Long-term biological effects of air ions and D.C. electric fields on Namru mice: First year report

This report describes for the first time the effects of long-term continuous exposures of animals to small air ions and D.C. electric fields. In this study we exposed 200 female NAMRU mice (25/cage) to the following conditions: ± high ions (2×10⁵/cm³), ± low ions (2×10³/cm³), ± field only and ground (ion depleted, no field). Specially designed cages provided a defined D.C. field of about 2 kV/meter in ionized environments, with somewhat lower values in the field only cages. Detailed mapping of ion flux originating from a tritium foil generating system (multiple sources in an overhead plate) indicated a well defined, but heterogenous pattern with eight peak areas. Using a 100 cm² probe, ion flux values ranged from 10^–12–10^–14 A/cm², with an average flux of 8.7±6.8×10^–13 A/cm² in high negative ion cages, with good reproducibility between cages.Measurements of serum glucose, cholesterol, and urea nitrogen (samples taken every three months) showed a number of small but consistent and statistically significant differences between animals maintained in different environments during the first year of exposure. Serum globulin and whole blood serotonin, however, did not show any significant environmental effects. Interestingly, pairwise comparisons between high negative and low negative ion conditions, or between high positive and low positive ion conditions, or between the two ground conditions, revealed no significant differences between cages. This argues for a similarity of environmental responses for the mice maintained in each of the compared conditions.The results of a multiple classification analysis for the entire first year showed a preponderence of effects for the ionized cages, although other conditions also had highly significant differences as compared to the grand mean value. While this study has shown effects of only small magnitude (compared to normal physiological variations) in the female NAMRU mice studied here, the significance of these results strongly validates the precept of long-term air ion effects in animals. Given the known variability of different species and strains, future studies will have to determine the actual range of responsiveness of animals exposed on a long-term basis to defined air ion and electromagnetic environments.Deceased December 8, 1982.     

Design and construction of cage environments for air ion and electric field research

This report describes the design and construction of cage environments suitable for chronic exposures of large groups of mice to air ions and electric fields. These environments provide defined and reproducible ion densities, ion flux, DC electric fields, sound levels, air temperature and air quality. When used during a 2 year study, these cage environments served as a durable and reliable continuous exposure system. Three environmental chambers (cubicles) housed a total of 12 cages and provided control of air temperature, air purity and lighting. Exposure cages had grounded metal exterior walls, a plexiglass door and interior walls lined with formica. An internal isolated field plate supplemented with guard wires, energized with ca 1000 VDC, created about a 2 kV/m electric field at the grounded cage floor. Air ions resulted from the beta emission of sealed tritium foils mounted on the field plate. Cages provided high ion (1.3×10⁵ ions/cc), low ion (1.6×10³ ions/cc) and field only (ion depleted < 50 ions/cc) conditions for both polarities with similar electric fields in ionized and field only cages. Detailed mapping of the floor level ion flux using 100 cm² flat probes gave average fluxes of 880 fA cm^–2 in high ion cages and 10 fA cm^–2 in low ion cages. Whole body currents measured using live anesthethized mice in high ion cages averaged 104±63 pA. Both ion flux and whole body currents remained constant over time, indicating no charge accumulation on body fur or cage wall surfaces in this exposure system.     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

Feasibility study on the use of hyperosmolar medium for improved antibody production of hybridoma cells in a long-term,repeated-fed batch culture

To test the feasibility of using hyperosmolar medium for improved antibody production in a long-term, repeated fed-batch culture, the influence of various culture conditions (serum concentration and cultivation method) on the hybridoma cells' response to hyperosmotic stress resulting from sodium chloride addition was first investigated in a batch culture. The degree of cell growth depression resulting from hyperosmotic stress was dependent on serum concentrations and cultivation methods (static and agitated cultures). Depression of cell growth was most significant in agitated cultures with low serum concentration. However, regardless of serum concentrations and cultivation methods used, the hyperosmotic stress significantly increased specific antibody productivity (q _MAb). Increasing osmolality from 284 to 396 mOsm kg^–1 enhanced the q_MAb in agitated cultures with 1% serum by approximately 124% while the similar osmotic stress enhanced the q _MAb in static cultures with 10% serum by approximately 153%. Next, to determine whether this enhanced q_MAb resulting from hyperosmotic stress can be maintained after adaptation, long-term, repeated-fed batch cultures with hyperosmolar media were carried out. The cells appeared to adapt to hyperosmotic stress. When a hyperosmolar medium (10% serum, 403 mOsmkg^–1) was used, the specific growth rate improved gradually for the first four batches and thereafter, remained constant at 0.040±0.003 (average ± standard deviation) hr^–1 which is close to the value obtained from a standard medium (10% serum, 284 mOsmkg^–1) in the batch culture. While the cells were adpating to hyperosmotic stress, the q_MAb was gradually decreased from 0.388×10^–6 to 0.265×10^–6 g cell hr^–1 and thereafter, remained almost constant at 0.272±0.014× 10^–6 g cell^–1 hr^–1. However, this reduced q _MAb after adaptation is still approximately 98% higher than the q_MAb obtained from a standard medium in the batch culture.The authors would like to thank Dr.M. Kaminski for providing the hybridoma cell line used in this study. This work was supported by the Korea Science and Engineering Foundation.     

Divinyl Sulfone as a Crosslinking Reagent for Oligomeric Proteins

The kinetics of the nucleophilic addition reactions of divinyl sulfone to amino groups of glycine and model proteins was studied in aqueous solution at 30°C. The rate constants for glycine, bovine serum albumin, and ₁-casein were (4.84 ± 0.58) × 10^–1, (2.97 ± 0.31) × 10^–2, and (2.38 ± 0.49) × 10^–2 M^–1 s^–1, respectively. Divinyl sulfone was proposed as a crosslinking reagent for the qualitative detection of protein association in solution. The crosslinking capacity of divinyl sulfone was compared to that of 1,3,5-triacryloylhexahydro-s-triazine.     

Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission spectrofluorometry

A method is described for monitoring intracellular ionized calcium (Ca²⁺) and determining kinetic and thermodynamic parameters of Ca⁺-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca²⁺ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca^2– that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca²⁺ with a rate constant of 0.031 ± 0.003 sec^–1, maximal rate of 23 ± 7 nM/sec, dissociation constant of 366 ± 63 nM, Hill coefficient of 2.3 ± 0.4, Q₁₀ of 2.58 ± 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca²⁺-extrusion system of lymphocytes and may be applicable to other blood cell types.Abbreviations DMSO dimethyl sulfoxide - HEPES [N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid],sodium salt - Indo-1/AM acetoxymethyl ester of indo-1 - IP₃ inositol 1,4,5-triphosphate - IP₄ inositol 1,3,4,5-tetrakisphosphate - RPMI Roswell Park Memorial Institute — 1640 culture medium - TPEN tetrakis-[2-pyridylmethyl]-ethylenediamine     

Caecal water and electrolyte absorption and the effects of acetate and glucose, in dehydrated, low-NaCl diet hens

Summary In vivo electrolyte transport and water absorption from the caeca of dehydrated, low-NaCl diet hens are reported. In the absence of luminal glucose or acetate, net electrolyte transport rates and water absorption are small. When physiological concentrations of acetate (40 mM) are included in the perfusate, Na⁺ transport and water absorption increase significantly (P<0.01): 38±7 eqNa⁺/caecum kg·h and 256±33 l H₂O/caecum · kg · h.A similar increase in water absorption occurs with the inclusion of 15 mM glucose in the perfusate (219±30 l H₂O/caecum · kg · h), however both net Na⁺ and Cl^– absorption increase: 28±6 eq Na⁺/caecum · kg · h and 21±5 eq Cl^–/caecum kg · h.These pronounced increases in electrolyte and water absorption are not accompanied by any significant increase in transmural potential difference.The data presented establish caeca as important sites in the recuperation of water and electrolytes in dehydrated, low-NaCl diet hens.Abbreviations ECPD electrochemical potential difference - PD (transmural) potential difference - PEG polyethylene glycol     

Diazepam metabolism and albumin secretion of porcine hepatocytes in collagen-sandwich after cryopreservation

Cryopreserved porcine hepatocytes in collagen cultures secreted albumin (up to 98 ± 3 g ml^–1; non-cryopreserved controls: 100 ± 9 g ml^–1) and metabolised diazepam (67.5% ± 7.5% of initially applied diazepam are metabolised; 77.5% ± 10% in non-cryopreserved controls) for up to 14 days after thawing. The cultures resembled the recently developed flat membrane bioreactors. Addition of 5% (v/v) serum did not effect diazepam metabolism. Hepatocytes in collagen-sandwich cultures offer a storable liver support system for potential clinical use.     

Adaptations to a terrestrial existence by the robber crab,Birgus latro L.

Summary The activity of carbonic anhydrase (CA), which catalyses the equilibrium CO₂H⁺+HCO ₃ ^- , was investigated in various tissues implicated in the excretion of CO₂ by Birgus latro. Carbonic anhydrase was detected in the water-soluble fraction of gill tissue but also occurred in association with lipids (membrane bound). This is consistent with a CO₂ excretory role and an ion regulation function for the gills. In the lungs (branchial chamber lining) CA activity was found in the membrane bound fraction but was not detected in the soluble fraction, suggesting that the lung CA is not important for ion regulation. The specific CA activity of gill tissue homogenate (A=1.8±0.7·mg^-1) was higher than that measured for lung homogenates (A=0.4±0.2·mg^-1), but when the whole organ was considered the total CA activity in the lungs was not significantly different from total CA activity in the gills. In comparison to aquatic and amphibious crustaceans the specific activity of carbonic anhydrase in the lungs was high (25% cf. gill activity). This CA activity in the lungs could be correlated with significant CO₂ excretion by the lungs. CA may be retained in the branchial tissue as an adjunct to ion reabsorption by the gills.     

Effect of Metabolic Inhibitors on Membrane Potential and Ion Conductance of Rat Astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin.2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13–20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique.3. After 2.0 min of incubation ATP decreased from the control level to 43%with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 M), and 69% with oligomycin (10 M).4. Under normal conditions V was –74.4±1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2±1.3 and 19.7±3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve.5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1±2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8±3.5 mV and increased g by 45.5±9.6%. The action of FCCP was not affected by the presence of TEA.6. Perfusion of the astrocytes with a Cl^– free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2±1.5mV in DNP and 3.7±0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl^–].7. Block of tentative K_ATP channels with tolbutamide (1 mM) or Cl^– channels with Zn²⁺ (1 mM) did not inhibit the depolarization caused by DNP or FCCP.8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl^– channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.     

Glucose-dependent respiration in suspensions of rabbit cortical tubules

Summary The effects of glucose on cellular respiration were examined in suspensions of rabbit cortical tubules. When glucose was removed from the bathing fluid, oxygen consumption (QO₂) decreased from 18.6±0.8 to 15.7±0.5 nmol O₂/mg protein·min (P<0.01). The transported but nonmetabolized analogue of glucose, -methyl-d-glucoside (MG), was found to support QO₂ to the same extent as glucose. These observations were also evident in the presence of butyrate, a readily oxidized substrate of the renal cortex. Additional studies with nystatin and ouabain indicated that glucose-related changes in QO₂ were the result of changes in Na, K-ATPase associated respiration. The effect of glucose was localized to the luminal membrane since phlorizin (10^–5 m), a specific inhibitor of liminalk glucose-sodium cotransport, also significantly reduced QO₂ by 10±1%. Phlorizin inhibition of QO₂ was also evident in the presence of MG but was abolished when glucose was removed from the bathing medium. Finally, measurement of NADH fluorescence showed that addition of glucose (5mm) to a tubule suspension causes an oxidation of NAD. These data are all consistent with glucose acting to increase respiration by stimulating sodium entry at the luminal membrane (via glucose-sodium cotransport) followed by increased sodium pump activity and its associated increase in mitochondrial respiration.     

The role of calcium in the regulation of glucose uptake in isolated working rat heart

Catecholamines or ischemia may increase myocardial glucose uptake by an increase in intracellular calcium. We tested the hypothesis that increasing or decreasing extracellular calcium supply would change glucose uptake. Hearts were perfused for 60 min at a physiological workload with Krebs-Henseleit buffer containing glucose (5 mM) and oleate (0.4 mM; bound to 1% BSA). Calcium concentration was 2.5 mM. In group A (control; n = 12), insulin (1 mU/ml) was added at 30 min. In Group B (n = 7), the calcium concentration was increased to 5.0 and 7.5 mM at 20 min and 40 min, respectively. In Group C (n = 7), verapamil was added at 20 min (0.25 M) and 40 min (1.0 M) to decrease calcium influx. In group D (n = 7), EDTA was added at 20 min (0.5 mM) and at 40 min (1.5 mM) to decrease the free extracellular calcium. Glucose uptake was measured by ³H₂O production from [2-³H]glucose and cardiac work was measured simultaneously. Cardiac power in group B was 8.24 ± 0.60 mW at 2.5 mM calcium, 9.45 ± 0.50 mW at 5 mM calcium and 7.99 ± 0.99 mW at 7.5 mM calcium (n.s.). The addition of verapamil decreased contractile function in a dose-dependent manner (8.50 ± 0.74 vs. 3.11 ± 0.84 vs. 1.48 ± 0.39 mW, p < 0.01) suggesting that verapamil decreased cytosolic calcium concentration. A similar dose-dependent reduction in contractile performance was observed in the EDTA group (8.44 ± 0.81 vs. 7.42 ± 0.96 vs. 4.03 ± 1.32 mW, p < 0.01). Glucose uptake was 1.35 ± 0.11 mol/min/g dry weight under control conditions. Glucose uptake increased threefold with the addition of insulin. Increasing extracellular [Ca²⁺] did not affect glucose uptake. Decreasing Ca²⁺ availability showed a trend towards a decrease in glucose uptake (n.s.), which was minor compared to the decrease in contractile function. We conclude that extracellular calcium does not regulate glucose uptake in the isolated working rat heart in the presence of glucose and fatty acids as substrates. The trend of decreased glucose uptake when calcium supply was limited may be due to dramatically reduced energy demand and not directly due to changes in calcium.     

Increased urinary zinc excretion in cancer patients is linked to immune activation and renal tubular cell dysfunction

Urinary zinc excretion is known to be increased in cancer patients, but the pathogenesis of this phenomenon remains uncertain. Both skeletal muscle catabolism and renal tubular cell dysfunction have been proposed to explain this observation. We have investigated urinary zinc and N-acetyl--d-glucosaminidase (NAG), an indicator of renal tubular cell dysfunction, as well as serum neopterin, an index of systemic immune activation, in 22 patients with cancer and seven controls. Both serum neopterin and urinary zinc were significantly elevated in cancer patients (15.8 ± 12.7 versus 7.3 ± 2.3 nmol l^–1 and 1.77 ± 0.80 versus 1.21 ± 0.41 mmol mol^–1 creatinine, P < 0 and P < 0.05, respectively), while NAG was similar in cancer patients and the controls (13.58 ± 13.80 versus 13.68 ± 12.19 kat mol^–1 creatinine). A significant correlation was observed between serum neopterin and urine zinc (rs = 0.5119, P < 0.02), serum neopterin and urine NAG (rs = 0.6761, P < 0.002), and urinary zinc and NAG (rs = 0.6348, P < 0.002). In conclusion, the present data indicate a link between urinary zinc excretion and immune activation as well as renal tubular cell dysfunction. In addition, renal tubular cell dysfunction appears to be linked to immune activation.     

The activity of the pyruvate dehydrogenase complex in heart muscle in the previously obese mouse model

Obese gold thioglucose injected mice were reduced to lean control weight by food restriction. When pair fed with lean controls these animals then gained weight (were metabolically more efficient). Serum glucose was also elevated in this group (14.5±0.4 (14)vs 12.1±0.3 mmol/L, p<0.001). If previously obese animals were weight maintained with lean controls (by mild food restriction), serum glucose remained at control levels. The activity of the pyruvate dehydrogenase complex in heart muscle was decreased in both obese and pair fed previously obese, whilst it was similar to that of lean controls in the weight maintained previously obese and in obese mice actually dieted. In all obese and previously obese animals serum insulin was elevated. In hearts from control animals subjected to mild food restriction the pyruvate dehydrogenase complex was activated (11.53±1.80 (5)vs 3.34±0.62 (9) U/g dry weight), despite a reduced serum insulin level (42±2vs 74±10 U/ml, p<0.01). These diverse changes in the proportion of the pyruvate dehydrogenase complex in the active form and insulin levels argue for a persistent alteration in the sensitivity of the pyruvate dehydrogenase complex to insulin in obesity, as well as indicating that glucose metabolism in obese animals is altered by both body weight and diet amount.To whom correspondence should be addressed.     

Structural characteristics of the saxitoxin receptor on nerve

Summary The effects of uranyl ion (UO ₂ ²⁺ ; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB⁺; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00±0.05 and 13.5mm Ca²⁺, addition of 10.0 m UO ₂ ²⁺ +5.0nm STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79±0.04 (viz., mean±sem) of control value] than did addition of 5.0nm STX alone (0.72±0.05). Under the same conditions of pH and Ca²⁺ concentration, 1.0mm MB⁺ had approximately the same effect: 1.0mm MB⁺+5.0nm STX, 0.76±0.03; 5.0nm STX alone, 0.70±0.04. However, at pH 7.00±0.05 and lower Ca²⁺ concentrations, 1.0mm MB⁺ significantly reduced STX potency. Using 6.0mm Ca²⁺: 1.0mm MB⁺+5.0nm STX, 0.92±0.01; 5.0nm STX alone, 0.68±0.08. Using 3.0mm Ca²⁺, the corresponding values were 0.94±0.03 and 0.67±0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.     

In vivo modulation of natural killer cell activity by tamoxifen in patients with bilateral primary breast cancer

Natural killer (NK) cell activity, the autologous mixed lymphocyte reaction (AMLR) and proportions of T cell subpopulations (CD3⁺/CD4⁺ and CD3⁺/CD8⁺) and NK cells (CD16⁺) were studied in 21 patients with bilateral primary breast cancer (BBC), 10 patients with single-breast cancer (SBC) and 20 healthy controls. All patients studied had no evidence of disease and had been off radiotherapy and/or chemotherapy for at least 1 year. Ten patients with BBC were also treated with tamoxifen. Patients with SBC had NK cell activity, AMLR responses and T cell subpopulations that were comparable to those of normal controls. In patients with BBC, a significant (P<0.01) increase in NK activity compared to that in normal controls (42±13% versus 21±10%, effector-to-target cell ratio, 251) and a significant (P<0.05) decrease in CD4⁺ T cell proportions (30±15% versus 49±13%) and absolute numbers (472±82/mm³ versus 953±131/mm³) were found. However, the proliferative response of BBC patients' T lymphocytes in AMLR was in the range of the normal controls. Lymphocytes derived from 10 BBC patients treated with tamoxifen exhibited NK cell activity that was comparable to that of normal controls and patients with SBC, and was significantly (P<0.01) reduced compared to the pretreatment period. BBC patients who received tamoxifen also show a reduction in the proportion of CD4⁺ T cells and in AMLR proliferative responses, which decreased compared to levels in normal controls. Taken together, these results indicate that long-term tamoxifen treatment modulates immune responses in BBC patients.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Calorimetric studies of the interaction between the insulin-enhancing drug candidate bis(maltolato)oxovanadium(IV) (BMOV) and human serum apo-transferrin

Bis(maltolato)oxovanadium(IV) (BMOV), and its ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), are candidate insulin-enhancing agents for the treatment of type 2 diabetes mellitus; in mid-2008, BEOV advanced to phase II clinical testing. The interactions of BMOV and its inorganic congener, vanadyl sulfate (VOSO₄), with human serum apo-transferrin (hTf) were investigated using differential scanning calorimetry (DSC). Addition of BMOV or VOSO₄ to apo-hTf resulted in an increase in thermal stability of both the C- and N-lobes of transferrin as a result of binding to either vanadyl compound. A series of DSC thermograms of hTf solutions containing different molar ratios of BMOV and VOSO₄ were used to determine binding constants; at 25 °C the binding constants of BMOV to the C- and N-lobes of apo-hTf were found to be 3 (±1) × 10⁵ and 1.8 (±0.7) × 10⁵ M⁻¹, respectively. The corresponding values for VOSO₄ were 1.7 (±0.3) × 10⁵ and 7 (±2) × 10⁴ M⁻¹. The results show that the vanadium species initially presented as either BMOV or VOSO₄ had similar affinities for human serum transferrin due to oxidation of solvated vanadyl(IV) prior to complexation to transferrin. Binding of metavanadate () was confirmed by DSC and isothermal titration calorimetry (ITC) experiments of the interaction between sodium metavanadate (NaVO₃) and hTf.     

Characterization of the reciprocal binding sites on human α-thrombin and factor XIII A-chain

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-a-thrombin interactions. -Thrombin activated Factor XIII more efficiently (Km = 0.83 ± 0.08 × 10^-7 M; V/K = 14.90 ± 3.20 × 10^-3 min^-1) than -thrombin (Km = 6.14 ± 1.26 × 10^-7 M; V/K = 3.30 ± 1.00 × 10^-3 min^-1) or -thrombin (Km = 6.25 ± 1.15 × 10^-7 M; V/K = 3.00 ± 0.80 × 10^-3 min^-1). Immobilized FPR--thrombin bound plasma Factor XIII (Kd = 0.17 ± 0.04 × 10^-7 M) > Factor XIIIa (Kd = 0.69 ± 0.18 × 10^-7 M) > liver transglutaminase (Kd = 4.73 ± 1.01 × 10^-7 M) > Factor XIII A-chain (Kd = 49.00 ± 9.40 × 10^-7 M). FPR--thrombin and -thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 × 10^-7 M and 13.6 × 10^-7 M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR--thrombin: IC₅₀ = 1.0 × 10^-7 M, 3.0 × 10^-6 M and 1.52 × 10^-4 M, respectively. Transglutaminase also inhibited Factor XIII binding to ×-thrombin (IC₅₀ = 2.0 × 10^-6 M). Thrombin-binding site was localized to G^-38-M^-731 fragment of Factor XIII A-chain, probably within homologous regions (N^-72-A^-493) of transglutaminase. R^-320-E^-579 of -thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in -thrombin were essential for binding but not catalytic H^-363 or residues R^-382-N^-394 and R^-443-G^-475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.     

β-Adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication

-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [³H]Dihydroalprenolol ([³H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of -adrenoreceptors with two different affinities. the dissociation constants (K_d) have been found to be 0.27±0.09×10^–9 M and 2.96±0.31×10^–9 M, respectively with the corresponding B_max of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [³H]DHA binding to the -receptor by atenolol (a ₁-antagonist) and ICI 118,551 (a ₁-antagonist) has suggested that the IC_50cor (=K_i) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10^–12 M and 0.25±0.08×10^–12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the ₁-selective antagonist atenolol is 3 times more potent than its ₂ counterpart, ICI 118,551. Displacement of [³H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of K_i for these agents as isoproterenol (0.56±0.19×10^–9 M)–9 M)>-norepinephrine (0.71±0.24×10^–9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10^–9 M, 6.21±0.86×10^–9 M and 5.90±0.82×10^–9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of -adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.Abbreviations DHA Dihydroalprenolol - cAMP 3:5 cyclic adenosine monophosphate - DTT dithiothreitol - MEM minimal essential medium - 8Br-cAMP 8-bromo-adenosine 3:5 cyclic monophosphate