Low incidence of hepatitis B virus reactivation and subsequent hepatitis in patients with chronic hepatitis C receiving direct-acting antiviral therapy.

To determine the clinical characteristics of hepatitis B virus (HBV) reactivation in patients undergoing interferon-free antihepatitis C virus (HCV) therapy, we examined HBV DNA in 25 HBV co-infected patients and 765 patients with resolved HBV infection during and after treatment with direct-acting antiviral agents (DAAs). Among those with HCV genotype 1, asunaprevir plus daclatasvir was administered to 160 patients, sofosbuvir (SOF) plus ledipasvir to 438 patients and paritaprevir plus ombitasvir and ritonavir to 25 patients. In total, 167 patients with genotype 2 were treated with SOF plus ribavirin. Three patients with an HBV DNA level ≥2000 IU/mL were treated with entecavir before anti-HCV therapy, without reactivation of HBV. In 3 of 22 (12%) HBV surface antigen (HBsAg)-positive patients with an HBV DNA level <2000 IU/mL, the viral load increased during treatment. However, hepatitis flare did not occur in these patients. There was no significant difference in clinical history between patients with and without HBV reactivation. Among 765 patients with resolved HBV infection, HBV reactivation occurred in 1 (0.1%) patient after initial resolution, whose HBV DNA level spontaneously decreased after DAA therapy. We compared anti-HBs titres at baseline with those at post-DAA therapy in 123 patients without HBsAg. There was no significant difference in anti-HBs levels between the two points (P = .79). In conclusion, HBV reactivation was rare in HBsAg-negative patients treated with DAA therapy. Additionally, hepatitis did not occur in HBV-reactivated patients with a baseline HBV DNA level <2000 IU/mL before DAA therapy.

Pharmacokinetics, efficacy and safety of daclatasvir plus asunaprevir in dialysis patients with chronic hepatitis C: pilot study.

The aim of this study was to evaluate the pharmacokinetic profile of daclatasvir (DCV) and asunaprevir (ASV) dual therapy in haemodialysis patients infected with hepatitis C virus (HCV). Eighteen haemodialysis patients and 54 patients with normal renal function were treated with DCV and ASV dual therapy for 24 weeks. We evaluated the pharmacokinetic profiles of DCV and ASV and examined the rate of sustained virological response 12 weeks after the end of treatment (SVR12 ) and incidence of adverse events during treatment of haemodialysis patients infected with chronic HCV genotype 1 infection. To adjust for potential differences in baseline characteristics between haemodialysis patients and patients with normal renal function, we used propensity scores case-control matching methods. Area under the plasma concentration time curve from 0 to 6 h (AUC0-6 h ) of DCV was slightly lower in haemodialysis patients than in patients with normal renal function (P > 0.6). AUC0-6 h of ASV was significantly lower in haemodialysis patients (P = 0.012). SVR12 rates were 100% (18/18) for haemodialysis and 96.2% (52/54) for patients with normal renal function. Changes in mean log10 HCV RNA levels and viral response were higher in haemodialysis patients compared to patients with normal renal function. No discontinuations due to adverse events occurred. In conclusion, DCV and ASV dual therapy for HCV infection is effective and safe with similar results in haemodialysis patients compared to patients with normal renal function.

Thermochromic properties of Sn, W co-doped VO2 nanostructured thin film deposited by pulsed laser deposition.

Tin (Sn) and tungsten (W) co-doped vanadium dioxide (VO2) nanostructured thin films with 50-nm thickness were deposited by pulsed laser deposition (PLD) to reduce the transition temperature and improve the IR transmittance. The crystal structure of the nanostructured thin films and the presence of elements were evaluated by XRD and XPS analysis. The transition temperature (T(c)) of 1 at% Sn-1 at% W co-doped VO2 nanostructured thin film was decreased to about 22 degrees C (from 70.3 to 48.5 degrees C) compared with the undoped VO2 nanostructured thin film. The transmittance width in the IR range of the co-doped nanostructured thin film decreased from 37.5% to 27% compared with the undoped VO2 nanostructured thin film. Also, the width of hysteresis was narrowed by Sn doping.

Factors associated with the presence of multiple Lugol-voiding lesions in patients with esophageal squamous-cell carcinoma.

Multicentric squamous dysplasia of the esophagus is characterized by multiple Lugol-voiding lesions (LVLs) on Lugol chromoendoscopy. Multiple LVLs are associated with a very high risk of multiple cancers arising in the esophagus as well as the head and neck. To gain insight into the pathogenesis of multiple LVLs of the esophageal mucosa, we studied risk factors for the development of such lesions in 76 patients who had a current or previous diagnosis of esophageal squamous cell carcinoma. All patients underwent Lugol chromoendoscopy of the esophageal mucosa. The history of tobacco and alcohol use was documented. Polymorphisms of the aldehyde dehydrogenase type 2 (ALDH2) gene were identified by polymerase chain reaction using sequence-specific primers. Clinical factors related to multiple LVLs were analyzed. All patients with multiple LVLs were drinkers. On univariate analysis, male sex (odds ratio [OR] 15, 95% confidence interval [CI] 1.84-122.45: P = 0.011), presence of the ALDH2-2 allele (OR 4.5, 95% CI 1.55-13.24: P = 0.006), and smoking index ≥1000 (OR 2.6, 95% CI 1.02-6.6: P = 0.045) were associated with multiple LVLs. On multivariate analysis, male sex (OR 10.02, 95% CI 1.13-88.44: P = 0.038) and presence of the ALDH2-2 allele (OR 4.56, 95% CI 1.4-14.82: P = 0.012) were associated with multiple LVLs. Among drinkers, a daily alcohol intake of ≥100 g pure ethanol with the ALDH2-2 allele (OR 17.5, 95% CI 1.97-155.59: P = 0.01) and a daily alcohol intake of <100 g pure ethanol with the ALDH2-2 allele (OR 8.85, 95% CI 1.68-46.69: P = 0.01) more strongly correlated with multiple LVLs than did a daily alcohol intake of <100 g pure ethanol without the ALDH2-2 allele, whereas a daily alcohol intake of ≥100 g pure ethanol without the ALDH2-2 allele (OR 4.0, 95% CI 0.54-29.81: P = 0.18) did not. In conclusion, male sex and the ALDH2-2 allele are associated with an increased risk for multiple LVLs of the esophageal mucosa in patients with esophageal squamous cell carcinoma. Among drinkers with the ALDH2-2 allele, the risk of multiple LVLs increased in parallel to the daily alcohol intake.

A novel triple secured technique for pancreatic reconstruction following pancreaticoduodenectomy for a soft pancreas.

BACKGROUND/AIMS: Soft pancreases are susceptible to developing pancreatic fistula following pancreaticoduodenectomy. To reduce the incidence of pancreatic fistula after pancreaticoduodenectomy in patients with a soft pancreas, we developed a triple secured technique. In this study, we describe the details of this technique and also report on the postoperative outcomes. METHODOLOGY: The triple secured technique employed an ultrasonic dissector for pancreatic transection with skeletonizing and ligating of the small pancreatic branch ducts, duct-invagination or duct-to-mucosa anastomosis for main pancreatic duct management, and, finally, four large stitches between the pancreatic stump parenchyma and the jejunal seromuscular layer to prevent minor pancreatic leakage. A total of 28 consecutive patients with a soft pancreas who underwent pancreaticoduodenectomy using our technique were included in this study. RESULTS: Postopetrative complications occurred in 16 patients. Grade B pancreatic fistula developed in 6 patients. However, no grade C pancreatic fistula occurred in this series. Neither any reoperation nor in-hospital mortality was observed in this series. CONCLUSIONS: Our triple secured technique after pancreaticoduodenectomy was feasible and safe, with an acceptable rate of grade B pancreatic fistula and no grade C pancreatic fistula for patients with a soft pancreas.

New flexible endoscopic full-thickness suturing device: a triple-arm-bar suturing system.

BACKGROUND AND STUDY AIM: A reliable full-thickness suturing device is necessary for pure natural orifice transluminal endoscopic surgery (NOTES). The present study focused on assessing the reliability of a new suturing device. METHODS: A total of 60 single sutures were tested to close 5-cm incisions in 8-cm square pieces of resected swine stomach. Each incision was sutured by an over-the-scope clip (OTSC; n = 20), a single hand-sewn stitch (n = 20), or a single triple-arm-bar suturing system (TBSS) stitch. The maximum pulling force durability (MPD) of each suture was tested. To assess the reliability of the TBSS for endoscopic full-thickness resection (EFTR), 60 EFTRs of 50 mm diameter were performed on excised swine stomachs. After EFTR, full-thickness sutures were made using 3-stitch OTSCs (n = 20), 10-stitch hand-sewn sutures (n = 20), or 10-stitch TBSS sutures (n = 20). Outcomes were the MPD test for both single stitch and multiple stitch applications and the suturing time for single-stitch sutures. RESULTS: In the single-stitch MPD tests, there were significant differences between OTSCs and hand-sewn sutures (P = 0.0002) and between OTSCs and TBSS sutures (P = 0.0001), but no significant difference between hand-sewn and TBSS sutures. The multiple-stitch sutures revealed significant differences between OTSCs and hand-sewn sutures (P = 0.0039), and between OTSCs and TBSS sutures (P = 0.013). There was no significant difference between hand-sewn and TBSS sutures. There were significant differences in suture times between OTSC, hand-sewn sutures, and TBSS sutures (P < 0.05). CONCLUSIONS: Both single-stitch and multiple-stitch sutures using TBSS have similar strength to hand-sewn sutures. TBSS is a reliable suturing device.

Aberrant activation of the intrarenal renin-angiotensin system in the developing kidneys of type 2 diabetic rats.

We have previously reported that intrarenal angiotensin II (Ang II) levels are increased long before diabetes becomes apparent in obese Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of type 2 diabetes. In this study, we examined the changes in intrarenal renin-angiotensin system (RAS) activity in the developing kidneys of OLETF rats. Ang II contents and mRNA levels of RAS components were measured in male OLETF and control Long-Evans Tokushima (LETO) rats at postnatal days (PND) 1, 5, and 15, and at 4-30 weeks of age. In both LETO and OLETF rats, kidney Ang II levels peaked at PND 1, then decreased during the pre- and post-weaning periods. However, Ang II levels and gene expression of RAS components, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), were not significantly different between LETO and OLETF rats. Intrarenal Ang IIcontents further decreased during puberty (from 7 to 11 weeks of age) in LETO rats, bur not in OLETF rats. At 11 weeks of age, kidney Ang II levels, urinary AGT excretion, and mRNA levels of AGT and renin were higher in OLETF rats than in LETO rats, while blood glucose levels were not significantly different between these groups of rats. These data indicate that continued intrarenal expression of Ang II during pubescence contributes to the increases in intrarenal Ang II levels in prediabetic OLETF rats, and is associated with increased intrarenal AGT and renin expression. Inappropriate activation of the intrarenal RAS in the prediabetic stage may facilitate the onset and development of diabetic nephropathy in later life.

Persistence of covalent bonding in liquid silicon probed by inelastic x-ray scattering.

Metallic liquid silicon at 1787 K is investigated using x-ray Compton scattering. An excellent agreement is found between the measurements and the corresponding Car-Parrinello molecular dynamics simulations. Our results show persistence of covalent bonding in liquid silicon and provide support for the occurrence of theoretically predicted liquid-liquid phase transition in supercooled liquid states. The population of covalent bond pairs in liquid silicon is estimated to be 17% via a maximally localized Wannier function analysis. Compton scattering is shown to be a sensitive probe of bonding effects in the liquid state.

Local steroid injection into the artificial ulcer created by endoscopic submucosal dissection for gastric cancer: prevention of gastric deformity.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) of large gastric lesions results in an extensive artificial ulcer that can lead to marked gastric deformity. The aim of the current study was to evaluate therapeutic efficacy in the prevention of gastric deformity of local triamcinolone acetonide (TCA) injection into the extensive artificial ulcer following ESD. PATIENTS AND METHODS: A total of 45 patients who were diagnosed with early gastric cancer were enrolled. Patients were randomly assigned by the sealed-envelope randomization method to either local TCA injections (n = 21) or sham-control (n = 20) groups. Two clips were placed at the two maximum outer edges of the artificial ulcer after the lesion had been resected (Day 0). Local TCA injections were performed on postoperative Day 5 and Day 12. The distance between the two clips was measured by endoscopic measuring forceps on Days 5, 12, 30, and 60. Granulation formation and gastric deformity were evaluated by visual analog scale (VAS) on Days 30 and 60. RESULTS: Local TCA injection did not alter clip-to-clip distance on postoperative Day 60, and formation of flat granulation tissue over the ulcer was followed by regenerative mucosa without any gastric deformity. The sham-control group showed significant shortening of clip-to-clip distance compared with the local steroid-injected group and protruded forms of granulation tissue with mucosal convergence. Histological evaluation revealed prominent growth of neovessels, swelling, and marked increases in endothelial cells in the local steroid-injected group compared with the sham-control group. CONCLUSIONS: Local steroid injection into the floor of a post-ESD artificial ulcer promotes the formation of granulation tissue at an early stage of the healing process leading to regeneration of gastric mucosa without mucosal convergence or gastric deformity.

Monocyte chemoattractant protein-1 is associated with silent cerebral infarction in patients on haemodialysis.

BACKGROUND: In patients with chronic renal failure undergoing haemodialysis (HD), silent cerebral infarctions (SCI) are associated with high mortality. Levels of monocyte chemoattractant protein-1 (MCP-1) increase with renal dysfunction and may be a novel predictor for cerebrovascular events. We tested the hypothesis that increased MCP-1 concentration correlate with the occurrence of SCI in HD patients. METHODS: Using cranial magnetic resonance imaging (MRI) findings, 52 Japanese patients undergoing HD were divided into two groups: with SCI (61 ± 7 years, mean ± SD, n= 28) and without SCI (60 ± 6 years, n= 24). The gender, metabolic profiles and MCP-1 concentration were compared between the two groups. RESULTS: The level of MCP-1 was higher in the with-SCI group than in the without-SCI group (P < 0.0001). The proportion of smokers was higher in the with-SCI group (P < 0.05) than in the without-SCI group. Plasma level of high-density lipoprotein cholesterol was lower, while uric acid level was higher, in the with-SCI group (P < 0.05 and P < 0.05 respectively) compared to the without-SCI group. Multiple logistic regression analysis identified MCP-1 level as being significantly associated with the presence of SCI (odds ratio 1.48, 95% confidence interval = 1.10-5.75, P < 0.0001). CONCLUSIONS: This study indicates that patients with chronic renal failure who are maintained on HD exhibit an increased prevalence of SCI, and that MCP-1 is significantly associated with the presence of SCI in HD patients.

Establishment of pure NOTES procedure using a conventional flexible endoscope: review of six cases of gastric gastrointestinal stromal tumors.

An increasing number of reports have recently been published on hybrid natural orifice transluminal endoscopic surgery (NOTES). These reports do not address how to complete an operation with a flexible endoscope alone (pure NOTES), but rather how to combine use of an endoscope and a laparoscope. Surgical procedures using flexible and rigid endoscopes have been developed using different processes and concepts. Recognizing this conceptual difference, we conducted a study to address how to establish a pure NOTES procedure. Six patients with gastric gastrointestinal stromal tumors (GISTs) underwent hybrid NOTES. Each case was retrospectively reviewed to determine the appropriateness of the treatment and the usefulness of the endoscopic submucosal dissection (ESD) method, double-scope method, spaced perforation method, duodenal balloon occlusion method, and loop clip technique. The development of operative procedures that take advantage of the characteristics of flexible endoscopes, even with conventional flexible endoscopic devices and conventional endoscopes alone, may contribute to the realization of pure NOTES.

Subdose of fasudil suppresses myocardial fibrosis in aldosterone-salt-treated uninephrectomized rats.

Rho/Rho kinase (ROCK) pathway plays an important role in pathological cardiovascular conditions. In the present study, the effect of a subdose of fasudil, a selective ROCK inhibitor, on systemic hypertension and myocardium fibrosis induced by aldosterone was investigated in uninephrectomized Sprague-Dawley rats (SD). Treatment with a fasudil (10 mg/kg x day, s.c.) for 5 weeks decreased the activity of ROCK activity for more than 53% as determined by the expression of phosphorylated Myosin phosphatase target subunit 1 (MYPT1). Although this dose of fasudil did not signifantly prevent hypertension, it remarkably alleviated myocardium hypertrophy and fibrosis. The elevated transcriptional expression of transforming growth factors beta1 (TGF-beta1), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and collagen I and III was also decreased. These results demonstrated that fasudil can protect the myocardium from injury by aldosterone at a subhypertensive dose.

Narrow band imaging for detecting superficial squamous cell carcinoma of the head and neck in patients with esophageal squamous cell carcinoma.

BACKGROUND AND STUDY AIMS: Narrow band imaging combined with magnifying endoscopy (NBI-ME) is useful for the detection of superficial squamous cell carcinoma (SCC) within the oropharynx, hypopharynx, and oral cavity. The risk of a second primary SCC of the head and neck is very high in patients with esophageal SCC. This prospective study evaluated the detection rate of superficial SCC within the head and neck region (superficial SCCHN) with NBI-ME in patients with esophageal SCC. PATIENTS AND METHODS: Between March 2006 and February 2008, 112 patients with a current or previous diagnosis of esophageal SCC were enrolled. All patients underwent endoscopic screening of the head and neck by NBI-ME. The primary end point was the detection rate for superficial SCCHN. Secondary end points were to compare demographic characteristics between patients with and without superficial SCCHN and to assess the clinical course of patients with superficial SCCHN. RESULTS: The detection rate for superficial SCCHN was 13 % (15/112). The prevalence of multiple Lugol-voiding lesions, observed endoscopically throughout the esophageal mucosa after application of Lugol dye solution, was significantly higher in patients with superficial SCCHN than in those without (100 % vs. 24 %, P < 0.0001). Minimally invasive curative treatment with organ preservation was feasible without severe complications in patients with superficial SCCHN after curative treatment of esophageal SCC. CONCLUSIONS: In patients with esophageal SCC, NBI-ME is useful for detecting superficial SCCHN, thereby facilitating minimally invasive treatment.

Visceral fat accumulation is a significant risk factor for white matter lesions in Japanese type 2 diabetic patients.

BACKGROUND: The presence of white matter lesions (WML) is an important prognostic factor for the development of stroke. The elevated visceral fat accumulation (VFA) has been reported to be closely related to the development of atherosclerosis. This preliminary study was therefore designed to test the hypothesis that the presence of WML correlates with VFA and insulin resistance in type 2 diabetic patients not receiving insulin treatment. MATERIAL AND METHODS: Based on brain magnetic resonance imaging (MRI), 95 type 2 diabetic patients were divided into two groups: WML-positive group (aged 59 +/- 7 years, mean +/- SD n = 37) and WML-negative group (aged 58 +/- 5, years, n = 58). The level of blood glucose was assessed by fasting plasma glucose (FPG), fasting immunoreactive insulin, homeostasis model assessment (HOMA) index, and haemoglobin A1c. The fat distribution was evaluated by measuring the visceral fat accumulation by abdominal computerized tomography at the umbilical level. RESULTS: The body mass index was higher in the WML-positive group than in the WML-negative group (P < 0.005). Plasma levels of triglycerides were higher while high-density lipoprotein cholesterol was lower in the WML-positive group than in the WML-negative group (P < 0.05 and P < 0.01, respectively). FPG (P < 0.01), insulin concentrations (P < 0.0001), HOMA index (P < 0.0001) and VFA (<0.0001) levels were higher in the WML-positive group than in the WML-negative group. Multivariate logistic analysis revealed that WML was independently predicted by the high VFA and insulin resistance (P < 0.001, P < 0.0001, respectively). CONCLUSIONS: The results of this preliminary study indicate that the presence of WML was associated with the high VFA and insulin resistance in Japanese patients with type 2 diabetes mellitus. Further larger cohort studies are warranted to confirm these findings.

Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture.

Antibodies to chicken fast skeletal muscle (pectoralis) alpha-actinin and to smooth muscle (gizzard) alpha-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard alpha-actinins ([C]anti-P alpha-An and [C]anti-G alpha-An), antibody specifically reactive with pectoralis alpha-actinin ([S]anti-P alpha-An), and antibody specifically reactive with gizzard alpha-actinin ([S]anti-G alpha-An). In indirect immunofluorescence microscopy, (C)anti-P alpha-An, (S)anti-P alpha-An, and (C)anti-G alpha-An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G alpha-An did not. Although (S)anti-G alpha-An and two common antibodies stained smooth muscle cells, (S)anti-P alpha-An did not. We used (S)anti-P alpha-An and (S)anti-G alpha-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal- and smooth-muscle-type alpha-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscle-type alpha-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type alpha-actinin was present diffusely in the cytoplasm and on membrane-associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletal-muscle-type alpha-actinin appears at the onset of fusion and that smooth-muscle-type alpha-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type alpha-actinin is under regulation coordination with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type alpha-actinin is closely related to alpha-actin, whereas smooth-muscle-type alpha-actinin is to gamma- and beta-actins; and (c) that skeletal- and smooth-muscle-type alpha-actinins have complementary distribution and do not co-exist in situ.

Characterization of the C-protein from posterior latissimus dorsi muscle of the adult chicken: heterogeneity within a single sarcomere.

Specific isoforms of myofibrillar proteins are expressed in different muscles and in various fiber types within a single muscle. We have isolated and characterized monoclonal antibodies against C-proteins from slow tonic (anterior latissimus dorsi, ALD) and fast twitch (pectoralis major) muscles of the chicken. Although the antibody against "fast" C-protein (MF-1) did not bind to the "slow" isoform and the antibody to the "slow" C-protein (ALD-66) did not bind to the "fast" isoform, we observed that both antibodies bound C-protein from the posterior latissimus dorsi (PLD) muscle. Here we demonstrate that in the PLD muscle the binding sites of these two antibodies reside in two different C-protein isoforms which have different molecular weights and can be separated by hydroxylapatite column chromatography. Since we have shown previously that both these antibodies stain all myofibers and myofibrils derived from PLD muscle, we conclude that all myofibers in this muscle contain both isoforms with all sarcomeres.

Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro.

Monoclonal antibodies (McAbs) against the myosin heavy chain (MHC) of adult chicken pectoralis muscle have been tested for reactivity with pectoralis myosin at selected stages of chick development in vivo and in vitro. Three such McAbs, MF 20 and MF 14, which bind to light meromyosin, and MF 30, which binds to myosin subfragment two (S2), were used to assay the appearance and accumulation of specific MHC epitopes with: (a) indirect, solid phase radioimmune assay (RIA), (b) immunoautoradiography, (c) immunofluorescence microscopy. McAb MF 20 bound strongly and equivalently to MHC at all stages of embryonic development in vivo. In contrast, the MF 30 epitope was barely detectable at 12 d of incubation but its concentration rose rapidly just before hatching. No detectable binding of MF 14 to pectoralis myosin could be measured during myogenesis in vivo until 1 wk after hatching. Immunofluorescence studies revealed that all three epitopes accumulate in the same myocytes of the developing pectoralis muscle. Since all three McAbs bound with high activity to native and denatured forms of myosin, it is unlikely that differential antibody reactivity can be explained by conformational changes in myosin during development in vivo. When myogenesis in vitro was monitored using the same McAbs, MF 20 bound to the MHC at all stages tested while reactivity of MF 30 and MF 14 with myosin from cultured muscle was never observed. Thus, this study demonstrates three different immunochemical states of the MHC during development in vivo of chick pectoralis muscle and the absence of later occurring immunochemical transitions in the MHC of cultured embryonic muscle.

Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies.

Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.