Morphological alterations and changes in cellular cations induced by Clostridium perfringens type a enterotoxin in tissue culture cells

The morphological alterations (bleb-balloon formation) induced by Clostridium perfringens type A enterotoxin in HeLa and Vero cells were studied under defined extracellular conditions. The action of enterotoxin was found to depend on the temperature but not on energy metabolism. The morphological alterations by the enterotoxin occurred in phosphate buffered saline containing Ca²⁺ and Mg²⁺. Of the constituents of the buffered saline, Ca²⁺ was essential for the morphological alterations and other ions were interchangeable. The morphological alterations by the enterotoxin occurred also in 10 mM Hepes-Na buffer, pH 7.2 containing NaCl, KCl or choline chloride at a concentration of over ca. 50 mM and in 10 mM Hepes-Ca buffer, pH 7.2 containing CaCl₂ at a concentration of over ca. 50 mM. Addition of sucrose to the medium prevented induction of the morphological alterations. The amount of sucrose necessary to protect the cells increased with increase in NaCl, KCl or CaCl₂ concentration in the medium. A calcium ionophore A23187 mimicked the action of enterotoxin. Examination of the cation contents of the cells by atomic absorption spectrophotometry showed early and rapid increase of Ca ²⁺ during intoxication with concomitant changes in Na⁺, K⁺ and Mg²⁺ that reduced the ion concentration gradients between inside and outside of the cell present before toxin treatment. The mechanism of action of C. perfringens type A enterotoxin is discussed on the basis of these findings.Corresponding Author.     

Inhibition of suicidal erythrocyte death by vitamin C

ObjectiveSimilar to apoptosis of nucleated cells, suicidal death of erythrocytes is paralleled by cell shrinkage and cell membrane disorganization with phosphatidylserine exposure at the erythrocyte surface. Triggers of suicidal erythrocyte death include cell shrinkage, energy depletion, and oxidative stress, challenges at least partially effective by increasing the cytosolic Ca²⁺ concentration. Apoptosis is inhibited by vitamin C. The present study thus explored whether vitamin C similarly influences suicidal erythrocyte death.MethodsThe cytosolic Ca²⁺ concentration was estimated from Fluo3 fluorescence, phosphatidylserine exposure from annexin V-binding, and cell volume from forward scatter in fluorescence activated cell sorting (FACS) analysis.ResultsEnergy depletion (48 h glucose removal) increased the cytosolic Ca²⁺ concentration, decreased the erythrocytic cell volume, and enhanced annexin V-binding. Similarly, cell shrinkage by 48 h replacement of extracellular chloride with gluconate and oxidative stress (30 min exposure to 0.3 mM tert-butylhydroperoxide) triggered suicidal erythrocyte death as evident from enhanced annexin V-binding. Vitamin C (up to 0.28 mM) did not significantly modify the cytosolic Ca²⁺ concentration, annexin V-binding, and cell volume in the absence of stressors stimulating suicidal erythrocyte death but significantly attenuated the suicidal erythrocyte death following cell shrinkage, energy depletion, and oxidative stress.ConclusionVitamin C is a potent inhibitor of suicidal erythrocyte death.     

Effects of ethanol exposure on neuropeptide-stimulated calcium mobilization in N1E-115 neuroblastoma

The effects of acute and chronic (100 mM for 7 days) ethanol exposures on resting intracellular free calcium, [Ca²⁺]_i, as well as bradykinin and neurotensin mediated [Ca²⁺]_i mobilization were determined in intact N1E-115 neuroblastoma. [Ca²⁺]_i was monitored fluorometrically with the calcium indicator, fluo-3/AM. Acute exposure to ethanol resulted in an inhibition of bradykinin mediated [Ca²⁺]_i mobilization with significant effects observed only at 400 mM ethanol. Neurotensin mediated [Ca²⁺]_i mobilization was not significantly affected by any of the ethanol concentrations tested. Similarly, resting [Ca²⁺]_i (64 ± 2 nM) was unaffected by either chronic or acute ethanol as high as 400 mM. However, chronic exposure to ethanol significantly reduced the magnitude of bradykinin mediated [Ca²⁺]_i mobilization both in the absence and presence of extracellular [Ca²⁺]. In contrast, [Ca²⁺]_i mobilization in the presence of various concentrations of neurotensin was not significantly affected by chronic ethanol exposure. The results suggest that neuropeptide mediated [Ca²⁺]_i mobilization is relatively insensitive to the acute presence of ethanol. In addition, chronic ethanol exposure appears to have selective effects on receptor mediated [Ca²⁺]_i mobilization because this response to bradykinin, but not neurotensin, was significantly reduced in cells exposed to ethanol. The results also suggest that the reduction in bradykinin stimulated [Ca²⁺]_i mobilization in chronically exposed cells is due in part to an inhibition of the release of intracellularly bound [Ca²⁺].     

Ethanol impairs calcium homeostasis following CCK-8 stimulation in mouse pancreatic acinar cells

Alcohol consumption has long been associated with cell damage, and it is thought that it is involved in approximately 40% of cases of acute pancreatitis. In the present study, we have investigated the early effects of acute ethanol exposure on cholecystokinin octapeptide (CCK-8)-evoked calcium (Ca²⁺) signals in mouse pancreatic acinar cells. Cells were loaded with fura-2 and the changes in fluorescence were monitorized using a spectrofluorimeter. Our results show that stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca²⁺]_c, which consisted of an initial increase followed by a decrease of [Ca²⁺]_c toward a value close to the prestimulation level. In the presence of 50 mM ethanol, CCK-8 lead to a greater Ca²⁺ mobilization compared to that obtained with CCK-8 alone. The peak of CCK-8-evoked Ca²⁺ response, the “steady-state level” reached 5 min after stimulation, the rate of decay of [Ca²⁺]_c toward basal values and the total Ca²⁺ mobilization were significantly affected by ethanol pretreatment. Thapsigargin (Tps) induced an increase in [Ca²⁺]_c due to its release from intracellular stores. After stimulation of cells with CCK-8 or Tps in the presence of 50 mM ethanol, a greater [Ca²⁺]_c peak response, a slower rate of decay of [Ca²⁺]_c, and higher values of [Ca²⁺]_c were observed. The effects of ethanol might result from a delayed or reduced Ca²⁺ extrusion from the cytosol toward the extracellular space by plasma membrane Ca²⁺adenosine triphosphatase (ATPase), or into the cytosolic stores by the sarcoendoplasmic reticulum Ca²⁺-ATPase. Participation of mitochondria in Ca²⁺ handling is also demonstrated. The actions of ethanol on CCK-8 stimulation of cells create a situation potentially leading to Ca²⁺ overload, which is a common pathological precursor that mediates pancreatitis.     

POTASSIUM INCREASES INTRACELLULAR CALCIUM SIMULATING PROGESTERONE ACTION IN HUMAN SPERM

Progesterone (P) and zona pellucida are known to induce acrosome reaction in human sperm by increasing cytosolic calcium. High concentrations of potassium ions (K⁺) improve the rate of acrosome reaction in human sperm in vitro. This article determined whether the effect of K⁺ on the acrosome in human sperm is mediated by increasing intracellular calcium ([Ca²⁺]i). The effect of K⁺ on [Ca²⁺]i was examined by using Fura 2 as the fluorescent indicator. The effect of K⁺ and P on [Ca²⁺]i in sperm and the involvement of ion channels was compared. Motile sperm were collected by the swim-up method from semen of healthy volunteers and capacitated overnight in BWW containing 0.5% BSA. Incubation of capacitated sperm with different concentrations of potassium chloride (1.25-20 mM) resulted in dose-dependent increase in [Ca²⁺]i similar to that observed with P. The increase in [Ca²⁺]i by K⁺ and P was blocked by the addition of EGTA, a Ca²⁺ chelator. K+-induced change in [Ca²⁺] was not altered by the addition of dihydropyridine derivatives. The combined treatment of K+ (20 mM) and P (0.75 mug/mL) caused an additive effect on the increase in [Ca²⁺]i. It would appear that human sperm plasma membrane possess different Ca2+ channels responsive to P and K⁺.     

In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells

Several recent studies have demonstrated that the direct precursor of vitamin D₃, the calcifediol [25(OH)D₃], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D₃ by binding the VDR could have a vitamin’s D₃ activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca²⁺ mobilization in response to 25(OH)D₃. Our result reveals the ability of 25(OH)D₃ to activate rapid, non-genomic pathways, such as an increase of intracellular Ca²⁺ levels, similar to what observed with the biologically active form of vitamin D₃. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca²⁺ concentration as a result of exposure to 10⁻⁵ M of 25(OH)D₃. In this work, we show for the first time the in vitro ability of 25(OH)D₃ to induce a rapid increase of intracellular Ca²⁺ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D₃ and its role in endocrine system.     

Combined Administration of l-Carnitine and Ascorbic Acid Ameliorates Cisplatin-Induced Nephrotoxicity in Rats

Objectives: Cisplatin (CIS) is an effective antitumor drug. However, its clinical use is limited due to nephrotoxicity. l-Carnitine and vitamin C are both natural antioxidant that can be obtained from diets. This study investigated the effects of l-carnitine and/or vitamin C in rats against cisplatin-induced nephrotoxicity.

Methods: Twenty-five male Wistar rats were divided into 5 groups of 5 rats each. Group 1, normal control. Group 2, positive control, received cisplatin (10 mg/kg/day intraperitoneally [i.p.]) for 3 days. Groups 3, 4, and 5 received cisplatin for 3 days and thereafter l-carnitine (50 mg/kg/day), vitamin C (100 mg/kg/day), or their combination, respectively, for 28 days. At the end of the study, a biochemical study was carried out in which nephrotoxicity markers, electrolytes, hematological indices, oxidative stress biomarkers, and renal histopathological alterations were evaluated.

Results: CIS-treated rats developed significant polyuria, increase in the plasma levels of creatinine, urea, and inorganic phosphate (P_i), alteration in hematological parameters, and decrease in plasma levels of Na⁺, Cl^?, K⁺, Ca²⁺, and Mg²⁺. Measurements of 24-hour urine output demonstrated markedly decreased creatinine and urea and increased Na⁺, Cl^?, K⁺, Ca²⁺, and Mg²⁺ levels in the CIS-treated group, whereas P_i levels were not changed. It also caused significantly decreased catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) levels in the rats' kidneys. Histopathological scores revealed renal tubular damage in CIS-treated rats. However, l-carnitine, vitamin C, or their combination significantly attenuated the alterations caused by CIS in the plasma and kidneys of the rats.

Conclusion: l-Carnitine and vitamin C administration ameliorated CIS-induced nephrotoxicity due to their antioxidant and anti-inflammatory effects.     

Surface Sorption Site and Complexation Structure of Ca2+ at the Goethite–Water Interface: A Molecular Dynamics Simulation and Quantitative XANES Analysis

The molecular-level surface complexation structure of Ca²⁺ at the goethite/water interface remains unclear. We investigated the sorption of Ca²⁺ on the surfaces of goethite using classical molecular dynamics (MD) simulations and Ca K-edge X-ray absorption near edge structure (XANES) spectroscopy. The XANES results showed that Ca²⁺ was sequestered by goethite dominantly via sorption at pH ≤ 9, whereas the Ca(OH)₂ surface precipitate formed at pH 10 for an initial Ca²⁺ concentration of 2 mM. The MD simulations showed that Ca²⁺ dominantly absorbed on the (100) and (110) surfaces of goethite via bidentate binuclear complexation by forming ≡(Fe-OH)₂Ca²⁺·5H₂O species, whereas little Ca²⁺ adsorbed on the (021) surface. The theoretical Ca K-edge XANES spectrum calculations gave a mean Ca-O interatomic distance of 2.34 Å (2.23–2.55 Å) and a Ca-Fe interatomic distance of 3.80 Å (3.79–3.81 Å) at goethite/water interface. Our results may shed some light on the geochemical cycling of calcium and other related cations and anions.

     

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

Aims: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/ pro-oxidant effects of vitamin C at the cellular level. Experimental approach: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. Results: Even at concentrations in the medium as high as 200 μM, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H₂O₂ or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 μM, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). Conclusion: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair.     

Soluble Mn2+-dependent Adenylate Cyclase Activity in the Testis of the Blue Fox (Alopex lagopus)

Soluble Mn²⁺ -dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35°C and for 2 hr after storage at 25°C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1—10 mM) of a reducing agent (β-mercapto ethanol, βME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p< 0.01) in activity. Substitution of Mn^2- in the assay medium with Mg^2- led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The K_m for Mn²⁺ was estimated to be 3.5 mM. The affinity of the enzyme for Mn²⁺ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn²⁺ appeared to increase the affinity of the enzyme for MnATP^2-. The K_m for MnATP^2- thus varied from 6 to 18 mM.     

Differential effects of ca2+ on proliferation of stomach,colonic, and pancreatic cancer cell lines in vitro

Calcium intake inhibits growth of colon cancer in vivo, the mechanisms of which are not fully elucidated. The objective of this study was to determine whether Ca²⁺ directly affects the growth of colon cancer cells in vitro and to compare the effects of Ca²⁺ on the growth of several gastroenteropancreatic cancer cells, including mouse colon cancer (MC‐26), human colon cancer (Lo Vo and WIDR), human gastric cancer (AGS and SII), and human pancreatic cancer (PANC‐1 and MIA) cells. All tumor cell lines tested grew in medium containing low concentration (approx 0.16 mM) of Ca²⁺. Higher concentrations of Ca²⁺ significantly inhibited the growth of all three colon cancer cell lines tested but had no significant effect on proliferation of the stomach and pancreatic cancer cell lines. Growth of AGS cells, in the presence of 0.1 or 0.5 mMEGTA (resulting in the loss of the extracellular Ca²⁺) was similar to that observed in the absence of EGTA, indicating that AGS cells were relatively insensitive to loss of extracellular Co²⁺. In the presence of TMB‐8, an inhibitor of intracellular Ca²⁺ release, the growth of colonic cancer cell lines was inhibited in a dose‐dependent manner, indicating that a minimum basal level of intracellular Ca²⁺ was required for continued proliferation of colon cancer cells. The stomach cancer cell lines (AGS) was once again less sensitive to the effects of TMB‐8 than were the colon cancer cells, indicating an inherent difference in Ca²⁺ requirements and sensitivity to Ca²⁺ for growth of different gastroenteropancreatic cancer cells in vitro. These results further demonstrate that in contrast to normal cells, all tumor cells tested can continue to proliferate in the presence of low concentrations of extracellular Ca²⁺ (approx <0.16 mM) and that higher concentrations of Ca²⁺ specifically and directly inhibit growth of human and mouse colon cancer cells. The latter results may provide an additional explanation for the mechanism by which intake of dietary calcium decreases the risk of developing colon cancers.     

The Increase in FGF23 Induced by Calcium Is Partially Dependent on Vitamin D Signaling

Background: Increased FGF23 levels are an early pathological feature in chronic kidney disease (CKD), causing increased cardiovascular risk. The regulation of FGF23 expression is complex and not completely understood. Thus, Ca²⁺ has been shown to induce an increase in FGF23 expression, but whether that increase is mediated by simultaneous changes in parathyroid hormone (PTH) and/or vitamin D is not fully known. Methods: Osteoblast-like cells (OLCs) from vitamin D receptor (VDR)^+/+ and VDR^−/− mice were incubated with Ca²⁺ for 18 h. Experimental hypercalcemia was induced by calcium gluconate injection in thyro-parathyroidectomized (T-PTX) VDR ^+/+ and VDR^−/− mice with constant PTH infusion. Results: Inorganic Ca²⁺ induced an increase in FGF23 gene and protein expression in osteoblast-like cells (OLCs), but the increase was blunted in cells lacking VDR. In T-PTX VDR ^+/+ and VDR^−/− mice with constant PTH levels, hypercalcemia induced an increase in FGF23 levels, but to a lower extent in animals lacking VDR. Similar results were observed in FGF23 expression in bone. Renal and bone 1α-hydroxylase expression was also modulated. Conclusions: Our study demonstrates that Ca²⁺ can increase FGF23 levels independently of vitamin D and PTH, but part of the physiological increase in FGF23 induced by Ca²⁺ is mediated by vitamin D signaling.     

Effects of Ca<Superscript>2+</Superscript> or Na<Superscript>+</Superscript> on Metallothionein Expression in Tilapia Larvae (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) Exposed to Cadmium or Copper

The objectives of this study were to try to determine the reasons of the external Ca²⁺ and Na⁺ enhancement of Cd²⁺ and Cu²⁺ resistance in fish. Tilapia larvae at 3 days posthatch were exposed to (A) 0 (control), 40 μg/L Cd²⁺, 40 μg/L Cd²⁺ + 2 mM Ca²⁺ (Cd/hyper-Ca), and 2 mM Ca²⁺ or (B) 0 (control), 75 μg/L Cu²⁺, 75 μg/L Cu²⁺ + 0.52 mM Na⁺ (Cu/hyper-Na), and 0.52 mM Na⁺. After 48 hours, results indicated that (1) Cd/hyper-Ca and Cu/hyper-Na treatments showed decreased growth inhibition induced by the metals; (2) metal accumulation in Cd/hyper-Ca–treated larvae was lower compared with those exposed only to Cd; and (3) metallothionein (MT) expression was significantly higher in Cu/hyper-Na–treated larvae than in the group treated with Cu only. Taking all of this into account, either supplementary Ca²⁺ or Na⁺ in ambient water may help fish to maintain Ca²⁺ or Na⁺ homeostasis, which could decrease metal accumulation and its detrimental effects. Consequently, the fish increase MT expression and retard the growth inhibition caused by metals.     

Differential Effects of Methanol on Rat Aortic Smooth Muscle

LI, W., B. T. ALTURA AND B. M. ALTURA. Differential effects of methanol on rat aortic smooth muscle. Alcohol 16(3) 221–229, 1998.—The effects of methanol on isolated segments of rat aorta were investigated. In the absence of any vasoactive agent, methanol (5–675 mM) failed to alter basal tension. In rat aortic rings precontracted with high K⁺ (30 mM), methanol elicited a concentration-related relaxation at concentrations of from 5 to 675 mM. The K⁺-induced contraction in the presence of endothelium was more strongly inhibited by methanol than in the absence of endothelium. The effective concentration producing approximately 50% of the maximal relaxation response (ED₅₀) to methanol was about 96 mM. Methanol-induced relaxations could not be abolished either by 5 × 10⁻⁵ M N-nitro-l-arginine methyl ester (l-NAME) or N^G-nitro-l-arginine (l-NNA), both selective inhibitors of nitric oxide (NO) formation; these relaxations were not potentiated by addition of excess l-arginine. An inhibitor of prostanoid synthesis, indomethacin (10⁻⁵ M), had no effects on methanol-induced relaxation. Removal of extracellular Ca²⁺ ([Ca²⁺]_o) resulted in almost complete inhibition of the relaxant effects of methanol on rat aortic ring segments. Marked attenuation of the relaxation responses of intact arteries to methanol was obtained after buffering intracellular Ca²⁺ ([Ca²⁺]_i) with 10 μM BAPTA-AM. In 5-hydroxytryptamine (5-HT, 2.5 μM)- or phenylephrine (PE, 0.1 μM)-precontracted rat aortic rings, methanol amplified contractile responses to 5-HT and PE; these increased responses were concentration dependent. No significant differences in these methanol potentiated responses were found between aorta with or without endothelial cells. The amplified rat aortic smooth muscle responses induced by methanol after PE could be modified only by phentolamine, an antagonist of PE, while responses to 5-HT could be inhibited by methysergide (an antagonist of 5-HT) and by phentolamine, diphenhydramine, and haloperidol. Pretreatment with 50, 200, and 500 mM methanol increased rat aortic contractile responses induced by 5-HT and PE. Our results suggest that: (a) acute methanol exposure relaxes rat aortic smooth muscle contractile responses induced by high K⁺, leading to vessel relaxation. This relaxation effect of methanol is endothelium-dependent, clearly Ca²⁺ dependent, and independent of endogenous vasodilators such as acetylcholine, histamine, catecholamines, serotonin, or PG. (b) Methanol seems to increase potassium current by shifting the potential towards more negative values in depolarized vascular muscle cell membranes, probably inducing hyperpolarization of the cell membranes leading to a repolarization. (c) In contrast to the relaxant responses, methanol protentiates contractile response of rat aorta to 5-HT and PE.     

Fusicoccin Counteracts the Toxic Effect of Cadmium on the Growth of Maize Coleoptile Segments

The effects of cadmium (Cd; 0.1–1000 μM) and fusicoccin (FC) on growth, Cd²⁺ content, and membrane potential (E _m) in maize coleoptile segments were studied. In addition, the E _m changes and accumulation of Cd and calcium (Ca) in coleoptile segments treated with Cd²⁺ combined with 1 μM FC or 30 mM tetraethylammonium (TEA) chloride (K⁺-channel blocker) were also determined. In this study, the effects of Ca²⁺-channel blockers [lanthanum (La) and verapamil (Ver)] on growth and content of Cd²⁺ and Ca²⁺ in coleoptile segments were also investigated. It was found that Cd at high concentrations (100 and 1000 μM) significantly inhibited endogenous growth of coleoptile segments and simultaneously measured proton extrusion. FC combined with Cd²⁺ counteracted the toxic effect of Cd²⁺ on endogenous growth and significantly decreased Cd²⁺ content (not the case for Cd²⁺ at the highest concentration) in coleoptile segments. Addition of Cd to the control medium caused depolarization of E _m, the extent of which was dependent on Cd concentration and time of treatment with Cd²⁺. Hyperpolarization of E _m induced by FC was suppressed in the presence of Cd²⁺ at 1000 μM but not Cd²⁺ at 100 μM. It was also found that treatment of maize coleoptile segments with 30 mM TEA chloride caused hyperpolarization of E _m and decreased Cd²⁺ content in coleoptile segments, suggesting that, in the same way as for FC, accumulation of Cd²⁺ was dependent on plasma membrane (PM) hyperpolarization. Similar to FC, TEA chloride also decreased Ca²⁺ content in coleoptile segments. La and Ver combined with Cd²⁺ (100 μM) significantly decreased Cd content in maize coleoptile segments, but only La completely abolished the toxic effect of Cd²⁺ on endogenous growth and growth in the presence of FC. Taken together, these results suggest that the mechanism by which FC counteracts the toxic effect of Cd²⁺ (except at 1000 μM Cd²⁺) on the growth of maize coleoptile segments involves both stimulation of PM H⁺-ATPase activity by FC as well as Cd²⁺-permeable, voltage-dependent Ca channels, which are blocked by FC and TEA chloride-induced PM hyperpolarization.     

The effects of food components on the digestion of DNA by pepsin

Recently, our study found that naked nucleic acids (NAs) can be digested by pepsin. To better understand the fate of dietary DNA in the digestive tract, in this study we investigated the effects of several food compositions on its digestion. The results showed that protein inhibited the digestion of DNA when the protein:DNA ratio was higher than 80:1 (m/m). DNA found in nucleoprotein (NA), which more closely resembles the state of DNA in food, was as efficiently digested as naked DNA. When the carbohydrate:DNA ratio was 50:1–140:1 (m/m), mono-, di- and polysaccharides did not inhibit DNA digestion. NaCl exhibited an inhibitory effect at 300?mM, whereas divalent cations (Ca^2+?and Mg²⁺) exerted a much stronger inhibitory effect even at 50?mM. The polycation compounds (e.g. chitosan and spermine) showed a significant inhibitory effect at N/P (NH₃⁺/PO₄^?)?=?10:1. The close relationship between food composition and DNA digestion suggests that dietary habits and food complexes are important for understanding the in vivo fate of the ingested DNA in the digestive tract.     

Increase of Intracellular free [CA2+] in Single Human Motile Spermatozoa Treated with Human Follicular Fluid

The intracellular free [Ca²⁺] concentration ([Ca²⁺]_i) in individual human sperm was measured using a fluorescent Ca²⁺ indicator. In 18 of 23 motile sperm (78.3%), [Ca²⁺]_i increased significantly and promptly after addition of 20% human follicular fluid (hFF), but in the others it did not increase. The mean resting [Ca²⁺]_i level of sperm in which [Ca²⁺]_i increased after addition of 20% hFF (the influx group) was significantly lower than those in which it did not increase (112.8 ± 40.1 nM vs. 156.9 ± 13.5 nM,p <. 05). After addition of 20% hFF, the mean [Ca²⁺]_i in the influx group reached a peak value of 210.7± 24.7 nM within 30 s and then decreased slowly; the mean [Ca²⁺]_i values 1, 5, 10, and 15 min after addition of 20% hFF were 179.3 ± 31.4, 174.3 ± 30.2, 172.5 ± 27.8, and 175.1 ± 27.2 nM, and all values were significantly higher than the resting level (p <. 01). The frequency distribution of [Ca²⁺]_i after addition of 20% hFF was shifted toward higher concentrations (p <. 01). However, the addition of 20% hFF did not increase the percentage of live acrosome reaction (before 3.8 ± 0.9% vs. after 2.9 ± 0.5%, respectively). Thus, hFF increased [Ca²⁺]_i in about 80% of the motile sperm. Relatively high [Ca²⁺]_i levels persisted for at least 10–15 min after its addition. However, hFF did not trigger a rapid response in acrosome reaction.     

Effect of Triglyceride Structure on Fecal Excretion of 13C-Labeled Triglycerides

Objective: The aim of this work was to determine the effects of specific changes in the structure of ¹³C-labeled triglyceride (TG*) on its fecal excretion relative to total stool fat excretion determined simultaneously in patients with reduced exocrine pancreatic function.

Methods: A series of 47 studies were conducted in 26 young cystic fibrosis (CF) patients and 11 adult patients with chronic pancreatitis over a five year period. Each test consisted of ingesting a single high fat test meal containing both ¹³C-labeled triglyceride (TG*) and dysprosium chloride (DyCl₃) a nonabsorbable marker of intestinal transit; in most studies the food colorant brilliant blue (FD&C blue #1) was administered along with the DyCl₃. The TG*s tested were: P*P*P* = TRIPALMITIN-1,1,1-¹³C₃; SO*S = 2-OCTANOYL-1,3-DISTEARIN-2-octanoyl-1,2-¹³C₂; and P*LP* = 2-LAURYL-1,3-DIPALMITIN-dipalmitoyl-1,1,2,2-¹³C₄. Ingestion of the test meal was followed by collection of individual stools for at least 72 hours. Stools were analyzed for ¹³C-Excess (¹³C*), total fat, and Dy.

Results: Excretion of P*LP* showed a high degree of linear correlation with stool fat (r² = 0.924) over a wide-range of fecal fat values. Excretion of SO*S was also significantly correlated with stool fat, but its excretion was less than 10% at all levels of steatorrhea and the slope of the regression line relating TG* excretion to stool fat was some four to five times smaller than observed for P*LP*. Fecal excretion of P*P*P* was highly correlated with stool fat (r² = 0.941) in patients with moderate steatorrhea (<25 g fat/24 hours) and the slope of the regression line (3.20) was considerably greater than for P*LP*. Only results from those studies in which stool collections were complete (Dy excretion >90%) were utilized in the statistical comparisons (36 of 47 studies).

Conclusions: The observed highly significant linear correlation between P*LP* and stool fat over the entire range of steatorrhea suggests that P*LP* excretion may be a suitable surrogate for fecal fat in patients with reduced exocrine pancreatic function. Because fecal excretion of TG* administered as described can be accurately determined by sampling only two visually marked stools, development of a noninvasive test to replace the current 72-hour stool fat test using this approach is possible. Use of other engineered TG*s and/or labeled fatty acids, may provide a method for non-invasive in vivo assessment of the specific defect(s) leading to steatorrhea in other patient groups.     

Effects of paraquat on Mg2+-ATPase activity in rat liver

Intraperitoneal injection of paraquat (70 mg/kg) elicited a significant decrease of 20% in activity of Mg²⁺-ATPase in hepatic mitochondria, which is always surrounded by a high concentration of oxygen, in rats. The decrease of mitochondrial Mg²⁺-ATPase activity was completely abolished by pretreatment with vitamin E, which is a scavenger of oxygen radicals. On the other hand, paraquat administration did not change the Mg²⁺-ATPase activity in endoplasmic reticulum, which exists in an anaerobic condition in living cells. When liver microsomes were incubated with 1 mM paraquat under aerobic conditions, the Mg²⁺-ATPase activity was decreased by 42%. The decrease of Mg²⁺-ATPase activity was completely eliminated by pretreatment with vitamin E. Furthermore, lipid peroxidation in microsomes was tremendously increased by the addition of 1 mM paraquat under aerobic conditions. The increase of lipid peroxidation was completely abolished by preadministration of vitamin E in rats. The results suggest that the inhibition of Mg²⁺-ATPase activity induced by paraquat may be mediated by active oxgen, which is produced by the reaction of paraquat radicals; molecular oxygen may be involved in the induction of hepatic cell injury.     

Effects of Magnesium on Cardiac Excitation-Contraction Coupling

Objective: Magnesium regulates a large number of cellular processes. Small changes in intracellular free Mg²⁺ ([Mg²⁺]_i) may have important effects on cardiac excitability and contractility. We investigated the effects of [Mg²⁺]_i on cardiac excitation-contraction coupling.

Methods: We used our ionic-metabolic model that incorporates equations for Ca²⁺ and Mg²⁺ buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na⁺-K⁺ pump, sarcolemmal and sarcoplasmic Ca²⁺ pumps).

Results: Model results indicate that variations in cytosolic Mg²⁺ level might sensitively affect diastolic and systolic Ca²⁺, sarcoplasmic Ca²⁺ content, Ca²⁺ influx through L-type channels, efficiency of the Na⁺/Ca²⁺ exchanger and action potential shape. The analysis suggests that the most important reason for the observed effects is a modified normal function of sarcoplasmic Ca²⁺-ATPase pump by altered diastolic MgATP levels.

Conclusion: The model is able to reproduce qualitatively a sequence of events that correspond well with experimental observations during cardiac excitation-contraction coupling in mammalian ventricular myocytes.