Crossbridge Recruitment Capacity of Wild-Type and Hypertrophic Cardiomyopathy-Related Mutant Troponin-T Evaluated by X-ray Diffraction and Mechanical Study of Cardiac Skinned Fibers.

X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without "troponin-T treatment," which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber elicited by contractile crossbridge formation and investigate the abnormality of hypertrophic cardiomyopathy-related troponin-T mutants. The intensity of the troponin reflection at 1/38.5 nm-1 was decreased significantly by ATP addition after treatment with wild-type or mutant troponin-T, indicating that crossbridge formation affected the conformation of troponin-T. In experiments on cardiac fibers treated with the hypertrophic cardiomyopathy-related mutants E244D- and K247R-troponin-T, treatment with K247R-troponin-T did not recruit contracting actomyosin to a greater extent than wild-type-troponin-T, although a similar drop in the intensity of the troponin reflection occurred. Therefore, the conformational change in K247R-troponin-T was suggested to be unable to fully recruit actomyosin interaction, which may be the cause of cardiomyopathy.

APOB codon 4311 polymorphism is associated with hepatitis C virus infection through altered lipid metabolism.

BACKGROUND: It has been reported that some single-nucleotide polymorphisms (SNPs) in lipid regulators such as apolipoproteins and cell surface molecules for hepatitis C virus (HCV) entry into hepatocytes are associated with HCV infection. However, it is unknown how HCV infection is affected by altered lipid metabolism resulting from the SNPs. We investigated the relationship between these SNPs and HCV infection status, and also analyzed the mechanism by which these SNPs mediate HCV infection via lipid metabolism alterations. METHODS: Serum lipid and apolipoprotein profiles were tested in 158 HCV-positive and 220 HCV-negative subjects. We selected 22 SNPs in five lipid regulator genes which were related to HCV entry into hepatocytes and to lipid metabolism (APOA1, APOB, SR-B1, LDLR, and APOE), and their polymorphisms were analyzed using the PCR-sequence-specific oligonucleotide probe-Luminex method. RESULTS: An APOB N4311S (g.41553a > g) SNP, rs1042034, was significantly associated with HCV positivity; the HCV positivity rate for the minor allele AA genotype was significantly higher than for genotype AG + GG (P = 0.016). Other SNPs except for APOB P2712L SNP rs676210, which is in linkage disequilibrium with rs1042034, showed no significant difference in genotype distribution. The serum level of low density lipoprotein-cholesterol (LDL-C) in the genotype AA group was significantly lower than in the genotype non-AA group (P = 0.032), whereas the triglyceride (TG) level was significantly higher (P = 0.007). CONCLUSION: An APOB SNP, rs1042034, is closely associated with HCV infection through lipid metabolism alteration. The minor allele AA genotype might contribute to facilitating serum LDL uptake into hepatocytes via LDLR by modifying their affinity and interaction and may have an influence on HCV infection by their entry to the liver through the LDLR.

Neuroendocrine carcinoma of the pancreas with similar genetic alterations to invasive ductal adenocarcinoma.

Neuroendocrine carcinoma (NEC) of the pancreas is very rare, and its origin is not fully elucidated. Here, we present a case of a small-size NEC of the pancreas that is genetically similar to invasive ductal adenocarcinoma (IDA). A 65-year-old man was referred to our hospital due to obstructive jaundice and found to have a 12-mm solid tumor in the pancreas head. The tumor exhibited low vascularity on enhanced computed tomography, and endoscopic retrograde pancreatographic imaging revealed an irregular obstruction in a branch duct of the pancreas. The patient was thereby diagnosed with a pancreatic ductal cancer, and stomach-preserving pancreaticoduodenectomy with regional lymph node resection was performed. Histochemical analysis of the resected tumor showed that the neoplastic cells with scanty cytoplasm and hyperchromatic nuclei strongly expressed chromogranin A and synaptophysin. The Ki-67 index was 40 % in the most proliferative tumor regions, and the tumor was diagnosed as a NEC of the pancreas. However, in the analysis of genetic alterations of the tumor tissue, the neoplastic cells showed altered KRAS, TP53, and SMAD4/DPC4, suggesting that the NEC in our case is genetically related to IDA. Our data suggest that poorly differentiated IDAs may transform into NECs.

Panitumumab in combination with irinotecan plus S-1 (IRIS) as second-line therapy for metastatic colorectal cancer.

BACKGROUND: Irinotecan plus S-1 (IRIS) is the only oral fluoropyrimidine-based regimen reported to be non-inferior to FOLFIRI and widely used in clinical practice for metastatic colorectal cancer (mCRC) patients. However, the combination of IRIS plus an anti-EGFR agent has not been evaluated previously. This study aimed to investigate the feasibility and efficacy of IRIS with panitumumab as second-line therapy for wild-type KRAS mCRC. METHODS: Main inclusion criteria were patients with wild-type KRAS mCRC refractory to one prior chemotherapy regimen for mCRC, ECOG PS 0-2, and age ≥20 years. Patients received panitumumab (6 mg/kg) and irinotecan (100 mg/m(2)) on days 1 and 15 and S-1 (40-60 mg according to body surface area) twice daily for 2 weeks, repeated every 4 weeks. The primary endpoint was the feasibility of the therapy. The secondary endpoints were response rate (RR), progression-free survival (PFS), and overall survival (OS). RESULTS: A total of 36 patients received protocol treatment in eight centers. Of these, 23 patients (63.9 %) completed protocol treatment, demonstrating achievement of the primary endpoint. The most frequent grade 3/4 toxicities were diarrhea (16.7 %), acne-like rash (13.9 %), and neutropenia (11.1 %). The overall RR was 33.3 % (12/36). Of these patients, five underwent conversion surgery. Median PFS and OS were 9.5 months (95 % CI 3.5-15.4 months) and 20.1 months (95 % CI 16.7-23.2 months), respectively. CONCLUSION: IRIS plus panitumumab has an acceptable toxicity profile and a promising efficacy in patients with previously treated wild-type KRAS mCRC. Accordingly, this regimen can be an additional treatment option for second-line chemotherapy in wild-type KRAS mCRC.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Unusual endoscopic findings of gastric neuroendocrine tumor.

Gastric neuroendocrine tumor (NET) is sometimes found as a submucosal tumor on upper gastrointestinal endoscopy. Gastric NET with malignant profile and neuroendocrine carcinoma (NEC) show various forms which are difficult to distinguish from gastric cancer and other disease. We report a case of a cauliflower-shaped NET of the stomach. A 61-year-old man was referred to our hospital with a complaint of abdominal fullness. Upper gastrointestinal endoscopic examination revealed an unusual, whitish cauliflower-shaped tumor that belongs to Borrmann type I on the lesser curvature of the gastric antrum. Histological examination of the biopsy specimen revealed NET G2, because the tumor cells were CD56- and synaptophysin-positive by immunohistochemical analysis. A distal gastrectomy with D2 lymphadenectomy was performed. A recurrence in the liver was revealed by follow up computed tomography after 11 months from operation. Combined chemotherapy with irinotecan (CPT-11) plus cisplatin (CDDP) was treated. The patient achieved a partial response, but he died after 31 months from gastrectomy. There is no independent, large-scaled prospective study and no standard treatment for gastric NETs with distant metastases. Our case is reported with a literature review of the treatment of metastatic gastric NET G2.

A phase I/II study of fixed-dose-rate gemcitabine and S-1 with concurrent radiotherapy for locally advanced pancreatic cancer.

PURPOSE: This study was conducted to identify the maximum-tolerated dose (MTD) of fixed-dose-rate gemcitabine (FDR-gem) administered concurrently with S-1 and radical radiation for locally advanced pancreatic cancer (LAPC) and to provide efficacy and safety data. METHODS: Patients with unrespectable pancreatic cancer confined to the pancreatic region were treated with FDR-gem (300-400 mg/m(2), 5 mg/m(2)/min) on days 1, 8, 22, and 29 and 60 mg/m(2) of S-1 orally on days 1-14, 22-35. A total radiation dose of 50.4 Gy (1.8 Gy/day, 28 fractions) was delivered concurrently. RESULTS: Twenty-five patients were enrolled; all were evaluable for toxicity assessment. In phase I, eight patients were treated in sequential cohorts of three to five patients per dose level. The MTD was reached at level 2, and dose-limiting toxicities were neutropenia and thrombocytopenia. The recommended doses were 300 mg/m(2) of gemcitabine and 60 mg/m(2) of S-1 daily. The overall response rate was 25% and disease control rate (partial response plus stable disease) was 92%. The progression-free survival was 11.0 months. The median overall survival and 1-year survival rates were 16.0 months and 73%, respectively. CONCLUSION: The combination of FDR-gem and S-1 with radiation is a feasible regimen that shows favorable antitumor activity with an acceptable safety profile in patients with LAPC.

Insulin-like growth factor II-producing metastatic colon cancer with recurrent hypoglycemia.

A 45-year-old man was referred to our hospital and found to have a tubular adenocarcinoma of the descending colon with multiple liver metastases. During hospitalization, the patient suffered recurrent hypoglycemic attacks that required intravenous 50% glucose infusion. He was diagnosed with non-islet cell tumor hypoglycemia (NICTH) because the colon cancer tissue obtained by biopsy was strongly stained for insulin-like growth factor-II (IGF-II) by immunohistochemistry. He received chemotherapy with oxaliplatin, 5-FU and leucovorin (FOLFOX) plus bevacizumab (Bmab), and showed a partial response. As the metastatic lesions decreased in size, the hypoglycemic attacks gradually disappeared. Subsequently, he received outpatient chemotherapy and maintained a high quality of life for about 10 months. Western blot analysis of IGF-II in serum at the time of admission showed a high-molecular-weight form of IGF-II, which was considered to have caused hypoglycemia. This patient presents a very rare case of colorectal cancer associated with NICTH syndrome due to production of high-molecular-weight IGF-II by cancer cells. It is important to investigate IGF-II expression in cancer tissues for establishing the diagnosis of NICTH in cases with intractable hypoglycemia complicated by advanced cancer.

Head and neck contrast-enhanced CT for identification of internal carotid artery stenosis progression on the affected side after treatment for oral squamous cell carcinoma.

OBJECTIVES: To determine the incidence of progressive internal carotid artery (ICA) stenosis by head and neck contrast-enhanced computed tomography (CT) in 82 patients who underwent surgery, chemotherapy, or combination therapy for oral squamous cell carcinoma (OSCC). METHODS: The study included 82 patients who underwent head and neck contrast-enhanced CT after surgery alone or combined surgery and chemotherapy for OSCC at the Department of Oral and Maxillofacial Surgery of Ichikawa General Hospital, Tokyo Dental College, or Tokyo Dental College Oral Cancer Center between December 2002 and March 2010. RESULTS: Comparison with previously obtained head and neck contrast-enhanced CT images revealed progressive arterial stenosis of the ICA in five patients with a mean age of 62.0 years. All five patients were male, and their OSCC sites were the tongue in two, the floor of the mouth in two, and the mandibular gingiva in one. Tumor resection and neck dissection were performed for four patients and tumor resection alone for one patient. Four patients underwent chemotherapy. ICA stenosis occurred on the same side as the tumor in all five patients. CONCLUSIONS: The results of this study suggest that, given the possibility of post-treatment vascular events, attention must be paid to subsequent changes in the ICA over time. The results also indicate the usefulness of head and neck contrast-enhanced CT in identifying such problems.

Clinical benefit of high-sensitivity KRAS mutation testing in metastatic colorectal cancer treated with anti-EGFR antibody therapy.

OBJECTIVE: We compared high-sensitivity KRAS mutation testing with direct sequencing for predicting the efficacy of antiepidermal growth factor receptor antibodies in patients with metastatic colorectal cancer (mCRC). METHODS: We analyzed the KRAS status in 61 tumors from cetuximab-treated mCRC patients by both direct sequencing and a high-sensitivity method: 2-step PCR restriction fragmentation length polymorphism (RFLP). Therapeutic effects in each mutational status were evaluated. RESULTS: The incidences of KRAS mutations determined by direct sequencing and 2-step PCR RFLP were 34.4 and 52.5%, respectively (p = 0.02). Patients were categorized into 3 groups [W/W, wild-type by both methods (n = 29); W/M, wild-type by direct sequencing, detected mutation by 2-step PCR RFLP (n = 11); M/M, mutant-type by both methods (n = 21)]. The response rate for cetuximab in the W/M group (0%) was the same as that in the M/M group, and was significantly lower than in the W/W group (41.4%) (p < 0.001). Progression-free survival in the W/M group (11.0 weeks) was similar to that in the M/M group (8.0 weeks), and was significantly shorter than in the W/W group (18.0 weeks) (p < 0.002). CONCLUSION: High-sensitivity KRAS mutation testing is useful for selecting true responders to cetuximab.

Intracellular phospholipase A1 and acyltransferase, which are involved in Caenorhabditis elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphatidylinositol.

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, -9, and -10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of Caenorhabditis elegans, the stearic acid content of PI is reduced, and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A(1) and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.

Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.

In multicellular organisms, the surface barrier is essential for maintaining the internal environment. In mammals, the barrier is the stratum corneum. Fatty acid transport protein 4 (FATP4) is a key factor involved in forming the stratum corneum barrier. Mice lacking Fatp4 display early neonatal lethality with features such as tight, thick, and shiny skin, and a defective skin barrier. These symptoms are strikingly similar to those of a human skin disease called restrictive dermopathy. FATP4 is a member of the FATP family that possesses acyl-CoA synthetase activity for very long chain fatty acids. How Fatp4 contributes to skin barrier function, however, remains to be elucidated. In the present study, we characterized two Caenorhabditis elegans genes, acs-20 and acs-22, that are homologous to mammalian FATPs. Animals with mutant acs-20 exhibited defects in the cuticle barrier, which normally prevents the penetration of small molecules. acs-20 mutant animals also exhibited abnormalities in the cuticle structure, but not in epidermal cell fate or cell integrity. The acs-22 mutants rarely showed a barrier defect, whereas acs-20;acs-22 double mutants had severely disrupted barrier function. Moreover, the barrier defects of acs-20 and acs-20;acs-22 mutants were rescued by acs-20, acs-22, or human Fatp4 transgenes. We further demonstrated that the incorporation of exogenous very long chain fatty acids into sphingomyelin was reduced in acs-20 and acs-22 mutants. These findings indicate that C. elegans Fatp4 homologue(s) have a crucial role in the surface barrier function and this model might be useful for studying the fundamental molecular mechanisms underlying human skin barrier and relevant diseases.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction.

To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibres towards the meridian and the resulting profile resembled that of masseter fibres. This suggests that the protruding mobile heads in the non-treated masticatory fibres are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibres during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibres, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

CH2-units on (poly-)ethylene glycol radially dehydrate cytoplasm of resting skinned skeletal muscle.

Observing the optical cross-section and electron micrographs of mechanically skinned fibres of frog skeletal muscle, we found that ethylene glycols (EGs) of small (mono-, di-, tri- and tetra-EGs; M(r) 62-194) and medium (poly-EGs; M(r) 900 and 3350) molecular weights efficiently dehydrate the fibres to shrink them radially without microscopic inhomogeneity. The medium-sized poly-EGs at 30% weight/weight concentration absorbed almost all the evaporable water from the fibre. Passive tension measurement at near slack sarcomere spacing indicated that this dehydration by EGs did not accompany longitudinal fibre shrinkage. Chemically relevant fully hydric alcohols (glycerol, threitol, ribitol and mannitol; M(r) 92-182) showed no appreciable dehydrating ability on fibres. An intimate correlation was found between fibre dehydration and CH(2)-concentration of the solutions. Viscosity measurements indicated that the hydrodynamic radii of the alcohols were comparable to those of the small EGs. Therefore, hydrodynamic radii are not a primary determinant of the dehydrating ability. Additionally, CH(2)-concentration of EGs but not alcohols was found to correlate intimately with the measured viscosity of the bulk solution of EGs. These results suggested that the interaction between water molecules and CH(2)-units in crowded cytoplasm of skeletal muscle affects cytoplasm as a whole to realize anisotropic fibre shrinkage.

CH2-units on (poly-)ethylene glycol radially dehydrate cytoplasm of resting skinned skeletal muscle.

Observing the optical cross-section and electron micrographs of mechanically skinned fibres of frog skeletal muscle, we found that ethylene glycols (EGs) of small (mono-, di-, tri- and tetra-EGs; M(r) 62-194) and medium (poly-EGs; M(r) 900 and 3350) molecular weights efficiently dehydrate the fibres to shrink them radially without microscopic inhomogeneity. The medium-sized poly-EGs at 30% weight/weight concentration absorbed almost all the evaporable water from the fibre. Passive tension measurement at near slack sarcomere spacing indicated that this dehydration by EGs did not accompany longitudinal fibre shrinkage. Chemically relevant fully hydric alcohols (glycerol, threitol, ribitol and mannitol; M(r) 92-182) showed no appreciable dehydrating ability on fibres. An intimate correlation was found between fibre dehydration and CH(2)-concentration of the solutions. Viscosity measurements indicated that the hydrodynamic radii of the alcohols were comparable to those of the small EGs. Therefore, hydrodynamic radii are not a primary determinant of the dehydrating ability. Additionally, CH(2)-concentration of EGs but not alcohols was found to correlate intimately with the measured viscosity of the bulk solution of EGs. These results suggested that the interaction between water molecules and CH(2)-units in crowded cytoplasm of skeletal muscle affects cytoplasm as a whole to realize anisotropic fibre shrinkage.

Chicken breast muscle connectin: passive tension and I-band region primary structure.

We performed cDNA cloning of chicken breast muscle connectin. Together with previous results, our analysis elucidated a 24.2 kb sequence encoding the amino terminus of the protein. This corresponded to the I-band region of the skeletal muscle sarcomere, which is involved in extension and contraction between the Z-line and the A-I junction. There were fewer middle immunoglobulin domains and amino acid residues in the PEVK segment of chicken breast muscle connectin than in human skeletal muscle connectin, but more than in human cardiac muscle connectin. We measured passive tension generation by stretching mechanically skinned myofibril bundles. This revealed that appreciable tension development in chicken breast muscle began at longer sarcomere spacings than in rabbit cardiac muscle, but at shorter spacings than in rabbit psoas and soleus muscles. We suggest that the chicken breast muscle sarcomere remains in a relatively extended state even in unstrained sarcomeres. This would explain why chicken breast muscle does not extend under force to the same degree as rabbit psoas and soleus muscles.

Skinning effects on skeletal muscle myowater probed by T2 relaxation of 1H-NMR.

To find the cause of the skinning-induced fragility of frog skeletal muscle, the transverse relaxation process of 1H-NMR signals from skinned muscle was observed. A set of four characteristic exponentials well described the process. Aside from the extremely slow exponential component (time constant T2 > 0.4 s) representing surplus solution, the process was generally slower than that in living muscle. It had larger amplitudes of slow (T2 approximately 0.15 s) and intermediate (0.03 < T2 < 0.06 s) exponentials and had smaller amplitude and faster T2 in the rapid one (T2 < 0.03 s), suggesting that skinned muscle is more sol-like than intact myoplasm. To resolve their causes, we traced the exponentials following a stepwise treatment of living whole muscle to an isolated skinned fiber. Osmotic expansion of living muscle comparable to skinned muscle increased the intermediate exponential and decreased the rapid one without affecting T2. Subsequent chemical skinning markedly increased the slow exponential, decreased the rapid one, and slowed the intermediate one. The fiber isolation had no appreciable effect. Because l-carnosine at physiological concentration could not recover the skinning-induced difference, the difference would reflect the dilution and efflux of larger macromolecules, which stabilize myoplasm as a gel.

Formulation of an ophthalmic lipid emulsion containing an anti-inflammatory steroidal drug, difluprednate.

Preparation of oil-in-water (o/w) type lipid emulsion is one of the approaches to formulate drugs that are poorly water-soluble but can be dissolved in the oil phase of the emulsions. A synthetic glucocorticoid medicine, difluprednate (DFBA), is a water-insoluble compound. We formulated DFBA (0.05%, w/v) ophthalmic lipid emulsion containing 5.0% (w/v) caster oil and 4.0% (w/v) polysorbate 80. The appearance of the emulsion was blue and translucent lipid emulsion, and the median particle size of the lipid emulsion was 104.4 nm. Neither separation nor change in particle size was observed after 6 months at 40 degrees C. Furthermore, when compared with DFBA (0.05%, w/v) ophthalmic suspension, the lipid emulsion showed 5.7-fold higher concentration of DFB that was an active metabolite of DFBA in aqueous humor at 1h after instillation. Ophthalmic lipid emulsion enhances the intraocular penetration of drugs, and it is useful as a delivery system for the ophthalmic preparations of lipophilic drugs.

Differential osmotic behavior of water components in living skeletal muscle resolved by 1H-NMR.

Using frog sartorius muscle, we observed transverse relaxation processes of (1)H-NMR signals from myowater. The process could be well described by four characteristic exponentials: the extremely slow exponential of relaxation time constant T(2) > 0.4 s, the slow one of T(2) approximately 0.15 s, the intermediate one of 0.03 s < T(2) < 0.06 s, and the rapid one of T(2) < 0.03 s. Addition of isotonic extracellular solution affected only the extremely slow exponential, linearly increasing its amplitude and gradually increasing its T(2) toward that of the bulk solution (1.7 s). Therefore, this exponential should represent extracellular surplus solution independently of the other exponentials. At two thirds to three times the isotonicity, the amplitude of the intermediate exponential showed normal osmotic behavior in parallel with the volume change of the myofilament lattice measured with x-ray diffraction. In the same tonicity range, the amplitude of the rapid exponential showed converse osmotic behavior. Lower tonicities increased the amplitude of only the slow exponential. Studied tonicities did not affect the T(2) values. The distinct osmotic behavior indicated that each characteristic exponential could be viewed as a distinct water group. In addition, the converse osmotic behavior suggested that the rapid exponential would not be a static water layer on the macromolecule surface.