Ultrastructural localization of adhalin in normal murine skeletal myofiber

The ultrastructural localization of adhalin and its relations to dystrophin, β-dystroglycan, and β-spectrin were studied in normal murine skeletal myofibers. The C-terminal peptides of adhalin and β-dystroglycan were synthesized based on their cDNAs, and the affinity-purified antibodies against these peptides were produced. Single-immunolabeling electron microscopy showed that the adhalin was located just inside the muscle plasma membrane or inside the myofiber a short distance from the plasma membrane. The adhalin signal was also noted at the sarcoplasmic side of plasmalemmd invaginations or at vesicular structures in subsarcolemmal areas. Double-immunogold-labeling electron microscopy disclosed a similar localization of dystrophin, β-dystroglycan, and β-spectrin. The close association of adhalin with dystrophin or β-dystroglycan was demonstrated by formation of doublets by signals of antibodies of adhalin with those of dystrophin or β-dystroglycan and was confirmed by statistical analyses. This study demonstrated that the location of adhalin is close to that of dystrophin and β-dystroglycan at the muscle plasma membrane.     

Non-viral in vivo thrombomodulin gene transfer prevents early loss of thromboresistance of grafted veins.

OBJECTIVE: Immediate loss of thrombomodulin activity in the endothelium of vein grafts has been demonstrated during 90 min exposure to arterial circulation; this loss of activity is ascribed as an important cause of early thrombosis. Conventional ex vivo gene transfection after vein harvest cannot cover this acute period immediately after implantation. We have established a highly efficient non-viral gene therapy protocol utilizing modified transferrin receptor-facilitated gene transfer. Using this technique, we examined whether in vivo thrombomodulin gene therapy, directed to the endothelium of rat veins 2 days prior to grafting, may prevent thromboresistance impairment of vein grafts under simulated arterial circulation. METHODS: Abdomen of SD rat was opened and cationic liposome:transferrin:thrombomodulin gene complexes or the vector without DNAs were applied to the inferior vena cava of rats while blood flow was reduced by proximal and distal clamping. After 2 days, the transfected veins were harvested and thrombomodulin expression and thromboresistance properties determined before and after exposure to an artificial circuit. RESULTS: The trial of gene transfection using variable doses of DNAs confirmed that 7.5 microg of total DNAs was the most efficient quantity for thrombomodulin gene transfection to IVCs, although accompanying an increase of gene expression in other downstream organs. By transfection of the thrombomodulin gene in IVCs, the generation capacity of activated protein C in venous endothelium increased three-fold compared with veins treated with vector alone (P<0.01). Under simulated arterial circulation, perfusion of veins treated with vector alone decreased thrombomodulin activity to 36% of preperfused levels (P<0.01), whereas transfected grafts preserved the activity at normal vein endothelium levels even after perfusion. Consequently, the increase in endothelial thrombin activity induced by simulated arterial circulation was markedly attenuated in transfected veins (P<0.01), while immunohistochemistry confirmed the preservation of endothelial lining. CONCLUSIONS: Transferrin receptor-facilitated in vivo gene transfer to the inferior vena cava resulted in sufficient thrombomodulin gene expression immediately after graft implantation and subsequent maintenance of thromboresistance even after exposure to arterial pressure. Although further studies are needed, the present results suggest the possibility of gene therapy targeting acute phases of vein graft disease.     

Effect of lead on electrophoretic mobility of rat erythrocytes

Lead often affects the erythrocyte membrane. The relationship between the changes in erythrocyte membrane and the anemia caused by lead is still unclear. Initially, the effect of lead injected intraperitoneally on the electrophoretic mobility of rat erythrocytes was investigated in order to study the relationship between them. As indices of lead exposure, hemoglobin (Hb) levels, hematocrits (Ht), δ-aminolevulinic acid dehydratase (ALA-D) activities and blood lead (blood Pb) levels in the injected rats were also examined. Exposure to lead significantly decreased the mobility of rat erythrocytes. The changes in mobility seemed to be less sensitive than those in ALA-D activity, however, the decreases in mobility were simultaneous with or prior to those in Hb level and Ht. The decreases in mobility were evident to some extent below a blood Pb level of 100 μg/100 ml and generally present at a level of 100 μg/100 ml and over. In the rats exposed to lead a significant negative correlation was found between the mobilities and the logarithms of blood Pb level.     

Aging gracefully: a retrospective analysis of functional status in Okinawan centenarians.

OBJECTIVE: This study retrospectively explored the late-life functional status of Okinawan centenarians. METHODS: Activities of daily living were measured retrospectively at five time points (10, 5, 3, and 1 year prior and present) for 22 centenarians in relation to seven physical, two sensory, and two cognitive functions using the Inoue Index. RESULTS: In all, 82% of individuals were still functioning independently at a mean age of 92 years and almost two-thirds were still functioning independently at a mean age of 97 years. CONCLUSION: Preliminary analyses suggest high functional status in Okinawan centenarians throughout their 90 s. The genetic and environmental factors contributing to this successful aging phenomenon deserve further investigation.     

PEUTZ‐JEGHERS SYNDROME ASSOCIATED WITH RENAL AND GASTRIC CANCER THAT DEMONSTRATED AN STK11 MISSENSE MUTATION

A 75‐year‐old male was admitted to the gastroenterology unit of Nagoya City University Hospital due to epigastralgia after surgical treatment for right renal cancer. Endoscopy revealed advanced type 1 gastric cancer in the corpus of the stomach and multiple polypoid lesions in the stomach and duodenum. X‐ray examination of the small intestine using barium showed multiple polyps in the upper jejunum. Faint pigmentation on the palm was also detected. Peutz‐Jeghers syndrome (PJS) was diagnosed, despite a lack of family history. Total gastrectomy, resection of part of the upper jejunum and intraoperative endoscopic polypectomy of duodenal polyps was performed. This is the second reported case of PJS associated with renal cancer. We also detected a missense mutation in the tumor suppressor gene STK11 that, when mutated, is causative for PJS.     

Leukocyte-Endothelium Interaction in the Rat Mesenteric Microcirculation during Halothane or Sevoflurane Anesthesia

Background: The effects of inhalational anesthetics on the microcirculation, including leukocyte dynamics, remain to be clarified. The authors investigated halothane and sevoflurane anesthesia to determine if these agents evoked leukocyte adhesion through endothelial cell-dependent mechanisms involving such adhesion molecules.

Methods: Rats were anesthetized with halothane or sevoflurane in 100% oxygen and the lungs were mechanically ventilated. Leukocyte behavior in mesenteric venules was recorded through intravital video microscopy under monitoring microvascular hemodynamics. To examine the mechanisms for leukocyte rolling and adhesion, these studies were repeated after animals were pretreated with a monoclonal antibody against P-selectin (MAb PB1.3) or against intracellular adhesion molecule-1 (ICAM-1; MAb 1A29): P-selectin required for rolling of circulating leukocytes and ICAM-1 for firm adhesive interactions with leukocyte integrins.

Results: Under baseline anesthetic conditions (1 minimum alveolar concentration [MAC]), venular wall shear rates, an index of the disperse force on marginating leukocytes, in the sevoflurane-treated rats were about two times higher than those with halothane. At 2 MAC, halothane caused a marked arteriolar constriction and decreasing shear rates concurrent with an increasing density of venular leukocyte adhesion. Sevoflurane at 2 MAC induced leukocyte rolling and adhesion, which were attenuated by PB1.3 and 1A29, without alterations in the wall shear rates. Halothane-induced leukocyte adhesion was not prevented by PB1.3 but it was by 1A29.     

Response of Müller cells following experimental lensectomy-vitrectomy

We used morphological, biochemical and immunohistochemical methods to assess the response of Müller cells after experimental lensectomy-vitrectomy in rabbits. We observed widened intercellular spaces between the Müller cells and nerve fibers of ganglion cells, and increased electron opacity in the Müller cells of eyes injected with silicone oil. No apparent morphological changes were detected in the Müller cells of air-injected eyes. The specific and total activities of Müller cell-marker enzymes (glucose 6-phosphatase and glutamine synthetase) showed an initial increase, followed by a decrease. Glial fibrillary acidic protein immunoreactivity was not found in the Müller cells of the normal rabbit retina but was exhibited after surgery. Our results showed that markers of Müller cells associated with glycogenolysis and/or gluconeogenesis, glutamate-glutamine cycle and cytoskeletal protein metabolism were affected by the experimental lensectomy-vitrectomy.