Occludin: a novel integral membrane protein localizing at tight junctions.

Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.

Carrier and spin dynamics of high-density exciton magnetic polarons in Cd0.8Mn0.2Te.

We investigated the carrier and spin dynamics of high-density exciton magnetic polarons (HD-EMPs) in Cd0.8Mn0.2Te based on the measurement of their time-resolved photoluminescence (PL) spectra and polarization states, and the utilization of photo-induced Faraday rotation techniques. The PL from the HD-EMPs were collected in a forward scattering configuration, and was observed as a pulsed emission of a few picoseconds duration, exhibiting a blue-shift with time evolution. The blue shift originated from the refractive-index dispersion of the sample. By excluding the influence of the refractive-index dispersion on the time profile, it was revealed that the ultra-short pulsed emission with a time width smaller than 1 ps was initially radiated with a time delay of ~2.4 ps after photoexcitation. From the results of time evolution of the polarization states, it is concluded that the exciton-Mn spin interactions occurs immediately after the excitation, which causes the Mn ion spins to align to follow the spin states of photoexcited excitons. The alignment of the Mn ion spins through the formation of the HD-EMPs was significantly faster than that of the localized EMP. On the other hand, the time evolution of the photo-induced Faraday rotation showed two decay components attributed to spin relaxations of the excitons and Mn ions within the HD-EMP. The observation of the Faraday rotation signal due to the Mn ion spins further confirms that these spins were aligned by the photo-excited spin-aligned excitons. Our findings suggest a novel mechanism for the effective optical control of spins in a semimagnetic semiconductor, which is associated with a multi-exciton system and its localized state.

Effects of a psychosocial intervention programme combined with exercise in community-dwelling older adults with chronic pain: A randomized controlled trial.

BACKGROUND: Although researchers have recommended exercise training and psychosocial intervention to manage chronic pain, an effective intervention for Japanese community-dwelling older adults with chronic pain has not been established. This randomized controlled trial examined whether exercise training combined with psychosocial intervention more effectively improves pain, psychological status and physical activity than does exercise training alone in this population. METHODS: We randomized 128 older adults with chronic pain to either an intervention group (n = 64) involving exercise training combined with psychosocial intervention, or a control group (n = 64) involving only exercise training. Exercise training comprised weekly 60-min sessions for 12 weeks. Psychosocial intervention involved changing participants' focus on pain using self-management education and cognitive behavioural therapy, and participants recorded their daily pain intensity and step counts. Pain intensity, psychological status and physical activity were assessed before and 12 weeks after the intervention. RESULTS: A time-by-group interaction emerged for psychological status (p = 0.003) and physical activity (p < 0.001), both favouring the intervention group. The intervention group also showed greater improvement in pain intensity at 12 weeks than did the control group (p = 0.007). CONCLUSIONS: Exercise training combined with psychosocial intervention improves key outcome indicators more effectively than does exercise training alone in older adults with chronic pain. SIGNIFICANCE: Although research has shown that combined exercise and psychosocial intervention is optimal for managing chronic pain, our study is the first, to the best of our knowledge, to test a specific intervention of this type in community-dwelling older adults with chronic pain in Japan.

Homodigital artery flap reconstruction for fingertip amputation: a comparative study of the oblique triangular neurovascular advancement flap and the reverse digital artery island flap.

This fingertip reconstruction study retrospectively compared sensory recovery and active range of motion outcomes in neurovascular island advancement and reverse digital artery island flaps. Seventeen oblique triangular flaps and 14 reverse digital artery island flaps were performed for nail bed level fingertip amputations (Ishikawa subzone II). There was no significant difference between the two procedures in the Semmes-Weinstein monofilament test and range of motion results. For static and moving two-point discrimination tests, however, those with a reverse digital artery island flap required a longer period for sensory recovery compared to those with an oblique triangular advancement flap. This trend equilibrated at 12 months after surgery showing no significant difference in both static and moving two-point discrimination tests between the procedures.

A 155-kDa undercoat-constitutive protein of cell-to-cell adherens junctions.

A fraction enriched in cell-to-cell adherens and tight junctions was isolated from the chick liver, and the undercoat-constitutive proteins were extracted from this isolated junctional fraction. Monoclonal antibodies (mAbs) were then obtained by injecting this extract into rats, and five antigens were identified to be concentrated in the isolated junctional fraction. We have characterized one mAb (E14 mAb) and its antigen (E14). By immunoblotting of the isolated junctional fraction the E14 mAb reacted strongly with a single band of approximately 155 kDa, and E14 was highly concentrated in the isolated junctional fraction. Immunofluorescence microscopy revealed that the E14 mAb exclusively stained the junctional complex region of the liver, renal epithelial cells, and the cell-cell border of endothelial cells in various tissues. The intercalated disc of the heart was also significantly stained. However, the E14 signal was hardly detected from intestinal epithelial cells. By immunoelectron microscopy using renal epithelial cells, E14 was mainly detected in the fibrous structures associated with the cell-to-cell adherens junction. We conclude that E14 is a novel undercoat-constitutive protein found in certain types of cell-to-cell adherens junctions.

A calcium channel blocker, benidipine, inhibits intimal thickening in the carotid artery of mice by increasing nitric oxide production.

OBJECTIVE: Recent studies suggest that several calcium channel blockers exert their protective effects against vascular disorders by increasing nitric oxide (NO) production from the endothelium. The purpose of this study was to clarify the effects of a long-lasting calcium channel blocker, benidipine, on vascular remodeling. METHODS: The left common carotid arteries of mice were completely ligated just proximal to the carotid bifurcation. Treatment with benidipine (3 mg/kg per day) or vehicle was started 1 week before the carotid ligation, and continued throughout the experiments. Four weeks after the carotid ligation, these mice were killed and vascular remodeling was analyzed. Moreover, NO production and endothelial NO synthase (eNOS) expression were assessed. RESULTS: At 4 weeks after ligation, the neointimal area in the vehicle-treated mice was 39,400 +/- 4,900 microm2 (n = 8), whereas that in the drug-treated mice was reduced to 18,300 +/- 3,800 microm2 (n = 10). Consequently, the luminal area was 35% larger in the drug-treated mice. Benidipine increased the basal as well as agonist-induced NO production from the endothelium, detected by Griess method or NOx analyzer. Endothelial NOS expression in vessels of the drug-treated mice was increased compared with that of the vehicle-treated mice. CONCLUSION: Our data provide evidence that benidipine increases NO production via increment of eNOS protein in vessels and prevents intimal thickening in mice. These results show the possibility of benidipine as a protective tool against vascular remodeling independent of its effect on blood pressure.

Hereditary heparin cofactor II deficiency and coronary artery disease.

We here present a Japanese family with type I hereditary heparin cofactor II (HC II) deficiency. The propositus (a 61-year-old man) suffered from angina pectoris and coronary artery disease was confirmed by coronary angiography. He underwent percutaneous transluminal coronary angioplasty (PTCA) four times in one year. Combined use of heparin, aspirin, and nitrates could not prevent the return of his symptoms and restenosis of segment 6 of the left anterior descending artery. His HC II activity and antigen levels were 49% and 50%, respectively, and his daughter also showed similar low levels. Cerebral infarction had occurred in two family members. Argatroban, a selective potent thrombin inhibitor, was administered after the fourth PTCA for the purpose of preventing reocclusion and achieved a successful outcome. A relationship between HC II deficiency and thrombosis has not yet been established. Our case suggests that standard heparin therapy is not effective in preventing restenosis in such individuals, in whom the process is accelerated by thrombin generation at the site where PTCA produces rupture of the atherosclerotic plaque. Argatroban may be more effective under low HC II conditions because of its potent inhibition of thrombin activity at sites of vascular wall damage.

Cerebrospinal fluid proteins in muscular dystrophy patients.

We analyzed and quantified the microcomponents of protein fractions in the cerebrospinal fluid of patients with various types of muscular dystrophy. The degenerative pattern is characterized by an increase in the prealbumin fraction and a decrease in the gamma-globulin fraction as shown in the Duchenne and congenital muscular dystrophy. The increase in CSF IgG, gamma-globulin fraction, and myelin basic protein is shown in the myotonic dystrophy. In addition to the lowness of IQ, and the abnormality of EEG and brain CT, abnormal CSF proteins obviously suggest the presence of CNS involvement in muscular dystrophy.

Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures.

Occludin is an integral membrane protein localizing at tight junctions with four transmembrane domains. When chicken occludin was overexpressed in insect cells by recombinant baculovirus infection, peculiar multilamellar structures accumulated in the cytoplasm. Partial isolation of these structures indicated that the introduced chicken occludin was highly enriched in these structures. Thin section electron microscopy revealed that each lamella was transformed from intracellular membranous cisternae whose luminal space was completely collapsed, and that in each lamella, outer leaflets of opposing membranes appeared to be fused with no gaps, like tight junctions. Furthermore, in the freeze-fracture replicas of these multilamellar structures, short tight junction-like intramembranous particle strands were occasionally observed, which were specifically labeled by anti-occludin mAb. These observations favor the idea that occludin plays a key role in the formation of tight junctions.

Acute myocardial infarction during pregnancy caused by coronary artery spasm.

A 33-year-old pregnant woman suffered from acute anteroseptal myocardial infarction at the 19th gestational week. Despite periodic attacks of myocardial ischaemia after admission, the coronary arteriograms under the use of nitroglycerin were normal. Thereafter, she remained free from the ischaemic events with diltiazem hydrochloride and delivered a healthy baby. The coronary arteriography at puerperium also showed no organic narrowing. However, the provocative test with acetylcholine chloride caused severe spasm of the left anterior descending coronary artery. These findings strongly suggest that acute myocardial infarction in this pregnant woman was caused by coronary artery spasm.

Protein phosphorylation and the regulation of cell-cell junctions in brain endothelial cells.

Endothelial cells lining the capillaries of the brain have two properties that distinguish them from their peripheral counterparts: (1) tight junctions of extremely low permeability; and (2) low rates of fluid-phase endocytosis. In combination, these features limit the nonspecific flux of ions, proteins, and other substances into the central nervous system environment, creating the blood-brain barrier. This barrier is not immutable. Tight junction permeability can be rapidly increased, which may play a role in the development of a variety of brain pathologies. We have therefore been interested in mechanisms regulating junctional permeability and strategies for interfering with this regulation. What is becoming increasingly apparent is that junctions are not passive mechanical entities; rather, they are targets for a variety of signaling pathways.

Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms.

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.