Relationship between the thromboxane A2 receptor gene and susceptibility to cerebral infarction.

The risk of cerebral infarction (CI) in an individual is dependent on the interplay between genetic risk factors and environmental influences. Binding of thromboxane A2 (TXA2) to its receptor (TP) modulates thrombosis/hemostasis and plays a significant role in the pathogenesis of CI. The aim of the present study was to investigate the relationship between human TP gene single nucleotide polymorphisms (SNPs) and haplotypes and CI in a Japanese population. A genetic association study was performed in 194 CI patients and 365 non-CI subjects by specifically characterizing 6 SNPs in the human TP gene (rs2271875, rs768963, rs2238634, rs11085026, rs4523 and rs4806942). Analysis demonstrated that there were significant differences in the overall distribution of genotypes and dominant or recessive models of rs2271875 and rs768963 between the CI and the non-CI groups. Multiple logistic regression analysis revealed that the C allele of rs768963 was significantly associated with CI (p = 0.029), even after adjusting for confounding factors (odds ratio: 2.41). Further, the C-T-C haplotype of rs768963-rs2238634-rs4806942 was significantly more frequent in the CI group (23.0%) than in the non-CI group (17.7%). These results suggest that specific SNPs and haplotypes may have utility as genetic markers for the risk of CI and that TP or a neighboring gene is associated with the increased susceptibility to CI.     

Whole-Blood Assay to Measure Oxidative Burst and Degranulation of Neutrophils for Monitoring Trauma Patients

AbstractBackground and Purpose: Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms, but excessive PMN activation after trauma causes tissue injury. Rapid monitoring of PMN function is critical for the assessment of the inflammatory state of trauma patients. Here, the authors adapted two simple and rapid methods to measure oxidative burst and degranulation of human PMNs in whole blood to avoid potential interference of cell isolation procedures with the assessment of PMN function.Material and Methods: Heparinized blood was drawn from healthy volunteers or trauma patients, preincubated at 37 °C for 5 min, and stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Four assays for oxidative burst were tested: (1) cytochrome C; (2) homovanillic acid (HVA); (3) Amplex^® Red; and (4) flow cytometry with dihydrorhodamine 123 (DHR). PMN degranulation was assessed with flow cytometry using antibodies to: (1) CD11b/Mac-1 (CD18); (2) CD63; and (3) CD66b (CD67).Results: With the exception of the DHR method, all methods to measure oxidative burst were found to be unsuitable in whole blood due to interference of plasma proteins and hemoglobin with the fluorimetric or photometric readouts. By contrast, all degranulation methods were suitable for whole-blood studies. However, for the assessment of formyl peptide-induced degranulation, anti-antibodies to CD11b/Mac-1 and CD66b were up to five times more sensitive than antibodies to CD63. Thus, the degranulation and DHR methods were optimized for increased sensitivity, speed, and specificity and their usefulness to measure PMN function in trauma patients was tested.Conclusion: The whole-blood methods based on flow cytometry with DHR, anti-CD11b/Mac-1, and anti- CD66b are rapid, simple, and reliable techniques to assess PMN function for trauma research.     

Local muscimol disinhibits mesolimbic dopaminergic activity as examined by brain microdialysis and Fos immunohistochemistry

Infusion of muscimol (5×10⁻⁵ M, 60 min) into the nucleus accumbens (NAC) through a dialysis membrane caused a significant increase in extracellular dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC). Fos-like immunoreactivity induced by intra-NAC infusion of muscimol was seen ipsilaterally in many accumbofugal target areas, but no Fos-positive neurons were seen in the vicinity of the dialysis membrane in the NAC. Sequential staining of Fos and tyrosine hydroxylase (TH) immunoreactivities revealed that a portion of A10 dopaminergic neurons were double-labelled. These results suggest that muscimol in the NAC disinhibits mesolimbic DA neuronal activity possibly through activity of the accumbofugal GABA neuron system.     

Techniques of nasotracheal intubation with the fiberoptic bronchoscope

When nasotracheal intubation with a fiberoptic bronchoscope is performed, the tube may be blocked in the nasal cavity or larynx, resulting in several complications including epistaxis and hoarseness. We review the causes and complications of tube blockage and discuss optimal techniques for minimizing it.     

Accelerated reversal of pancuronium blockade with divided administration of neostigmine

Key words  reversal of neuromuscular blockade pancuronium - neostigmine     

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1α,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1α,25(OH)₂D₃ were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1α,25(OH)₂D₃, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that (1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1α,25(OH)₂D₃ exhibit non-functional basic features of osteoclast morphology, and (2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.     

Invasive fungal infection following reduced-intensity cord blood transplantation for adult patients with hematologic diseases.

Invasive fungal infection (IFI) is a significant complication after allogeneic hematopoietic stem cell transplantation (HSCT); however, we have little information on its clinical features after reduced intensity cord blood transplantation (RICBT) for adults. We reviewed medical records of 128 patients who underwent RICBT at Toranomon Hospital between March 2002 and November 2005. Most of the patients received purine-analogbased preparative regimens. Graft-versus-host disease (GVHD) prophylaxis was a continuous infusion of either tacrolimus 0.03 mg/kg or cyclosporine 3 mg/kg. IFI was diagnosed according to the established EORTC/NIH-MSG criteria. IFI was diagnosed in 14 patients. Thirteen of the 14 had probable invasive pulmonary aspergillosis and the other had fungemia resulting from Trichosporon spp. Median onset of IFI was day 20 (range: 1-82), and no patients developed IFI after day 100. Three-year cumulative incidence of IA was 10.2%. Four of the 13 patients with invasive aspergillosis (IA) developed grade II-IV acute GVHD, and their IA was diagnosed before the onset of acute GVHD. The mortality rate of IFI was 86%. Multivariate analysis revealed that the use of prednisolone >0.2 mg/kg (relative risk 7.97, 95% confidence interval 2.24-28.4, P = .0014) was a significant risk factor for IA. This study suggests that IFI is an important cause of deaths after RICBT, and effective strategies are warranted to prevent IFI.     

Calcium-phosphate-hybridized tendon directly promotes regeneration of tendon-bone insertion

We developed a novel technique to improve tendon-bone attachment by hybridizing calcium phosphate (CaP) with tendons using an alternate soaking process. We characterized the deposited CaP on or in tendons and determined the healing process of anterior cruciate ligament (ACL) grafts by implanting CaP-hybridized free tendons in bone tunnels intra-articularly. Tendons to be implanted were alternately soaked 10 times in a Ca-containing solution and a PO(4)-containing solution for 30 s each. Treated tendons had ash contents threefold that of untreated tendons. Low-crystallinity apatite was found on or in treated tendons. In animal experiments, the CaP-hybridized tendon exhibited osteoclasts at the tendon-bone interface at 5 days after operation. At 2 weeks after operation, there were more osteoclasts and osteoblasts around the tendon than at 5 days after operation. Directly bonded areas were partially found between the implanted tendon and newly formed bone. The formation of a cartilage layer was partially apparent at 3 weeks after operation. The newly formed bone was observed almost around the tendon. We conclude that CaP-hybridized tendons clearly enhance the healing process of ACL grafts at the tendon-bone interface and regenerate a direct insertion-like formation of tendons similar to a normal healthy ACL insertion within 3 weeks after operation.     

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.