Nonlinear pharmacokinetics of oral quinidine and verapamil in healthy subjects: a clinical microdosing study.

Microdosing studies are effective in enabling the early identification of the pharmacokinetic properties of compounds administered to humans. However, the nonlinearity of the pharmacokinetics between microdose and therapeutic dose, attributable to the saturation of metabolic enzymes and transporters, is a major concern. Verapamil and quinidine are good substrates of both the multidrug resistance 1 transporter (MDR1) and the cytochrome P450 (CYP) 3A4 enzyme (CYP3A4). We investigated their dose-dependent pharmacokinetics in healthy subjects. Four different doses of verapamil or quinidine were administered orally to each subject, and the plasma concentrations of the parent drugs and their major metabolites were measured. The dose-normalized area under the plasma concentration-time curve (AUC) values of quinidine and verapamil increased in a dose-dependent manner and were 2.6- and 2.3-fold higher, respectively, at the therapeutic dose than at microdose. These results suggest that the nonlinear pharmacokinetics of these drugs is caused mainly by the saturation of MDR1 and/or CYP3A4 in the small intestine.

Detecting early oral cancer: narrowband imaging system observation of the oral mucosa microvasculature.

The aim of this study was to analyze and describe the intrapapillary capillary loops (IPCL), which are a feature of early oral neoplastic lesions, using a narrowband imaging (NBI) system. Forty-one patients (26 men, 15 women; mean age, 52.34 years; range, 23-83 years) presenting with non-neoplastic or neoplastic lesions, and normal cases, were examined using the prototype Evis Lucera Spectrum (Olympus Co.). The images were analyzed and an IPCL classification was devised. All normal cases (n=10) had regularly distributed capillary loops of the same shape (type I). Non-neoplastic lesions (n=8) had mild changes of the capillary loops (types II and III) and neoplastic lesions (n=23) were irregularly distributed and had several loop shapes (types III and IV). The microvascular organization of non-neoplastic lesions was notably different from that of neoplastic lesions. A brownish area was found in five cases of early carcinoma. The narrowband imaging system is a potential approach for clinically analyzing microvascular organization and IPCL. It could be useful for diagnosing oral squamous cell carcinoma at an earlier stage and for determining the margin of resection.

Overcoming depolarizing resonances with dual helical partial Siberian snakes.

Acceleration of polarized protons in the energy range of 5 to 25 GeV is challenging. In a medium energy accelerator, the depolarizing spin resonances are strong enough to cause significant polarization loss but full Siberian snakes cause intolerably large orbit excursions and are also not feasible since straight sections usually are too short. Recently, two helical partial Siberian snakes with double pitch design have been installed in the Brookhaven Alternating Gradient Synchrotron (AGS). With a careful setup of optics at injection and along the energy ramp, this combination can eliminate the intrinsic and imperfection depolarizing resonances otherwise encountered during acceleration to maintain a high intensity polarized beam in medium energy synchrotrons. The observation of partial snake resonances of higher than second order will also be described.

Epstein-Barr virus-associated gastric carcinoma in Lima, Peru.

We examined 254 gastric carcinomas (GCs) diagnosed in four hospitals in Lima, Peru, and its suburban area during the period between 1994-2001. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) was identified by the in situ hybridization (ISH) technique to detect EBV-encoded small RNA (EBER) in gastric tissue. EBVaGCs, where EBER ISH staining was observed in all carcinoma cells, accounted for 3.9% (10/254) of gastric adenocarcinomas, the lowest frequency ever reported in Latin American countries. EBVaGC incidence rates in Peru, which we estimated on the basis of the present study and cancer incidence in Lima, were 0.8 per 100,000 among men and 0.5 per 100,000 among women. These estimates are much lower than those reported in our previous studies in Colombia (4.1 and 1.4 per 100,000 among men and women, respectively), a neighboring country, and in Japan (6.4 and 1.1 per 100,000 among men among women, respectively). Interestingly, EBVaGC in Peru showed no evident male predominance, as opposed to the findings reported in a majority of studies. Other clinicopathological features of EBVaGC in Peru were similar to those found in literature: EBVaGC showed no age dependence, a predominance in the non-antrum part of the stomach, and high frequencies in histological subtypes of moderately differentiated tubular adenocarcinoma and solid poorly differentiated adenocarcinoma. There was a case of well-differentiated adenocarcinoma showing a partial EBER-1-positive staining. In this carcinoma, the tumor in the body (middle third of the stomach) was EBER-1 positive but the tumor in the stomach antrum showed no noticeable EBER-1 ISH staining. We suspect this was a case of synchronous double carcinomas. Further studies are needed to identify the cause of the low frequency and lack of male predominance of EBVaGC in Peru.

Detection of B virus infection in cynomolgus monkeys by ELISA using simian agent 8 as alternative antigen.

The use of simian agent 8 (SA8) as an antigen for B virus (BV) antibody detection was evaluated in cynomolgus monkeys. Seventy-two sera judged as positive using BV antigen were all positive when the SA8 antigen was used. Out of 28 BV-negative sera 2 were positive against the SA8 antigen and one serum was classified as indeterminate. The present data indicates that detection of BV antibody can be achieved accurately and safely by enzyme-linked immunosorbent assay (ELISA) using SA8 antigen.

Highly repetitive DNA families restricted to germ cells in a Japanese hagfish (Eptatretus burgeri): a hierarchical and mosaic structure in eliminated chromosomes.

It is known that in eight hagfishes chromosome elimination occurs during early embryogenesis. The eliminated chromosomes are mostly C-band positive, so that none of the somatic cells have any C-band-positive chromatin. Recently, some highly repetitive DNA sequences have been reported as eliminated elements in these hagfishes based on molecular biological methods. However, no germline-restricted repetitive DNA have been directly isolated from the Japanese hagfish Eptatretus burgeri, from which approximately 21% of the total DNA is eliminated from presumptive somatic cells. Through electrophoretic investigation after digestion with restriction endonucleases, two DNA families that are restricted to germline DNA were isolated. Molecular cloning and sequence analysis revealed that these families are composed of closely related sequences of 64 and 57bp in length, respectively. Southern blot hybridization revealed that the two DNA families are restricted to germline DNA and were thus named EEEb1 and EEEb2, respectively. Moreover, these eliminated elements were highly and tandemly repeated, and it is predicted that they might amplify by saltatory replication and have evolved in a concerted manner. By densitometric scanning, EEEb1 and EEEb2 were found to amount to make up approximately 18.5 and 0.024% of the total germline genomic DNA, accounting for 88.6% of the total eliminated DNA. A fluorescence in situ hybridization experiment demonstrated that EEEb1 is located on all C-band-positive chromosomes that are limited to germ cells, suggesting that EEEb1 is the primary component of eliminated DNA of E. burgeri.

Quantitative prediction of in vivo drug interactions between nevirapine and antifungal agents from in vitro data in rats.

We investigated the effects of ketoconazole (KCZ) and fluconazole (FCZ) on rat liver microsomal nevirapine (NVP) metabolism in vitro and on NVP plasma profiles in vivo in order to determine whether the in vivo drug interactions could be predicted quantitatively from the in vitro data. The Ki values of KCZ and FCZ for NVP 12-hydroxylation were 1.59 microm and 11.5 microM, respectively, indicating that KCZ inhibited this activity more strongly than FCZ in vitro. In contrast, FCZ orally pre-administered at 20 mg/kg to rats increased the area under the plasma concentration-time curve (AUC) of NVP 7.4-fold, whereas KCZ increased it 2.1-fold, compared to the vehicle. We next investigated the inhibitory potency and unbound concentrations of KCZ and FCZ in microsomal mixtures with or without rat albumin. In the presence of albumin, the inhibition by KCZ was greatly decreased. Further, the unbound fraction of KCZ was decreased dramatically to around 3%, whereas more than 90% of FCZ remained in unbound form. When the increase in the AUC for NVP was calculated based on the concentrations of unbound inhibitors in the portal vein, good agreement with the observed in vivo values was obtained.

Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues.

Calpastatin, a specific inhibitor of calpain, consists of a unique N-terminal domain (domain L) and four repetitive protease-inhibitor domains (domains 1-4). The isolated cDNAs from various mammalian species have conspicuous differences in the regions encoding the N-terminal sequences and can be classified into four types. Mouse and bovine calpastatins (Type I and Type II, respectively), which also differ from each other in the uttermost N-terminal regions, possess longer domain L sequences than those of rabbit, pig, and human inhibitors (Type III). A sequence of a shorter isoform, registered in a DNA data bank, starts from a part of domain 2 with a different N-terminal sequence (Type IV). To clarify the source of this molecular diversity, we investigated the entire exon-intron organization of the mouse calpastatin gene. The previously obtained mouse calpastatin cDNA is encoded by as many as 31 exons including the first exon, designated 1xa. Three additional exons specifying the N-terminal sequences of the other types (designated exons 1xb, 1u, and 14t, respectively) were identified in the mouse genomic DNA sequence. While the mRNAs for Types I and III were expressed at high levels in liver, the Type II mRNA was abundant in heart and skeletal muscle and expressed at lower levels in liver, brain and testis. The Type IV mRNA was specifically expressed in testis among the tissues examined. These results suggest that the calpastatin isoforms possessing different N-terminal sequences are generated by alternative transcription initiation from their own promoters and skipping of the mutually exclusive exons.

Calpastatin domain L is involved in the regulation of L-type Ca2+ channels in guinea pig cardiac myocytes.

We have found previously that L-type Ca2+ channel run-down in cell-free patches is partially (10-28%) reversed by calpastatin (CS) and have suggested that CS, an endogenous inhibitor of calpain, has a Ca2+-channel-regulating function. CS is composed of repetitive domains 1-4 (calpain-inhibitory domain) and domain L (a domain whose function is unknown). We therefore investigated which domain of CS was involved in the regulation of Ca2+ channel activity in guinea pig cardiac myocytes using the patch-clamp technique. After the patches were excised into inside-out mode in basic internal solution, the Ca2+ channel activity ran down to 0.45% of the control level recorded in the cell-attached mode. Application of human recombinant full-length CS (25 microM) and domain L (25 microM) restored the Ca2+ channel activity to 13 and 19% of the control level, respectively, while the channel activity was not restored by CS domain 1 (25 microM) (0.66%). Mouse CS domain XLL (25 microM), a complex of domain XL and domain L, restored the calcium channel activity to 11% of the control level. These results suggested that the Ca2+ channel-regulating function of CS is located in domain L. This study is the first description of the function of CS domain L.

Structure of mouse calpastatin isoforms: implications of species-common and species-specific alternative splicing.

Mouse calpastatin cDNAs were cloned by the method of RT-PCR using RNA isolated from myoblast C2C12 cells. Nucleotide sequencing of the isolated clones revealed an in-frame ATG codon upstream of the previously assigned translation initiation methionine. Except for the N-terminal segment, the new translatable region (domain XL) was similar to the sequence of bovine calpastatin in which domain XL was first identified. Among the isolated mouse calpastatin cDNA clones, three isoforms (mCS-a, mCS-b, and mCS-c) were identified. In domain L, mCS-b had a deletion of the region corresponding to exon 3 of the human calpastatin gene. RT-PCR analyses of various mouse tissues revealed that mCS-b was the major form and that the content of mCS-a, nondeleted form, was 5-10% in tissues including skeletal muscle, liver, brain, etc. and about 30% in the myoblast C2C12 cells. Unlike human and rat cDNAs, no other deletions were detected in mouse calpastatin domain L. Isolation of the cDNA clone of mCS-c, which lacked regions corresponding to exons 3 and 12, was obtained by chance because its expression level was under the detectable level in the mouse tissues and even in C2C12 cells.

Heterozygous gsp mutation renders ion channels of human somatotroph adenoma cells unresponsive to growth hormone-releasing hormone.

Ionic mechanisms play an important role in the regulation of hormone secretion. The GHRH-induced GH release by human GH-secreting cells is transmitted through protein kinase A (PKA), which activates nonselective cation current (NSCC) and induces membrane depolarization, intracellular Ca2+ increase, and GH secretion. To evaluate whether ionic mechanisms have pathophysiological significance in GH oversecretion of GH-secreting pituitary adenomas, we examined four adenomas with constitutively active Gs alpha mutation (gsp mutation) and compared with three gsp-negative adenomas. In primary-cultured cells of gsp-positive adenomas, GHRH did not increase the NSCC under voltage-clamp experiments. Detailed examination showed that NSCC was maximally activated at the basal level and application of GHRH did not increase the current in these adenomas. Furthermore, by using single-cell RT-PCR method, we demonstrated for the first time at the single cell level that gsp mutation is heterozygous in GH-secreting pituitary adenomas. These indicate that heterozygous gsp mutation fully activates NSCC at the basal level, which may account for the GH oversecretion in gsp-positive GH-secreting pituitary adenomas.

Effect of Ca2+ and cAMP on capacitance-measured hormone secretion in human GH-secreting adenoma cells.

Membrane capacitance (Cm) was measured as an index of exocytosis in human growth hormone-secreting adenoma cells using the perforated whole cell, patch-clamp technique; the effects of membrane depolarization, growth hormone-releasing hormone, and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) were examined. Cm was increased by membrane depolarization to potentials beyond the threshold necessary to open voltage-gated Ca2+ channels. These voltage-dependent changes in Cm varied as a function of both depolarization amplitude and duration and were blocked in the presence of the Ca2+ channel antagonist nitrendipine (10(-6) M). When membrane potential was clamped at the holding potential (-78 mV), voltage-gated Ca2+ channels were closed, and neither application of growth hormone-releasing hormone nor 8-BrcAMP affected Cm. However, when these agents were applied to depolarized cells, where the voltage-gated Ca2+ channels were open, the increases in Cm were augmented. From these data, it was concluded that elevation of intracellular cAMP, per se, did not stimulate exocytosis. Rather, Ca2+ influx through voltage-gated channels was a prerequisite for cAMP-induced exocytosis.

Different G proteins mediate somatostatin-induced inward rectifier K+ currents in murine brain and endocrine cells.

1. Types of G proteins (G protein alpha-subunit subtypes) which mediate the activation of inward rectifier K+ currents by somatostatin (somatotrophin release-inhibiting factor, SRIF) were determined in cultured locus coeruleus neurones from newborn rats and in AtT-20 cells (a mouse pituitary cell line). 2. The whole-cell patch clamp technique was used together with injection of antibodies against pertussis toxin (PTX)-sensitive G protein alpha-subunits or with injection of antisense (or sense) oligonucleotides against these G proteins. 3. In locus coeruleus neurones, the SRIF-induced activation of inward rectifier K+ currents was inhibited by anti-G alpha i1/G alpha i2 antibody injection, but not by anti-G alpha i3 or by anti-G alpha o/G alpha i3 antibody injection, suggesting that the SRIF response is mediated through G alpha i1 and/or G alpha i2. 4. The SRIF-induced activation of the inward rectifier was suppressed in locus coeruleus neurones after injection of antisense oligonucleotides against G alpha i2, but not by injection of sense oligonucleotides against G alpha i2. Injection of antisense (or sense) oligonucleotides against G alpha i1, G alpha i3 and G alpha O (common) had no effect. These results suggest that G alpha i2 is involved in this SRIF response. 5. In AtT-20 cells, the SRIF-induced activation of inward rectifier K+ currents was suppressed by injection of anti-G alpha i3 antibody, but not by injection of anti-G alpha i1/G alpha i2 antibody. 6. The above results indicate that Gi mediates the SRIF effects on inward rectifier K+ currents. However, different subtypes of Gi are involved in the brain neurones and in the endocrine cells: Gi2 in locus coeruleus neurones and Gi3 in AtT-20 cells.

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs.

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN(-)-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca2+]i (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN(-)-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN(-)-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2, induced the envelope formation, acid production, augmentation of CN(-)-insensitive and sensitive respiration, but did not cause any increase in [Ca2+]i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN(-)-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca(2+)-triggered sequential reactions other than Ca(2+)-caused activation of phospholipase A2.

Gi3 mediates somatostatin-induced activation of an inwardly rectifying K+ current in human growth hormone-secreting adenoma cells.

SRIF activates an inwardly rectifying K+ current in human GH-secreting adenoma cells. Activation of this K+ current induces hyperpolarization of the membrane and abolishment of action potential firing. This mechanism is an essential mechanism for SRIF-induced decrease in intracellular Ca2+ concentration and inhibition of GH secretion. The activation of the inwardly rectifying K+ current is mediated by a pertussis toxin-sensitive G protein. In this article, the expression of the pertussis toxin-sensitive G protein alpha-subunits in the human GH-secreting adenoma cells were analyzed by RT-PCR, and the G protein transducing the SRIF-induced activation of this inwardly rectifying K+ current was investigated. RT-PCR of the messenger RNA from two human GH-secreting adenomas revealed that all G alpha(i1), G alpha(i2), G alpha(i3), and G alpha(o) were expressed in these adenomas. Primary cultured cells from these two adenoma cells were investigated under the voltage clamp of the whole-cell mode. Specific antibodies against the carboxyl terminus of G protein alpha-subunits were microinjected into the cells. Microinjection of antibody against the carboxyl terminal sequence of G alpha(i3) attenuated the SRIF-induced activation of the inwardly rectifying K+ current, whereas antibody against the common carboxyl terminal sequence of G alpha(i1) and G alpha(i2) did not. These data indicate that the G protein transducing the SRIF-induced activation of the inwardly rectifying K+ current is Gi3.

Differences in spatial localization and chromatin pattern during different phases of cell cycle between normal and cancer cells.

We studied differences in chromatin patterns and the spatial localization of centromeres of chromosome 11 during the cell cycle between normal peripheral blood lymphocytes (PBL) and human promyelocytic leukemia cells (HL-60) using fluorescence in situ hybridization. The pericentromeres in both cells were located at the periphery during Gq (quiescent) phase, but moved towards the nuclear center in G1 and mid-S phase. During G2, the pericentromeres of PBL continued to move towards the nuclear center whereas those of HL-60 returned to the periphery. The angle defining the spatial location of two pericentromeres, in reference to the center of the nucleus, increased in PBL cells from a mean of 67 degrees during Gq phase to 106 degrees during G1 phase (P < 0.01), and the two pericentromeres remained wide apart throughout the entire cell cycle. In HL-60, the angle also increased during G1, but then decreased during mid-S and G2 phases. Both cells exhibited pericentromeric signals during Gq that were round and compact, and the entire chromatin was loosely condensed. The signal became more loose and dispersed during the G1 and mid-S phases. The pericentromere signal varied during G2 and was generally rod-like or bipartite with condensation of the entire chromatin or chromosome-like. Our results suggest that subtle but important differences in spatial localization of pericentromeres are present during the interphase between normal PBL and HL-60 cells.

Corticotropin-releasing hormone excites adrenocorticotropin-secreting human pituitary adenoma cells by activating a nonselective cation current.

The mechanisms of corticotropin-releasing hormone (CRH) induced excitation of ACTH-secreting adenoma cells were investigated using the perforated whole-cell clamp technique and intracellular Ca2+ concentration ([Ca2+]i) measurement. CRH depolarized ACTH-secreting adenoma cells by activating a nonselective cation current that showed slight inward rectification. This channel did not seem to be a member of the Ca(2+)-activated cation currents because it was activated even when the [Ca2+]i was chelated below 50 nM. The activation of the current was induced by protein kinase A-mediated pathways. By [Ca2+]i measurement, CRH increased [Ca2+]i of these cells dependently on voltage-gated Ca2+ current. This CRH-induced [Ca2+]i increase was abolished in Na(+)-free extracellular solution, but was not abolished by the addition of 5 microM tetrodotoxin to the extracellular solution. CRH-induced ACTH secretion from the cultured adenoma cells was also abolished in Na(+)-free extracellular solution, but not in tetrodotoxin-containing extracellular solution. These data indicate that a Na+ current (maybe the nonselective cation current) other than voltage-gated Na+ current plays an important role in CRH-induced [Ca2+]i increase and ACTH secretion. CRH also activated a nonselective cation current in nonadenoma human corticotrophs, suggesting that the activation of a nonselective cation current is a physiological mechanism of CRH-induced excitation in human corticotrophs.