Abnormal cell proliferation in the p75NTR-positive basal cell compartment of the esophageal epithelium during squamous carcinogenesis.

The low affinity neurotrophin receptor p75NTR is known to be expressed in the mitotically quiescent basal layer (BL) of the normal esophageal epithelium. The aim of the present study was to detect oncogenic changes in the p75NTR-positive BL during esophageal squamous carcinogenesis. The normal epithelium (NE), low-grade intraepithelial neoplasia (LGN), high-grade intraepithelial neoplasia (HGN), and esophageal squamous carcinoma (SCC), in which invasion was limited to the muscularis mucosa, were obtained from surgically removed esophagi. The expression of p75NTR, the proliferation marker ki67, hTERT, p53, and p63 was examined immunohistochemically. The expression of p75NTR was detected in these tissues with average staining indexes (number of stained cells/100 nucleated cells; SI) of 1.00, 0.99, 0.81, and 0.73, respectively. The expression of ki67 in the BL significantly increased with the progression from LGN to HGN. The expression of hTERT and p53 significantly increased with the progression from NE to LGN, and then increased in a stepwise manner in HGN and SCC, with SI (hTERT/p53) of 0.10/0.11, 0.32/0.45, 0.50/0.72, and 0.65/0.61, respectively. The expression of p63 showed no significant difference among NE, LGN, HGN, and SCC, with SI of 0.82, 0.77, 0.85, and 0.87, respectively. A correlation was observed between the expression of ki67 and p53 (P = 0.005), while a negative correlation was found between p75NTR and hTERT (P = 0.01). Our results demonstrated that phenotypic changes from quiescent to active proliferation in the p75NTR-positive BL occurred during the progression from LGN to HGN. The altered expression of hTERT and p53 in the BL was detected in LGN, which suggested that additional oncogenic events that disrupt mitotic regulation in the p75NTR-positive quiescent BL may play a crucial role in malignant transformation. Further investigations using the isolation and tracing of p75NTR-positive cells in precancerous epithelia may provide us with a better understanding of squamous carcinogenesis.

Role of aquaporin-5 in gallbladder carcinoma.

BACKGROUND/PURPOSE: Aquaporins (AQPs) are important in controlling bile formation. However, the exact role in human gallbladder carcinogenesis has not yet been defined. METHODS: AQP-5-expressing gallbladder carcinoma (GBC) cell lines (NOZ) were transfected with anti-AQP-5 small interfering RNA (siRNA). Growth, migration, invasion assay, and drug susceptibility tests were performed. Next, microRNA (miRNA) expression was analyzed by miRNA oligo chip (3D-Gene®). AQP-5 and AQP-5-related miRNA target gene expressions were also analyzed using tissue microarray (TMA) in 44 GBC samples. RESULTS: Treatment with AQP-5 siRNA decreased cell proliferation, migration, and invasion. On the other hand, those cells increased IC50 of gemcitabine. By performing miRNA assays, miR-29b, -200a, and -21 were shown to be highly overexpressed in cells treated with AQP-5 siRNA NOZ. When focusing on miR-21, phosphatase and tensin homolog (PTEN) was found to be a target of miR-21. In the TMA, AQP-5/PTEN coexpression was significantly associated with the depth of invasion and MIB-1 index (p = 0.003, 0.010). Survival of patients with a high AQP-5/PTEN coexpression was longer than that of patients with a low coexpression (p = 0.003). CONCLUSIONS: Our result suggested that miR-21 and PTEN may contribute to the role of AQP-5 in GBC. AQP-5 and PTEN cascades are favorable biomarkers of GBC.

The role of activators in assembly of RNA polymerase II transcription complexes.

The past year has provided new insights into the biochemical mechanism of gene activation. Key discoveries include the finding that TFIIA plays an important regulatory role in transcription complex assembly, the TBP-associated factors are direct targets of at least two classes of activator, and a largely pre-assembled transcription complex has been isolated from yeast cells, challenging the step-wise assembly pathway. This review also presents an update on the argument that TFIIB is the target of VP16 and insights into the energetic role of ATP in RNA polymerase II initiation.

Transcription: TRF walks the walk but can it talk the talk?

The TBP-related factor TRF binds to a TATA box promoter sequence and mediates basal transcription in vitro. A central question is: does TRF function in vivo to transduce the effects of activators in a tissue-specific fashion?

Role of chloride on carrier-mediated transport of p-aminohippurate in rat renal basolateral membrane vesicles.

Effect of inorganic anions on p-amino[3H]hippurate transport in renal basolateral membranes has been studied using the vesicles preloaded with unlabeled p-aminohippurate (countertransport condition). The uptake of p-amino[3H]hippurate was stimulated by the outward gradient of unlabeled p-aminohippurate and the labeled substrate was accumulated into the vesicles against its concentration gradient in the presence of Cl-. The substitution of SCN- and SO4(2-) for Cl- in both sides of the vesicles depressed the initial rate and the overshoot magnitude of p-amino[3H]hippurate uptake. These results suggest that Cl- may play an important role for the carrier-mediated transport system of organic anion in renal basolateral membranes.

Transport of organic cation in renal brush-border membrane from rats with renal ischemic injury.

Transport of tetraethylammonium, an organic cation has been studied using renal brush-border membrane vesicles isolated from rats with ischemic and ischemia-reperfusion injury. H+ gradient-dependent uptake of tetraethylammonium slightly, but significantly, decreased in brush-border membrane vesicles from ischemic kidneys. When the kidney was reperfused after ischemia, the extent of the decrease of tetraethylammonium uptake was much greater than that after ischemia alone. The Vmax value of tetraethylammonium uptake by brush-border membrane vesicles from reperfused kidneys was decreased compared with control, without any change in the Km value. The tetraethylammonium uptake by the vesicles from reperfused kidneys was decreases both in the presence and absence of the outward H+ gradient (driving force). Uptake of D-glucose in renal brush-border membrane vesicles was also decreased by ischemia and again, reperfusion caused a further decrease of the uptake. Reperfusion also induced marked changes in the enrichment and recovery of marker enzymes in the isolated brush-border membrane fraction compared with ischemia. These findings suggest that renal ischemic injury altered the transport properties of tetraethylammonium as well as D-glucose, and that reperfusion after ischemia induced further damages on these functions in the brush-border membrane.

Differential sensitivity of organic anion transporters in rat renal brush-border membrane to diethyl pyrocarbonate.

The effect of various chemical modifiers on p-aminohippurate (PAH) uptake by a potential-sensitive system and by an anion exchanger was studied in rat renal brush-border membrane vesicles. Among various chemical modifiers, diethyl pyrocarbonate (DEPC) selectively inhibited potential-sensitive PAH uptake but not the uptake by the anion exchanger. The inhibitory effect of DEPC on potential-sensitive PAH uptake was not due to the facilitated dissipation of membrane potential, which was evidenced by the studies with a potential-sensitive fluorescence dye diS-C3(5). The potential-sensitive PAH uptake was inhibited by DEPC in a concentration-dependent manner, and kinetic analysis showed that the decreased uptake of PAH in DEPC-treated vesicles was due to the decrease of Vmax. The inhibition of the PAH uptake was protected by the presence of organic anions during the DEPC treatment. These findings indicate that PAH transport by the potential-sensitive system and by the anion exchanger is mediated by structurally distinct transporters. Amino acid residue(s) modified by DEPC, most likely a histidine residue, should play an important role in the potential-sensitive transport of PAH in rat renal brush-border membrane.

Transport of procainamide in a kidney epithelial cell line LLC-PK1.

Transport of procainamide, an anti-arrhythmic drug, was investigated in LLC-PK1 kidney epithelial cell line. The uptake of procainamide by LLC-PK1 monolayers cultured in plastic dishes was temperature-dependent, saturable and inhibited by organic cations such as cimetidine and N-acetylprocainamide. An aminocephalosporin antibiotic, cephalexin, also inhibited procainamide uptake, but an organic anion, p-aminohippurate, did not. The uptake of procainamide was greater at an alkaline external pH than at an acidic pH. In addition, procainamide uptake increased when intracellular pH was decreased and the uptake decreased when the intracellular pH was increased by ammonium chloride treatment, indicating the involvement of an H+/procainamide antiport system in apical membrane. The basolateral to apical flux of procainamide across LLC-PK1 monolayers cultured on permeable supports was 2.5-times larger than the apical to basolateral flux, and only the former process was inhibited by other organic cations. These findings suggest that LLC-PK1 cells can transport procainamide by the organic cation transport system and that procainamide is transported unidirectionally from basolateral to apical side across the cell monolayers.

Mechanisms of p-aminohippurate transport by brush-border and basolateral membrane vesicles isolated from rat kidney cortex.

The uptake of [3H]-labeled p-aminohippurate by brush-border and basolateral membrane vesicles isolated from rat renal cortex has been studied by a rapid filtration technique. Some characteristics of carrier-mediated transport for p-aminohippurate were demonstrated in basolateral membrane vesicles: the uptake was inhibited by probenecid or 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS), was saturable, was stimulated by the countertransport effect, and showed discontinuity in the Arrhenius plot. In contrast, brush-border membrane vesicles failed to display saturability of p-aminohippurate uptake and stimulation by the countertransport effect, although probenecid and DIDS reduced the uptake. Furthermore, p-aminohippurate uptake by brush-border membrane vesicles was influenced more sensitively by alteration in the membrane potential compared with that by basolateral membrane vesicles.

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex. Enzyme activities and some properties of glucose transport.

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of D-[3H] glucose into these vesicles was studied by a rapid filtration technique. (Na+ + K+)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of D-glucose uptake was almost unaffected by Na+ gradient (no overshoot).

Carrier-mediated transport of cephalexin via the dipeptide transport system in rat renal brush-border membrane vesicles.

Carrier-mediated transport of aminocephalosporin antibiotics by renal brush-border membrane vesicles has been studied in relation to the transport systems for dipeptides and amino acids. Dipeptides such as L-carnosine (beta-alanyl-L-histidine) and L-phenylalanylglycine competitively inhibited the uptake of cephalexin, but amino acids did not. Cephalexin uptake was stimulated by the countertransport effect of L-carnosine in the normal and papain-treated vesicles, and by the effect of L-phenylalanylglycine only in the papain-treated vesicles. In the papain-treated vesicles, the hydrolysis of dipeptides was markedly decreased, and the specific activity for cephalexin transport was increased approx. 2-fold because of the partial removal of membrane proteins. These results suggest that carrier-mediated transport of cephalexin can be transported by the system for dipeptides in renal brush-border membranes.

Enzyme activities and sodium-dependent active D-glucose transport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK1).

Apical membrane vesicles were isolated from the confluent LLC-PK1 cells by nitrogen cavitation and Mg/EGTA precipitation methods. The specific activities of marker enzymes for apical membranes were enriched 8- to 18-fold relative to those in the homogenate. D-[3H]Glucose uptake into the vesicles was stimulated in the presence of Na+ gradient (overshoot phenomenon), and the values of apparent Km and Vmax for Na+-dependent component of D-glucose uptake were 0.3 mM and 5.8 nmol/mg protein per min, respectively.

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles.

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.

Characteristics of organic cation transporter in rat renal basolateral membrane.

Characteristics of organic cation transport system were studied in rat renal basolateral membrane and compared with those in brush-border membrane. We first examined the effect of various chemical modifiers on tetraethylammonium uptake by the membrane vesicles. Treatment with N,N'-dicyclohexylcarbodiimide and phenylglyoxal (carboxyl groups and arginine residues specific reagent, respectively) resulted in inhibition of tetraethylammonium transport in both basolateral and brush-border membranes. Tetraethylammonium uptake by brush-border, but not by basolateral, membrane vesicles was decreased by diethyl pyrocarbonate, histidine residues specific reagent, treatment. Treatment of sulfhydryl groups with HgCl2 decreased tetraethylammonium transport in both membranes. However, in contrast to brush-border membrane, unlabeled tetraethylammonium failed to protect against the inhibition of [14C]tetraethylammonium uptake by p-chloromercuribenzene sulfonate in basolateral membrane. We next examined the inhibitory effect of various organic cations on tetraethylammonium uptake. The order of inhibitory potency of organic cations was somewhat different between two membranes. These findings suggest that the characteristics of organic cation transport systems in basolateral and brush-border membranes were different in regard to essential amino acid residues and the affinity of substrates.

Ampicillin and cephalexin in renal insufficiency.

We analyzed the relationship between functional damage and transport processes in the kidney in patients with glomerulonephritis and renal failure by a new analytic method. In renal failure patients, there was substantial diminution of maximum transport of secretion in renal tubules. This reduction affected the urinary excretion of ampicillin and cephalexin substantially because both drugs depend on active renal tubular secretion. Our results indicate that dosage adjustment based on creatinine clearance is not appropriate for patients receiving drugs requiring active tubular secretion for urinary excretion. Our data point to a need for a prolongation of the dosage interval of cephalexin to 20 times that in normal subjects, while five times is recommended by the creatinine clearance. In these patients, it is therefore suggested that a dosage adjustment method that involves both factors--glomerular and renal tubular functions--is required.

A new dosing regimen in renal insufficiency: application to cephalexin.

We describe a new method of drug dosage adjustment. The method simultaneously considers glomerular and tubular functions as parameters, because nonparallel decreases in both functions limit the use of the conventional endogenous creatinine clearance (CLCR) method for dosage adjustment. In the new method, CLCR and the 15-minute phenolsulfonphthalein (PSP15') test were used and applied to patients with renal insufficiency with cephalexin (CEX) as a model drug for renal tubular secretion. The results clearly demonstrate good control of plasma CEX concentrations by the CLCR-PSP15' method, whereas there were marked changes in plasma CEX levels with the CLCR method alone. Our method appears to be more useful for patients with renal impairment than the conventional CLCR method for CEX, which is mainly excreted in urine by renal tubular secretion. A nomogram for the CEX dosing interval is proposed for application to clinical practice.

P-glycoprotein-mediated transcellular transport of MDR-reversing agents.

Understanding of the interactions between P-glycoprotein and multidrug resistance (MDR) reversing agents is important in designing more effective MDR modulators. We examined transcellular transport of several MDR modulators by using a drug-sensitive epithelial cell line, LLC-PK1, and its transformant cell line, LLC-GA5-COL300, which expresses human P-glycoprotein on the apical surface. Basal-to-apical transports of azidopine and diltiazem across the LLC-GA5-COL300 monolayer were increased and apical-to-basal transports were decreased compared to those across the LLC-PK1 monolayer, indicating that P-glycoprotein transports azidopine and diltiazem. Movements of nitrendipine and staurosporine across the epithelial monolayer were not affected by P-glycoprotein. These results suggests that some MDR modulators exert their inhibitory effect not only by blocking the initial binding of anticancer drugs but throughout the course of the transport process.

Regional myocardial ajmaline concentration and antiarrhythmic activity for ischaemia- and reperfusion-induced arrhythmias in rats.

1. Antiarrhythmic actions of ajmaline against ischaemia (left coronary artery occlusion for 15 min) and subsequent reperfusion-induced arrhythmias were investigated in anaesthetized rats. 2. Ajmaline (2 mg kg-1, i.v.) was effective in suppressing ischaemia-induced arrhythmias whether given pre- or post-occlusion. 3. Ajmaline diminished the reperfusion-induced arrhythmias completely when given pre-occlusion but had little effect when given post-occlusion. 4. Reperfusion-induced increases in plasma enzyme activities of lactate dehydrogenase, glutamate-oxaloacetate transaminase and creatine phosphokinase were prevented more effectively when ajmaline was given pre-occlusion rather than post-occlusion. 5. Fifteen min post-occlusion, the ajmaline concentrations in the ischaemic ventricle were 18.42 +/- 1.66 and 1.18 +/- 0.15 micrograms g-1 for pre- and post-occlusion administration, respectively. However, ajmaline concentrations in whole blood and normal ventricle were not significantly different between pre- and post-occlusion administration. 6. We suggest that the beneficial effect of ajmaline against reperfusion-induced arrhythmias is related to the ischaemic myocardial concentration of ajmaline which is markedly affected by the time of drug administration (i.e. pre- and post-occlusion).

Pharmacokinetics and antiarrhythmic activity of ajmaline in rats subjected to coronary artery occlusion.

The pharmacokinetics and the antiarrhythmic action of intravenous ajmaline were investigated in anaesthetized rats subjected to coronary artery occlusion. Ajmaline (0.125-2 mg kg-1, i.v. given just after occlusion) suppressed arrhythmias in a dose-dependent manner, judged by the reduction of premature ventricular complexes. The incidence of malignant arrhythmias (ventricular tachycardia and fibrillation) was preferentially suppressed at the higher doses of ajmaline (1 and 2 mg kg-1). Coronary occlusion induced a change in pharmacokinetics of ajmaline (2 mg kg-1) and its total body blood clearance was significantly decreased from 56.6 ml min-1 kg-1 in sham-operated rats to 43.1 ml min-1 kg-1 in rats after coronary occlusion. Ajmaline exhibited a significantly increased negative dromotropic action (increased PQ interval) in rats after coronary occlusion compared with that in sham-operated rats. The difference seems to be due to the pharmacokinetic change since the concentration-effect relationship was similar in the two groups of rats. We suggest that the measurement of drug levels is important in the assessment of antiarrhythmic agents.

Pharmacokinetics and dromotropic activity of ajmaline in rats with hyperthyroidism.

1. The pharmacokinetics and the dromotropic action (increased PQ interval) of intravenously administered ajmaline (2 mg kg-1) were studied in hyperthyroid rats with sinus tachycardia. The hyperthyroidism was induced by intraperitoneal injection of 3,5,3'-triiodo-L-thyronine (0.5 mg kg-1) for 4 days. 2. The change in the ajmaline concentration in whole blood could be described by a biexponential equation. The steady state distribution volume of ajmaline decreased from 4.81 l kg-1 in control rats to 3.80 l kg-1 in hyperthyroid rats and the total body blood clearance was slightly higher in hyperthyroid rats than in control rats. 3. Ajmaline exhibited a saturable binding to rat plasma proteins, and one kind of binding site was found in the observed range of concentrations. The binding capacity was 2 fold higher in hyperthyroid rats than in control rats. 4. On the basis of the plasma unbound concentration, ajmaline exhibited an increased negative dromotropic activity in hyperthyroid rats compared with control rats. 5. A positive correlation was found between the pacing rate and the dromotropic action of ajmaline on atrioventricular conduction in isolated perfused hearts. There was no significant difference in the rate-dependence of the effect of ajmaline on the heart between control and hyperthyroid rats. 6. Our findings suggest that the increased dromotropic activity of ajmaline is mainly due to the increased heart rate in hyperthyroid rats.