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Hepatic adipogenesis has common mechanisms with adipocyte differentiation such as PPARγ involvement and the induction of adipose tissue-specific molecules. A previous report demonstrated that integrator complex subunit 6 (INTS6) is required for adipocyte differentiation. This study aimed to investigate INTS6 expression and its role in hepatic steatosis progression. The expression of INTS6 and PPARγ was examined in the liver of a mouse model of steatohepatitis and in paired liver biopsy samples from 11 patients with severe obesity and histologically proven metabolic dysfunction associated steatohepatitis (MASH) before and one year after bariatric surgery. To induce hepatocellular steatosis in vitro, an immortalized human hepatocyte cell line Hc3716 was treated with free fatty acids. In the steatohepatitis mouse model, we observed hepatic induction of INTS6, PPARγ, and adipocyte-specific genes. In contrast, β-catenin which negatively regulates PPARγ was reduced. Biopsied human livers demonstrated a strong positive correlation (r 2  = 0.8755) between INTS6 and PPARγ mRNA levels. After bariatric surgery, gene expressions of PPARγ, FABP4, and CD36 were mostly downregulated. In our in vitro experiments, we observed a concentration-dependent increase in Oil Red O staining in Hc3716 cells after treatment with the free fatty acids. Alongside this change, the expression of INTS6, PPARγ, and adipocyte-specific genes was induced. INTS6 knockdown using siRNA significantly suppressed cellular lipid accumulation together with induction of β-catenin and PPARγ downregulation. Collectively, INTS6 expression closely correlates with PPARγ. INTS6 suppression significantly reduced hepatocyte steatosis via β-catenin-PPARγ axis, indicating that INTS6 could be a novel therapeutic target for treating MASH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Context: The efficacy and tolerability of high-flow nasal cannula (HFNC) for relieving dyspnea in advanced cancer patients with limited prognosis requires elucidation., Objectives: The primary aim of this trial was to assess the efficacy and tolerability of HFNC regarding dyspnea including severe as well as moderate for longer durations in patients under palliative care., Methods: In this prospective study, hospitalized patients with advanced cancer who had dyspnea at rest (numeric rating scale, NRS≥3) and hypoxemia were enrolled. They were treated with HFNC for five days in the respiratory unit. Primary endpoint was mean change of modified Borg scale at 24 hours. Key secondary endpoints consisted of mean changes in modified Borg scale during the study period and feasibility (Trial Identifier, UMIN000035738)., Results: Between February 2019 and February 2022, 25 patients were enrolled and 21 were analyzed. Twenty patients used inspired oxygen and the mean fraction of inspired oxygen (FiO2) was 0.34 (range, 0.21-1.0). At baseline, mean NRS (dyspnea) was 5.9 (range, 3-10). Median survival time was 19 days (range, 3-657). The mean change of modified Borg scale was 1.4 (80% confidence interval [CI]: 0.8-1.9) at 24 hours, 12 patients (57%) showed 1.0 points improvement of modified Borg scale. Within two hours, 15 patients showed 1.0 points improvement of modified Borg scale and such early responders were likely to maintain dyspnea improvement for 24 hours. Nineteen patients could continue HFNC for 24 hours and 11 patients completed five days of HFNC., Conclusion: To our knowledge, this trial is the first prospective study to assess the five-day efficacy and tolerability of HFNC for dyspnea in patients under palliative care. Although this did not reach the prespecified endpoint, about half of the patients showed 1.0 point improvement, a minimally clinically important difference (MCID) in the chronic lung disease. HFNC can be a palliative treatment option in advanced cancer patients with dyspnea., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Combination therapy with glecaprevir and pibrentasvir (PIB) has high efficacy for patients with hepatitis C virus (HCV) infection except among those who experienced NS5A-P32 deletion (del) mutation during prior DAA treatment failure. However, some patients fail to achieve SVR through combination treatment even in the absence of NS5A-P32del. We analyzed emergence of NS5A resistance-associated substitutions (RASs) against PIB using HCV-infected mice. Male human hepatocyte transplanted mice were infected with genotype 1b wild-type HCV. Mice were treated with PIB, resulting in a transient decrease in serum HCV RNA levels but followed by relapse during the treatment. Direct sequence analysis showed emergences of various mutations in the NS5A region, including L31V/P32del, L31F/P32del/Y93H, NS5A-P29del/Y85C, and NS5A-F37Y. PIB was less effective in mice with NS5A-F37Y mutations compared to mice with wild-type HCV. NS5A-F37Y showed 5.4-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. The present in vivo and in vitro studies identified NS5A-F37Y as a novel RAS against PIB and showed the possibility of emergence of various NS5A RASs including P29del, P32del and F37Y following PIB treatment. These mutations might emerge and lead to failure to respond to DAA therapies including PIB-based regimens in chronic hepatitis C patients., Competing Interests: Declaration of competing interest M.I. has received research funding from Bristol-Myers Squibb and AbbVie. H.A. has received honoraria from Eisai and Bayer. K.C. has received honoraria from Bristol-Myers Squibb and MSD K.K., AbbVie, Gilead Science, Dainippon Sumitomo Pharma and Mitsubishi Tanabe Pharma and research funding from Gilead Science, Dainippon Sumitomo Pharma, MSD K.K., AbbVie, Eisai, TORAY, Otsuka Pharma, Chugai Pharma, Takeda Pharma and Roche., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sofosbuvir (SOF) is a nucleotide prodrug which has been used as a backbone for the clinical treatment of hepatitis C viral infection. Because sofosbuvir undergoes complex first pass metabolism, including metabolic activation to form its pharmacologically active triphosphate (GS-331007-TP) to inhibit the viral RNA polymerase in the liver, it is difficult to project the human dose for clinical evaluation based on preclinical data. Selecting an appropriate animal model for drug exposure in the target tissue is challenging due to differences in absorption, stability, hepatic uptake, and intracellular activation across species. Efficient liver delivery has been established in human liver following administration in a clinical trial of patients receiving sofosbuvir prior to liver transplantation. Using the clinical liver exposure as a benchmark, we assessed and compared the pharmacokinetic profile in mouse, rat, hamster, dog and monkey. Liver accumulation was also assessed in the PXB mouse model in which the liver is mostly populated with human hepatocytes. At human equivalent dose, the hepatic concentrations of GS-331007-TP in dog and PXB mouse were comparable to those observed in the human livers. In these species, high and sustained levels of GS-331007-TP were observed in both primary hepatocytes in vitro and the liver in vivo., Competing Interests: Declaration of competing interest All the authors are current or former employee of Gilead Sciences., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Objective: Pain at or around the posterior superior iliac spine (PSIS) is characteristic of sacroiliac joint (SIJ) -related pain. This pain can be treated by either a peri- or intra-articular injection into the joint, with the former being much easier to perform. We investigated whether peri- or intra-articular injections were more frequently effective in patients with SIJ-related pain, and aimed to create an efficient treatment strategy for SIJ-related pain at or around the PSIS., Design: Prospective case-control study., Patients and Methods: We evaluated 85 patients with pain at or around the posterior superior iliac spine as indicated by the one finger test. First, we performed a peri-articular sacroiliac joint injection. If it was ineffective, an intra-articular injection was later given. Groin pain, sitting pain, sacroiliac joint shear test results, and posterior superior iliac spine and sacro-tuberous ligament tenderness were also compared between patients for whom a peri- or intra-articular injection was effective., Results: Seventy-two (85%) of 85 patients had an effective injection. Out of these 72 patients, 58 (81%) had a positive peri-articular injection and 14 (19%) had a positive intra-articular injection. Four items, excluding tenderness of the sacro-tuberous ligament had no significant difference between these two injection types., Conclusion: To treat sacroiliac joint-related pain at or around the posterior superior iliac spine, a peri-articular injection should be performed first, and only if it is not effective should an intra-articular injection be administered. Using this strategy, we expect that most patients with sacroiliac joint-related pain will be efficiently diagnosed and treated., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: We investigated the prevalence of groin pain in patients with sacroiliac joint (SIJ) dysfunction, lumbar spinal canal stenosis (LSS), and lumbar disc herniation (LDH) who did not have hip disorders, and evaluated the clinical features that distinguished SIJ dysfunction from LSS and LDH., Patient and Methods: We evaluated 127 patients (57 men, 70 women, average age 55 years) with SIJ dysfunction, 146 (98 men, 48 women, average age 71 years) with LSS, and 124 (83 men, 41 women, average age 50 years) with LDH. The following data were retrospectively collected from the patients' medical charts: (1) the prevalence of groin pain for each pathology; (2) corresponding spinal level of LSS and LDH in the patients with groin pain; (3) the pain areas in the buttocks and back; pain increase while in positions such as sitting, lying supine, and side-lying; an SIJ shear test; and four tender points composed of the posterior superior iliac spine (PSIS), long posterior sacroiliac ligament (LPSL), sacrotuberous ligament (STL), and iliac muscle., Results: Fifty-nine (46.5%) patients with SIJ dysfunction, 10 (6.8%) with LSS, and 10 (8.1%) with LDH reported groin pain. Of the 10 patients with LSS, five presented with cauda equina symptoms, two had stenosis of L2-L3, and three had stenosis below L3-L4. The other five presented with radiculopathy: the corresponding nerve root was L2, L3, and L4 in one patient each, and L5 in two. Of the 10 patients with LDH, eight presented with radiculopathy: the corresponding nerve root was L2 and L4 in three patients each, and L5 in two. Two patients presented with L4-L5 discogenic pain without radiculopathy. In patients with groin pain, pain provoked by the SIJ shear test and the tenderness of the PSIS and LPSL were significant physical signs that differentiated SIJ dysfunction from LSS and LDH. (Fisher's exact test, P<0.05) CONCLUSION: The prevalence of groin pain in patients with SIJ dysfunction was higher than in those with LSS or LDH. When patients who do not have hip disorders complain of groin and lumbogluteal pain, not only lumbar disorders but also SIJ dysfunction should be considered., (Copyright © 2017. Published by Elsevier B.V.)

Objective: The symptoms of sacroiliac joint (SIJ) disorders are usually detected in the buttock and groin, and occasionally referred to the thigh and leg. However, lumbar disorders also cause symptoms in these same body regions. The presence of a characteristic, symptomatic pattern in the legs would be useful for diagnosing SIJ disorders. This study aimed to identify specific leg symptoms in patients with SIJ pain originating from the posterior sacroiliac ligament and determine the rate of occurrence of these symptoms., Patients and Methods: The source population consisted of 365 consecutive patients from February 2005 to December 2007. One hundred patients were diagnosed with SIJ pain by a periarticular SIJ injection (42 males and 58 females, average age 46 years, age range, 18-75 years). A leg symptom map was made by subtracting the symptoms after a periarticular SIJ injection from the initial symptoms, and evaluating the rate of each individual symptom by area., Results: Ninety-four patients reported pain at or around the posterior-superior iliac spine (PSIS). Leg symptoms comprised pain and a numbness/tingling sensation; ≥60% of the patients had these symptoms. Pain was mainly detected in the back, buttock, groin, and thigh areas, while numbness/tingling was mainly detected in the lateral to posterior thigh and back of the calf., Conclusions: Leg symptoms associated with SIJ pain originating from the posterior sacroiliac ligament include both pain and numbness, which do not usually correspond to the dermatome. These leg symptoms in addition to pain around the PSIS may indicate SIJ disorders., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: Single nucleotide polymorphisms within the interferon lambda 4 (IFNL4) locus are strongly associated with spontaneous clearance of hepatitis C virus (HCV) infection and early viral response to interferon therapy. Interaction between host genotype and amino acid substitutions might also influence the risk of antiviral resistance in interferon-free direct acting antiviral (DAA) therapies., Methods: The relationship between IFNL4 genotype and HCV substitutions was analyzed in 929 patients with chronic HCV genotype 1b infection. Ultra-deep sequencing and quasispecies reconstruction was performed on the N-terminal region of NS5A in 57 patients., Results: IFNL4 genotype was strongly associated with HCV NS5A Y93 and core protein substitutions, and the number and diversity of predicted quasispecies was marginally greater in IFNL4 TT/TT patients compared to TT/ΔG, ΔG/ΔG patients. RNA secondary structure prediction of the NS5A region suggests that variable sites are more likely to occupy unpaired, high entropy positions., Conclusions: HCV infection is proposed to induce a more efficient antiviral response in individuals with the IFNL4 TT/TT genotype that results either in viral clearance or selection for viral adaptations. The association between IFNL4 TT/TT genotype and Y93 substitutions may impact the risk of antiviral resistance in NS5A inhibitors in DAA therapy., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA-SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA-SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA-SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8weeks after injection) was similar in both TK-NOG mice and uPA-SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The sacroiliac joint (SIJ) can be a source of low back and lower limb pain. The SIJ pain can originate not only from the joint space but also from the ligaments supporting the joint. Its diagnosis has been difficult because the physical and radiological examinations have proved less than satisfactory. Thus, to know the specific sites of SIJ pain, if these exist, could be very useful for making the diagnosis. The purpose of the present study was to identify the main site of SIJ pain according to the patient's pointing with one finger and to confirm the site by a pain-provocation test and periarticular lidocaine injection., Methods: Forty-six of 247 consecutive patients with low back pain at our outpatient clinic, who could indicate with one finger the main site of the pain, which presented at only one site and was reproducible, were the subjects of this study. The main site of pain was anatomically confirmed by fluoroscopy. Then, a periarticular SIJ injection was performed. The patients were blindly assessed and a diagram of the main site of the SIJ pain was made., Results: There were 19 males and 27 females and the age averaged 50 years. Eight patients showed a positive placebo response and were excluded from this study. Twenty-five of the remaining 38 patients indicated the main site of pain at the posterior-superior iliac spine (PSIS) or within 2 cm of the PSIS, and 18 of these patients showed a positive effect with periarticular SIJ block. The other 13 patients, including 2 patients with a positive response to the periarticular block, did not show the PSIS as the main site of pain., Conclusions: Our study clearly indicated that when patients point to the PSIS or within 2 cm of it as the main site of low back pain, using one finger, the SIJ should be considered as the origin of their low back pain.

Background: The aetiology of ulcerative colitis is inadequately understood, and drug therapy has been empirical rather than based on sound understanding of disease aetiology. This has been a major factor for refractoriness and adverse drug effects as additional complications. However, ulcerative colitis by its very nature is exacerbated and perpetuated by inflammatory cytokines, which are released by peripheral granulocytes and monocytes as well. Additionally, active ulcerative colitis is often associated with elevated peripheral granulocytes and monocytes with activation behaviour and are found in vast numbers within the colonic mucosa. Hence, from the clinicopathologic viewpoint, granulocytes and monocytes are appropriate targets for therapy in ulcerative colitis. Based on this thinking, an Adacolumn has been developed for depleting excess granulocytes and monocytes by adsorption., Methods: By colonoscopy, biopsy and histology, we investigated the impact of granulocyte and monocyte adsorption (GMA) on the mucosal level of granulocytes and monocytes in patients with active ulcerative colitis. Forty-five patients (26 steroid naïve and 19 steroid-dependent), mean age 44.7 yr, were included. Twenty patients had total colitis and 25 had left-sided colitis. Each patient was given up to 11 GMA sessions over 12 weeks. No patient received additional medications within 4 weeks (steroid) to 8 weeks (other immunosuppressants) prior to entry or during the GMA course. Colonoscopy together with biopsy was done at entry and within 2 weeks after the last GMA session., Results: At entry, the mean clinical activity index was 12.6; range 10-16. A total of 400 colonic biopsies were examined, which revealed massive infiltration of the colonic mucosa by granulocytes, and GMA was associated with striking reduction of granulocytes in the mucosa. At week 12, 33 of 45 patients (73.3%, P<0.01) had achieved clinical remission (the mean clinical activity index

Interleukin-5 (IL-5) plays a key role in the pathogenesis of bronchial asthma. Thrombin is a procoagulant factor that has been also reported to participate in the inflammatory response by stimulating the secretion of cytokines. Interaction of inflammatory cells with airway epithelial cells may also promote the secretion of cytokines. However, the role of thrombin and cell-to-cell interaction in pathogenesis of allergic inflammation is unclear. In this study, we evaluated the role of thrombin and cell-to-cell interaction in the secretion of IL-5 from basophils. The human basophil cell line KU-812 was used in the assays. Thrombin and co-culture with alveolar epithelial cells significantly stimulated the secretion of IL-5 from KU-812 cells as compared to controls. Secretion of IL-5 was synergistically stimulated when KU-812 cells were incubated in the presence of both thrombin and alveolar epithelial cells. Co-culture of KU-812 cells with epithelial cells significantly increased the expression of tissue factor, an activator of coagulation activation, in a cell dose-dependent manner. Secretion of IL-5 from KU-812 basophils co-cultured with epithelial cells was significantly inhibited by LY294002, an inhibitor of phosphatidylinositol 3-kinase. These results suggest that thrombin and cell interaction with lung epithelial cells may augment the inflammatory response in allergic diseases by stimulating the secretion of IL-5 from basophils.

Background: Accurate diagnosis and appropriate therapy for low back pain are based on knowledge of the site of the patient's pain. In clinical settings, patients generally indicate the site of low back pain using the hand, but Chapman and Syrjala as well as Macnab recommended that the patient should indicate the site with the finger. Whether any differences emerge from these methods is an important fundamental question. The purpose of this study was to compare low back pain sites identified by the patient's finger versus the hand. Specifically, we conducted a prospective randomized controlled comparison and investigated the surface area and the accuracy of the sites indicated by the two identifications., Methods: Subjects were 137 low back pain patients meeting the following criteria: (1) age 18-65 years; (2) low back pain induced by lumbar flexion, extension, or lateral bending while standing; (3) absence of leg pain; and (4) pain at only one site. Subjects indicated the site of pain by hand and by index finger immediately after provocation of pain. A demonstrably reproducible site was outlined on the skin. The outlines of the sites were recorded and verified by digital images. The body surface area of the sites was determined using NIH Image Version 1.61. Sites identified by hand versus index finger were compared on the surface area. Additionally, to compare the accuracy of the two identifications, patients were asked which of two sites more accurately represented the pain site in outline form after the two identification procedures., Results: The surface area of sites identified by the finger was significantly smaller than that of sites identified by the hand (P < 0.05). Furthermore, a considerably higher proportion, approximately 80%, of patients answered that the site of the pain was identified more accurately by finger than by hand., Conclusions: The indication by a finger resulted in more accurate, localized identification of the site of low back pain than the indication by a hand. The identification of the pain site with a patients' finger indication does not directly clarify the etiology of low back pain, but it has the potential to provide important information useful for making an accurate diagnosis and for determining the appropriate therapy.

Background: The sacroiliac joint (SIJ) can be a source of low back pain. Previous studies indicated that SIJ pain could originate from both the joint capsule and the posterior ligamentous tissues. It has not been clarified as to whether an intraarticular or periarticular injection procedure is more effective for this type of pain. The purpose of this study was to evaluate the effect of two injection procedures prospectively., Methods: After a pain provocation test, an intraarticular injection of local anesthetic (2% lidocaine) was performed on the first 25 consecutive patients with SIJ pain and a periarticular injection on another 25. The periarticular injections were given to one or more sections of the posterior periarticular area of the SIJ and to another section in the extracranial portion. The effect of these injections was assessed using the "restriction of activities of daily life" scoring system from the Japanese Orthopaedic Association., Results: The periarticular injection was effective in all patients, but the intraarticular one was effective in only 9 of 25 patients. An additional periarticular injection was performed in 16 patients who experienced no effect from the initial intraarticular injection and was considered effective in all of them. The injection into the middle of the periarticular area was more effective for SIJ pain. The improvement rate after the periarticular injection was 96%, which was significantly higher than that after the intraarticular injection, which was 62%., Conclusions: For patients with SIJ pain, periarticular injection is more effective and easier to perform than the intraarticular injection and should be tried initially.

Nitric oxide (NO) plays a critical role in the pathogenesis of endotoxin-induced uveitis (EIU). Since NO is a labile free radical, it is difficult to examine the dynamics of NO directly in vivo. In this study, we established a system for direct monitoring of the dynamics of NO and partial pressure of oxygen (pO(2)) in EIU in rabbits. The currents (calculated concentrations) of NO and pO(2) in the vitreous were monitored after the intravitreal injection of lipopolysaccharide (LPS). In addition, the protein concentrations and nitrite levels in the aqueous humor were analyzed. The eyes were enucleated, and a histologic study was performed on their posterior segments. The tissue slices were also immunostained with anti-nitrotyrosine as a marker of peroxinitrite and/or nitrogen-related oxidants. The NO level decreased temporarily after LPS injection and then increased from 1 to 7 h. pO(2) increased temporarily for about 30 min after LPS injection. The change in NO current was inversely proportional to pO(2) after LPS injection and vice versa. The protein concentration and nitrite level after LPS injection increased significantly. These changes were suppressed by pretreatment with N(G)-nitro-l-arginine-methyl-ester. Immunohistochemical study showed enhanced immunoreactivity of nitrotyrosine in the inflamed retina. Since nitrotyrosine was detected, it appears that NO readily reacts with oxygen to produce cytotoxic species, peroxynitrite, and/or nitrogen-related oxidants. This process may be related to the retinal injury in EIU. This monitoring system can provide useful information on dynamic changes in intravitreal NO and pO(2) for understanding EIU.

A novel non-chain terminating nucleoside analog anti-HIV inhibitor, KP-1212 has been designed to form base pairs with multiple bases that may lead to mutagenesis in the HIV-1 viral genome. After multiple replication cycles, the accumulation of mutations surpasses a crucial threshold beyond which the virus can no longer replicate. HIV-1 reverse transcriptase (RT) incorporates the KP-1212 monophosphate into the genome during viral replication after metabolic activation of the KP-1212 nucleoside to the triphosphate. The propensity for forming alternate base pairs with the KP-1212 nucleotide leads to mismatched nucleotides and the subsequent misincorporation is the basis for the inhibitory activity. The results showed that HIV-1 RT and human mitochondrial DNA polymerase (Pol gamma) incorporated KP-1212-TP with a significant level of efficiency, whereas mouse DNA polymerase beta (Pol beta) did not. Misincorporation studies suggest that both HIV-1 RT and Pol gamma may cause mutations at significantly high rates. These in vitro data confirm the mechanistic basis of KP-1212 as a viral mutagen but suggest that there may be a potential for toxicity to the mitochondria.

Enantiomers of beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D/L-D4FC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) currently under investigation as antiviral agents. One of the major problems of NRTIs is toxicity to mitochondria. It has been shown that mitochondrial toxicity of NRTIs can correlate with incorporation and removal of these compounds by mitochondrial DNA polymerase (Pol gamma). Mechanistic studies have shown that, if activated, NRTIs are incorporated more efficiently by HIV-1 reverse transcriptase (RT) and less efficiently by Pol gamma, the corresponding nucleosides are considered to be more selective. In the present study, in order to predict potential DNA Pol gamma-related mitochondrial toxicity of D- and L-D4FC, the incorporation and removal of the monophosphate form of these compounds by Pol gamma were studied using transient kinetic methods. Our cell-free results showed that Pol gamma incorporated the natural D-isomer significantly more efficiently than the unnatural L-isomer. However, the removal rates of these enantiomers from the chain-terminated primers were almost identical. While these results suggest that D-D4FC may present more mitochondrial toxicity than L-D4FC in cell-free assays, we have previously shown that HIV-1 RT prefers D-D4FC-TP as a substrate over the L-isomer, particularly in the case of mutant forms of RT associated with nucleoside drug resistance such as M184V. Since the effectiveness of NRTIs is a balance between efficiency of incorporation by wild-type and drug-resistant forms of HIV-1 RT and mitochondrial toxicity, our kinetic results suggest that both enantiomers may show promise as potential therapeutics.

Using a Langendorff-perfused rat heart preparation and selective electrodes, we determined nitric oxide (NO) and oxygen levels in cardiac tissue. An NO-selective electrode that was calibrated by electron spin resonance (ESR) spectroscopy was inserted into the middle of the myocardium in the left ventricle. Simultaneously, we used an O2-selective electrode to measure the partial pressure of oxygen (pO2) in the perfusate, Krebs-Henseleit (K-H) solution, that was ejected from the heart. After 30 min of aerobic control perfusion, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Under ischemic conditions, with a gradually decreasing pO2, NO detected by an NO-sensitive electrode within the myocardium was gradually increased. The maximum concentration increases in NO and decreases in pO2 during global ischemia were +10.200 +/- 1.223 microM and -58.608 +/- 4.123 mmHg, respectively. NO and pO2 levels both recovered to pre-ischemia baseline values when perfusion was restarted after global ischemia (reperfusion). The presence of Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mM), a NOS inhibitor, prevented ischemia/reperfusion-induced changes in NO. This study shows that an NO-selective electrode that is calibrated by ESR can provide accurate, real-time monitoring of cardiac NO in normal and ischemic myocardium.

Both the beta-D-(+) and beta-L-(-)-enantiomers of 2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D4FC) are clinically relevant compounds because of their potent anti-HIV and anti-HBV activities. Cross-resistance to L-D4FC with HBV containing a mutation in the conserved polymerase YMDD region has been observed. In order to better understand the effects of stereochemistry on planar 5-fluorinated cytidine analogs and to gain insight into resistance caused by YMDD mutations in HIV-1 reverse transcriptase (RT), a combination of transient kinetic studies and computer modeling were employed. In contrast to studies with the (+) and (-) isomers of 3TC-TP and FTC-TP, it was found that wild type RT had a high enantiomeric selectivity between the D-(+) and L-(-) isomers of D4FC-TP. While no resistance was conferred by the methionine 184 to valine mutation to D-D4FC-TP, L-D4FC-TP was incorporated 50- to 70-fold less efficiently. The kinetic parameters of incorporation in the presence of L-D4FC-TP by RT(WT) and the mechanism of resistance by RT(M184V) were found to be distinct from those seen with the corresponding L-isomers containing an oxathiolane ring: (-)-3TC-TP and (-)-FTC-TP. Molecular modeling suggests that L- and D-D4FC-TP are positioned in the active site favorably for incorporation by RT(WT) and that L-D4FC-TP, but not D-D4FC-TP, is sterically hindered by the addition of a beta branched amino acid at position 184 of RT(M184V).

Study Objectives: We sought to assess the incidence of transient U-wave inversion during vasospasm of the left anterior descending coronary artery (LAD) with ST-segment depression as opposed to that with ST-segment elevation., Design: Retrospective study., Setting: Cardiology division of acute-care hospitals., Patients: We studied 49 patients with vasospastic angina whose vasospasm was induced in the LAD, not in the left circumflex coronary artery, by intracoronary injection of acetylcholine., Measurements and Results: The ECG traces obtained during acetylcholine-induced vasospasm of the LAD were examined. Based on the direction of ST-segment shift, the patients were categorized into two groups: the ST-segment elevation group (n = 27) and the depression group (n = 22). There were no differences in age, gender, or cardiovascular risk factors between the two groups. The distribution of the spastic site in the LAD was also similar. A total reduction in luminal diameter during a provoked attack was more often observed in the ST-segment elevation group than in the ST-segment depression group (37% vs 9%, p = 0.02). Collateral circulation to the LAD was found in only one patient in each group. There were no differences between the two groups in heart rate, systolic BP, and double product of heart rate and systolic BP during the attack. The incidence of acetylcholine-induced anginal attack with U-wave inversion in the ST-segment depression group was nearly as high as that in the ST-segment elevation group (77% vs 78%, p > 0.99)., Conclusions: The development of transient U-wave inversion during vasospasm of the LAD induced by intracoronary injection of acetylcholine does not depend on the magnitude of myocardial ischemia as judged by the direction of ST-segment shift.

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.

Background: There has been no report of ECG changes during anginal attacks in patients with coexistent hypertrophic cardiomyopathy (HCM) and vasospastic angina., Study Objectives: To elucidate the change in ST-segment during anginal attacks in patients with coexistent HCM and vasospastic angina (the HCM group) in comparison with that of patients with vasospastic angina and no left ventricular hypertrophy (the non-HCM group)., Design: Retrospective study., Patients: Twelve patients in the HCM group, and 28 patients in the non-HCM group., Measurements: The direction of ST segment shift, either ST-segment elevation or depression, on the ECGs recorded during vasospastic anginal attacks with severe vasoconstriction in the epicardial coronary artery after intracoronary injection of acetylcholine., Results: Age, male gender, and distribution of coronary arteries in which the vasospasm occurred were similar between the two groups. Collateral circulation to the affected arteries was absent in all the study patients. The prevalence of anginal attacks associated with ST-segment elevation was 2.7 times higher in the non-HCM group than in the HCM group (51. 5% [17 of 33 attacks] vs 18.8% [3 of 16 attacks], respectively; p = 0.03)., Conclusions: In the HCM group, myocardial ischemia associated with a transmural injury pattern seen on the ECG, which is represented as ST-segment elevation, seldom develops during vasospastic anginal attacks because of marked left ventricular hypertrophy.

Comparative studies concerning glycoform analysis of sialoglycoproteins by capillary electrophoresis were performed using a few separation modes hitherto reported. Glycoprotein samples examined in the present study were successfully separated to their respective glycoforms using surface-modified capillaries commercially available for capillary gas chromatography in the running buffer near their isoelectric points. The analysis times were less than 50 min and reproducibilities in migration times were excellent (less than 2.0% RSD for both run-to-run and day-to-day analyses). We present a method for the glycoform analysis of alpha1-acid glycoprotein in sera by simple pre-treatment as an application. The present technique will become one of the general methods for the evaluation of glycosylation heterogeneity of commercially available glycoprotein drugs.

Nicotinic acid, nicotinamide and their possible metabolites were successfully separated within 17 min by micellar electrokinetic chromatography using 50 mM borate buffer (pH 9.0) containing 150 mM sodium dodecyl sulfate as the running buffer. Calibration curves for all compounds showed good linearity in a range of 5 microg/ml and 250 microg/ml with good correlation. The present method did not require any clean-up procedures and made it possible to determine all metabolites without interference on a photodiode array detector. Urine samples collected from Wistar male rats were analyzed after high-dose oral or intravenous administration of nicotinic acid or nicotinamide. Metabolic pathways of nicotinic acid in male Wistar rats are also discussed.

Background: For high-risk Fontan candidates, the introduction of a bidirectional Glenn shunt before total cavopulmonary connection (a two-staged strategy) may extend the indications for the Fontan procedure. The clinical results of the two-staged and one-staged Fontan procedure were thus reviewed and compared., Methods: Between November 1991 and July 1996, the two-staged strategy was performed in 40 high-risk Fontan candidates with a mean interval of 17.2 months after introducing the bidirectional Glenn shunt (staged group). We considered a young age (<2 years), high mean pulmonary arterial pressure (> or =20 mm Hg), high pulmonary vascular resistance (> or =3 Wood units), small pulmonary artery (Nakata index <200 mm2/m2), atrioventricular valve incompetence (> or = moderate), distortion of pulmonary artery, anomalous pulmonary venous return, and poor ventricular function as risk factors for the successful completion of Fontan circulation. During the same period, 68 patients underwent the modified Fontan procedure in a one-step fashion (primary group)., Results: In the staged group after the bidirectional Glenn shunt, the mean pulmonary arterial pressure and ventricular end-diastolic pressure were both found to have decreased significantly to the same level as those in the primary group, whereas the pulmonary artery demonstrated a significantly smaller size than that in the primary group. Operative morbidity was similar in both groups. Operative mortality was also similar and low in both groups (1.5% in the primary group and 0% in the staged group)., Conclusions: A bidirectional Glenn shunt was found to be a useful interim palliation in high-risk Fontan candidates. This two-staged strategy may extend the operative indications for the Fontan procedure.

25,26,27,28-tetrakis(N,N-diethylaminocarbonylmethoxy)-5,11,17,23-tetrakis(1,1,3,3-tetramethylbutyl)calix[4]arene, a macrocyclic extraction reagent, and p-(1,1,3,3-tetra-methylbutyl)phenoxymethyl-N,N-diethylamide, an acyclic extraction reagent corresponding to the former one, were synthesized to investigate their extraction behavior for silver(I), gold(III), palladium(II), and platinum(IV) from highly acidic solution into chloroform. In the extraction of silver and gold from hydrochloric acid solution, a completely different extraction behavior was observed between these two types of the reagents. The extraction behavior was examined in detail for silver and was found to be dependent on whether silver ion was extracted as a cationic species or a anionic species complexed with chloride ion. This was supported by proton nuclear magnetic resonance study of the calix[4]arene derivative. As a result, the extraction of silver ion with calix[4]arene derivative was very peculiar which was attributable to the fitting between cyclic size of calix[4]arene and ionic radius of silver.

In order to directly detect nitric oxide (NO) liberated from isolated tissue, a practical and convenient method using a nitric oxide-sensitive electrode is described. To avoid the nonselective signal caused by ionic substances, the electrode was covered with three layers but remains permeable for gaseous substances. In a solution bubbled with 20% oxygen (pO2, approximately 150 mm Hg), administration of S-nitroso-N-acetyl-d, l-penicillamine (SNAP) at concentrations greater than 10(-7) mol/L elicited an electrode response. Based on a comparison with the chemical determination of NO released from SNAP, the electrode may be able to detect nitric oxide around nmol/L. At least 30 nmol NO per liter in anoxic conditions was reported to be detected by this electrode (Matsui, 1995). In a specially designed small chamber, the electrode was attached on the surface of endothelial side of the isolated aorta of the guinea pig. When carbachol was added to the chamber, the electrode responded when the solution was bubbled with 20% but not with 40% or 95% of oxygen, suggesting a much faster decomposition of nitric oxide in the presence of higher concentrations of oxygen. The electrode response to carbachol was abolished in the presence of NG-monomethyl-L-arginine or nitro arginine. These results suggest that the electrode method described in this manuscript is suitable for detecting nitric oxide liberated from isolated tissues when comparatively low oxygen levels are present in the physiological salt solution.

The effects of early reperfusion on the infarct area were evaluated by measuring the plasma creatine phosphokinase (CPK) activity and myosin light chain I (LCI) in 30 patients with acute myocardial infarction. Twenty of these patients underwent coronary angiography, of whom 9 had reperfusion with successful intracoronary thrombolysis, which revealed significant correlations between peak values of LCI and peak values of CPK or CPK-MB activity (r = 0.775, p < 0.01 or r = 0.783, p < 0.01). Similarly, peak value of LCI correlated with left ventricular ejection fraction (r = -0.729, p < 0.01) and the infarct size which was estimated according to the extent and severity scores measured by Tl-201 myocardial SPECT (vs extent score, r = 0.439, p < 0.05; vs severity score, r = 0.429, p < 0.05). The time activity curves of plasma CPK activity and LCI differed in patients with and without reperfusion; in the former, mean peak values of CPK activity and LCI were 1,170 +/- 321 U/L (mean +/- SD) and 10.7 +/- 3.5 ng/ml, respectively, while in the latter, they were 5,430 +/- 3,315 U/L and 25.2 +/- 12.9 ng/ml, respectively. The times to peak values of LCI and CPK did not differ between these 2 patient groups. We concluded that the early reperfusion suppresses the progressive extension of irreversible injury in the infarct area and significantly diminishes the infarct size.

To examine the efficacy of magnetic resonance imaging (MRI) in diagnosing hypertrophic cardiomyopathy (HCM), 16 patients with HCM and 14 hypertensives with left ventricular hypertrophy (LDH) were studied using a 0.5 Tesla Siemens MRI apparatus equipped with cardiac gating. In HCM, left ventricular hypertrophy was localized to the septal wall in four, to the apical wall in two, to both the septal and apical walls in two, and to the apical and inferior walls in one, and it was diffuse in seven patients. In hypertensives, LVH was localized to the septal wall in three, to both the septal and anterior walls in two, to the free wall in one, and it was diffuse in eight patients. The distribution of the hypertrophic portion was nearly equal in both groups. The thickest portion of the left ventricular wall was 24.6 +- 4.8 mm in HCM and 21.6 +- 5.4 mm in hypertension, and there was no significant difference between them. The T2 relaxation time of the hypertrophic portion was 52.2 +- 4.8 msec in HCM and 45.3 +- 6.1 msec in hypertension, and there was a significant difference between them (p less than 0.01). However, there were no significant differences between the T2 relaxation times of the hypertrophic and non-hypertrophic portions in both groups. In conclusion, it may be difficult to differentiate HCM from hypertension based on the distribution of hypertrophic portions, but measurements of the T2 relaxation times may be useful for making the differential diagnosis.

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17 beta estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000-65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.