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At least 775 records · Page 43Linked to original sources
PubMed1990

Molecular mechanisms of glial cell swelling in acidosis.

The mechanism of glial swelling in acidosis were investigated in an in vitro model. C6 glial cells were exposed to pH levels between 7.4 and 6.2. The cell volume response was determined by flow cytometry. Cell swelling of 110% to 115% was observed if pH was reduced to 6.8 or below. This effect was independent of the length of exposure to acidosis. Swelling induced by pH could be attenuated by (a) inhibition of the Na+/H+ antiporter by amiloride, (b) replacement of bicarbonate by HEPES, and (c) inhibition of carbonic anhydrase by acetazolamide. Absence of Na+ ions from the incubation medium completely prevented acidosis-induced glial swelling. Inhibition of the Cl-/HCO3- antiporter reduced swelling only in its first phase. The results suggest that glial swelling in acidosis may be from an activation of anion and cation antiporters as an attempt to maintain a normal intracellular pH. It is concluded that swelling of glial cells in the ischemic penumbra zone evolves along similar mechanisms.

Acetazolamide↗
PubMedDate not supplied

Aminodiphosphonate or AI preincubation inhibits calcification of aortic homografts in the rat subdermal model.

Calcification is frequently noted in valved and non-valved aortic homografts, predominantly in the aortic wall portion. This study was designed to assess the individual anticalcification effects of the trivalent cation, aluminum (AI ), and the compound aminopropanehydroxydiphosphonate (APDP) when used to pretreat aortic wall homograft tissue before subdermal implantation in the rat model. Thoracic aortas of male rats (CD, Sprague-Dawley, 350 to 400 g) were harvested distal to the aortic valve and incubated in the following solutions: 1) APDP (0.004 M), 2) APDP (0.004 M) + glutaraldehyde (GLUT) (0.02 M), 3) GLUT (0.02 M), 4) ALCL3 (0.01 M), 5) HCL (0.001 M), and 6) HEPES (0.05 M). The homograft tissue was then implanted subcutaneously for 21 days in weanling male rats (50 to 60 gm) of the same strain. Explant results showed that calcification (CALC) was markedly inhibited in the APDP and 0.01 M ALCL3 groups, compared to the control and GLUT pretreated groups. No diphosphonate or aluminum related side effects were seen.

Aluminum↗
PubMed1988

[Effect of application of oxytocin on neurons of Helix pomatia: depolarization effects].

Depolarization of the neuronal membrane in the snail neurons induced by oxytocin application to the soma of these neurons have been examined under voltage clamp conditions. Replacement of external chloride by HEPES led to an increase its amplitude and a shift of its current-voltage characteristic in the depolarizing direction. External application of furosemide inhibited the current evoked by oxytocin application. Extracellular application of theophylline increased its amplitude. Imidazole, tolbutamide and temperature decrease had an opposite effect on this current. It is suggested that the surface membrane of some snail neurons probably possesses oxytocin receptors whose activation leads to the increase of its chloride permeability. This process is probably mediated through the system of cyclic nucleotides.

Animals↗
PubMed1985

Apparent binding of ionized calcium by various buffers.

Using different substance concentrations in an aqueous 1.25 mmol/L solution of CaCl2 plus NaCl to a final solution ionic strength of 160 mmol/L, we tested six buffers for their effect on measurements of ionized calcium (Ca2+). Measured Ca2+ decreased with increasing ionic strength and pH. Increasing concentrations of Tris caused a positive Ca2+ electrode bias; the other five buffers caused a negative Ca2+ electrode bias with increasing concentration. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid ("HEPES") at 10 mmol/L maintained pH 7.39 in the aqueous reference solutions containing 0.75 to 2.50 mmol of CaCl2 per liter, and showed a linear Ca2+ response among three different suppliers of Ca2+ instruments; it is thus a suitable buffer for a reference solution.

Buffers↗
PubMed1984

Phenotypical stability of a human hepatoma cell line, HuH-7, in long-term culture with chemically defined medium.

A chemically defined medium containing selenium was further improved, and the improved medium showed excellent ability to support growth and colony formation of a human hepatoma cell line, HuH-7. The improved defined medium (designated as IS-RPMI) contained small amounts of unsaturated fatty acids, inorganic trace elements including selenium, and HEPES buffer. Many of these additions exert only a small individual effect on the growth of cells, but their cumulative effects were substantial. HuH-7 cells replicated continuously with a doubling time of 47-51 hr in IS-RPMI. A conditioned medium from a semiconfluent population increased the colony-forming ability of HuH-7 cells in IS-RPMI. The addition of insulin to the medium gave good colony growth, thus duplicating the effect of the conditioned medium. Although some alterations of morphological and growth characteristics of HuH-7 cells were observed, the differentiated liver functions, as revealed by production of plasma proteins including high levels of alpha 1-fetoprotein, were maintained for a period of more than 3 years and through 70 passages under the above culture conditions.

Animals↗
PubMed1983

Studies on the role of serum in long-term bone marrow cell cultures.

The effect of fetal calf serum (FCS) concentration on hemopoiesis in long-term bone marrow cell cultures (supplemented with 10(-6) M hydrocortisone) was investigated. The duration of hemopoiesis was directly related to the concentration of serum in the cultures. At each concentration, marked differences were seen between different batches of FCS. The longevity of cultures established with a suboptimum concentration of serum was increased by the addition of HEPES buffer. Removal of all non-adherent cells and growth medium for the weekly feedings increased the overall production of hemopoietic cells when compared to the standard feeding procedure of replacing one-half of the growth medium.

Animals↗
PubMedDate not supplied

Effects of buffers and pH on rabbit red blood cell hexokinase.

The kinetic properties of rabbit red blood cell hexokinase in different buffer systems have been studied. At pH 8.0 the reaction velocity (v) is about 30% higher in glycylglycine compared to Tris, Tea, Hepes or ammonium acetate buffers. The enzyme stability, heat-dependence and spectral properties of the enzyme are also affected by the buffer utilized. None of the following kinetic properties of red blood cell hexokinase varies with pH in the range 6.8-8.5: Km of glucose; Km of ATP and Ki of glucose 6-phosphate.

Acetates↗
PubMed1994

Emergence of a chloride permeability after internal perfusion with calcium in squid giant axons.

In the resting condition, the membrane of the squid giant axon is highly permeable to K+, and shows large changes in membrane potential following an increase in external or internal K-concentration. When 3-5 mM Ca2+ was applied internally for 3 min, the membrane became highly permeable to Cl- as compared to K+. The permeability ratio PCl/PK was about 0.77 in control experiments, rising to a ratio of 5.5 after Ca2+ administration. The membrane conductance also increased 6-fold over controls. The permeability ratios for other anions, Cl-, F-, methanesulfate (MS), and HEPES (PCl, PF, PMS, and PHEPES) were 1.00, 0.54, 0.23, and 0.13, respectively.

Animals↗
PubMed1994

Interaction of sex steroids and oxytocin on term human myometrial contractile activity in vitro.

OBJECTIVE: To investigate the interactions of progesterone, estradiol (E2), and oxytocin in regulating the contractile activity of term human myometrium in vitro. METHODS: Myometrial tissues from 12 term pregnant women were obtained from the lower uterine segment at elective cesarean and immediately immersed in Hepes buffer with and without progesterone and progesterone plus E2. Oxytocin was freshly dissolved in solutions when the superfusion was started. Muscle biopsies were dissected along the bundles of smooth muscle under a stereomicroscope, and the preparations were mounted in six tissue baths superfused with different hormonal solutions. The isometric tension was recorded during the experimental time. RESULTS: During the 2-hour contraction period, increased frequency (P < .01 for each) and decreased activity area (P < .01 for each) and duration of contractions (P < .01 for each) resulted from superfusion of muscle strips with progesterone plus oxytocin, progesterone plus E2, and a combination of progesterone, E2, and oxytocin, compared to that found with buffer alone. The higher concentration of progesterone (5 micrograms/mL) plus oxytocin and progesterone plus E2 elevated the tonus (P < .05 and P < .01), whereas progesterone in combination with E2 and oxytocin decreased it (P < .05). The lower concentration of progesterone (0.5 microgram/mL) plus E2, or both in conjunction with oxytocin, had no significant effect on tonus as compared with buffer. CONCLUSION: The results suggest that progesterone and E2 have a direct effect on uterine excitability and a regulating influence on the oxytocin sensitivity of term human myometrium.

Adult↗
PubMed1982

Evaluation of endothelial viability in human donor corneas.

The endothelial viability of 45 paired, isolated human corneas that were removed 7, 17, and 41 hours after death was determined using in vitro perfusion with a modified HEPES (N'-2-hydroxyethylpiperazine-N'-ethanesulfonic acid)-buffered, glutathione-bicarbonate Ringer's solution. During the test period of four hours, a significant decrease in corneal thickness, measured by specular microscopy, occurred within the first two hours. The improved endothelial visualization allowed simultaneous cell-density measurements. Perfused corneas showed an almost intact endothelial ultrastructure compared with their 45 partners that were examined immediately after removal. Control corneas were perfused with isotonic saline. They failed to reverse their postmortem swelling and continued to increase in thickness at a rate of 45 +/- 54 micrometer/hr.

Cell Survival↗
PubMed1986

A medium-term corneal preserving medium (K-Sol).

A new corneal preserving solution, K-Sol, was prepared by combining TC 199, 100 mg/L of gentamicin sulfate, 0.025M HEPES buffer to a pH of 7.4, and various concentrations (2.0%; 2.25%; 2.5%) of chondroitin sulfate to a final osmolality of 310 mOsm. Rabbit corneas stored at 4 degrees C for 12 days in each solution, as well as fresh controls, were used for penetrating keratoplasty. Three weeks later, one graft in the 2.0% group was opacified; the rest were clear and showed minimal thickening. There were no significant differences between controls in terms of pachymetry and endothelial cell loss. This study supports the finding that K-Sol with 2.5% chondroitin sulfate is a simple, effective, medium-term preserving solution for donor tissue for penetrating keratoplasty in rabbits.

Animals↗
PubMed1996

Growth cone calcium elevation by GABA.

Cytoplasmic calcium plays a key role in neurite growth. In contrast to previous work suggesting that gamma aminobutyrate's (GABA) role in regulating growth cone calcium is primarily to antagonize the effects of glutamate, we report that GABA can act in an excitatory manner on developing hypothalamic neurites, independently raising calcium in growing neurites and their growth cones. Time-lapse digital video and confocal laser microscopy with the calcium-sensitive dyes fluo-3 and fura-2 were used to study the influence of GABA on neurite calcium levels. GABA (10 microM) evoked a calcium rise in both bicarbonate- and Hepes-based buffers. The calcium rise was greatly reduced after chloride transport was blocked. GABA raised calcium by stimulating the cell body, resulting in an increase in calcium throughout the neuronal cell body and dendritic arbor. GABA also acted locally, stimulating a neuritic calcium rise only in a single dendrite or growth cone. In some neurites and growth cones during early development, GABA generated a greater calcium rise than did glutamate. Bicuculline, a GABAA receptor antagonist, reduced calcium levels in neurites of young synaptically coupled neurons, indicating that ongoing synaptic release of GABA raised neuritic calcium. These data suggest that during early development, GABA may play a significant role in regulating process growth and modulating the formation of early connections in the hypothalamus. Our data support the hypothesis that GABA receptors are functionally active and may play a calcium regulating role similar to that of glutamate in neuronal development. This is particularly true in early development, as later in development GABA's role becomes more inhibitory, and glutamate plays the primary excitatory role.

Animals↗
PubMed1999

Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate.

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space.

The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.

Animals↗
PubMed1996

Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblasts cultured within collagen matrices.

Electroporation provides a useful method for loading fibroblasts with fluorescent probes for the cytoskeleton, but the possible deleterious effects of this loading technique on cell motility are unknown. We have used conventional and confocal microscopy of living cells and immunohistochemistry to examine the migration and cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured within collagen gels. Fibroblasts cultured in collagen (1 mg/ml) are successfully electroloaded (0.5-1.0 kVcm-1/960 microF in DMEM/F12/20 mM Hepes, pH 7.2) with dextran (4-150 kDa) and immunoglobulin, but subsequently display uncoordinated pseudopodia and hence are unable to migrate effectively in any one direction. The lack of directed movement is due to depolymerization of microtubules and/or a perinuclear collapse of vimentin filaments, seemingly caused by millimolar levels of Ca2+ ions derived from culture medium following electroporation. Fibroblasts loaded in a buffer which resembles intracellular fluid (< or = 10 microM Ca2+) maintain their cytoskeleton and continue to migrate, when returned to culture medium within 10 min. Using this novel approach, we have loaded fibroblasts migrating through extracellular matrix (ECM) with rhodamine phalloidin and monitored the behavior of the labeled actin cortex by confocal microscopy. During migration phalloidin-actin accumulates near the base of pseudopodia and at the rear of the cell where it is subsequently left behind. We conclude that electroporation is a valuable technique for loading fibroblasts to study migration within ECM, provided that the conditions used support stability of the tubulin cytoskeleton.

Actins↗
PubMed1998

Slow rate of free radical scavenging in the gastric antral mucosa of male Wistar rats: a possible mechanism of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.

We previously suggested that hydroxyl free radical (-OH) production may play a role in carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG-induced gastric cancer in rats and human gastric carcinoma occur most often in the antral mucosa and rarely in the normal fundic mucosa. We hypothesized that regional differences in anti-oxidant activity may be responsible. In the present study, we examined anti-oxidant activity by comparing the relative rates of reduction of a nitroxide free radical, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol), in the antral and fundic mucosa of male Wistar rats using ESR. The relative rate of Tempol reduction was significantly slower in the antral portion of the wall than in the fundic portion when Tempol [4 x 10(-6) mole/mg wet weight of gastric wall] in HEPES buffer (pH 7.4) was spread over the mucosal surface of a section of the gastric wall. Addition of a sulfhydryl group modulator, N-ethylmaleimide, to the mucosal surface before treatment with Tempol removed the significant difference observed in the rates of reduction in the antral and fundic portions of the gastric wall. No signals were detected in the muscle layer. Our results indicate that the relative rate of free radical reduction by sulfhydryl groups was significantly slower in the antral mucosa than in the fundic mucosa. We therefore conclude that a regional difference in the rates of reduction of free radicals by sulfhydryl groups may result in the site susceptible to development of MNNG-induced gastric cancer.

Animals↗
PubMed1996

Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.

A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively independent of cell type. Electroporation with three pulses of 300 volts at 540 microF capacitance at 4 degrees C is a good starting point for many cell types. Electrode distance from the adherent cells was critical at 1.0 +/- 0.15 mm. Suitable poration medium includes calcium-magnesium free phosphate buffered saline (PBS), PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, or unbuffered sucrose. Potential use in fluorescence imaging and metabolic studies is shown with DNA synthesis, cell replication, cell substratum attachment, 32P-ATP phosphorylation, and insulin-mediated increases in glucose uptake and its suppression by antiphosphotyrosine and antiglucose transporter protein antibodies. The ability to load foreign molecules into large numbers of adherent cells provides a means of studying these cells individually via microscopic approaches, such as fluorescence spectroscopic imaging, as well as with conventional biochemical and physiological techniques.

3T3 Cells↗
PubMed1999

Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.

BACKGROUND: The analysis of the subcellular distribution of proteins is essential for the understanding of processes such as signal transduction. In most cases, the parallel analysis of multiple components requires fixation and immunofluorescence labeling. Therefore, one has to ascertain that the fixation procedure preserves the in vivo protein distribution. Fusion proteins with the green fluorescent protein (GFP) are ideal tools for this purpose. However, one must consider specific aspects of the fluorophore formation or degradation, i.e. reactions that may interfere with the detection of GFP fusion proteins. METHODS: Fusion proteins of the epidermal growth factor receptor (EGFR) with GFP as well as free, soluble GFP stably or transiently expressed in adherent cultured cells served as test cases for comparing the distribution in vivo with that after fixation by conventional epifluorescence and laser scanning microscopy. Indirect immunofluorescence was employed to compare the distributions of the GFP signal and of the GFP polypeptide in the fusion protein. RESULTS: Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinuclear region. The redistribution was confirmed with the GFP and EGFR immunofluorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, which also retained the fluorescence of soluble GFP. The anti-GFP antiserum was negative for the N-terminal fusion protein. CONCLUSIONS: The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP.

Animals↗