 OS cells were investigated in vitro. Fisetin reduced the viability of cells at different concentrations (2.5, 5 and 10 μM) as measured by flow cytometric assay. Fisetin suppressed cell mobility, migration and invasion of U-2 OS cells, as shown by wound healing assay and transwell filter chambers, respectively. The gelatin zymography assay showed that fisetin inhibited MMP-2 activity in U-2 OS cells. Results from western blotting indicated that fisetin reduced the levels of pEGFR, SOS-1, GRB2, Ras, PKC, p-ERK1/2, p-JNK, p-p-38, VEGF, FAK, RhoA, PI3K, p-AKT, NF-ĸB, uPA, MMP-7, MMP-9, and MMP-13, but increased GSK3β and E-cadherin in U-2 OS cells after 48 h of treatment. Fisetin can be used in the future, as a target for the treatment of metastasis of human osteosarcoma cells.

Effects on Groundwater Microbial Communities of an Engineered 30-Day In Situ Exposure to the Antibiotic Sulfamethoxazole

Effects upon microbial communities from environmental exposure to concentrations of antibiotics in the μg L(-1) range remain poorly understood. Microbial communities from an oligotrophic aquifer (estimated doubling rates of only once per week) that were previously acclimated (AC) or unacclimated (UAC) to historical sulfamethoxazole (SMX) contamination, and a laboratory-grown Pseudomonas stutzeri strain, were exposed to 240-520 μg L(-1) SMX for 30 days in situ using filter chambers allowing exposure to ambient groundwater, but not to ambient microorganisms. SMX-exposed UAC bacterial communities displayed the greatest mortality and impairment (viable stain assays), the greatest change in sensitivity to SMX (dose-response assays), and the greatest change in community composition (Terminal Restriction Fragment Length Polymorphism; T-RFLP). The sul1 gene, encoding resistance to SMX at clinically relevant levels, and an element of Class I integrons, was not detected in any community. Changes in microbial community structure and SMX resistance over a short experimental period in previously nonexposed, slow-growing aquifer communities suggest concentrations of antibiotics 2-3 orders of magnitude less than those used in clinical applications may influence ecological function through changes in community composition, and could promote antibiotic resistance through selection of naturally resistant bacteria.

Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering

The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.

Performance of a filtration system equipped with filter media for parking lot runoff treatment

Stormwater runoff from automobile trafficked areas introduces significant loads of pollutants to receiving water bodies leading to an adverse long term effect. To control runoff pollutants from parking lot, a filtration treatment system with a layered filter media activated carbon, composite, vermiculite and zeolite was installed. The removal of the pollutant constituents was evaluated in this study. During a monitoring period of eighteen months eleven runoff samples were collected from the influent and effluent sections and analyzed for the mentioned pollutant parameters. The mean influent annual load of TSS, Cu and Zn were 2600, 2.1 and 3.5kg respectively. For the monitoring period mean removal efficiencies of 85% for TSS, 75% for Cu, 73% for Zn, 83% for 16 EPA PAHs, 70 to 98% for individual PAHs, 93% for mineral oil, 71% for NH4–N and 52% for TOC were achieved. Greater than 60% of the Cu load was removed within the filter chambers, but >60% of Zn and TSS loads were removed in the sedimentation tank, oil separator and geotextile filter mainly due to filtration, precipitation and sorption to sediments. Furthermore, except for chrysene heavier molecular weight PAH compounds were effectively removed than lighter molecular weight PAH compounds. The treatment mechanisms include: filtration, precipitation, adsorption and cation exchange.

Identification and characterization of functional aquaporin water channel protein from alimentary tract of whitefly, Bemisia tabaci

Some hemipteran xylem and phloem-feeding insects have evolved specialized alimentary structures or filter chambers that rapidly transport water for excretion or osmoregulation. In the whitefly, Bemisia tabaci, mass movement of water through opposing alimentary tract tissues within the filter chamber is likely facilitated by an aquaporin protein. B. tabaci aquaporin-1 (BtAQP1) possesses characteristic aquaporin topology and conserved pore-forming residues found in water-specific aquaporins. As predicted for an integral transmembrane protein, recombinant BtAQP1 expressed in cultured insect cells localized within the plasma membrane. BtAQP1 is primarily expressed in early instar nymphs and adults, where in adults it is localized in the filter chamber and hindgut. Xenopus oocytes expressing BtAQP1 were water permeable and mercury-sensitive, both characteristics of classical water-specific aquaporins. These data support the hypothesis that BtAQP1 is a water transport protein within the specialized filter chamber of the alimentary tract and functions to translocate water across tissues for maintenance of osmotic pressure and/or excretion of excess dietary fluid.

Nck Adapters Are Involved in the Formation of Dorsal Ruffles, Cell Migration, and Rho Signaling Downstream of the Platelet-derived Growth Factor β Receptor

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFbeta receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFbeta receptor.

Design and evaluation of a filter-based chairside amalgam separation system

This study evaluated the ability of a chairside filtration system to remove particulate-based mercury (Hg) from dental-unit wastewater. Prototypes of the chairside filtration system were designed and fabricated using reusable filter chambers with disposable filter elements. The system was installed in five dental operatories utilizing filter elements with nominal pore sizes of 50μm, 15μm, 1μm, 0.5μm, or with no system installed (control). Daily chairside wastewater samples were collected on ten consecutive days from each room and brought to the laboratory for processing. After processing the wastewater samples, Hg concentrations were determined with cold vapor atomic absorption spectrometry (USEPA method 7470A). Filter systems were exchanged after ten samples were collected so that all five of the configurations were evaluated in each room (with assignment order balanced by a Latin Square). The numbers of surfaces of amalgam placed and removed per day were tracked in each room. In part two, new filter systems with the 0.5μm filter elements were installed in the five dental operatories and vacuum levels at the high-velocity evacuation cannula tip were measured with a vacuum gauge. In part three of the study, the chairside filtration system utilizing 0.5μm and 15μm filter elements was evaluated utilizing the ISO 11143 testing protocol, a laboratory test of amalgam separator efficiency utilizing amalgam samples of known particle size distribution. Mean Hg per chair per day (no filter installed) was 1087.38mg (SD = 993.92mg). Mean Hg per chair per day for the 50μm, 15μm, 1μm, 0.5μm filter configurations was 79.13mg (SD = 71.40mg), 23.55mg (SD = 23.25mg), 17.68mg (SD = 17.35mg), and 4.25mg (SD = 6.35mg), respectively ( n = 50 for all groups). Calculated removal efficiencies from the clinical samples were 92.7%, 97.8%, 98.4%, and 99.6%, respectively. ANCOVA on data from the four filter groups, with amalgam-surfaces-removed included as a significant covariate, was statistically significant ( P < 0.0001). Tukey post-hoc comparisons ( P ≤ 0.05) indicated that the 50μm filter removed less mercury than all other filters and the 0.5μm removed more mercury than the 50μm and 15μm filters. Chairside vacuum measured on chairs with the 0.5μm filters installed were minimally affected at the time of installation, and then gradually diminished as the filters became loaded with debris. The 0.5μm configuration passed the ISO 11143 testing protocol at 96.8% efficiency.

Conjugal Transfer of Large Plasmids Conferring Oxytetracycline (OTC) Resistance: Transfer between Environmental Aeromonads, Fish-Pathogenic Bacteria, andEscherichia coli

The ability of environmental, motile Aeromonas species and fish-pathogenic bacteria to transfer oxytetracycline resistance to various bacterial species was assessed in vitro and in situ. Oxytetracycline-resistant motile aeromonads and A. salmonicida with large plasmids were used as donors; the recipients were a rifampicin-resistant Escherichia coli, the fish pathogens Yersinia ruckeri and Flavobacterium psychrophilum, a Pseudomonas putida strain, and motile aeromonads originating from aquatic environments. Transfers of oxytetracycline resistance were seen in both laboratory and environmental settings. In vitro transfers from motile Aeromonas spp. to E. coli using filter mating yielded transfer frequencies ranging from 1.4 × 10−9 to 6.8 × 10−5 transconjugants per recipient. Tetracycline resistance determinants A and D and unidentified tetracycline resistance determinants were transferred, usually on plasmids of approximately 150 kilo-base-pairs in size. In vitro transfer from motile Aeromonas spp. to Y. ruckeri was seen with frequencies of 1.7 × 10−7 to 2.1 × 10−5 transconjugants per recipient and to motile Aeromonas spp. with frequencies of 1.0 × 10−6 to 1.9 × 10−3 transconjugants per recipient, but no transfer to P. putida or F. psychrophilum was detected. Transfer to E. coli was detected in membrane filter chambers in aquaria and in situ at the outlet of a freshwater fish farm. Two motile Aeromonas isolates were used as donors for in situ experiments, and transfer frequencies ranged from 5.9 × 10−8 to 5.5 × 10−4. No transfers from motile Aeromonas spp. to Y. ruckeri or other motile Aeromonas spp. were detected in membrane filter chambers. The fish pathogen A. salmonicida transferred R plasmids to E. coli, but no transfer was detected to a motile Aeromonas spp. in membrane filter chambers.

Real-time MR Properties of Particulate Embolic Agents Tested in a Dynamic Flow Model

Interventional magnetic resonance (MR)-guided transcatheter embolization could potentially limit radiation exposure and improve visualization of target organs. The feasibility of monitoring injection and distribution of embolic agents was assessed in a dynamic flow model with real-time MR imaging. MR-compatible flow models were constructed with use of clear plastic chambers containing 170-microm polyethylene tubular filters. Gadolinium (Gd)-impregnated polyvinyl alcohol (PVA) particles (355-500 and 500-710 microm in size) and Gd-impregnated microspheres (Embospheres, 300-500 and 500-700 microm in size) were injected into the flow circuit under real-time dynamic T1-weighted fast field echo guidance at four images per second. A dynamic steady-state free precession sequence at four images per second was used to monitor the injection of unmodified Embo-Gold 700-900- microm particles. High-resolution scans were obtained before and after each particle injection. MR signal enhancement on the dynamic T1-weighted fast field echo sequence was visible during the injection of Gd-impregnated microspheres. Gd-impregnated PVA particles were not detected by this sequence. After injection, microsphere and PVA localization to the filter chambers was confirmed by the high-resolution scans. On the high-resolution sequences, relative MR signal enhancement of the microspheres was higher than that of the PVA particles. The Embo-Gold particles were minimally detectable on the dynamic sequence and undetectable by the high-resolution scan. After particle injection, direct inspection of the filter chamber showed trapping of all particle types and sizes. Real-time MR tracking of Gd-impregnated embolic agents is possible in vitro.

Larox Pressure Filters in Filtration and Washing of Non-Ferrous Minerals Concentrates and Metallurgical Slurries

Automatic pressure filter with horizontally positioned filtration chambers and one endless filter cloth was first introduced in the late 1960s. Actual product development and process adaptation was done during the 1970s. During the next decade this type of dewatering equipment established a firm position in a wide range of applications in mining, metallurgical, and chemical industries worldwide. More than 320 of these first generation LAROX PF filters are in operation in many types of dewatering applications. The maximum unit size has been 35 m2. In many cases, particularly in mining applications, the size of one unit has not been enough. For large production capacities in mining and mineral processing LAROX introduced in 1992 the big PF filter with maximum filtering area up to 144 m2, the second generation PF filter. The big 144 has 24 filter plates (filter chambers) and all mechanical operations are performed hydraulicaly. The normal size PF filters (below 38 m2) are also available with hydraulic operation devices. The second generation PF and big PF filters considerably increase the application range of this type of dewatering equipment. There are many processes in the mining, mineral and metallurgical industries, where improved efficiency in the solid/liquid separation would help achieve significant savings in energy consumption, affluent treatment costs, capital cost by simplifying the process, operation costs and especially labor costs. In most cases the efficiency can be improved by replacing the existing solid/liquid separation process with pressure filtration.

X-ray exposure system for induced chemistry and dry processes in microlithography

A new x-ray microlithography exposure system has been designed and built at the Center for Advanced Microstructures and Devices (CAMD) to meet the specific demands of synchrotron radiation assisted processing for microfabrication and high aspect ratio micromachining. The system consists of a broadband transmission (1.2–6.5 Å) beamline and a multichamber x-ray exposure station which can simultaneously provide x-ray exposure and activation, plasma, or reactive ion etching (RIE). The beamline is operational at the CAMD 1.3–1.5 GeV synchrotron storage ring. The x-ray exposure station includes filter and exposure chambers. A filter chamber is incorporated into the exposure system to optimize the radiation spectra for thick resist (in excess of 1 mm) exposures and to protect the Be window from reactive gases during exposure. Two assisting agents, x-ray irradiation and rf (13.53 MHz) induced plasma for RIE, can be used separately or in combination. Samples of ∼500-μm-thick PMMA resist have been exposed in neutral and reactive atmospheres and successfully developed. The preliminary results indicate that He gas acts as a ‘‘catalyst’’ for PMMA losses during exposure.

A novel method to study the hyphal phase of Candida albicans and to evaluate its hydrophobicity.

As little is known about the surface properties of the hyphal phase of the dimorphic fungus Candida albicans due to the difficulty in examining the latter phase in isolation, a novel method was designed to compartmentalize the two phases of the yeast using a commercially available filter-type device-Chemotaxicell. When yeast cells in the blastospore phase were incubated in filter chambers of Chemotaxicell submerged in hyphal-induction media, hyphae traversed through the pores of the filter into the exterior of the chamber enmeshing the entire outer filter surface, after 72 h of incubation. However, the inner or the chamber surface of the filter comprised mainly blastospores. The hydrophobicity of the two morphologic phases of the yeast was then compared using this method, by contact angle measurement. When the 72-h specimens with matted hyphal elements were evaluated, the contact angles varied depending on the incubation medium (such as TC 199 or serum), and in each case hydrophobicity of the hyphal phase was significantly higher than the blastopore phase yeasts. This simple, reproducible method should help not only in evaluating the properties of the hyphal elements of C. albicans but also in studying parameters such as saliva and serum, which are known to affect hyphal formation in vivo.

Permeabilization of MDCK cells with cholesterol binding agents: dependence on substratum and confluency

We studied systematically the susceptibility of Madin-Darby canine kidney (MDCK) cells to permeabilization by two cholesterol binding agents, digitonin and streptolysin-O (SLO), under different culture conditions. Monolayers grown on polycarbonate filter chambers (Transwells) required twice the concentration of digitonin effective on monolayers grown on glass or plastic (80 vs. 40 micrograms/ml) to allow antibody penetration or the release of 90% of the cytosolic protein lactate dehydrogenase (LDH). Neither the apical nor the basolateral surface showed preferential susceptibility to digitonin. Confluent MDCK cells, cultured either on filters or on impermeable substrates, showed poor antibody permeability after addition of commercial SLOs, even when used at concentrations 100 times higher (20 U/ml) than those effective on nonepithelial Chinese hamster ovary cells. Surprisingly, culture conditions that prevent tight junction formation and the acquisition of a polarized phenotype (< 10 microM Ca2+) increased dramatically the susceptibility to permeabilization by SLO. On restoration of normal Ca2+ levels, susceptibility to SLO quickly decreased. Thus conditions that lead to the full establishment of polarity result in decreased sensitivity to disruption by digitonin and SLO.

A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial ( Caco-2) cells grown in permeable filter chambers

A linear relationship between apparent permeability coefficients ( P app) and agitation rate was used to determine cell permeability coefficients ( P c) and the thickness of the aqueous boundary layer ( h aq) in monolayers of Caco-2 cells. Drugs with different physical/chemical properties were studied. P app values for ‘highly permeable drugs’ (testosterone, corticosterone, propranolol, metoprolol, warfarin) were dependent on the agitation rate, i.e. on the h aq. The permeation of testosterone was studied in detail. In the absence of agitation, the measured P app value of testosterone was 35.7 ± 3.3 × 10 −63 cm/s while the P c value was 137.4 ± 8.1 X 10 −6 cm/s, i.e. 3.8 times higher. P app values for ‘less permeable drugs’ (hydrocortisone, salicylic acid, sulphasalazine, mannitol) were not dependent on the agitation conditions. In the absence of agitation, h aq for testosterone was 1544 ± 142 μm and the contribution of the resistance of the aqueous boundary layer to the total diffusional resistance was 70%. h aq was reduced to 128 + 10 μm when the highest agitation rate was used. Under these conditions, the contributions of the resistances of the aqueous boundary layer and the Caco-2 cell monolayers were 16 and 73%, respectively. Similar results were obtained for the other highly permeable drugs. The results indicate that the P app values for highly permeable drugs are controlled not only by the aqueous boundary layer but also by the Caco-2 cell monolayers.