Ketamine Suppresses Platelet Aggregation Possibly by Suppressed Inositol Triphosphate Formation and Subsequent Suppression of Cytosolic Calcium Increase

Background: Ketamine has been shown to suppress platelet aggregation, but its mechanisms of action have not been defined. The purpose of the current study is to clarify the effects of ketamine on human platelet aggregation and to elucidate the underlying mechanisms of its action.

Methods: Platelet aggregation was measured using an eight-channel aggregometer, and cytosolic free calcium concentration was measured in Fura-2/AM-loaded platelets using a fluorometer. Inositol 1,4,5-triphosphate (IP3) was measured with use of a commercially available IP3 assay kit. To estimate thromboxane A2 (TXA2) receptor binding affinity and expression, Scatchard analysis was performed using [3H]S145, a specific TXA2 receptor antagonist. TXA2 agonist binding assay was also performed. The membrane-bound guanosine 5'-triphosphatase activity was determined using [[gamma]-32P]guanosine triphosphate by liquid scintillation analyzer.

Results: Ketamine (500 [mu]m) suppressed aggregation induced by adenosine diphosphate (0.5 [mu]m), epinephrine (1 [mu]m), (+)-9,11-epithia-11,12-methano-TXA2 (STA2) (0.5 [mu]m), and thrombin (0.02 U/ml) to 39.1 +/- 30.9, 46.3 +/- 4.3, -2.0 +/- 16.8, and 86.6 +/- 1.4% of zero-control, respectively. Ketamine (250 [mu]m-1 mm) also suppressed thrombin- and STA2-induced cytosolic free calcium concentration increase dose dependently. Although ketamine (2 mm) had no effect on TXA2 receptor expression and its binding affinity, it (1 mm) suppressed intracellular peak IP3 concentrations induced by thrombin and STA2 from 6.60 +/- 1.82 and 4.39 +/- 2.41 to 2.41 +/- 0.98 and 1.90 +/- 0.86 pmol/109 platelets, respectively, and it suppressed guanosine triphosphate hydrolysis induced by thrombin (0.02 units/ml) and STA2 (0.5 [mu]m) to 50.3 +/- 3.2 and 67.5 +/- 5.5%versus zero-control, respectively.     

The infeasibility of using ten-ring irradiated grafts for tracheal allotransplantation even with omentopexy

In our previous study, we demonstrated that high-dose ⁶⁰Co irradiation was able to prevent rejection of canine tracheal allografts. To determine the maximum possible length of these grafts, in the present study we attempted to transplant five-ring and ten-ring tracheal allografts in two groups of five dogs each. Either five or ten rings were excised from donor tracheas and irradiated with 100,000 cGy of ⁶⁰Co. The irradiated tracheal grafts were transplanted to replace either five- or ten-ring sections of the mediastinal tracheas removed from the recipient dogs. The grafts were covered with omental pedicles and no immunosuppressants were used. Graft incorporation was achieved in four of the five dogs in the five-ring group, and three of these dogs survived for more than 700 days. However, four of the five animals in the ten-ring group died from tracheostenosis accompanied by ischemia within 3 weeks. These findings demonstrate the impossibility of performing ten-ring tracheal allotransplantation using irradiated grafts, even with omentopexy.     

Fine structure of the mouse portal vein in relation to its peristaltic movement

The hepatic portal vein has been known to make a spontaneous peristaltic movement in some mammals, including the mouse and rat. To investigate the fine structure of the portal vein in relation to its physiological characteristics, we observed the mouse portal vein by using various histological techniques including conventional light microscopy, videomicroscopy, transmission and scanning electron microscopy, and real-time confocal laser scanning microscopy. The mouse hepatic portal vein was provided with a spiral fold which was produced by the inner layer, i.e. the endothelium and smooth muscles of the wall protruding into the lumen. Longitudinal smooth muscle cells spanned the interval of the fold, like a spirally arranged palisade around the vessel wall. The longitudinal muscle fibers ended at the spiral fold, being partly connected with a network of irregularly shaped smooth muscle cells. This network, hitherto unknown, was recognized to be restricted to the fold in distribution and characterized by numerous gap junctions connecting the muscle cells. Real-time confocal laser scanning microscopy using a Ca2+ sensitive fluorescent dye revealed that a transient and periodic increase in Ca2+ concentration occurred in the longitudinal smooth muscle cells and was transmitted spirally from the intestinal to the hepatic side. These findings indicate that, during the peristaltic movement, the contraction of smooth muscle cells is transmitted along the longitudinal smooth muscles of the portal vein wall toward the liver, presumably controlled by the network of the irregularly-shaped smooth muscle cells in the fold of the portal vein. Light microscopic observation in some specimens indicated an occurrence of cardiac muscle cells outside the smooth muscle layer. Restricted to the site of the porta hepatis in distribution, their involvement in the peristaltic contraction of the portal vein seemed unlikely.     

Bcl11b is required for differentiation and survival of alphabeta T lymphocytes

The gene Bcl11b, which encodes zinc finger proteins, and its paralog, Bcl11a, are associated with immune-system malignancies. We have generated Bcl11b-deficient mice that show a block at the CD4-CD8- double-negative stage of thymocyte development without any impairment in cells of B- or gammadelta T cell lineages. The Bcl11b-/- thymocytes showed unsuccessful recombination of V(beta) to D(beta) and lacked the pre-T cell receptor (TCR) complex on the cell surface, owing to the absence of Tcrb mRNA expression. In addition, we saw profound apoptosis in the thymus of neonatal Bcl11b-/- mice. These results suggest that Bcl11b is a key regulator of both differentiation and survival during thymocyte development.     

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

Background: Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit–subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II.
Results: The rpb5 ⁺ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe . The polypeptide predicted from DNA sequence of the rpb5 ⁺ gene consists of  210 amino acids with a calculated molecular weight of 23 914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far-Western blot analysis of S. pombe RNA polymerase II using ³²P-labelled recombinant Rpb5 fused to glutathione S-transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane-immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15-kDa subunit (Rpb8 or Rpb11). In agreement with this result, the ³²P-labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5–Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein-immobilized beads.
Conclusion: Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.     

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.     

Chorioamnionitis caused by Serratia marcescens in a non-immunocompromised host

A 26 year old pregnant woman with antithrombin III deficiency developed recurrent septicaemia with Serratia marcescens. In spite of the administration of antibiotics, high grade fever persisted. She subsequently manifested lower abdominal pain, and spontaneous abortion occurred. After the abortion, she became completely afebrile. The amnion was turbid, and microscopic examination of the placenta showed haemorrhage and massive infiltration of neutrophils, suggestive of infectious chorioamnionitis. Pulsed field gel electrophoresis showed that isolates from the blood, urine, and vaginal discharge were genetically identical. Intravenous pyelography revealed that she had a bilateral completed double ureter. It was thought that a urinary tract anomaly caused infection with S marcescens, and the pathogen spread to the chorioamnion via the bloodstream. This is the first report of chorioamnionitis caused by S marcescens in a non-immunocompromised host. In addition, these findings indicate that the chorioamnion can serve as a site for persistent infection in normal pregnancies.     

Early induction of neuronal lipocalin-type prostaglandin D synthase after hypoxic-ischemic injury in developing brains

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is up-regulated in oligodendrocytes (OLs) in mouse models for genetic neurological disorders including globoid cell leukodystrophy (twitcher) and GM₁ and GM₂ gangliosidoses and in the brain of patients with multiple sclerosis. Since L-PGDS-deficient twitcher mice undergo extensive neuronal death, we concluded that L-PGDS functions protectively against neuronal degeneration. In this study, we investigated whether L-PGDS is also up-regulated in acute and massive brain injury resulting from neonatal hypoxic-ischemic encephalopathy (HIE). Analysis of brains from human neonates who had died from HIE disclosed that the surviving neurons in the infarcted lesions expressed L-PGDS. Mouse models for neonatal HIE were made on postnatal day (PND) 7. Global infarction in the ipsilateral hemisphere was evident at 24 h after reoxygenation in this model. Intense L-PGDS immunoreactivity was already observed at 10 min after reoxygenation in apparently normal neurons in the cortex, and thereafter, in neurons adjacent to the infarcted area. Quantitative RT-PCR revealed that the L-PGDS mRNA level of the infarcted hemisphere was 33-fold higher than that of the sham-operated mouse brains at 1 h after reoxygenation and that it decreased to the normal level by 24 h thereafter. Furthermore, in both human and mouse brains, many of L-PGDS-positive cells were also immunoreactive for p53; and some of these expressed cleaved caspase-3. The expression of L-PGDS in degenerating neurons implies that L-PGDS functions as an early stress protein to protect against neuronal death in the HIE brain.     

Simple method for the identification of oxidative fibers in skeletal muscle

Skeletal muscle is composed of several different types of myofiber: slow oxidative (SO), fast glycolytic oxidative and fast glycolytic. However, the classification is usually determined by myosin heavy chain typing rather than by metabolic index. In this study, the oxidative metabolic index was investigated as a possible method of myofiber typing. Myoglobin, which is involved in oxygen transport and storage in myofibers, and mitochondria, which are the central organelles for oxidative metabolism, were studied. High levels of myoglobin and mitochondria are believed to exist in SO fibers, but the current study showed that they are considerably richer in some fast type fibers. As myofiber typing using the oxidative metabolic index is important physiologically, an attempt was made to find a simple method for this purpose. Some mitochondrial proteins have been observed to auto-fluoresce but until now this effect was too faint to detect easily. Owing to the recent advances in cooling charge-coupled device technology, such auto-fluorescence can now be used for myofiber typing, and the simple and rapid method for doing so is reported here.