Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Synergistic effects of injection of bone marrow cells into both portal vein and bone marrow on tolerance induction in transplantation of allogeneic pancreatic islets.

We have established a new method for allogeneic pancreatic islet (PI) transplantation: relatively low doses of irradiation followed by simultaneous transplantation of PIs and bone marrow cells (BMCs) via the portal vein (PV). In the present study, we have compared this method with intra-bone marrow (IBM)-bone marrow transplantation (BMT), and with a combination of both methods. Streptozotocin (STZ)-induced diabetic-recipient rats, Fischer 344 (F344, RT1A(l), RT1B(l)), were irradiated 1 day before transplantation. PIs of Brown Norway rats (BN, RT1A(n), RT1B(n)) were transplanted into the liver of the diabetic F344 rats via the PV. BMCs from BN rats were injected into the recipients' bone marrow (IBM), PV or intravenously (IV) or by a simultaneous combination of PV plus IBM (PV+IBM). We compared graft survival among the groups of '9 Gy+IBM'(10/10 accepted), '9 Gy+PV'(7/10 accepted), '9 Gy+IV'(0/7 accepted), '9 Gy+PV+IBM'(8/8 accepted), '8.5 Gy+IBM'(4/9 accepted), '8.5 Gy+PV'(0/7 accepted), '8.5 Gy+IV'(0/7 accepted), '8.5 Gy+PV+IBM'(9/12 accepted), '8 Gy+IBM'(2/10 accepted) and '8 Gy+PV+IBM'(2/8 accepted). As we reported previously, PV-BMT is more effective in inducing the acceptance of allogeneic PIs than IV-BMT. However, IBM-BMT requires less pretreatment than PV-BMT. (PV+IBM)-BMT was found to be the most effective in inducing the acceptance of allogeneic PIs. These results suggest that allogeneic PI-transplantation in conjunction with (PV+IBM)-BMT could become a viable strategy.

Protection of T cells from radiation-induced apoptosis by Cepharanthin.

Cepharanthin (CE) is a medicine that contains several biscoclaurine alkaloids. We examined the effects of CE on radiation-induced T cell apoptosis. Radiation induced apoptosis on T cells in a dose-dependent manner, while CE inhibited radiation-induced apoptosis. CE also attenuated the cytotoxic effects of radiation on the proliferative response of T cells. CE inhibited not only the loss of mitochondrial transmembrane potential but also the activation of caspase 3 in irradiated T cells. Radiation plus CE induced the up-regulation of Bax and the down-regulation of Bcl-2 in T cells in comparison with radiation alone. These results suggest that CE inhibits the signal transduction pathway of apoptosis induced by radiation, regardless of the expression of Bcl-2 or Bax.

The clinical importance of the trimethadione tolerance test as a method for quantitative assessment of hepatic functional reserve in patients with biliary atresia.

BACKGROUND: The trimethadione (TMO) tolerance test was performed to evaluate its usefulness in the assessment of hepatic functional reserve in patients with biliary atresia. METHOD: Nineteen patients with biliary atresia after hepatic portoenterostomy (age range: 2 months to 25 years; sex: 6 males and 13 females) were studied. The study was performed in the morning after a 12-h fast. TMO was given orally, at a dose of 4 mg/kg, with 5 mL of 5% glucose 2 h before breakfast. Blood samples (0.5 mL) were collected to determine serum TMO and dimethadione (DMO), a metabolite of TMO, levels 4 h after the administration of TMO. TMO and DMO were measured by a gas-liquid chromatographic method. RESULTS: A higher total bilirubin level (over 1 mg/dL) in patients with jaundice was reflected in the smaller serum DMO/TMO ratio 4 h after the oral administration of TMO. In addition, these patients with total bilirubin levels of 1 mg/dL or less had a significantly lower DMO/TMO ratio than the control group (healthy subjects). The serum DMO/TMO ratio showed a close correlation with the Child-Pugh score, which is used for overall evaluation of severity of cirrhosis and Mayo risk scores for primary biliary cirrhosis in adults (0.856, P < 0.01 and 0.788, P < 0.01, respectively). The TMO tolerance test shows the benefit of performing a relatively early test of dynamic liver function to evaluate hepatic functional reserve in pre- and post-operative biliary atresia patients.

Dioxin detection based on immunoassay using a polyclonal antibody against octa-chlorinated dibenzo-p-dioxin (OCDD).

An enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody against octa-chlorinated dibenzo-p-dioxin (OCDD) is presented. This method is based on a competitive reaction between OCDD and OCDD-HRP (horseradish peroxidase) conjugate against the antibody, whereby OCDD-HRP is detected colorimetrically at 450 nm. The detection limit of OCDD was 0.78 pg mL(-1). Optimizing the reaction conditions of the assay, cross reactivities of some dioxins against the antibody are discussed.

Proteasome inhibitor 1 enhances paclitaxel-induced apoptosis in human lung adenocarcinoma cell line.

Paclitaxel is a chemotherapeutic drug that induces apoptosis in tumor cells by stabilizing microtubules, prevents normal mitosis, and blocks the cell cycle at the G2/M phase. We have previously reported that the activation of caspase-3 and caspase-8 plays a crucial role in paclitaxel-induced apoptosis. Anti-tumor reagents including paclitaxel, irradiation, and other stimuli activate the transcription factor NF-kappaB, which has the ability to suppress the apoptotic potential of those stimuli. Using a human lung adenocarcinoma cell line (LC-2-AD), we therefore examined whether the inhibition of NF-kappaB activity by proteasome inhibitor 1 (PS1) could become a new adjuvant therapy for cancer. A synergistic effect on apoptosis induction was observed with the combination of more than 0.1 microg/ml paclitaxel and 0.5 microM PS1. An increase in the cell number of apoptotic cells is correlated with the loss of Deltaphim and the activation of caspase-3 and caspase-8. Furthermore, augmented apoptosis is related to NF-kappaB activation. Based on these findings, we propose that the combination of paclitaxel with PS1 could be a new strategy for cancer treatment.

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques.

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.

An autopsy case of giant cell myocarditis probably due to a non-steroidal anti-inflammatory drug.

An autopsy case of giant cell myocarditis (GCM) in a 74-year-old woman is presented. She suffered from hepatic dysfunction, skin eruption and disseminated intravascular coagulation due to the side-effects of a non-steroidal anti-inflammatory drug. After admission, heart failure progressed rapidly, and the patient died suddenly. At autopsy, her heart was slightly enlarged and the heart muscle was thickened with many small whitish nodules. She was diagnosed with GCM because of the infiltration of multinuclear giant cells, histiocytes, eosinophils and lymphocytes into the heart. We did not find any similar lesions in any other organs. Giant cell myocarditis, the etiology of which is not defined, is a rare disease with unfavorable prognosis. This case suggests the possibility of drug-induced GCM.

A fluorescent nitrate sensing system using a reaction cartridge and titanium trichloride.

A simple and highly sensitive cartridge type nitrate sensing system was developed using titanium trichloride (TiCl(3)) in hydrochloric acid to reduce nitrate to ammonium ion. The system primarily consisted of a nitrate reduction section using titanium trichloride and an ammonia detection section. The nitrate was reduced in a simply made cartridge equipped with filter units and the resulting ammonium ion solution was directly introduced into a flow injection system, where it was neutralized to ammonia and allowed to react with o-phthalaldehyde (OPA). The isoindole thus formed was detected by virtue of its fluorescence, allowing quantitation of the nitrate in the initial sample. Our sensing system has a detection limit of 0.01 mg l(-1) and a dynamic linear range from 0.05 to 2.5 mg l(-1) with response times of less than 5 min for the entire procedure. The system had a relative standard deviation (RSD) of less than 2.56% after more than 30 consecutive measurements of 0.5 mg l(-1) NO(3)(-). The system is unaffected by FeCl(3), Na(2)SO(4) and NaCl at concentrations of 200 mg l(-1) or by biological oxygen demand (BOD) values as high as 110 mg O l(-1). The effects of reaction time and titanium trichloride concentration were also investigated. Furthermore, several river water samples were examined.

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR).

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.

A new method for bone marrow cell harvesting.

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new "perfusion method" for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4(+) and CD8(+) T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this "perfusion method" are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the "perfusion method" is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-host-disease in allogeneic BM transplantation.

Bioassay of bile acids using an enzyme-linked DNA aptamer.

A new analytical method for the detection of bile acids has been developed by adopting an alkaline phosphatase-linked DNA oligomer that binds to bile acids. A 5'-biotin-labeled DNA oligomer with a 40-nucleotide length that is defined by the in vitro selection method was connected with alkaline phosphatase through an avidin-biotin linkage and applied to an enzyme immunoassay format. Sample solutions were incubated with small aliquots for a cholic acid-immobilized agarose matrix, on which the alkaline phosphatase-linked DNA oligomer had been bound prior to carrying out the assay. The amount of the alkaline phosphatase-linked DNA oligomer dissociated from the cholic acid-immobilized agarose matrix, which was detected using a fluorogenic substrate for alkaline phosphatase, indicated the amount of bile acids in the samples. The results suggest that the DNA aptamer directly linked with the reporter enzyme is applicable as a detector ligand for the immunoassay format. A linear calibration range was obtained for cholic acid between 0.1 to 5 mmol l-1 with a limit of detection of 10 mumol l-1. The %RSD was 7 at 5 mmol l-1 of cholic acid.

In vitro selection of DNA aptamers which bind to cholic acid.

DNA aptamers which bind to cholic acid have been identified by in vitro selection from a pool of approximately 9x10(14) DNA molecules. After 13 rounds of selection, 19 clones with 95-100 nucleotide length were sequenced. Deletion-mutant experiments and computational sequence analysis suggested that all clones contained cholic acid binding sequences which could fold into three-way junction structures. By comparing the sequences involved in the predicted three-way junction structure of these 19 clones, it was determined that the nucleotide sequences and lengths of three stem and loop regions have no similarity. The most conserved structure seems to have three base pairs flanking the junction of the three stems and they may form a hydrophobic cavity in which they interact with cholic acid.

Photocatalytic sensor for chemical oxygen demand determination based on oxygen electrode.

The construction and performance evaluation of a novel Chemical Oxygen Demand (COD) sensor is described. The sensor measures, using an oxygen electrode, a decrease of dissolved oxygen of a given sample resulting from photocatalytic oxidation of the organic compounds therein. As the photocatalyst, titanium dioxide (TiO2) fine particles adsorbed on a translucent poly(tetrafluoroethylene) (PTFE) membrane was used. The oxygen electrode with the membrane attached on its tip was used as the sensor probe. The operation characteristics of the sensor are demonstrated using an artificial wastewater and real water samples from lakes in Japan. This method is considered to be reliable, in that the observed parameter is close to the theoretical definition of chemical oxygen demand (COD), the amount of oxygen consumed for oxidation of organic compounds.

Detection of atrazine based on the SPR determination of P450 mRNA levels in Saccharomyces cerevisiae.

We describe a novel method for quantification of atrazine based on detection of P450 mRNA levels in Saccharomyces cerevisiae. The selected oligonucleotide probe exhibited specificity against P450 mRNA and was successfully immobilized on the sensor chip. The mRNA was subsequently quantified by RU change using a SPR system. When the cells were disrupted by boiling, mRNA could be measured without further purification at reduced sensitivity. This simple technique permits the detection of atrazine within 15 min. This rapid and highly sensitive method can be used for the detection of atrazine.

Application of polymer-embedded proteins to fabrication of DNA array.

A plasma-polymerized film (PPF) of hexamethyldisiloxane [HMDS; (CH(3))(3)SiOSi(CH(3))(3)] was used to immobilize streptavidin on a glass substrate. Another layer of HMDS-PPF was also applied to the protein, which was first adsorbed to an underlayer of the same kind of film. As the result, the streptavidin was "embedded" between the two layers of HMDS, whereby biotinylated molecules could be efficiently captured. The second layer of approximately 30 to 45 A PPF was sufficient to allow the binding of biotinylated molecules, whereas thicknesses of >90 A significantly hindered the streptavidin-biotin interactions. Fluorescence analysis revealed that the absence of an HMDS plasma-polymer (HMDS-PP) layer on either side of the streptavidin film resulted in a decrease in biotin binding. This immobilization technique was used to bind biotinylated oligonucleotides in sequence-specific DNA-DNA interactions. The hydrophobic properties of the plasma-polymerized HMDS thin film acted to minimize nonspecific DNA binding to the glass substrate. A DNA array was fabricated using this procedure and showed greatly decreased nonspecific DNA binding compared with a poly-L-lysine coated substrate.

Morphological change, loss of deltapsi(m) and activation of caspases upon apoptosis of colorectal adenocarcinoma induced by 5-FU.

Apoptosis is clearly distinguished from necrosis, morphologically and chemically. Morphologically, apoptosis is characterized by a condensed nucleus and the disappearance of microvilli without disruption of the cytoplasm. In this report, we demonstrate that 5-fluorouracil (5-FU)-induced early apoptotic cells are characterized by (i) ultracondensed mitochondria, (ii) no change in the microvilli or nucleus, (iii) a high mitochondrial transmembrane potential (Deltapsi(m)), and (iv) being annexin V(negative). The early apoptotic cells also show the active forms of caspase 8 and caspase 9. They rapidly lose Deltapsi(m) after further incubation. Therefore, we conclude that the ultracondensation of mitochondria precedes the loss of Deltapsi(m) and the exposure of phosphatidylserine to the outer leaflet of the cell membrane.

Interaction of three-way DNA junctions with steroids.

DNA aptamers that bind to cholic acid were previously isolated by an in vitro selection method. Secondary structural prediction and deletion-mutant experiments suggested that the cholic-acid binding regions of 19 sequenced clones could form three-way-junction structures. In this article, the secondary structures of the sequenced clones and the structural requirements for binding to cholic acid were evaluated. A course of mutational-analysis and chemical-modification experiments provided strong support for the predicted secondary structure and also indicated that the binding site is located at the branching point of the three-way junction. Sequence analysis revealed that the sequences of the three base pairs flanking the junction of the three stems are highly conserved among selected clones. The evaluation of the relative binding of several bile acids and structurally related steroids with the aptamer was also carried out. The results revealed a broad range of selectivity and preference for hydrophobic steroids rather than for cholic acid upon binding, indicating that the binding is driven by a hydrophobic interaction. The experimental results reported here allowed us to propose a structural model of a binding site formed by three Watson-Crick base pairs.

Effect of incident angle of light on sensitivity and detection limit for layers of antibody with surface plasmon resonance spectroscopy.

The effect of the incident angle of light on sensitivity and the detection limit for surface-plasmon resonance spectroscopy were examined. The sensitivities and the detection limit were experimentally measured using an antibody as a modeled analyte in the incident angles of a light region of 66-76 degrees. The results showed that the sensitivity of a smaller incident angle was higher than that of a larger one. For instance, the sensitivity of a 66 degree incident angle was three times higher than that of a 76 degree incident angle. The detection limit with a 66 degree incident angle was one-tenth of that with a 76 degree incident angle. These sensitivities and detection limits were compared with those of a commercially produced surface-plasmon resonance instrument. This comparison demonstrated that a wavelength resolution of the order of less than 10(-2) nm was necessary to obtain satisfactory sensitivities and detection limits. In addition, the refractive index and thickness of the antibody layer formed on a sensor surface was proposed by experimental results and theoretical calculation.