Electrophysiologic study of rabbit proximal tubular cell monolayers in primary culture

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7-10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 omega cm2, a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of -75 mV when cells were bathed in 0.4 mM K and -49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 micrograms/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCl gradient) of -2.6 mV with respect to the serosa. These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.     

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.     

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.     

A Plasmodium falciparum exo-antigen alters erythrocyte membrane deformability

Here we describe a reduced membrane deformability of human erythrocytes when aspirated into 0.6 microns diameter in polycarbonate sieves, after exposure of uninfected cells to spent parasite-culture supernatant. This, taken in concert with a previous observation that intra-erythrocytic development of the parasite P. falciparum decreases host localised membrane deformability, may indicate a biological role for such parasite-induced changes in the rheological properties of the erythrocyte.     

Micropatterned polymer surfaces improve retention of endothelial cells exposed to flow-induced shear stress

The use of synthetic polymeric vascular grafts is limited by the thrombogenecity of most biomaterials. Efforts to reduce thrombogenicity by seeding grafts with endothelial cells, the natural non-thrombogenic lining of blood vessels, have been thwarted by flow-induced cell detachment. We hypothesized that by creating well-defined micro-textured patterns on a surface, fluid flow at the surface can be altered to create discrete regions of low shear stress. We further hypothesized that, due to reduced shear stress, these regions will serve as sanctuaries for endothelial cells and promote their retention. To test these hypotheses, well-defined micro-textured polyurethane (PU) surfaces consisting of arrays of parallel 95-micron wide and 32-micron deep channels were created using an etched silicon template and solvent casting techniques. Based on computational fluid dynamics, under identical bulk flow conditions, the average local shear stress in the channels (46 dyn/cm2) was 28% lower than unpatterned surfaces (60 dyn/cm2). When PU surfaces pre-seeded with endothelial cells (EC) were exposed to the same bulk flow rate, EC retention was significantly improved on the micropatterned surfaces relative to un-patterned surfaces (92% vs. 58% retention).     

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.     

Malaria and ovalocytosis--molecular mimicry?

Two recently published reports have described findings which will have a profound impact on the understanding of molecular mechanisms of human resistance to malaria infection. In Melanesian ovalocytosis, a genetic polymorphism found in Papua New Guinea and parts of South East Asia, the red cells are highly resistant to invasion by various species of malaria parasite. The molecular nature of the defect in ovalocytic erythrocytes was not known. Recent reports by Liu et al. (Liu, S.-C., Zhai, S., Palek, J., Golan, D., Amato, D., Hassan, K., Nurse, G., Babona, D., Coetzer, T., Jarolim, P. Zaik, M. and Borwein, S. (1990) N. Engl. J. Med. 323, 1530-1538.) and Jones et al. (Jones, G.L., Edmundson, H.M., Wesche, D. and Saul, A. (1991) Biochim. Biophys. Acta 1096, 33-40.) have now identified the abnormality in the band 3 protein of ovalocytic red cell membranes. A major discovery in the Jones et al. study is the presence of an extended peptide at the N-terminus of ovalocyte band 3 protein. This novel 13 amino acid extended sequence is not found in the primary structure of normal band 3 protein and was suggested to be the cause of band 3 defect in ovalocytes. We have analyzed this extended sequence through Genbank using SWISS-PROT database and found that an almost identical sequence exists in a malaria parasite protein called RESA.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks.

The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5'-bromo-2'-deoxyuridine increased 6- to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were grown in identical polycarbonate culture flasks sterilized by autoclaving.     

Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms.

Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria. Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent. This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853). Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates. The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples. Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples. Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction. However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples. Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment. The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficiency of several micro-fiber glass filters for recovery of poliovirus from tape water.

Micro-fiber glass filters from Gelman, Filterite, Johns-Manville, and Whatman were compared with Millipore membrane filters on the basis of their virus adsorbancy, flow rate, clogging resistance, and virus concentration efficiency by using tap water at 2 nephelometric turbidity units. As virus adsorbants the Johns-Manville D39, Filterite 0.25-micron, Filterite 0.45-micron, and Millipore 0.45-micron filters were the most efficient, retaining more than 99% of the added virus in water at pH 3.5 and 0.0005 M aluminum chloride. The Johns-Manville D79 and D49 filters retained 92 and 96% of the virus, respectively, whereas the Whatman GF-D, Whatman GF-F, Gelman A-E, and Millipore AP-20 filters retained only 28, 78, 56, and 34% of the virus, respectively. The best flow rate and clogging resistance were obtained with the Johns-Manville D79 filter or with this filter acting as a prefilter to the Johns-Manville D49, Johns-Manville D39, or Filterite 0.45-micron filter. Finally, poliovirus experimentally seeded in 20 liters of tape water was recovered from Johns-Manville D79-Johns-Manville D39 or Johns-Manville D79-Filterite 0.45 micron 142-mm filter combinations was a efficiencies of 86 and 85%, respectively.     

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.     

Malaria and ovalocytosis — molecular mimicry?

Two recently published reports have described findings which will have a profound impact on the understanding of molecular mechanisms of human resistance to malaria infection. In Melanesian ovalocytosis, a genetic polymorphism found in Papua New Guinea and parts of South East Asia, the red cells are highly resistant to invasion by various species of malaria parasite. The molecular nature of the defect in ovalocytic erythrocytes was not known. Recent reports by Liu et al., (Liu, S.-C., Zhai, S., Palek, J., Golan, D., Amato, D., Hassan, K., Nurse, G., Babona, D., Coetzer, T., Jarolim, P. Zaik, M. and Borwein, S. (1990) N. Engl. J. Med. 323, 1530–1538.) and Jones et al. (Jones, G.L., Edmundson, H.M., Wesche, D. and Saul, A. (1991) Biochim. Biophys. Acta 1096, 33–40.) have now identified the abnormality in the band 3 protein of ovalocytic red cell membranes. A major discovery in the Jones et al, study is the presence of an extended peptide at the N-terminus of ovalocyte band 3 protein. This novel 13 amino acid extended sequence is not found in the primary structure of normal band 3 protein and was suggested to be the cause of band 3 defect in ovalocytes. We have analyzed this extended sequence through Genbank using SWISS-PROT database and found that an almost identical sequence exists in a malaria parasite protein called RESA.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks

Summary The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5′-bromo-2′-deoxyuridine increased 6-to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were growth in identical polycarbonate culture flasks sterilized by autoclaving.     

Endothelial Cells from Human Fetal Brain Microvessels May Be Cholinoceptive, but Do Not Synthesize Acetylcholine

Brain homogenate, cerebral microvessels, and endothelial cells (ECs) were prepared from 15-18-week-old human fetuses and analyzed biochemically for the presence of elements of the cholinergic system [acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and butyrylcholinesterase]. The ECs were cultured, and their purity was checked by light microscopic immunohistochemistry with the application of anti-human factor VIII and glial fibrillary acidic protein. The highest activity of ChAT was found in the brain homogenate and the lowest in the microvessel fraction. No ChAT activity could be detected in the cultured ECs, despite the presence of high AChE activity. It is suggested that human brain ECs may be under the control of acetylcholine released from cholinergic nerve terminals but that the cells do not produce the transmitter itself. In coculture experiments, when ECs were plated on the upper surface of a polycarbonate filter and glial cells were seeded on the lower surface, the electric resistance was measured. During the culture period, the resistance first increased up to 5 days in vitro (297 +/- 17 ohm.cm2) but later gradually declined. These results demonstrate that human ECs cocultured with glial cells provide a useful model for study of the function of the blood-brain barrier in vitro.     

2-micron circle plasmids do not reduce yeast life span

Extrachromosomal rDNA circles (ERCs) and recombinant origin-containing plasmids (ARS-plasmids) are thought to reduce replicative life span in the budding yeast Saccharomyces cerevisiae due to their accumulation in yeast cells by an asymmetric inheritance process known as mother cell bias. Most commonly used laboratory yeast strains contain the naturally occurring, high copy number 2-micron circle plasmid. 2-micron plasmids are known to exhibit stable mitotic inheritance, unlike ARS-plasmids and ERCs, but the fidelity of inheritance during replicative aging and cell senescence has not been studied. This raises the question: do 2-micron circles reduce replicative life span? To address this question we have used a convenient method to cure laboratory yeast strains of the 2-micron plasmid. We find no difference in the replicative life spans of otherwise isogenic cir⁺ and cir⁰ strains, with and without the 2-micron plasmid. Consistent with this, we find that 2-micron circles do not accumulate in old yeast cells. These findings indicate that naturally occurring levels of 2-micron plasmids do not adversely affect life span, and that accumulation due to asymmetric inheritance is required for reduction of replicative life span by DNA episomes.     

DNA damage repair in quiescent murine mammary carcinoma cells in culture

Murine mammary carcinoma cells (line 67) were grown in unfed cultures for up to 9 days. In cultures (day 2-3) in which cells were proliferatively active and in day 3-5 (transition) cells, a large fraction of nuclear DNA was retained on polycarbonate filters when assayed by the alkaline filter elution technique. In contrast, the fraction of DNA retained on filters was significantly reduced for nonproliferating (Q, quiescent) cells from unfed 7-9 day cultures. The increase in endogenous DNA breaks followed both the decrease in proliferative state and clonogenicity in these cells. When day 7 Q cells were refed these endogenous DNA breaks were removed with a half-time of about 2.5 h. When the cells were exposed to X-irradiation and the integrity of their nuclear DNA measured by the alkaline filter elution assay, as much as a 2-fold greater frequency of radiation-induced DNA breaks was produced in Q versus P cells. DNA breaks were also removed from irradiated Q cells at a rate which was 0.23 that observed in P cells. We suggest that the depressed capacity for DNA damage removal in Q cells is responsible for their greater radiosensitivity, and the impaired DNA damage repair is probably due to a reduced level of energy sources in these unfed Q cell cultures.     

Trophic cascades in an invaded ecosystem: native keystone predators facilitate a dominant invader in an estuarine community

Determining how various factors contribute to the invasibility of systems is essential for both understanding community formation and informing management of natural areas. Research demonstrating that predators can provide biotic resistance to invasions by consuming invasive species has led to the presence of healthy predator populations being associated with reduced invasion potential of ecosystems. However, predators structure communities in many ways and their presence could also potentially facilitate invasions if they decrease populations of native species that compete with or consume an invader. We considered these two impacts of predators on invasion by analyzing the effects of two keystone predators (Pisaster spp. and Enhydra lutris nereis) on two foundation species (a native mussel Mytilus californianus and the invasive exotic bryozoan Watersipora subtorquata, a putative competitor for space with Mytilus californianus). Both native predators were found to facilitate the invasion of the exotic bryozoan, and the rate of invasion was highest when both predators were present. Facilitation of W. subtorquata occurred via indirect mechanisms that partly involved the removal of a competitor (mussels) via predation. These results illustrate that although predators can provide biotic resistance to invasion, healthy predator populations do not always confer this advantage and in fact may facilitate invasions. Therefore, implementation of management actions to enhance populations of top predators could also potentially increase the invasibility of some ecosystems.     

Control of rat C6 glioma cell proliferation: uncoupling of the inhibitory effects of hydrocortisone hormone in suspension and monolayer cultures

We undertook a comparative study of the effects of the hormone hydrocortisone (Hy) on C6 glioma cells grown in monolayer and in suspension in cultures. We found Hy reversibly renders C6 cells anchorage- and serum-dependent for their growth. In monolayer cultures, Hy was found to inhibit cell cycle traversing exclusively at G1 phase. In agarose suspension, Hy was found to block colony development. Hy-resistant variants were selected and isolated in agarose suspension. Examination of these variants showed that cells selected for Hy-resistance in suspension can be Hy sensitive when anchored to a solid substrate. We conclude that resistance to Hy in suspension and resistance to it in monolayer culture are two independent phenotypes.