Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli

Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10⁻⁶ M levorphanol to 92% at 10⁻³ M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM d-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the K_m of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (K_i) for levorphanol inhibition of proline transport is approximately 3 × 10⁻⁴ M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with [¹⁴C]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed.     

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells: SELECTIVE INHIBITION BY CLOFIBRIC ACID

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-¹⁴C]leucine to ¹⁴CO₂ or to the [1-¹⁴C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of ¹⁴CO₂ from [1-¹⁴C]leucine was saturable with a K_m = 6.3 mM and a maximum oxidation rate (V_max) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-¹⁴C]leucine (K_i = 0.85 mM) and [U-¹⁴C]isoleucine, but had no effect on the oxidation of [U-¹⁴C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-¹⁴C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of ¹⁴CO₂ and had relatively little effect on the production of [1-¹⁴C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD⁺ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.     

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na⁺/H⁺ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP₃) receptor-binding protein released with IP₃ (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2–dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.     

The lymphocyte Na+/H+ antiport: activation in primary hypertension and during chronic NaCl-loading

Abstract. Increased activity of the Na⁺/H⁺ antiport may be a major abnormality in essential hypertension. The activity of this transport system was investigated in lymphocytes from 13 patients with untreated essential hypertension (Ht) and 13 normotensive control subjects (Nt) on an ad libitum (130–170 mmol d^-1) NaCl intake. Furthermore, the effects of different states of NaCl balance on lymphocyte Na⁺/H⁺ antiport were evaluated in two groups of Nt volunteers receiving 20 vs. 300 mmol d^-1 (n= 8) and 85 vs. 200 mmol d^-1 (n= 14) of NaCl for 1 week each and in seven Ht patients (20 vs. 300 mmol NaCl d^-1 for 1 week each). Additionally, during the 20 and 300 mmol/d NaCl intake red blood cell membrane transport was studied in eight subjects. For the determination of lymphocyte antiport activity, cells were loaded with the cytosolic pH (pH_i) indicator bis-carboxyethyl carboxyfluorescein (BCECF-AM) and acidified by addition of different amounts of Na⁺-propionate (5–40 mM). Initial pH_i-recovery was taken as the activity of the antiport system and plotted against pH_i-values after acidification. Non-linear regression analysis yielded higher ’apparent’ maximal transport rates in Ht than Nt (Nt: 2·00 pL 0·22; Ht: (3·81 pL 0·59)·10^-3 s^-1; P < 0·025). In contrast, baseline pH_i-values and pH_i-values at half-maximal activity (pK) were identical in Nt and Ht. In normotensive control subjects on an NaCl intake of 20, 85, 200 and 300 mmol d^-1 for 7 d, ’apparent’ maximal transport rates averaged 2.75 0·20, 2·89 0·17, 2·81 ± 0·18 and (3·62 ± 0·25) · 10^-3 s^-1, respectively. Thus, antiport activity was significantly (P < 0·05) stimulated on the 300 mmol d^-1 intake as compared to the three other NaCl intakes. The extreme intakes of NaCl (20 vs. 300 mmol d^-1) in normotensive volunteers did not affect the erythrocyte Na⁺/K⁺ pump, Na⁺/K⁺ cotransport and Na⁺/Li⁺ countertransport. Our study supports the concept that a group of patients with primary hypertension exhibit an activated Na⁺/H⁺ antiport. Furthermore, our data demonstrate that a chronic high intake of NaCl is associated with an increase in lymphocyte antiport activity towards the high values observed in primary hypertension.     

Direct effect of metolazone on sodium-dependent transport across the renal brush border membrane

Studies with clearance and micropuncture techniques indicate that metolazone inhibits transport of sodium and phosphate in the proximal tubule. The present study is focused on transport across the luminal BBM of the proximal tubule to determine whether metolazone has any direct effect on this initial step in transtubular reabsorption. Addition of metolazone (0.01 to 1.00 mM) to isolated renal BBM vesicles caused dose-dependent inhibition (30% to 70%) of the initial uphill phase of Na+ gradient-dependent phosphate transport but did not inhibit the uptake at equilibrium. There were no significant changes in Na+-independent phosphate transport and phosphate transport under nongradient conditions when metolazone was present at 1.0 mM. The initial Na+ gradient-dependent BBM transport of both D-glucose and L-proline was markedly inhibited by 1.0 mM metolazone, indicating the nonspecific inhibitory action of the drug. Metolazone also inhibited efflux of D-glucose and L-proline from vesicles. Neither acetazolamide nor chlorothiazide at 0.1 to 1.0 mM inhibited BBM transport of phosphate, D-glucose, or L-proline. Metolazone did not change significantly BBM transport of Na+, suggesting that inhibition of Na+-dependent transport was not due to major changes in Na+ flux. These in vitro data indicate that metolazone inhibition of phosphate reabsorption in vivo may be due, in part, to a direct effect of metolazone on transport across the BBM of the proximal tubule.     

Kinetic Study of the Effect of Histidines 240 and 164 on TEM-149 Enzyme Probed by β-Lactam Inhibitors

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149^H240, and TEM-149^H164-H240 versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149^H240, and TEM-149^H164-H240 β-lactamases, and the K_i (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k₊₂ and k₊₃ (first-order acylation and deacylation constants, respectively), and k₊₂/K values were calculated.     

In vitro Characterization of Uptake Mechanism of l-[methyl-3H]-methionine in Hepatocellular Carcinoma

Purpose

Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with l-[methyl-¹¹C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of l-[methyl-¹¹C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of l-[methyl-¹¹C]-Met in HCC cells.

Procedures

A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with l-[methyl-³H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively.

Results

Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na⁺ dependent, and low affinity with partial energy dependence. The total uptake of l-[methyl-³H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of MAT2A expression (the duration of SAM treatment). The incorporated ³H was mostly distributed in the protein phase and, to a lesser degree, in the lipid phase via PE methylation pathway in HCC cells with SAM treatment. The downregulated MAT2A expression led to the decreased uptake in protein and water-soluble phases. In addition, an increased uptake in the lipid phase was observed in WCH17 cells transfected with PEMT2 expression vector.

Conclusions

The amino acid transport processes may be responsible for the rapid accumulation of radiolabeled Met after the intravenous injection of tracers for the imaging of HCC. Upregulated MAT2A expression and impaired PEMT2 activities in HCC are associated with the specific metabolic pattern of l-[methyl-¹¹C]-Met detected by PET.     

23Na chemical shift imaging and Gd enhancement of myocardial edema

Myocardial edema can arise in several disease states. MRI contrast agent can accumulate in edematous tissue, which complicates differential diagnosis with contrast-enhanced (CE)-MRI and might lead to overestimation of infarct size. Sodium Chemical Shift Imaging (²³Na-CSI) may provide an alternative for edema imaging. We have developed a non-infarct, isolated rat heart model with two levels of edema, which was studied with ²³Na-CSI and CE-MRI. In edematous, but viable tissue the extracellular sodium (Na _e ⁺ ) signal is hypothesized to increase, but not the intracellular sodium (Na _i ⁺ ) signal. Isolated hearts were perfused at 60 (n = 6) and 140 mmHg (n = 5). Dimethyl methylphosphonate (DMMP) and phenylphosphonate (PPA) were used to follow edema formation by ³¹P-MR Spectroscopy. In separate groups, Thulium(III)1,4,7,10 tetraazacyclododecane-N,N′,N″,N′′′-tetra(methylenephosphonate) (TmDOTP^5?) and Gadovist were used for ²³Na-CSI (n = 8) and CE-MRI (n = 6), respectively. PPA normalized signal intensity (SI) was higher at 140 versus 60 mmHg, with a ratio of 1.27 ± 0.12 (p < 0.05). The (DMMP-PPA)/dry weight ratio, as a marker of intracellular volume, remained unchanged. The mid-heart cross sectional area (CSA) of the left ventricle (LV) was significantly increased at 140 mmHg. In addition, at 140 mmHg, the LV Na _e ⁺ SI increased with a 140 mmHg/60 mmHg ratio of 1.24 ± 0.18 (p < 0.05). Na _i ⁺ SI remained essentially unchanged. With CE-MRI, a subendocardially enhanced CSA was identified, increasing from 0.20 ± 0.02 cm² at 60 mmHg to 0.31 ± 0.02 cm² at 140 mmHg (p < 0.05). Edema shows up in both CE-MRI and Na _e ⁺ . High perfusion pressure causes more edema subendocardially than subepicardially. ²³Na-CSI is an attractive alternative for imaging of edema and is a promising tool to discriminate between edema, acute and chronic MI.     

Pancreatic Beta Cell Mass PET Imaging and Quantification with [11C]DTBZ and [18F]FP-(+)-DTBZ in Rodent Models of Diabetes

Purpose

The aim of this study is to compare the utility of two positron emission tomography (PET) imaging ligands ((+)-[¹¹C]dihydrotetrabenazine ([¹¹C]DTBZ) and the fluoropropyl analog ([¹⁸F]FP-(+)-DTBZ)) that target islet ??-cell vesicular monoamine transporter type II to measure pancreatic ??-cell mass (BCM).

Procedures

[¹¹C]DTBZ or [¹⁸F]FP-(+)-DTBZ was injected, and serial PET images were acquired in rat models of diabetes (streptozotocin-treated and Zucker diabetic fatty) and ??-cell compensation (Zucker fatty). Radiotracer standardized uptake values (SUV) were correlated to pancreas insulin content measured biochemically and histomorphometrically.

Results

On a group level, a positive correlation of [¹¹C]DTBZ pancreatic SUV with pancreas insulin content and BCM was observed. In the STZ diabetic model, both [¹⁸F]FP-(+)-DTBZ and [¹¹C]DTBZ correlated positively with BCM, although only ??25% of uptake could be attributed to ??-cell uptake. [¹⁸F]FP-(+)-DTBZ displacement studies indicate that there is a substantial fraction of specific binding that is not to pancreatic islet ?? cells.

Conclusions

PET imaging with [¹⁸F]FP-(+)-DTBZ provides a noninvasive means to quantify insulin-positive BCM and may prove valuable as a diagnostic tool in assessing treatments to maintain or restore BCM.     

Molecular Mode of Action of the Antifungal β-Amino Acid BAY 10-8888

BAY 10-8888 is a cyclic β-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H⁺-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a K_T (Michaelis constant of the transport reaction) of 0.95 mM and a V_max of 18.9 nmol × min⁻¹ × 10⁷ cells⁻¹. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.     

Heteroleptic Re(CO)2+ and Re(CO)3+ complexes with α-diimines: similarities and differences in their luminescence properties

The photophysical properties of two series of phosphorescent rhenium(i) complexes, [Re(CO)₂(N^N)(tpp)₂]⁺ and [Re(CO)₃(N^N)(tpp)]⁺ with carbon monoxide (CO), triphenylphosphine (tpp) and α-diimine (N^N) ligands have been investigated in deoxygenated acetonitrile solution at room temperature and in solid methanol/ethanol 1 : 1 matrices at 77 K. The complexes display moderate to strong phosphorescence which is related to the N^N ligand modulated metal-to-ligand charge-transfer S₀ ← ³*MLCT or intraligand S₀ ← ³*LC transitions. Luminescence properties of the investigated series have been found to be very similar but some intrinsic differences between them are clearly seen. Whereas the [Re(CO)₂(N^N)(tpp)₂]⁺ series shows MLCT emission in both temperature regimes studied, the [Re(CO)₃(N^N)(tpp)]⁺ series exhibits intrinsic changes in its emission character when the measurement temperature is lowered from 298 to 77 K. In both investigated series, their emission characteristics are strongly affected by the nature of coordinated α-diimine N^N ligands. The observed trends, changes in the radiative k_r and non-radiative k_nr deactivation rate constants, have been compared with those found for the previously investigated [Re(CO)₃(N^N)(Cl)], [Re(CO)₃(N^N)(CH₃CN)]⁺, and [Re(CO)₂(N^N)(dppv)]⁺ series (dppv = cis-1,2-bis(diphenylphosphino)-ethene). Similarities and differences in the spectroscopic and photophysical properties of five series of the Re(CO)₃⁺ and Re(CO)₂⁺ complexes have been analyzed in the view of results from DFT and TD-DFT computation and the emission band-shape analyses performed according to the Marcus–Jortner formalism.

We report results from comparative studies of luminescence properties of five series of α-diimine rhenium(i) complexes.     

Cytoplasmic calcium regulation and the electrocardiogram in patients with primary hyperparathyroidism

Summary. Primary hyperparathyroidism (HPT) is a disease caused by an abnormal cytosolic regulation of calcium concentration [Ca⁺⁺]_i leading to an increased secretion of parathyroid hormone and thereby increased levels of extracellular calcium. It is well known that the QT-interval measured at electrocardiography (ECG) is shortened in HPT subjects. Whether this is due to an abnormal intracellular handling of calcium also in the heart or to the raised extracellular calcium levels is not known. In order to study the extent to which the deranged extra- and intracellular levels of calcium in HPT patients were related to ECG characteristics, [Ca⁺⁺]_i was determined in vitro by microfluorometry in surgery-removed parathyroid cells at extracellular calcium concentrations of 0–5 mM and 30-0 mM and ECG was recorded preoperatively in 42 HPT patients and in 15 subjects operated on for atoxic goitre. Serum calcium and plasma-ionized calcium also were measured preoperatively. The QT-interval and ST-segment duration were both shortened in the HPT patients compared to controls (P < 0–001). [Ca⁺⁺]_i at 3-0 mM extracellular calcium divided by that at 0–5 mM was correlated to the QT-interval, when measured at the onset of the T-wave (QoT, r= 0–39, P < 0–03) and early diastolic phase (end of T-wave to onset of p-wave, r= -0–34, P= 0–05). Plasma ionized calcium was related inversely to the QT-interval, when measured to the apex of the T-wave (QaT, r= -0–48, P < 0–04), while the non-ionized form of calcium in serum was correlated to the length of the PQ-interval (r= 0–53, P< 002). The study described here showed the altered cytosolic calcium levels in the HPT subjects to be related to QoT-interval and early diastolic phase implying that a defect intracellular handling of calcium could be present also in the heart to cause these ECG changes. On the other hand, the levels of plasma ionized and serum non-ionized calcium were related to ECG characteristics suggesting that the extracellular levels of this ion are important.,     

Mechanisms of toxicity of Cleistanthus collinus: Vacuolar ATPases are a putative target

Ingestion of Cleistanthus collinus, a shrub native to South India, either intentionally or accidentally, is a common cause of death in the area. Consumption of a boiled decoction of leaves is highly toxic, but medical management of patients is mainly supportive because the molecular mechanisms of toxin action are unknown. Distal renal tubular acidosis is one of the symptoms of poisoning in patients and adenosine triphosphate (ATP) requiring proton pumps is important for acid secretion in the kidney. Hence, we hypothesized that these may be putative targets for C. collinus action and we tested this by exposing rat renal brush border membrane (BBM) as well as cultured kidney cells to a boiled decoction of C. collinus. Exposure to the C. collinus decoction resulted in significant inhibition of vacuolar type H⁺-ATPase (V-ATPase) activity in renal BBM as well as blocking of the proton pump in renal BBM vesicles. C. collinus decoction was also found to inhibit acidification of intracellular organelles in cells in culture, similar to the effect seen with either bafilomycin or concanamycin – specific inhibitors of the V-ATPase. This was accompanied by a decrease in V-ATPase activity, but an increase in protein levels. These results demonstrate that the V-ATPase in renal cells is a putative target for the toxins in C. collinus and the inhibition of this important proton pump probably plays a role in the development of distal renal tubular acidosis and subsequent renal failure seen in poisoned patients.     

Initial Experience with the Radiotracer Anti-1-amino-3-[18F]Fluorocyclobutane-1-Carboxylic Acid (Anti-[18F]FACBC) with PET in Renal Carcinoma

Purpose

Assessment of renal masses with conventional imaging may be challenging. Anti-1-amino-3-[¹⁸F]fluorocyclobutane-1-carboxylic acid (anti-[18F]FACBC) is a synthetic l-leucine analog with relatively little renal excretion. The present study examines anti-[¹⁸F]FACBC positron emission tomography uptake in patients with renal masses.

Procedures

Six patients with seven renal lesions were imaged dynamically for 2 h after injection of 10–10.9 mCi (370–403 MBq) anti-[¹⁸F]FACBC. Lesions were evaluated qualitatively and quantitatively and correlated with histology.

Results

Four clear cell and one Rosai–Dorfman lesion were hypo/isointense to normal cortex; two papillary lesions in the same patient were hyperintense. Mean SUV_max?±?SD at 30 min was 2.8?±?0.24 for clear cell carcinomas and 4.5?±?1.7 for papillary cell lesions. Mean SUV_max/SUV_mean ratios?±?SD of lesion to normal cortex at 30 min was 1.15?±?0.19 for the clear cell carcinomas and 2.3?±?0.84 for papillary cell.

Conclusions

In this small patient sample, relative amino acid transport compared with renal cortex is elevated in renal papillary cell carcinoma but not in clear cell carcinoma.     

Spatiotemporal Effects of Sonoporation Measured by Real-Time Calcium Imaging

To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca²⁺]_i) was determined using real-time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to a 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison^™ microbubbles. At extracellular [Ca²⁺]_o of 0.9 mM, ultrasound application generated both nonoscillating and oscillating (periods 12 to 30 s) transients (changes of [Ca²⁺]_i in time) with durations of 100–180 s. Immediate [Ca²⁺]_i transients after ultrasound application were induced by ultrasound-mediated microbubble–cell interactions. In some cases, the immediately affected cells did not return to pre-ultrasound equilibrium [Ca²⁺]_i levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca²⁺]_i in different cells formed a calcium wave that was observed to propagate outward from the immediately affected cells at 7–20 μm/s over a distance >200 μm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca²⁺ for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison^™ microbubbles can generate transient [Ca²⁺]_i changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane. (E-mail address: cxdeng@umich.edu)     

Chloride Uptake by Brush Border Membrane Vesicles Isolated from Rabbit Renal Cortex: COUPLING TO PROTON GRADIENTS AND K+ DIFFUSION POTENTIALS

Brush border membrane vesicles were isolated from rabbit renal cortex by Mg⁺⁺-precipitation and differential centrifugation. ³⁶Cl⁻ and [³H]glucose uptakes were simultaneously determined by a rapid filtration technique. Lysis of the vesicles with distilled water abolished 90-95% of the radioactivity on the filters, suggesting that nearly all of the ³⁶Cl⁻ and [³H]glucose counts represented uptake into an osmotically reactive intravesicular space. Inwardly directed K⁺ gradients plus valinomycin stimulated ³⁶Cl⁻ uptake, demonstrating a conductive pathway for chloride uptake into brush-border membrane vesicles. ³⁶Cl⁻ uptake could also be stimulated by inwardly directed proton gradients (pH_outside < pH_inside). This effect was seen in the absence of sodium, as well as in the presence of valinomycin when the vesicles had equal K⁺ concentrations inside and out. An “overshoot” phenomenon was observed when external ³⁶Cl⁻ was 2 mM and the external pH was lowered from 7.5 to 6.0 or to 4.5. The effect of the proton gradient was presumed to be different from the conductive mechanism because (a) the stimulation of ³⁶Cl⁻ uptake by inwardly directed K⁺ diffusion potentials was additive to the proton gradient effect, and (b) competition studies revealed statistically significant effects of thiocyanate on the conductive pathway, but not on the proton-driven pathway.     

Comparative study of gold and silver interactions with amino acids and nucleobases

Metal nanoclusters (NCs) have gained much attention in the last decade. In solution, metal nanoclusters can be stabilized by proteins, and, thus, exhibit many advantages in biocatalysis, biosensing, and bioimaging. In spite of much progress in the synthesis of polypeptide-stabilized gold (Au) clusters, their structure, as well as amino acid-cluster and amino acid–Au⁺ interactions, remain poorly understood. It is not entirely clear which amino acid (AA) residues and sites in the protein are preferred for binding. The understanding of NC-protein interactions and how they evolve in the polypeptide templates is the key to designing Au NCs. In this work, binding of gold ion Au⁺ and diatomic neutral gold nanocluster Au₂ with a full set of α-proteinogenic amino acids is studied using Density Functional Theory (DFT) and the ab initio RI-MP2 method in order to find the preferred sites of gold interaction in proteins. We demonstrated that the interaction of gold cations and clusters with protonated and deprotonated amino acid residues do not differ greatly. The binding affinity of AAs to the Au₂ cluster increases in the following order: Cys(−H⁺) > Asp(−H⁺) > Tyr(−H⁺) > Glu(−H⁺) > Arg > Gln, His, Met ≫ Asn, Pro, Trp > Lys, Tyr, Phe > His(+H⁺) > Asp > Lys(+H⁺) > Glu, Leu > Arg(+H⁺) > Ile, Val, Ala > Thr, Ser > Gly, Cys, which agrees with the available experimental data that gold cluster synthesis occurs in a wide range of pH – amino acid residues with different protonation states are involved in this process. The significant difference in the binding energy of metal atoms with nucleobases and amino acids apparently means that unlike on DNA templates, neutral metal atoms are strongly bound to amino acid residues and can''t freely diffuse in a polypeptide globula. This fact allows one to conclude that formation of metal NCs in proteins occurs through the nucleation of reduced Au atoms bound to the neighboring amino acid residues, and the flexibility of the amino acid residue side-chains and protein chain as a whole plays a significant role in this process.

Our calculations showed that amino acids stabilize gold nanoclusters; binding energy between organics and gold is higher than between organics and silver.     

Differences in Transport Mechanisms of trans-1-Amino-3-[18F]Fluorocyclobutanecarboxylic Acid in Inflammation,Prostate Cancer,and Glioma Cells: Comparison with l-[Methyl-11C]Methionine and 2-Deoxy-2-[18F]Fluoro-d-Glucose

Purpose

We aimed to elucidate trans-1-amino-3-[¹⁸F]fluorocyclobutanecarboxylic acid (anti-[¹⁸F]FACBC) uptake mechanisms in inflammatory and tumor cells, in comparison with those of l-[methyl-¹¹C]methionine ([¹¹C]Met) and 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG).

Procedures

Using carbon-14-labeled tracers, in vitro time-course, pH dependence, and competitive inhibition uptake experiments were performed in rat inflammatory (T cells, B cells, granulocytes, macrophages), prostate cancer (MLLB2), and glioma (C6) cells.

Results

Anti-[¹⁴C]FACBC uptake ratios of T/B cells to tumor cells were comparable, while those of granulocytes/macrophages to tumor cells were lower than those for [¹⁴C]FDG. Over half of anti-[¹⁴C]FACBC uptake by T/B and tumor cells was mediated by Na⁺-dependent amino acid transporters (system ASC), whereas most [¹⁴C]Met transport in all cells was mediated by Na⁺-independent carriers (system L).

Conclusions

The low anti-[¹⁸F]FACBC accumulation in granulocytes/macrophages may be advantageous in discriminating inflamed regions from tumors. The significant anti-[¹⁸F]FACBC uptake in T/B cells may cause false-positives in some cancer patients who undergo FACBC-positron emission tomography (PET).