The temporal and spatial effects of reconstructive surgery on the atrophy of hindlimb muscles in anterior cruciate ligament transected rats.

After anterior cruciate ligament (ACL) injury, a decrease in muscle strength associated with muscle atrophy is frequently observed. The temporal and spatial effects of reconstructive surgery on muscle atrophy have not been examined in detail. This study aimed to 1) reveal the short and mid-term effects of reconstructive surgery on muscle atrophy, and 2) investigate the differences in the degree of atrophy after ACL reconstruction in the hindlimb muscles. ACL transection with or without reconstructive surgery was performed unilaterally on the knees of rats. Untreated rats were used as controls. At one or four weeks post-surgery, the relative muscle wet weights (wet weight/body weight) of the hindlimb muscles were calculated to assess atrophy. At one week post-surgery, muscle atrophy was induced by ACL transection and further aggravated by reconstructive surgery. Reconstructive surgery facilitated recovery from muscle atrophy in some muscles compared with those without reconstructive surgery (ACL transection alone) at four weeks post-surgery. Muscle atrophy after ACL reconstruction was greater in the rectus femoris and plantar flexors than in the semitendinosus and plantar extensors at one week post-surgery. These results indicate that reconstructive surgery exacerbates muscle atrophy in the first week post-surgery, while facilitating recovery between the first and fourth week post-surgery. After reconstructive surgery, muscle atrophy was observed not only in the quadriceps and hamstrings, but also in the lower leg muscles, suggesting the need for muscle strengthening interventions for the lower leg muscles as well as the quadriceps and hamstrings.

Low-level laser therapy attenuates arthrogenic contracture induced by anterior cruciate ligament reconstruction surgery in rats.

Therapeutic approaches to treat joint contracture after anterior cruciate ligament (ACL) reconstruction have not been established. Arthrofibrosis accompanied by joint inflammation following ACL reconstruction is a major cause of arthrogenic contracture. In this study, we examined whether anti-inflammatory treatment using low-level laser therapy (LLLT) can prevent ACL reconstruction-induced arthrogenic contracture. Rats underwent ACL transection and reconstruction surgery in their right knees. Unoperated left knees were used as controls. After surgery, rats were reared with or without daily LLLT (wavelength: 830 nm; power output: 150 mW; power density: 5 W/cm2; for 120 s/day). We assessed the passive extension range of motion (ROM) after myotomy at one and two weeks post-surgery; the reduction in ROM represents the severity of arthrogenic contracture. ROM was markedly decreased by ACL reconstruction at both time points; however, LLLT partially attenuated the decrease in ROM. One week after ACL reconstruction, the gene expression of the proinflammatory cytokine interleukin-1beta in the joint capsule was significantly upregulated, and this upregulation was significantly attenuated by LLLT. Fibrotic changes in the joint capsule, including upregulation of collagen type I and III genes, shortening of the synovium, and thickening were caused by ACL reconstruction and seen at both time points. LLLT attenuated these fibrotic changes as well. Our results indicate that LLLT after ACL reconstruction could attenuate the formation of arthrogenic contracture through inhibition of inflammation and fibrosis in the joint capsule. Thus, LLLT may become a novel therapeutic approach for ACL reconstruction-induced joint contracture.

Using a quality of life (QoL)-monitor: preliminary results of a randomized trial in Dutch patients with early breast cancer.

PURPOSE: The diagnosis and treatment of cancer negatively affect patients' physical, functional and psychological wellbeing. Patients' needs for care cannot be addressed unless they are recognized by healthcare providers (HCPs). The use of quality of life (QoL) assessments with feedback to HCPs might facilitate the identification and discussion of QoL-topics. METHODS: 113 patients with stage I-IIIB breast cancer treated with chemotherapy were included in this randomized controlled trial. Patients were randomly allocated to receive either usual care, or usual care with an intervention consisting of a QoL-monitor assessing QoL, distress and care needs before every chemotherapy cycle visit. Patients completed questionnaires regarding QoL, illness perceptions, self-efficacy, and satisfaction with communication. From the 2nd visit onwards, patients in the intervention arm and their HCPs received a copy of the QoL overview and results were shown in patients' medical files. Audio-recordings and patients' self-reports were used to investigate effects on communication, patient management and patient-wellbeing. A composite score for communication was calculated by summing the number of QoL-topics discussed during each consultation. RESULTS: Use of the QoL-monitor resulted in a higher communication score (0.7 topics increase per visit, p = 0.04), especially regarding the disease-specific and psychosocial issues (p < 0.01). There were no differences in patient management, QoL, illness perceptions or distress. Patients in the experimental arm (n = 60) had higher scores on satisfaction with communication (p < 0.05). CONCLUSIONS: Use of a QoL-monitor during chemotherapy in patients with early breast cancer might result in a more frequent discussion of QoL-topics, associated with high levels of patients' satisfaction.

Modern precipitation of hydrogenetic ferromanganese minerals during on-site 15-year exposure tests.

Redox-sensitive metallic elements, Mn and Fe, are oxidized in deep sea waters and form abundant ferromanganese crusts and nodules on the world's ocean floors at ultraslow rates of growth. This process of oxidation and the mechanism of precipitation are yet unknown. In this paper, the results of the first successful, long-term, on-site experiment of mineral precipitation that ascertains modern, ongoing hydrogenetic deposition of oxide materials from normal seawaters at water depths of 900-4500 m of geologically active and inactive environments are presented. We succeeded in the in-situ precipitation experiment on the sea floor and characterized the precipitates using high-resolution and submicron-scale chemical, mineralogical, and structural analyses. The installed artificial plates of glass, ceramics, and plastic yielded spread-out particles of sizes varying from one to a few micrometers in diameter, of coccoid-like irregular shapes, with a maximum of 1,000-10,000 individual particles/mm2/year after 12-15 years of exposure. The results indicated a continuous substantial growth of the hydrogenetic minerals if both Mn and Fe are supplied to the bottom waters. The mineralogical, chemical, and structural properties of the precipitates are similar to those of the natural precipitates on the seabed that are made up of hydrogenetic ferromanganese crusts and nodules, together with settling sediments, suspended hydrothermal particles, or microbial precipitates from cultivated Mn-oxidizing bacteria. Our work presents new realistic insight into proposed genetic models of marine hydrogenetic ferromanganese deposits in modern diverse ocean environments.

Intra-articular injection of mitomycin C prevents progression of immobilization-induced arthrogenic contracture in the remobilized rat knee.

This study tested whether cell cycle inhibitor mitomycin C (MMC) prevents arthrogenic contracture progression during remobilization by inhibiting fibroblast proliferation and fibrosis in the joint capsule. Rat knees were immobilized in a flexed position to generate flexion contracture. After three weeks, the fixation device was removed and rat knees were allowed to freely move for one week. Immediately after and three days after fixator removal, rats received intra-articular injections of MMC or saline. The passive extension range of motion (ROM) was measured before and after myotomy of the knee flexors to distinguish myogenic and arthrogenic contractures. In addition, both cellularity and fibrosis in the posterior joint capsule were assessed histologically. Joint immobilization significantly decreased ROMs both before and after myotomy compared with untreated controls. In saline-injected knees, remobilization increased ROM before myotomy, but further decreased that after myotomy compared with that of knees immediately after three weeks of immobilization. Histological analysis revealed that hypercellularity, mainly due to fibroblast proliferation, and fibrosis characterized by increases in collagen density and joint capsule thickness occurred after remobilization in saline-injected knees. Conversely, MMC injections were able to prevent the remobilization-enhanced reduction of ROM after myotomy by inhibiting both hypercellularity and joint capsule fibrosis. Our results suggest that joint capsule fibrosis accompanied by fibroblast proliferation is a potential cause of arthrogenic contracture progression during remobilization, and that inhibiting fibroblast proliferation may constitute an effective remedy.

Cross-cultural comparison of breast cancer patients' Quality of Life in the Netherlands and Japan.

PURPOSE: Cultural differences are hypothesized to influence patients' Quality of Life (QoL) reports. However, there is a lack of empirical cross-cultural studies comparing QoL of patients with cancer. This study aims to compare QoL of women with breast cancer in the Netherlands and Japan, and to investigate the association of QoL with sociodemographic, clinical, and psychological variables (illness perceptions). METHODS: Dutch (n = 116) and Japanese (n = 148) women with early breast cancer undergoing chemotherapy completed the EORTC QLQ-C30 and Brief Illness Perception Questionnaire immediately before their second cycle of chemotherapy. RESULTS: Dutch women reported poorer Physical, Role, Emotional, and Cognitive functioning than Japanese women. Additionally, illness perceptions were significantly different in Japan and the Netherlands, but these did not vary across treatment type. In Japan, QoL of women receiving AC-chemotherapy was better than that of women receiving FEC-chemotherapy, whereas in the Netherlands, QoL did not vary as a function of chemotherapy. Illness perceptions about symptom severity, adverse consequences, and emotional representations were negatively related to most domains of patients' QoL in both countries. Adding illness perceptions as covariates to the ANOVA analyses rendered the effects of country and treatment type on QoL non-significant. CONCLUSIONS: Comparing Dutch and Japanese women with early breast cancer revealed important differences in treatment modalities and illness perceptions which both appear to influence QoL. Perceptions about cancer have been found to vary across cultures, and our study suggests that these perceptions should be considered when performing cross-cultural studies focusing on patient-reported outcomes.

Increased Syk phosphorylation leads to overexpression of TRAF6 in peripheral B cells of patients with systemic lupus erythematosus.

OBJECTIVE: Activation of B cells is a hallmark of systemic lupus erythematosus (SLE). Syk and TRAF6 are key signaling molecules in B-cell activation through BCR and CD40/TLR, respectively. Nevertheless, whether expression of Syk and TRAF6 is altered in SLE B cells remains unknown. METHODS: Phosphorylation and/or expression of Syk and TRAF6 were analyzed by flow cytometry in peripheral blood mononuclear cells isolated from SLE patients. RESULTS: Pronounced phosphorylation and expression of Syk were noted in B cells from SLE patients compared with healthy donors. Levels of Syk phosphorylation correlated with the disease activity score. TRAF6 was significantly over-expressed in B cells of SLE patients as compared with healthy donors, and significant correlation of levels of TRAF6 expression and Syk phosphorylation was observed in SLE patients. Levels of TRAF6 expression were more pronounced in CD27+ memory B cells than in CD27-naïve B cells. In vitro treatment of SLE B cells with a Syk inhibitor (BAY61-3606) reduced Syk phosphorylation as well as TRAF6 expression. CONCLUSION: Our results suggest that the activated Syk-mediated TRAF6 pathway leads to aberrant activation of B cells in SLE, and also highlight Syk as a potential target for B-cell-mediated processes in SLE.

Preventive and curative effects of radon inhalation on chronic constriction injury-induced neuropathic pain in mice.

BACKGROUND: Radon therapy is clinically useful for the treatment of pain-related diseases. However, there have been no studies regarding the effects of radon inhalation on neuropathic pain. In this study, we aimed to determine whether radon inhalation actually induced a remission of neuropathic pain and improved the quality of life. METHODS: First, we investigated the antinociceptive effects of radon inhalation in the chronic constriction injury (CCI) model of neuropathic pain. We evaluated pain behaviour in mice before and after CCI surgery, using von Frey test. Pretreated mice received CCI surgery immediately after 24-h inhalation of radon at background (BG) concentration (c. 19 Bq/m(3) ), or at a concentration of 1000 or 2000 Bq/m(3) , and post-treated mice inhaled similar levels of radon 2 days after CCI surgery. RESULTS: CCI surgery induced mechanical allodynia and hyperalgesia on a plantar surface of mice, as assessed using von Frey test, and 2000 Bq/m(3) radon inhalation alleviated hyperalgesic conditions 22-37% compared to BG level concentration. Concurrently, CCI surgery increased norepinephrine (NE), tumour necrosis factor-alpha (TNF-α) and nitric oxide (NO) concentrations in plasma, and leukocyte migration in paws. Furthermore, CCI-induced neuropathy reduced superoxide dismutase (SOD) activity. Treatment with radon inhalation, specifically at a concentration of 2000 Bq/m(3) , produced antinociceptive effects, i.e., lowered plasma TNF-α, NE and NO levels and restored SOD activity, as well as pain-related behaviour. CONCLUSIONS: This study showed that inhalation of 2000 Bq/m(3) radon prevented and alleviated CCI-induced neuropathic pain in mice.

Suppression of streptozotocin-induced type-1 diabetes in mice by radon inhalation.

We examined the protective effect of radon inhalation on streptozotocin (STZ)-induced type-1 diabetes in mice. Mice inhaled radon at concentrations of 1000, 2500, and 5500 Bq/m3 for 24 hours before STZ administration. STZ administration induced characteristics of type-1 diabetes such as hyperglycemia and hypoinsulinemia; however, radon inhalation at doses of 1000 and 5500 Bq/m3 significantly suppressed the elevation of blood glucose in diabetic mice. Serum insulin was significantly higher in mice pre-treated with radon at a dose of 1000 Bq/m3 than in mice treated with a sham. In addition, superoxide dismutase activities and total glutathione contents were significantly higher and lipid peroxide was significantly lower in mice pre-treated with radon at doses of 1000 and 5500 Bq/m3 than in mice treated with a sham. These results were consistent with the result that radon inhalation at 1000 and 5500 Bq/m3 suppressed hyperglycemia. These findings suggested that radon inhalation suppressed STZ-induced type-1 diabetes through the enhancement of antioxidative functions in the pancreas.

In vitro and in vivo analysis of a JAK inhibitor in rheumatoid arthritis.

Multiple cytokines play a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The appropriate intracellular signalling pathways must be activated via cytokine receptors on the cell surface, and the tyrosine kinases transduce the first 'outside to in' signals to be phosphorylated after receptor binding to its ligand. Among them, members of the Janus kinase (JAK) family are essential for the signalling pathways of various cytokines and are implicated in the pathogenesis of RA. The in vitro, ex vivo and in vivo effects of a JAK inhibitor CP-690,550 (tofacitinib) for the treatment of RA are reported. In vitro experiments indicated that the effects of tofacitinib were mediated through suppression of interleukin 17 (IL-17) and interferon γ production and proliferation of CD4 T cells, presumably Th1 and Th17. A treatment study was conducted in the severe combined immunodeficiency (SCID)-HuRAg mice, an RA animal model using SCID mice implanted with synovium and cartilage from patients. Tofacitinib reduced serum levels of human IL-6 and IL-8 in the mice and also reduced synovial inflammation and invasion into the implanted cartilage. A phase 2 double-blind study using tofacitinib was carried out in Japanese patients with active RA and inadequate response to methotrexate (MTX). A total of 140 patients were randomised to tofacitinib 1, 3, 5, 10 mg or placebo twice daily and the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12, a primary end point, was significant for all tofacitinib treatment groups. Thus, an orally available tofacitinib in combination with MTX was efficacious and had a manageable safety profile. Tofacitinib at 5 and 10 mg twice a day appears suitable for further evaluation to optimise the treatment of RA.

Relationship between pelagic larval duration and abundance of tropical fishes on temperate coasts of Japan.

The influence of pelagic larval duration (PLD) and egg type dispersal capabilities of 35 demersal and pelagic-spawning tropical fish species is examined in relation to their abundance on the temperate coasts of Japan. The PLDs of pelagic spawners were significantly longer than those of demersal spawners, and a high occurrence of pelagic spawners on the temperate coasts suggests that these fishes are more easily transported to temperate coasts than demersal spawners. For demersal spawners, the common species on the temperate coasts had significantly longer PLDs than the rare species; this suggests that PLD is a major factor influencing the distribution patterns of tropical demersal spawners on temperate coasts. Moreover, a negative correlation between PLD and the abundance of some species of pelagic and demersal spawners suggests the presence of reproductively active fishes in northern subtropical and even in temperate waters.

Radioactivity of 210Pb in Japanese cigarettes and radiation dose from smoking inhalation.

It is well known that cigarette tobaccos contain naturally occurring radioactive nuclides such as (210)Pb and (210)Po. In many countries, the radioactivity of tobaccos has been measured to estimate the effective dose from smoking inhalation. The present study covered 24 cigarette brands including the top 20 of sales in Japan between April 2008 and March 2009. The activity concentrations of (210)Pb were measured by gamma-ray spectrometry, and then those of its progeny ((210)Po) were evaluated assuming the radioactive equilibrium between the two nuclides. Their concentrations were in the range of 2-14 mBq cigarette(-1) with an arithmetic mean of 8±3 mBq cigarette(-1). The annual committed effective doses were also calculated, based on the scenario that a smoker consumes 20 cigarettes a day. The average doses from (210)Pb and (210)Po inhalations were 22±9 and 68±27 µSv y(-1), respectively.

Morphological changes in hind limb muscles elicited by adjuvant-induced arthritis of the rat knee.

We investigated qualitative and quantitative changes in rat hind limb muscles caused by complete Freund's adjuvant (CFA)-induced knee joint pain. One week after CFA injection, muscle atrophy was induced only on the CFA-injected side. Wet weight of the rectus femoris (RF) and soleus (SOL) muscles were significantly decreased by 20% and 19%, respectively. The reduction in cross-sectional areas by CFA was similar for fast and slow muscle fibers in the RF (10% vs 15%, respectively) and SOL muscles (16% vs 16%, respectively). At the light microscopic level, pathological changes were not found in the RF muscles on both sides, although the infiltration of mononuclear cells and muscle regeneration were found in the SOL muscles on CFA-injected and contralateral control sides. On the other hand, electron microscopy revealed degenerative changes in the RF and SOL muscles on the CFA-injected side. Interestingly, sarcomere hypercontraction, indicating overexercise, was observed to a limited extent in the SOL muscles on the control side. In conclusions, knee joint pain can trigger the rapid development of muscle atrophy with degenerative changes not only in thigh but also calf muscles. This indicates that early interventions to inhibit joint pain or inflammation may prevent muscle atrophy.

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes.

OBJECTIVE: Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation. MATERIALS AND METHODS: We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA). RESULTS: FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-beta), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-beta expression, suggesting a decrease in responsiveness of TGF-beta to FGF18 stimulation in the dedifferentiated chondrocytes. CONCLUSION: FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.

Conceptual equivalence and health-related quality of life: an exploratory study in Japanese and Dutch cancer patients.

Research into the equivalence of Western and Japanese conceptualizations of health-related quality of life (HR-QOL) is scarce. We used the Western (European Organization for Research and Treatment of Cancer, EORTC-QLQ-C30) and the Japanese (HRQoL-20) questionnaire in order to analyze the conceptual similarity of HR-QOL factors, and the associations between specific symptom items with overall HR-QOL in Japanese (n=265) and Dutch (n=174) patients with various types of cancer. Both populations completed both instruments. In both patient groups, the overall health scale of the EORTC-QLQ-C30 correlated highly (r=0.59; p<0.001) with the HRQOL-20 composite average score, indicating substantial conceptual comparability. Relationships between all EORTC-QLQ-C30 symptom items with HR-QOL were examined by ranking their correlations with the two overall measures of HR-QOL. Comparable patterns in the Japanese and Dutch samples were observed. The results suggest a considerable conceptual equivalence of HR-QOL in Japanese and Dutch cancer patients, and indicate a satisfactory structural and cross-cultural equivalence for the EORTC-QLQ-C30 with regard to items measuring functioning and specific symptoms. Longitudinal studies are needed to examine the impact of specific symptoms on general quality of life.

Na+ channel pharmacology and molecular mechanisms of gating.

Electrogenesis of efficiently propagated action potentials requires synchronized opening of transmembrane Na+ channels possessing a sodium selectivity-filter, a high-throughput ion-conductance pathway, and voltage-dependent gating functions. These properties of the Na+ channel have long been the target of molecular analysis. Several toxins and drugs, known to selectively bind to Na+ channels, have been used as pharmacological tools to investigate Na+ channel properties either electrophysiologically or chemically. Recent analyses of the protein crystal structure of bacterial voltage-dependent K+ channels have provided important clues to the identity of mobile structures involved in channel gating. The new information may be applicable to Na+ channels, and may well require a total revision of our understanding of gating mechanisms of sodium channels. Several experiments challenge the emerging view that channel gating by S6 transmembrane segments is triggered by signals from voltage sensors floating in membrane lipid. Herein, we review the various toxin and drug molecules that affect the gating behavior of Na+ channels in this new structural framework, by characterizing the binding sites of these toxins, and assessing the pharmacological effects resulting from changes in the structure of the toxin or sodium channel.

Pulmonary adenocarcinoma with micropapillary component: an immunohistochemical study. Case report.

Micropapillary carcinoma has been described in various organs, including the breast, urinary bladder, ovary and lung. We here present a case of pulmonary micropapillary carcinoma in a 72-year-old Japanese man who died of respiratory failure and septic shock, following which autopsy was performed. A mass measuring 2.5 x 2.5 x 2.5 cm was observed in the left lower lobe of the lung. The tumor showed moderately differentiated papillary adenocarcinoma with a focal micropapillary component. Carcinomatous lymphangiosis was also observed in the left lung and metastatic lesions were observed in the bilateral lung, liver, vertebra, muscle layer of the urinary bladder, right adrenal gland, spleen and lymph nodes. The micropapillary component was predominant at some metastatic sites. Immunohistochemically, both the adenocarcinoma and micropapillary components were positive for cytokeratin (CK) 7, CK19, TTF (thyroid transcription factor)-1, carcinoembryonic antigen (CEA) and surfactant apoprotein A (SP-A), and negative for CK20, estrogen receptor, progesterone receptor, uroplakin III, and CA125. The invasive area of the conventional adenocarcinoma component contained a large number of myofibroblasts, whereas the stroma of the micropapillary component contained a small number of myofibroblasts. However, no myofibroblasts were observed in the stroma of the central core of the non-invasive micropapillary carcinoma. Several lymphatic invasions by neoplastic cells were identified in the peripheral area of the micropapillary component using D2-40 antibody. The immunohistochemical profile may be helpful in determining the primary location of the neoplasm containing micropapillary features. Myofibroblasts are present in the stroma of the invasive neoplastic nests in the micropapillary component as well as the conventional adenocarcinoma component, and D2-40 monoclonal antibody may be useful for evaluating the lymphatic invasion of pulmonary micropapillary carcinoma.

Novel mutations in the a3 subunit of vacuolar H(+)-adenosine triphosphatase in a Japanese patient with infantile malignant osteopetrosis.

A case of infantile malignant osteopetrosis is described. The patient died from respiratory hemorrhage at 7 months of age despite treatment that included high doses of active vitamin D and administration of interferon-gamma. A postmortem examination revealed the presence of many osteoclasts in the bone, which lacked ruffled borders. This observation was consistent with the histology of bone reported in Atp6i-knockout mice, which lack the gene encoding the a3 subunit of vacuolar-type H(+)-adenosine triphosphatase (ATPase). Sequence analysis of the TCIRG1 gene encoding the a3 subunit revealed two novel mutations: a deletion/insertion mutation in exon 9 and a T-to-C transition at the splice donor site of intron 19. The former mutation caused a frame shift and premature stop codon. The latter was associated with abnormal splicing, which was confirmed by sequencing the products amplified by reverse transcription-polymerase chain reaction (RT-PCR), using total RNA from the liver specimen as template. Although several mutations in the TCIRG1 gene in infantile malignant osteopetrosis have been reported in other populations, this is the first case of a Japanese patient with a mutation identified in this gene. These results support the important role of the subunit in the function of the proton pump.

Introduction of recirculatory analysis into portal and systemic concentration difference method.

Recirculatory analysis was introduced into the portal and systemic concentration difference method with double dosing (PS-DD method), which is an evaluation system for the local intestinal and hepatic first-pass effect. 5-Fluorouracil (5-FU) and cephalexin (CEX) were selected as model drugs. A new recirculatory system was constructed to predict the time courses of a drug concentration in the systemic and portal bloods. Bioavailability (F), local absorption ratio (Fa), hepatic recovery ratio (FH), and local mean absorption time (ta) estimated by recirculatory analysis were close to those calculated by moment analysis with numerical integration. Using recirculatory analysis, the sampling period was considerably shortened and the sampling number was also reduced, which demonstrates that recirculatory analysis is useful in PS-DD method.

Novel site on sodium channel alpha-subunit responsible for the differential sensitivity of grayanotoxin in skeletal and cardiac muscle.

We searched for sites on the alpha-subunit of the fast Na(+) channel responsible for the difference in GTX (grayanotoxin) sensitivity of the skeletal- and cardiac-muscle Na(+) current. cDNA clones, encoding the skeletal or cardiac isoforms of the alpha-subunit, were inserted into a mammalian expression vector and transiently transfected into human embryonic kidney cells. The expressed channels were measured using whole-cell patch-clamp techniques and examined for GTX sensitivity. As a measure of GTX sensitivity, we used relative chord conductance (ratio of maximum chord conductance of noninactivating GTX-modified Na(+) currents to that of unmodified peak currents). Wild-type channels from skeletal muscle (mu 1) were more sensitive to GTX modification than wild-type cardiac channels (rH1) by a factor of 1.6. To facilitate exploration of alpha-subunit sites determining GTX sensitivity, we used SHHH, a chimera of skeletal muscle (S) domain D1 and heart muscle (H) domains D2D3D4 with supernormal sensitivity to GTX I (1.5-fold of wild-type mu 1). Successive replacement of Ser-251 (D1S4-S5 intracellular loop) and Ile-433 (D1S6 transmembrane segment), with corresponding rH1 residues Ala and Val, reduced, in a stepwise manner, the GTX sensitivity of the chimera and related mutants to that of wild-type rHl. We concluded that, in addition to Ile-433, known as the GTX-binding site, Ser-251 represents a novel site for GTX modification.