Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Characterization of a K+-ATPase from Lactobacillus helveticus ATCC 15009

Lactobacillus helveticus ATCC 15009 (wild-type) membrane preparations hydrolyzed Mg²⁺-ATP as a function of K⁺ concentration (2–200 mM). Mg²⁺-ATP hydrolysis by L. helveticus membranes was strongly inhibited in the absence of exogenous K⁺, while it amounted to 6 nmol ATP hydrolyzed min^–1 (mg membrane protein)^–1 at 50 mM KCl (saturating conditions) and pH 7.2. The K⁺-dependent ATPase of L. helveticus displayed a relatively high affinity for potassium ions (K _m = 800 μM) and was not affected by pretreatment of membranes with N,N’-dicyclohexylcarbodiimide. Membrane preparations were subjected to hypotonic shock to obtain a maximum yield of open profiles. The formation of a maximum level of enzyme-phosphate complex with a molecular mass of approximately 82 kDa was induced upon treatment of L. helveticus membrane preparations with low concentrations of [γ-³²P]ATP in the presence of K⁺ and La³⁺ ions and was visualized by acidic SDS-PAGE. It was concluded that L. helveticus membranes contain an inwardly directed K⁺ pump whose presence is discussed in terms of its putative role in cytoplasmic pH regulation. Received: 16 December 1996 / Accepted: 14 May 1997     

Growth of Bradyrhizobium sp. SEMIA 6144 in Response to Methylglyoxal: Role of Glutathione

We previously showed the important role of glutathione (GSH) in the protection mechanism against different stresses, such as acid pH, saline, and oxidative stress, using a GSH-deficient mutant of Bradyrhizobium sp. (peanut microsymbiont). In this work, we studied the role of GSH in the protection mechanism against methylglyoxal (MG) toxicity. MG is a naturally occurring toxic electrophilic compound, and it has been shown that GSH is involved in the detoxification of MG in Escherichia coli. One recognized component of this detoxification process is the formation of a GSH adduct, which in turn transports potassium (K⁺) out of bacterial cells. Our results showed that growth of wild-type strain Bradyrhizobium sp. SEMIA 6144 was not affected at a MG concentration of 0.5 mM in the yeast extract–mannitol culture medium. However, a reduction of growth, at concentrations of 1.5 and 2.5 mM MG and reaching complete growth inhibition at 3.0 mM MG, was observed. In wild-type strain, intracellular GSH content decreased, and intracellular K⁺ content was unchanged, whereas GSH-deficient mutant SEMIA 6144-S7Z was unable to grow at 1.5 mM MG. The addition of external GSH to the incubation medium did not restore the growth rate either in wild-type or mutant strains. Our findings showed that GSH has not proven to be protective against the cell-growth inhibiting activity of MG. Therefore, the response of Bradyrhizobium sp. growth to MG is different from that reported in E. coli and other Gram-negative bacteria.     

Mitochondrial ATPase in the gills of the shore crabCarcinus maenas

Posterior gills (No. 7 and 8) of shore crabsCarcinus maenas were homogenized and fractionated by means of differential and density gradient centrifugation. Employment of marker enzymes Na-K-ATPase and carbonic anhydrase for plasma membranes and cytochrome oxidase for mitochondria showed that these structural elements were separated. Ultramicroscopic investigations of combined fractions confirmed the presence of the respective mitochondrial and vesicular plasma membrane structures. An ATPase which did not depend on the presence of sodium (20 mM) ions in the incubation medium but on the presence of potassium (20 mM) ions only was found in the mitochondrial fractions. The mitochondrial ATPase was tightly bound to cellular particulates and activated approximately threefold by bicarbonate (20 mM) ions. The activity of this ATPase was nearly completely inhibited by oligomycin (1 μg ml⁻¹) and greatly inhibited by low levels (5 mM) of thiocyanate and calcium ions, the K_i for Ca²⁺ being ca 4 mM. The results obtained confirm literature data on high mitochondrial densities in crab gills and allow the assumption of significant rates of energy metabolism in these organs. Considering its properties the mitochondrial ATPase is clearly distinct from crab gill Na-K-ATPase and can be measured specifically in samples containing Na-K-ATPase. Mitochondrial ATPase is therefore considered a suitable and reliable marker enzyme for mitochondria.     

Reversible immobilization of uricase on conductive polyaniline brushes grafted on polyacrylonitrile film

Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film containing 2.4% PANI was about 216 μg/cm². The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K _m values for free and immobilized uricase were found to be 94 and 138 μM, respectively. V _max values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity within 4 weeks.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Pharmacological properties of the potassium-activated inward current in pyramidal hippocampal neurons

Elevation of the external potassium concentration induced a two-phase inward current in freshly isolated pyramidal hippocampal neurons. This current was voltage-dependent and demonstrated strong inward rectification. The current consisted of a leakage current and a time-dependent current (τ=40–50 msec at 21°C); the latter was designated asI _ΔK. As was shown earlier, K⁺ is a major charge carrier in the development of slow potassium-activated current. The pharmacological properties ofI _ΔK were studied using a patch-clamp technique.I _ΔK was completely blocked by external 10 mM TEA or 5 mM Ba²⁺ (IC₅₀=480±90mM) and exhibited low sensitivity to extracellular Cs⁺ (2 mM). This current was not affected by 1 mM 4-aminopyridine and was insensitive to a muscarinic agonist, carbachol (50 μM), and to 1 mM extracellular Cd²⁺. Elevation of external Ca²⁺ from 2.5 mM to 10 mM did not changeI _ΔK. Our data indicate that the pharmacological properties ofI _ΔK differ from those of other voltage-gated potassium currents, but more specific blockers must be used to make this evidence conclusive.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Chloride transport across the integumentary epithelium ofManduca sexta (Lepidoptera: Sphingidae)

Summary Moulting fluid ofManduca sexta contains high concentrations of potassium and bicarbonate (100 mM) and low concentrations of chloride (5 mM). This fluid begins to disappear from the exuvial space approximately 9–10 h before the actual shedding of the integument. During this time, the integument can be isolated in an Ussing cell and electrical properties measured in vitro. In a normal 32 mM KHCO₃ saline, potential difference (PD) is around 10 mV, exuvial side positive, and short-circuit current (SCC) is 15–20 A cm^–2. Substitution of chloride slightly reduces both PD and SCC, although resistance does not change significantly. Measurement of chloride transport in the absence of K⁺ indicates that 100% of the SCC can be accounted for by the net chloride flux (2 A cm^–2). TheK _m andJ _max for transepithelial chloride transport are 14 mM and 0.1 Eq cm^–2 h^–1. Bilateral potassium addition stimulates chloride transport, doubling net chloride flux as potassium concentration increases from 2 to 5 mM. Chloride net flux is not inhibited by the presence of furosemide (1 mM), nor in HCO ₃ ^– -free saline by thiocyanate (1 or 10 mM) or acetazolamide (0.1 mM), but is inhibited by 100% N₂. The pattern of chloride transport inM. sexta is similar to that previously reported for the rectum of locusts. As chloride is normally at low concentrations in the moulting fluid, it is suggested that this transport system acts to maintain low intracellular concentrations which may be necessary for enzymatic functions in the epidermal cells and has little importance in fluid transport.Abbreviations PD potential difference - PPI pharate pupal integument - SCC short circuit current In the time since this research was performed, A.M. Jungreis passed away. He will be missed by his friends and colleagues     

Electrophysiological properties of cellular and paracellular conductive pathways of the rabbit cortical collecting duct

Summary Microelectrode techniques were applied to the rabbit isolated perfused cortical collecting duct to provide an initial quantitation and characterization of the cell membrane and tight junction conductances. Initial studies demonstrated that the fractional resistance (ratio of the resistance of the apical cell membrane to the sum of the resistances of the apical and basolateral membranes) was usually independent of the point along the tubule of microelectrode impalement—implicating little cell-to-cell coupling—supporting the application of quantitative techniques to the cortical collecting duct. It was demonstrated that in the presence of amiloride, either reduction in the luminal pH or the addition of barium to the perfusate selectively reduced the apical membrane potassium conductance. From the changes inG ^te and fractional resistance upon reducing the luminal pH or addition of barium to the perfusate, the transepithelial, apical membrane, basolateral membrane and tight junction conductances were estimated to be 9.3, 6.7, 8.1 and 6.0 mS cm^–2, respectively. Ninety to ninety-five percent of the apical membrane conductance reflected the barium-sensitive potassium conductance in the presence of amiloride with an estimated potassium permeability of 1.1×10^–4 cm sec^–1. Reduction in the perfusate pH to 4.0 caused a 70% decrease in the apical membrane potassium conductance, implying a blocking site with an acidic group having a pK _a near 4.4. It is concluded that both the transcellular and paracellular pathways of the cortical collecting tubule have high ionic conductances, and that the apical membrane conductance primarily reffects a high potassium conductance. Furthermore, both reduction in the perfusate pH and addition of barium to the perfusate selectively block the apical potassium channels, although the site of inhibition likely differs since the two ions display markedly different voltage-dependent blocks of the channel.     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis     

Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD⁺-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (ΩKm) or gentamycin (ΩGm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3′-O-phosphotransferase gene and the gentamycin- 3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by ΩKm or ΩGm, and the functional vdh gene was replaced by vdhΩKm or vdhΩGm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HRvdhΩKm, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase. Received: 19 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells

Summary A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special “epithelial cell” medium. Confluency was proved with an epithelial volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 Ω×cm². A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an l-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated. Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated transport processes across the epithelial cells and their plasma membranes.     

Some electrophysiological and permeability properties of the mouse egg

Certain electrophysiological and ionic properties of the mouse egg (CF-1 and BDF 12–18 hr post ovulation) have been investigated. Membrane potential (?14 ± 0.4 mV, ± SE, inside negative), membrane resistance (2610 ± 38 ohm·cm²), and membrane capacitance (1.6 ± 0.03 μF cm^?2) have been determined by means of intracellular microelectrode recording techniques. Membrane potential and related parameters are stable for extended periods of time upon impalement and the magnitude of the cell membrane potential has been demonstrated to be sensitive to alteration in external sodium. The electrophysiological studies in conjunction with measurements of unidirectional potassium fluxes using isotope tracer-techniques have allowed determination of membrane permeability to potassium (8 × 10^?8 cm sec^?1) and membrane potassium conductance (25 μmho cm^?2). Furthermore, the use of tracer flux techniques has indicated that the exchangeable fraction of intracellular potassium is 204 ± 14 mM. This represents the bulk of egg potassium (222 ± 19 mM as determined from flame photometry). Studies of unidirectional potassium efflux have indicated that its movement out of the egg is made up of at least two components; an external potassium-independent potassium efflux and external potassium-dependent efflux, the latter possibly representing a potassium exchange mechanism. The combined electrophysiological and tracer-flux data indicate that only a small portion of the total membrane conductance is composed of potassium conductance at this stage of development. This and the fact that the membrane potential is far from the potassium equilibrium potential are similar to observations made on mature eggs of several other species.     

Active and passive properties of rabbit descending colon: A microelectrode and nystatin study

Summary The electrical properties of the basolateral membrane of rabbit descending colon were studied with microelectrode methods in conjunction with the polyene antibiotic nystatin. Two problems were examined: (i) the relative distribution of tight junctional, apical membrane and basolateral membrane resistances, and (ii) the ionic basis of the basolateral membrane potential. Intracellular K⁺ activity (K⁺) was measured using liquid ion exchanger microelectrodes ((K⁺)=76±2mm) and was found not to be in equilibrium with the basolateral membrane potential. In order to measure membrane resistances and to estimate the selective permeability of the basolateral membrane, the apical membrane was treated with nystatin and bathed with a K₂SO₄ Ringer's solution which was designed to mimic intracellular K⁺ composition. This procedure virtually eliminated the resistance and electromotive force of the apical membrane. Shunt resistance was calculated by two independent methods based on microelectrode and transepithelial measurements. Both methods produced similar results (R _s =691±63 cm² and 770±247 cm², respectively). These findings indicate that the shunt has no significant selectivity, contrary to previous reports. Native apical membrane resistance was estimated as 705±123 V cm² and basolateral membrane resistance was 95±14 V cm².To estimate basolateral membrane selectivity, the serosa was bathed in a NaCl Ringer's solution followed by a series of changes in which all or part of the Na⁺ was replaced by equimolar amounts of K⁺. From measures of bi-ionic potentials and conductance during these replacements, we calculated potassium permeability and selectivity ratios for the nystatin-treated colon by fitting these results to the constant field equations. By correcting for shunt conductance, it was then possible to estimate the selective permeability of the basolateral membrane alone. Selectivity estimates were as follows:P _Na/P _K=.08 andP _Cl/P _K=.07 (uncorrected for shunt) andP _Na/P _K=.04 andP _Cl/P _K=.06 (basolateral membrane alone).In a second set of experiments, evidence for an electrogenic Na⁺ pump in the basolateral membrane is presented. A small ouabain-sensitive potential could be generated in the nystatin-treated colon in the absence of chemical or electrical gradients by mucosal, but not serosal, addition of NaCl. We conclude that this electrogenic pump may contribute to the basolateral membrane potential; however, the primary source of this potential is passive: specifically, a potassium gradient which is maintained by an active transport process.An appendix compares the results of nystatin experiments to amiloride experiments which were conducted separately on the same tissues. The purpose of this comparison was to develop a comprehensive model of colonic transport. The analysis reveals a leak conductance in the apical membrane and the presence of an amiloride-insensitive conductance pathway.     

Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps

Summary The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K⁺)=72mm and (Cl^–)=15.8mm. Cl^– but not K⁺ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (P _Na/P _K) was 0.044, and the chloride to potassium permeability ratio (P _Cl/P _K) was 1.17. Since K⁺ was not in electrochemical equilibrium, intracellular (K⁺) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K⁺ and Cl^– the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na⁺. An equivalent electrical circuit for Na⁺ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.     

Oxidative stress as a component of chromium-induced cytotoxicity in rat calvarial osteoblasts

It has been documented that medical prosthetic alloys release metal ions into surrounding tissues and cause cytotoxicity, but the mechanisms remain undefined. In that regard the cellular oxidative stress may be a common pathway in cellular responses to metal ions. The objective of this study was to approach the hypothesis that oxidative stress mediates chromium-induced cytotoxicity in rat calvarial osteoblasts. Osteoblasts were exposed to different concentrations of Cr⁶⁺ or Cr³⁺ (5–20 μM) in the presence or absence of the antioxidant N-acetyl-cysteine (NAC; 1–5 mM). Cellular viability, differentiation, and intracellular ultrastructural alterations were evaluated by MTT assay, alkaline phosphatase (ALP) activity assay, and transmission electron microscopy. Cellular oxidative stress was evaluated by intracellular reactive oxygen species (ROS) production. ROS production was monitored by the oxidation-sensitive fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA). A time- and concentration- dependent increased cytotoxicity, time-dependent increased intracellular ROS production were indicated on exposure to Cr⁶⁺. Pretreatment of osteoblasts with 1–5 mM NAC afforded dose-dependent cytoprotective effects against Cr⁶⁺-induced cytotoxicity in osteoblasts. NAC decreased the level of intracellular ROS induced by Cr⁶⁺, too. While Cr³⁺ and NAC did not have any significant effects on osteoblasts (5–20 μM). These results suggest that oxidative stress is involved in Cr⁶⁺-induced cytotoxicity in osteoblasts, and NAC can provide protection for osteoblasts against Cr⁶⁺-induced oxidative stress. Cr³⁺ (5–20 μM) have no significant cytotoxicity in osteoblasts based on the results of this study.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Anin vitro model for endothelial permeability: Assessment of monolayer integrity

Summary An essential component of anyin vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9±11.4 Ω · cm², 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to³H-inulin demonstrated a linear relationship between the luminal concentration of³H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of³H-inulin, was 2.01±0.076 × 10⁻⁴ ml/min (r²=.9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17±0.427×10⁻⁶ cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07±0.617×10⁻⁶ cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to³H-inulin (1.43±0.445×10⁻⁵ cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.     

Tuning of electron delocalization in polynuclear mixed-valence clusters by super-exchange and double exchange

 Values for the exchange-coupling constant J and the double-exchange parameter B have been estimated for dimeric and hexameric mixed-valence iron clusters. For sulfur-bridged species the range of J values is 300–450 cm^–1, and B values vary between 320 and 400 cm^–1. For an OH-bridged diiron cluster B is as large as 1300 cm^–1. Received and accepted: 23 January 1996