Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames.

AIMS: To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. METHODS AND RESULTS: Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D=0.989) was equal to that of PFGE subtyping (D=0.986). CONCLUSIONS: MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. SIGNIFICANCE AND IMPACT OF THE STUDY: MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks.

Nicotine suppresses the P13 auditory evoked potential by acting on the pedunculopontine nucleus in the rat.

We identified a potential novel site of action for nicotine (NIC) since (a) systemic injection of NIC led to a dose-dependent decrease in the amplitude of the sleep state-dependent, vertex-recorded, P13 midlatency auditory evoked potential (generated by the reticular activating system, RAS), (b) localized injections of a nicotinic receptor antagonist into the pedunculopontine nucleus (PPN, the cholinergic arm of the RAS) blocked the effects of systemic NIC on the P13 potential (a measure of level of arousal), and (c) localized injection of a nicotinic receptor agonist into the PPN also led to a decrease in the amplitude of the P13 potential, an effect blocked by PPN injection of a nicotinic receptor antagonist. There were minor changes in the manifestation of the startle response (SR) at the concentrations used; however, NIC did decrease the hippocampal N40 potential, although its effects were not affected by antagonist or agonist injections into the PPN. These results suggest a potential mechanism underlying the anxiolytic effects of NIC-suppression of the cholinergic arm of the RAS.

The P50 midlatency auditory evoked potential in patients with chronic low back pain (CLBP).

OBJECTIVE: Patients with Chronic Low Back Pain (CLBP) show arousal, attentional and cognitive disturbances. The sleep state-dependent P50 midlatency auditory evoked potential was used to determine if patients with CLBP [with and without co-morbid depression (DEP)] show quantitative disturbances in the manifestation of the P50 potential. METHODS: P50 potential latency, amplitude and habituation to repetitive stimuli at 250, 500 and 1000ms interstimulus intervals (ISIs) was recorded, along with the McGill Pain Questionnaire-Short Form (MPQ-SF). CLBP subjects (n=42) were compared with Controls (n=43), and with subjects with DEP only (n=6). Of the CLBP subjects, 20/42 had clinical depression (CLBP+DEP); 8/20 were taking anti-depressant medication (CLBP+DEP+med), the others were not (CLBP+DEP-med). RESULTS: There were no differences (ANOVA) in age, sex or P50 potential latency, although there was a trend towards increased latencies in CLBP groups. P50 potential amplitude was lower in CLBP groups, but not in sub-groups, again indicating a trend. P50 potential habituation was decreased in the DEP only subjects at the 250m ISI, and decreased in CLBP+DEP-med subjects at the 500ms ISI. This difference was not present in CLBP+DEP+med subjects. The MPQ-SF revealed that patients with CLBP and CLBP+DEP-med showed lower pain scores than CLBP+DEP+med patients. CONCLUSIONS: There is decreased habituation of the P50 potential habituation in unmedicated patients with CLBP+DEP compared to Controls. SIGNIFICANCE: Patients with CLBP+DEP-med may be less able to disregard incoming sensory information, including painful sensations, but anti-depressant medications help correct this deficit. However, their perception of pain may be increased by medication.

Effects of rotation on the sleep state-dependent midlatency auditory evoked P50 potential in the human.

Sopite syndrome, characterized by loss of initiative, sensitivity to normally innocuous sensory stimuli, and impaired concentration amounting to a sensory gating deficit, is commonly associated with Space Motion Sickness (SMS). The amplitude of the P50 potential is a measure of level of arousal, and a paired-stimulus paradigm can be used to measure sensory gating. We used the rotary chair to elicit the sensory mismatch that occurs with SMS by overstimulating the vestibular apparatus. The effects of rotation on the manifestation of the P50 midlatency auditory evoked response were then assessed as a measure of arousal and distractibility. Results showed that rotation-induced motion sickness produced no change in the level of arousal but did produce a significant deficit in sensory gating, indicating that some of the attentional and cognitive deficits observed with SMS may be due to distractibility induced by decreased habituation to repetitive stimuli.

Scirrhous cancer of the stomach which survived for more than five years after neoadjuvant chemotherapy with UFT (uracil and tegafur) and cisplatin.

A 68-year-old man was diagnosed as having a scirrhous cancer of the stomach. Carcinomatous peritonitis was suspected on abdominal CT examination. Three courses of uracil and tegafur (UFT)/cisplatin (CDDP) chemotherapy were administered. The primary foci were reduced in size, then total gastrectomy was performed. Histological findings revealed a poorly differentiated adenocarcinoma with scirrhous invasion into the subserosa. Histological efficacy of the chemotherapy was judged to be grade 2. The patient has been alive without disease for more than five years after total gastrectomy. Neoadjuvant chemotherapy with UFT and CDDP may have contributed to the favorable clinical outcome in this patient.

Hepatitis C virus core protein binds to a DEAD box RNA helicase.

Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation.

Orthotopic transplantation of a partial hepatic autograft in dogs.

The purpose of this study was to investigate the availability of an orthotopic transplantation of partial hepatic autograft in dogs as a means of surgical training. Male mongrel dogs weighting 10-15 kg were used. The left lobe of the liver was harvested while preserving the left branches of the portal vein, hepatic artery and bile duct, and the left hepatic vein. The remnant liver was removed while preserving the inferior vena cava using a veno-venous bypass. Orthotopic transplantation of the autograft was performed while anastomosing the left hepatic vein to the inferior vena cava, portal and arterial reconstruction, and external biliary drainage. Thirteen out of 29 dogs survived more than 48 h after transplantation. However, 6 out of 13 dogs were sacrificed after developing bile peritonitis due to a dislodgement of the biliary catheter, and only two dogs were able to survive for 7 days after transplantation. The arterial ketone body ratio recovered to 1.0 within 1 h after reperfusion, and the ratio of the dogs that survived for more than 48 h remained above 1.0 until sacrifice. Orthotopic transplantation of a partial hepatic autograft is a useful and simple procedure to train surgeons for partial liver transplantation.

Isoform-specific intracellular vesicle formation by recombinant annexin XI-A in Sf9 cells.

Annexins are a group of structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and have the ability to self-aggregate and to promote vesicle aggregation and membrane fusion. Two isoforms of annexin XI, termed XI-A and XI-B, were previously identified by screening a bovine chondrocyte cDNA library. But little is known about differences in their biological function. In the present study, we therefore examined the results of expression of the two proteins in Spodoptera frugiperda (Sf9) insect cells, and in mammalian COS-7 cells. Annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. Recombinant annexin XI-A but not XI-B caused formation of spherical "annexin XI-associated vesicles, " in the cytoplasm of Sf9 cells. Furthermore, indirect immnocytofluorescence studies showed similar phenomenon, that of local aggregation, with transfected annexin XI-A in COS-7 cells, whereas annexin XI-B remained diffusely distributed throughout the cytoplasm. Since annexin XI isoforms differ in amino acid sequence only in the alternative splicing region of the N-terminal domain, these findings suggest that this domain has distinct biological significance in terms of aggregation and vesicle formation.

Gilbert's syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDP-glucuronosyltransferase.

Gilbert's syndrome, which is characterized by chronic, non-hemolytic unconjugated hyperbilirubinemia, is caused by a reduction in the activity of hepatic bilirubin UDP-glucuronosyltransferase (UGT). Here, we report that all examined patients with this disease carried missense mutations in the gene for UGT and that the mutations were heterozygous. An expression study in COS cells in vitro, using the expression vector pcDL that carried the mutated gene for UGT from a patient, indicated that approximately 14% of the normal UGT activity was expressed. However, the UGT activity of the patient with Gilbert's syndrome was unexpectedly < 50% of the normal, perhaps as the result of the dominant negative nature of the mutation.

Development-related and cell-type specific nuclear localization of annexin XI: immunolocalization analysis in rat tissues.

Subcellular distribution of a novel annexin, annexin XI, was examined in rat tissues. As we have previously reported, annexin XI mainly localizes in nuclei of 3Y1, the rat embryonic fibroblast cell line; however, immunoperoxidase staining was not particularly nuclear in most of the tissues examined in adult rats. Nuclear localization of annexin XI was uncommon in adult rat tissues. By contrast, in the day-14 rat embryo, the undifferentiated mesenchymal cells were labeled mainly in the nuclei. The connective tissues of 18-day fetus, however, did not show predominantly nuclear staining any longer. In addition, the developing gray matter of the embryonic rat spinal cord exhibited primarily nuclear localization of annexin XI while the annexin XI immunoreactivity diminished and became absent from the nuclei in the adult spinal cord. On the other hand, the endodermal cells never displayed nuclear annexin XI at any developmental stages examined. All these findings suggested that subcellular localization of annexin XI is regulated depending on developmental stages and the cell types.

Inhibition of acid secretion in gastric parietal cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93.

A novel Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) inhibitor, KN-93 potently inhibits gastric acid secretion from parietal cells. As previously reported (1), treatment of parietal cells with a selective inhibitor of CaM kinase II, KN-62 resulted in the inhibition of cholinergic-stimulated rabbit parietal cell secretion, whereas it failed to inhibit the histamine and forskolin response. In contrast effects of carbachol, histamine and forskolin were significantly inhibited by KN-93 with an IC50 of 0.15, 0.3 and 1 microM, respectively; these effects occurred without any changes in intracellular cyclic AMP and Ca2+ levels. In the present study we investigated the mechanism by which KN-93 acts upon the acid-secreting machinery of gastric parietal cells. Neither redistribution of the proton pump activity nor the morphological transformation were affected by KN-93. The drug only weakly inhibited the H+, K(+)-ATPase activity but strongly dissipated the proton gradient formed in the gastric membrane vesicles and reduced the volume of luminal space. Thus KN-93 acts at pH gradient formation whereas KN-62 acts only at CaM Kinase II.

Reconstruction of the four major ligaments in an unstable knee joint after dislocation by solvent-preserved human fascia lata transplantation. A case report.

Once the opportunity for primary repair of injured knee ligaments after traumatic dislocation has been lost, ligamentous reconstruction is difficult using only autogenic tissues because of the risk of loss of function at the donor site, so other substitutes are needed. The four major ligaments in the unstable knee of a 35-year-old man were reconstructed by solvent-preserved human fascia lata three months after traumatic open dislocation. The clinical results were satisfactory. Arthroscopic examination one year later showed that the reconstructed ligaments had good thickness and tension and were composed of autologous connective tissue without evidence of rejection. The literature on dislocation of the knee and on cruciate ligament reconstruction by allograft was reviewed, and a brief introduction to solvent-preserved human fascia lata was presented. The commercialization of this material has solved some common problems concerned with using allogenic tissues.