Somatic gene transfer of tagged K+ channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes.

To evaluate the roles of the C-termini of K + channels in subcellular targeting and protein-protein interactions, we created fusion constructs of the cell-surface antigen CD8 and the C-termini of Kv4.3, Kv1.4 and KvLQT1. Using a Cre-lox recombination system, we made 3 adenoviruses containing a fusion of the N-terminal-and transmembrane segments of CD8 with the C-termini of each of the 3 K + channels. Expression in polarized Opossum Kidney (OK) epithelial cells led to localization of CD8-Kv4.3 and CD8-Kv1.4 into the apical and basolateral membranes, while CD8-KvLQT1 remained in the endoplasmic reticulum (ER), even when co-expressed with MinK. When expressed in rat cardiac myocytes in culture, all the 3 constructs were diffusely targeted to the surface membrane. The ER retention of CD8-KvLQT1 in OK cells but not in cardiomyocytes thus reveals functional differences in trafficking between these two cell types. To probe functional roles of C-termini, we studied K + currents in cardiac myocytes expressing CD8-Kv4.3. Patch-clamp recordings of transient outward current revealed a hyperpolarizing shift of steady-state inactivation, implying that CD8-Kv4.3 may be disrupting the interaction of Kv4.x channels with one or more as-yet-undefined regulatory subunits. Thus, expression of tagged ion-channel fragments represents a novel, generalizable approach that may help to elucidate assembly, localization and function of these important signaling proteins.

Induction of hepatic tissue-type plasminogen activator and type 1 plasminogen activator-inhibitor gene expressions and appearance of their translation products in the bile following acute liver injury in rats.

BACKGROUND/AIMS: The plasminogen activator (PA)-plasmin system is primarily involved in fibrinolysis, but is also in the patho/physiological events in which breakdown of extracellular matrices is evoked topically. In this present study, we examined the expression of fibrinolytic factors, tissue-type PA (t-PA) and Type 1 PA inhibitor (PAI-1), in acute liver injury. METHODS: Acute liver injury was produced in rats by the intraperitoneal administration of carbon tetrachloride (CCl(4)). Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured to verify the hepatocellular damage. t-PA and PAI-1 gene expressions were measured by Northern blotting, and the cell type(s) expressing these genes was identified by in situ hybridization. t-PA and PAI-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: A single intraperitoneal administration of CCl(4) caused severe acute parenchymatous liver injury. Both t-PA and PAI-1 gene expressions were induced by the acute liver injury, and plasma t-PA and PAI-1 concentrations were also increased. In situ hybridization studies demonstrated that the hepatocytes were the cells expressing t-PA and PAI-1 genes during the acute liver injury. t-PA was also augmented in the bile, whereas PAI-1 was decreased there. CONCLUSIONS: t-PA and PAI-1 gene expressions are induced in the hepatocytes of rats with acute liver injury. These fibrinolytic factors induced by liver injury may play important roles in liver regeneration.

Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca(2+) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection.

Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca(2+) concentration ([Ca(2+)](m)), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoK(ATP) channel opener diazoxide attenuated the accumulation of [Ca(2+)](m) during simulated I/R (EC(50)=18 micromol/L). These effects of diazoxide were blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca(2+)](m) accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, DeltaPsi(m), in permeabilized myocytes revealed that diazoxide depolarized DeltaPsi(m) (by 12% at 10 micromol/L, P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca(2+) overload, as a consequence of partial mitochondrial membrane depolarization by mitoK(ATP) channels, underlies cardioprotection. Furthermore, mitoK(ATP) channels and the MPT differentially affect mitochondrial calcium homeostasis: mitoK(ATP) channels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion.

Morphological character of crystalline components present in saiga horn.

The purpose of this study was to investigate the ultrastructure of saiga-antelope (Saiga tatarica) horn for proposing the mechanism of the initial mineralization. Horn is derived from horny tooth of Cyclostomata. The minerals in saiga horn were identified crystallographically using electron microscopy and X-ray diffraction techniques. Soft X-ray photographs revealed the degree of the mineralization pattern. However, the number of rings did not indicate the age of saiga. Mineral deposites were observed among well banded keratin fibers and composed of powder like crystals. This deposited crystals were found by the X-ray diffraction method to be octacalcium phospate (OCP) by comparing these periodic lattice fringes to JCPDS card data. The chemical formula of OCP is Ca8H2(PO4)6.5H2O. Evidences for the presence of OCP in mature hard tissues have never been obtained. This phenomenon described here may be characteristic of saiga horn because we have found no reports on this type of OCP mineralization in any other animal species. It is possible that OCP is the precursor in the initial mineralization step, indicating in a nucleation of mineral on the keratin fibers.

Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells.

Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential (DeltaPsi) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK(ATP) channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 micromol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of DeltaPsi. Pharmacological opening of mitoK(ATP) channels by diazoxide (100 micromol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented DeltaPsi depolarization in a concentration-dependent manner (EC(50) approximately 40 micromol/L, with saturation by 100 micromol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent DeltaPsi indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK(ATP) agonist, and blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (500 micromol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK(ATP) channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.

Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini-plasmin. Mini-plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad-spectrum influenza A viruses and Sendai virus, even that of the plasmin-insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini-plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini-plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.

Clinical pathway for impalpable or small lung lesions treated with coil marking and thoracoscopy.

OBJECTIVES: Advances in computed tomography are detecting increasingly impalpable or small pulmonary lesions. We propose a clinical pathway for managing such lesions. METHODS: We conducted a retrospective study in a community teaching hospital to describe the hospital schedules of 18 patients having 19 lesions 10 mm or less and ground glass attenuation. Under computed tomography, a coil (Complex Helical Fibered Platinum Coil-18) was placed at the proximal side of the lesion. Using thoracoscopy and radiographic fluorography, we conducted partial lung resection targeting the coil the next day, adding lobectomy, if required. RESULTS: Final diagnosis included primary and metastatic lung cancer (n = 14), atypical adenomatous hyperplasia (n = 1), and benignancy (n = 4). Patients were admitted 2* days before surgery (*Numbers are medians). On postoperative day 3, chest tubes were removed. Epidural analgesia was continued for 5 days. On postoperative day 7, patients were discharged. Their admission charge was a total of yen 979,610. CONCLUSIONS: The hospital course above may be applied to the clinical pathway for managing impalpable or small lung lesions.

Penetration of the duodenum by an ingested needle with migration to the pancreas: report of a case.

A case of a penetration of the duodenum by a needle with migration to the pancreas in a 50-year-old man is reported herein. The patient was referred to us with a chief complaint of diarrhea. An abdominal plain roentgenogram showed a needle in the upper abdominal area. An abdominal computed tomography scan and contrast X-ray revealed the foreign body to be located outside of the duodenum and in the head of the pancreas. An emergency operation was therefore performed on the first day and the needle in the head of the pancreas was thus extirpated safely. A perforation of the gastrointestinal tract by an ingested foreign body is difficult to accurately and quickly diagnose when no peritonitis or abscess formation is observed. Therefore, the use of contrast X-ray is considered to be useful in the diagnosis of such a perforation.

Cluster analysis on multiple drugs susceptibility supplements genotyping of methicillin-resistant Staphylococcus aureus.

OBJECTIVE: To evaluate the typing power of cluster analysis of antimicrobial susceptibility. METHODS: Results of pulsed-field gel electrophoresis in 71 strains of methicillin-resistant Staphylococcus aureus were compared with cluster analysis of the diameter of growth inhibition in 11 drugs. Subjects were a consecutive series of patients (n = 71) from the wards and outpatient units of a community teaching hospital. RESULTS: The cluster analysis took 2 to 3 seconds once the data were entered into a computer. The sensitivity, specificity, and accuracy of the cluster analysis were 76.3%, 58.3%, and 73.2%, respectively, using genotyping as the reference. CONCLUSIONS: The cluster analysis offered real-time epidemiologic data at minimal cost and labor, warranting its cost-effective role.

Angiotensin II type 1 receptor blockade abolishes specific K(ATP)channel gene expression in rats with myocardial ischemia.

The cardiac ATP-sensitive potassium (K(ATP)) channel is potentially composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the cardiac type of sulfonylurea receptor (SUR2A). We reported that cardiac Kir6.1 mRNA and protein are specifically upregulated in the non-ischemic as well as the ischemic regions in rats with myocardial ischemia, suggesting that humoral and/or hemodynamic factors are responsible for this regulation. In the present study, pretreatment with TCV-116, an angiotensin (Ang) II type 1 receptor antagonist, completely inhibited the upregulation of Kir6.1 mRNA and protein expression in both regions of rat hearts subjected to 60 min of coronary artery occlusion followed by 24 h of reperfusion; whereas pretreatment with lisinopril, an Ang converting enzyme (ACE) inhibitor, partly inhibited this upregulation. Except for rats pretreated with TCV-116, Kir6.1 mRNA levels were positively correlated with those for brain natriuretic peptide (BNP), a molecular indicator of regional wall stress, in both the non-ischemic and the ischemic regions. Plasma Ang II levels were not elevated in rats with control myocardial ischemia compared with sham rats. Thus, the stress-related induction of cardiac Kir6.1 mRNA and protein expression under myocardial ischemia is inhibited by pretreatment with an AT1 antagonist, but also in part by an ACE inhibitor, suggesting that activation of local renin-angiotensin system may play a role.

The properties of the Kir6.1-6.2 tandem channel co-expressed with SUR2A.

Functional ATP-sensitive K (KATP) channels have an octameric subunit structure with four pore-forming subunits (Kir6.x) and four sulfonylurea receptors (SURx). In the present study, the properties of the heteromeric KATP channel whose pore subunits are composed of Kir6.1 and Kir6.2 were examined using a heterologous expression system. In COS7 cells co-transfected with Kir6.1, Kir6.2 and SUR2A at a ratio of 1:1:2, KATP channels showed various unitary conductances between those of Kir6.1/SUR2A (33.6+/-4.2 pS) and Kir6.2/ SUR2A (67.1+/-1.6 pS). Kir6.1-6.2 tandem protein, constructed by fusing the C-terminus of Kir6.1 to the N-terminus of Kir6.2 with a ten glutamine linker sequence, also formed a channel with an intermediate conductance (58.9+/-1.5 pS). Kir6.2 and Kir6.1-6.2 showed similar sensitivity to ATP4-: half-maximal inhibition (IC50) was obtained at 14.1+/-12.8 microM and 17.6+/-9.6 microM, respectively. In the presence of Mg2+, Kir6. 1-6.2 was significantly less sensitive than Kir6.2 to MgATP (IC50=95.5+/-49.6 microM versus 18.9+/-5.0 microM). These results suggest that Kir6.1 and Kir6.2 are endowed with the potential to form a heteromeric KATP channel, which has a low sensitivity to MgATP.

Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus by vapour contact.

The inhibitory effect of seven essential oils on the apical growth of hyphae of Aspergillus fumigatus was studied using a bio cell tracer by vapour contact in a sealed vessel. Based on the inhibitory pattern, these essential oils were classified into three groups. The first group, composed of citron, lavender and tea tree oils, stopped the apical growth in a loading dose of 63 micrograms ml-1 air, but allowed the regrowth of the hyphae after removal of the vapour, indicating fungistatic action. The second group, consisting of perilla and lemon-grass oils, stopped the apical growth in a loading dose of 6.3 micrograms ml-1 air, and did not allow the regrowth after gaseous contact at 63 micrograms ml-1 air, indicative of fungicidal action. The third group, consisting of cinnamon bark and thyme oils, retarded the growth in a dose of 6.3 micrograms ml-1 air, stopped it in a dose of 63 micrograms ml-1 air, and incompletely suppressed regrowth of the hyphae. Gas chromatographic analysis revealed that vapours of essential oils were absorbed on fungal mycelia and agar medium most abundantly by the first group, followed by the second and third groups, reflecting the volatility of the respective groups. Suppression of the apical growth by vapour contact was ascribed to the direct deposition of essential oils on fungal mycelia, together with an indirect effect via the agar medium absorbed.

Hemopneumothorax and hemoperitoneum in a case with large cell carcinoma of the lung.

Hemopneumothorax and hemoperitoneum coincide rarely in nontraumatic cases. Here, a 70-year-old male presented a left axillary lymph node and was diagnosed as having metastatic squamous cell carcinoma. Under the same diagnosis, another lesion developed in the right femur and was resected. One year later, computed tomography detected another tumor in the left adrenal gland. Shortly afterwards, left pneumothorax developed and a chest operation revealed hemopneumothorax due to a ruptured cavitary form of large cell carcinoma. The serum showed a human chorionic gonadotropin-beta level of 1,100 ng/ml. At three-months later, he died of hemoperitoneum. The autopsy demonstrated hepatic metastases and a ruptured adrenal metastasis; microscopy showed marked trophoblastic and squamous cell changes in these organs. This patient was unique in that the rupture of the pulmonary and the adrenal lesions caused clinical manifestation.

Alteration of the membrane lipid environment by L-palmitoylcarnitine modulates K(ATP) channels in guinea-pig ventricular myocytes.

Sarcolemmal adenosine 5'-triphosphate-sensitive K+ channels (K(ATP)) are dramatically up-regulated by a membrane phospholipid, phosphatidyl-inositol-4,5-bisphosphate (PIP2). During ischaemia, L-palmitoylcarnitine (L-PC), a fatty acid metabolite, accumulates in the sarcolemma and deranges the membrane lipid environment. We therefore investigated whether alteration of the membrane lipid environment by L-PC modulates the K(ATP) channel activity in inside-out patches from guinea-pig ventricular myocytes. L-PC (1 microM) inhibited KATP channel activity, without affecting the single channel conductance, through interaction with Kir6.2. L-PC simultaneously enhanced the ATP sensitivity of the channel [concentration for half-maximal inhibition (IC50) fell from 62.0+/-2.7 to 30.3+/-5.5 microM]. In contrast, PIP2 attenuated the ATP sensitivity (IC50 343.6+/-54.4 microM) and restored Ca2+-induced inactivation of KATP channels (94.1+/-13.7% of the control current immediately before the Ca2+-induced inactivation). Pretreatment of the patch membrane with 1 microM L-PC, however, reduced the magnitude of the PIP2-induced recovery to 22.7+/-6.3% of the control (P<0.01 vs. 94.1+/-13.7% in the absence of L-PC). Conversely, after the PIP2-induced recovery, L-PC's inhibitory action was attenuated, but L-PC partly reversed the PIP2-mediated decrease in the ATP sensitivity (IC50 fell from 310+/-19.2 to 93.1+/-9.8 microM). Thus, interaction between L-PC and PIP2 in the plasma membrane appears to regulate K(ATP) channels.

Dissolution of dense carbonate apatite subcutaneously implanted in Wistar rats.

Hydroxyapatite (HA) is used as a biomaterial in orthopedic applications because it is similar in composition to bone mineral; however, carbonate apatite (CHA) is closer in chemical composition to bone mineral because bone mineral contains significant amounts of carbonate, yet there have been few reports comparing biological responses to HA and CHA. It is generally agreed that bone forms a bond of some kind to HA, and there is conflicting evidence as to whether HA is resorbed in vivo or not. However, comparative reports generally agree that beta-tricalcium phosphate (beta-TCP) is removed faster than HA from an implant site by an undetermined combination of resorption and/or dissolution. The work reported here attempts to provide the first directly comparable subcutaneous dissolution data for dense sintered HA, beta-TCP, and CHA in rats. The weight losses of HA, beta-TCP, and 3.2 wt % CHA were approximately 60, 520, and 320 mg m(-2) day(-1), respectively. Histological sections did not show any evidence of giant cells, and all of the samples were encapsulated with fibrous tissue. beta-TCP and (to a lesser degree) CHA were found to be resistant to dissolution around the edges of the sample. An X-ray analysis did not indicate that any phase transformation had occurred in the dissolution resistant region.

Computerized antibiogram for methicillin-resistant Staphylococcus aureus in chest surgery.

BACKGROUND: An increasing number of cases of postoperative morbidity involving methicillin-resistant Staphylococcus aureus have been reported in thoracic surgery. To prevent its outbreak, cluster analysis using a personal computer was employed. METHODS: A total of 120 patients undergoing operations on the lung and mediastinum were included into this study. Materials were isolates of methicillin-resistant Staphylococcus aureus newly recovered from across the hospital. The cluster analysis used antimicrobial susceptibility in 12 drugs, which were categorically valued to produce Euclidean distance to form clusters of similarity. RESULTS: Six of the 120 patients were found to be positive for the microbe before or after thoracotomy. A total of two patients (1.7%) became symptomatic postoperatively, i.e., one of four preoperatively-positive patients and one of two postoperatively-positive cases. The analysis suggested that preoperative patients shared the strains in the same non-surgical ward. DISCUSSION: A computerized antibiogram does not always strictly type Staphylococcal strains but has advantages in typing with ease and at decreased cost. The current analysis suggested that patient harboring the strains migrated across wards. CONCLUSION: Computerized antibiograms for Staphylococcal strains may assist to prevent an outbreak of their infection in chest surgery.

[A patient operated for occlusion of the superior mesenteric artery].

A 40-year-old woman presented with fever, dysarthria, and left hemiparesis. Diagnostic imaging detected an infarction in the right cerebrum; lupus anticoagulant was positive. A T4N3M0 adenocarcinoma coexisted in the left lung, which was irradiated once. Two days later, the superior mesenteric artery was occluded. The bowel was widely resected but she died of hepatic infarction. The lung cancer may have triggered catastrophic antiphospholipid syndrome.