An ultrashort-duration, high-repetition-rate pulse source for laser ionization/mass spectrometry.

This paper describes a sample inlet system with several advantages over other pulsed valves, as applied to resonance-enhanced multiphoton ionization/time-of-flight mass spectrometry. The nozzle is based on online concentration by analyte adsorption/laser desorption (online COLD), where a capillary column with a narrowly synthesized tip is employed for sample introduction. The analyte molecules adsorbed at the tip are desorbed by a pulsed laser and are injected into a mass spectrometer as a packet. The online COLD nozzle can produce very short gas pulses on the order of 1 µs. Moreover, this nozzle is capable of operating over a wide range of repetition rates from 1 Hz to 1 kHz. In addition, this nozzle intrinsically possesses several unique characteristics; for instance, it can be heated to very high temperatures and has nearly zero dead volume. Therefore, the present sample introduction technique offers an ideal and versatile nozzle for laser ionization/mass spectrometry.

Population pharmacokinetic-pharmacodynamic analysis of istradefylline in patients with Parkinson disease.

This model-based analysis quantifies the population pharmacokinetic-pharmacodynamic efficacy and safety/tolerability relationships of orally administered istradefylline, a selective adenosine A(2A) receptor antagonist, in healthy participants and patients with Parkinson disease. Data from 6 phase 2/3 clinical trials comprised the population database, with 1760 and 1798 patients contributing to the efficacy and safety/tolerability analyses, respectively. The relationship between istradefylline area under the curve at steady state and percentage OFF time was described by a nonlinear model (Emax) based on time for the disease progression/placebo response component and an Emax model for the effect of istradefylline. The typical maximum decrease in percentage OFF time due to istradefylline exposure would be 5.79% (95% confidence interval = 4.09%-7.49%) with one-half of the maximum effect reached at an exposure of 1690 ng × hr/mL (95% confidence interval = 199-3180 ng × hr/mL). The pharmacokinetic-pharmacodynamic relationships for dyskinesia and dizziness were described by an Emax model, and for nausea, a power model was used. The probabilities of dyskinesia and dizziness are expected to plateau at a dose of 40 mg/d, and the probability of nausea is expected to continually rise as the dose is increased. Collectively, these results support a starting istradefylline dose of 20 to 40 mg/d.

A pair of concentric capillaries as an interface for gas chromatography and supersonic jet/multiphoton ionization/mass spectrometry.

A pair of concentric capillaries was developed to mix helium, which was used as the carrier gas for gas chromatography, with argon for efficient molecular cooling by supersonic jet expansion. A simple instrument was constructed for the evaluation of nozzle diameter using the Hagen-Poiseuille equation. The effects of nozzle diameter, type of expansion gas, flow rate, and the distance from the nozzle to the observation region were investigated. Mixing argon gas with the carrier gas helium resulted in efficient molecular cooling from 30 to 10 K and the complete disappearance of the background signal from the multiphoton ionization spectrum. Consequently, the spectral selectivity was significantly improved and the nozzle was successfully applied to isomer-selective analysis of dichlorotoluenes. Since the dead volume in the nozzle was negligible, it was suitable as an interface for gas chromatography and supersonic jet/multiphoton ionization/mass spectrometry.

A study of the pharmacokinetic interaction of istradefylline, a novel therapeutic for Parkinson's disease, and atorvastatin.

The effect of steady-state istradefylline, an agent for Parkinson's disease with P-glycoprotein and CYP3A inhibitory activity, on the pharmacokinetics of atorvastatin and its metabolites was evaluated in healthy volunteers. A single 40-mg dose of atorvastatin was administered to 20 subjects. After a 4-day washout, subjects received a single 40-mg atorvastatin dose following 40 mg istradefylline (n=16) or placebo (n=4) daily for 14 days. Plasma samples collected for 96 hours after atorvastatin administration, alone and in combination, were analyzed for atorvastatin, orthohydroxy atorvastatin, and parahydroxy atorvastatin. Istradefylline increased atorvastatin C(max) (53%), AUC(0-infinity) (54%), and t((1/2)) (27%); and increased AUC(0-infinity) for orthohydroxy atorvastatin (18%), but had no significant effect on its C(max) or t((1/2)); and had minimal effect on parahydroxy atorvastatin AUC(0-infinity). The lack of inhibition by istradefylline on metabolite systemic exposure, combined with increased atorvastatin systemic exposure, suggests a predominant P-glycoprotein inhibitory effect of istradefylline.

Evaluation of Langerhans' cells in human papillomavirus-associated squamous intraepithelial lesions of the uterine cervix.

Current research has been evaluating morphological modifications and density of Langerhans' cells in women with histopathological HPV lesions of the uterine cervix. Fourteen women with subclinical HPV infections underwent clinical, colpocytological colposcopy examinations, and paired biopsies of the uterine cervix. Histopathological, HPV hybrid capture and S-100 immunohistochemical examinations were performed in biopsy specimens. Groupings of viral lesions and normal tissue were analyzed by the Wilcoxon rank-sum test. Langerhans' cells in the specimens were frequently located in the intermediate and basal layers of the epithelium. A significant reduction of cytoplasmic profiles occurred in viral lesions (144.08 profiles/mm2) when compared to normal tissue (256.27 profiles/mm2) of the epithelium. An inverse modulation occurred in the cytoplasmatic profiles/nuclei ratio with 2.80 in viral lesions and 4.89 in normal tissue of the stroma. A local immunodeficiency based on cytoplasmic changes of Langerhans' cells has been postulated as a mechanism by which HPV could be involved in the genesis of neoplasia.

Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products.

AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan. METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers. Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so. The bacteriocin-producing isolates were divided into nine groups by phenotypic tests. As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes. The nine representative strains from each group were identified by 16S rRNA analysis. These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group'. The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles. The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila. CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes. They were considered to play an important role in preventing the growth of undesirable bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.

Potential value of CML-Hb in predicting the progression of bone cysts in dialysis-related amyloidosis.

BACKGROUND/AIM: Carboxymethyllysine is one of the common advanced glycation end products in vivo. In a previous report, we were the first to describe the increase of circulating carboxymethyllysine-hemoglobin (CML-Hb) levels in hemodialysis patients, particularly in patients with dialysis-related amyloidosis (DRA). The aim of this study was to investigate the predictive value of CML-Hb in the progression of DRA using computed tomography images of 2-year or 3-year follow-up periods of patients with amyloid bone cysts at the hip joint. METHODS: Circulating CML-Hb levels were measured as previously reported, and computed tomography scanning was conducted from 1996 to 1998 or 1999 in 57 hemodialysis patients whose original renal disease had been confirmed to be nondiabetic. Patients who showed a new growth of cysts or a growth rate of 30% or more were classified as progressive cases, while the other patients were classified as nonprogressive cases. RESULTS: (1) The circulating CML-Hb levels showed a strong correlation with the DRA score by multiple regression analysis, and (2) patients with progression of amyloid cysts showed a significantly higher circulating CML-Hb level than patients without progression. CONCLUSION: Based upon these results, we conclude that measurement of circulating CML-Hb levels has a potential value in both judgment of the clinical state and prediction of progression of DRA in hemodialysis patients.

Elevation of N-(carboxymethyl)valine residue in hemoglobin of diabetic patients. Its role in the development of diabetic nephropathy.

OBJECTIVE: Advanced glycation end products (AGEs) are a risk factor for diabetic complications. We have developed an assay method for N-(carboxymethyl)valine (CMV) of the hemoglobin (CMV-Hb), which is an AGE generated from HbA1c. Herein we describe the clinical utility of CMV-Hb measurement for the diagnosis of diabetic nephropathy RESEARCH DESIGN AND METHODS: BALB/c mice were immunized with carboxy-methylated Hb and monoclonal antibody raised against CMV-Hb. This antibody was characterized by a surface plasmon resonance. We developed a latex immunoassay using the antibody and measured CMV-Hb from erythrocytes in type 2 diabetic patients and healthy control subjects (age 64.6 +/- 12.0 vs. 61.1 +/- 13.2 years, NS: HbA1c 69 +/- 1.5 vs. 5.2 +/- 0.4%, P < 0.0001). RESULTS: A monoclonal antibody against CMV-Hb beta-chain NH2-terminal and an assay method for measurement for CNMV-Hb were both developed in our laboratory. CMV-Hb levels were significantly greater in the diabetic patients than in the control subjects (18.2 +/- 6.9 vs. 12.7 +/- 0.9 pmol CMV/mg Hb, P < 0.0001). No correlation was found between CMV-Hb and HbA1c or CMV-Hb and glycated albumin. Levels of CMV-Hb increased as the diabetic nephropathy progressed. CONCLUSIONS: We established an assay method for CMV-Hb and confirmed the presence of CMV-Hb in circulating erythrocytes. CMV-Hb was more prevalent in diabetic patients than in healthy subjects. Furthermore, it was significantly higher in patients with diabetic nephropathy, suggesting that the presence of CMV-Hb may be a valuable marker for the progression of diabetic nephropathy.

Asaia siamensis sp. nov., an acetic acid bacterium in the alpha-proteobacteria.

Five bacterial strains were isolated from tropical flowers collected in Thailand and Indonesia by the enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located within the cluster of the genus Asaia. The isolates constituted a group separate from Asaia bogorensis on the basis of DNA relatedness values. Their DNA G+C contents were 58.6-59.7 mol%, with a range of 1.1 mol%, which were slightly lower than that of A. bogorensis (59.3-61.0 mol%), the type species of the genus Asaia. The isolates had morphological, physiological and biochemical characteristics similar to A. bogorensis strains, but the isolates did not produce acid from dulcitol. On the basis of the results obtained, the name Asaia siamensis sp. nov. is proposed for these isolates. Strain S60-1T, isolated from a flower of crown flower (dok rak, Calotropis gigantea) collected in Bangkok, Thailand, was designated the type strain ( = NRIC 0323T = JCM 10715T = IFO 16457T).

Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin in rats.

The pharmacokinetics and pharmacodynamics of recombinant human erythropoietin (rh-EPO; CAS for EPO: 11096-26-7) after repeated intravenous and subcutaneous administrations in rats were studied. Administration of rh-EPO by both routes caused significant increases in hematocrit. The pharmacokinetics of rh-EPO after intravenous and subcutaneous administration exhibited nonlinearity. The pharmacodynamics of rh-EPO was analyzed using the maximum effect (Emax) and sigmoid maximum effect (sigmoid Emax) models. Both models involved the assumption that rh-EPO in plasma would stimulate the proliferation of erythroid progenitor cells. Akaike's information criterion for the Emax model was lower than that for the sigmoid Emax model, suggesting that the Emax model might be an optimal model. The rh-EPO concentration at which the effect is half of the maximum was 0.383 ng/ml. This pharmacodynamic analysis suggests that the maintenance of effective plasma concentration might be important for the efficacy of rh-EPO.

Emendation of Pseudomonas straminea Iizuka and Komagata 1963.

The description of Pseudomonas straminae lizuka and Komagata 1963 was emended with data newly obtained. The spelling of the name of this taxon is also corrected as Pseudomonas straminea. Strains that were previously named 'Pseudomonas ochracea' were identified as P. straminea.

Supersonic jet/multiphoton ionization/mass spectrometry of dioxins formed by the thermal reaction of phenols in the absence and presence of an FeCl3 catalyst.

Dioxins, which are thermally produced from several precursor molecules, were investigated by supersonic jet/multiphoton ionization/time-of-flight mass spectrometry (SSJ/MPI/TOF-MS). Dibenzofuran and dibenzo-p-dioxin were efficiently generated from o-chlorophenol and also from phenol after a chlorination reaction with FeCl3. The present technique was employed for the continuous monitoring of a specified isomer, e.g., m-chlorophenol, which is formed at relatively low temperatures by chlorination of phenol with FeCl3. A dimerization reaction that forms a dibenzo-p-dioxin, e.g., dichlorodibenzo-p-dioxin from 2,4-dichlorophenol, at relatively high temperatures was also investigated. The number of chlorine atoms in the dioxin molecule was largely correlated with the number of chlorine atoms in the precursor molecule. However, some unexpected compounds, which probably occur by dechlorination and rearrangement reactions, were also found. Thus, the SSJ/MPI/TOF-MS technique represents a sensitive, as well as selective, analytical method for monitoring thermally generated dioxins.

Asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the alpha-Proteobacteria.

Eight Gram-negative, aerobic, rod-shaped and peritrichously flagellated strains were isolated from flowers of the orchid tree (Bauhinia purpurea) and of plumbago (Plumbago auriculata), and from fermented glutinous rice, all collected in Indonesia. The enrichment culture approach for acetic acid bacteria was employed, involving use of sorbitol medium at pH 3.5. All isolates grew well at pH 3.0 and 30 degrees C. They did not oxidize ethanol to acetic acid except for one strain that oxidized ethanol weakly, and 0.35% acetic acid inhibited their growth completely. However, they oxidized acetate and lactate to carbon dioxide and water. The isolates grew well on mannitol agar and on glutamate agar, and assimilated ammonium sulfate for growth on vitamin-free glucose medium. The isolates produced acid from D-glucose, D-fructose, L-sorbose, dulcitol and glycerol. The quinone system was Q-10. DNA base composition ranged from 59.3 to 61.0 mol% G + C. Studies of DNA relatedness showed that the isolates constitute a single species. Phylogenetic analysis based on their 16S rRNA gene sequences indicated that the isolates are located in the acetic acid bacteria lineage, but distant from the genera Acetobacter, Gluconobacter, Acidomonas and Gluconacetobacter. On the basis of the above characteristics, the name Asaia bogorensis gen. nov., sp. nov. is proposed for these isolates. The type strain is isolate 71T (= NRIC 0311T = JCM 10569T).

Development of a sensitive liquid chromatography-electrospray ionization mass spectrometry method for the measurement of KW-5139 in rat plasma.

A sensitive method for the determination of a prokinetic peptide, KW-5139 (Leu(13)-motilin), in rat plasma has been developed utilizing liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). KW-5139 was separated by reversed-phase HPLC, with a mixture of 75 mM ammonium formate (pH 3.0) and acetonitrile (4:1, v/v), and monitored by single ion recording (SIR)-ESI-MS at m/z 894 ([M+3H](3+)). Simple protein precipitation and the LC-ESI-MS analysis allowed the determination of KW-5139 in rat plasma with the mean precision and accuracy at the lower limit of quantitation (LLOQ, 0.5 ng/mL) of 5.7 and 11.2%, respectively. The method was applied to the monitoring of the plasma time-concentration profile of KW-5139, intravenously administered to rats at a dose of 1 microg/kg.

N epsilon-(carboxymethyl)lysine in blood from maintenance hemodialysis patients may contribute to dialysis-related amyloidosis.

BACKGROUND: Recent studies demonstrated not only that advanced glycation end product could be found in amyloid tissue from patient with dialysis related amyloidosis, but also that amyloid beta2-microglobulin was modified with N(epsilon)-(carboxymethyl)lysine (CML). We wanted to determine if CML could be a biomarker in these patients. METHODS: To raise polyclonal anti-carboxymethyllysine antibody, human serum albumin was carboxymethylated by glyoxylic acid and was immunized to rabbits as antigen. Carboxymethyllysine-hemoglobin (CML-Hb) levels were measured by the dot blotting method using this antibody. RESULTS: The levels of CML-Hb were 6.68 +/- 3.10 nmol CML/mg Hb in nondiabetic hemodialysis patients (N = 70), 6.39 +/- 3.43 nmol CML/mg Hb in diabetic hemodialysis patient (N = 21), and 3.13 +/- 0.88 nmol CML/mg Hb in 47 healthy volunteers. For clinical signs of dialysis-related amyloidosis, 70 nondiabetic hemodialysis patients were scored according Gejyo's criteria. The CML-Hb levels in patients with a high amyloid score as well as a low amyloid score were significantly higher than in patients with negative amyloid score (8.89 +/- 3.53 nmol CMLmg Hb, 7.28 +/- 2.32 nmol CML/mg Hb vs. 5.11 +/- 2.09 nmol CML/mg Hb, P < 0.001, P < 0.05). Furthermore, the CML-Hb levels correlated significantly with serum values of the methylguanidine over creatinine ratio and hyaluronate. CONCLUSIONS: We suggest that CML-Hb is increased in blood from patients on maintenance hemodialysis and is thus a potential biomarker of oxidative damage in these patients. Moreover, CML-modification of protein may play a pathogenic role in the development of dialysis related amyloidosis.

Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148.

An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.

Effect of non-insulin dependent diabetes on cyclosporin A disposition in Goto-Kakizaki (GK) rats.

We previously reported that the pharmacokinetics of cyclosporin A (CyA), particularly absorption, were altered in diabetic rats treated with streptozotocin. In the present study, the effect of diabetes on pharmacokinetics of CyA after intravenous and oral administration of CyA using the blood and lymph and the gastrointestinal transit were examined in Goto-Kakizaki (GK) rats, a genetic model of non-obese non-insulin-dependent diabetes mellitus (NIDDM), and compared to non-diabetic Wistar rats. Although the systemic and lymphatic availability after intravenous administration of CyA to the GK rats was not significantly different from those of the control Wistar rats, those availability after oral administration of CyA to GK rats was markedly reduced in comparison. These results suggest that the pharmacokinetics of CyA, particularly absorption, was altered in GK rats. Studies on the gastrointestinal transit in GK rats showed that the gastric emptying rate was lower than that of Wistar rats, suggesting that a change in gastrointestinal transit in GK rats may influence the absorption of CyA. The gastric emptying rate in GK rats altered not only the systemic availability but also the lymphatic availability, suggesting that the altered systemic availability may cause adverse effects and that altered lymphatic availability may influence the immunosuppressive effects.

Receptor-mediated clearance of G-CSF derivative nartograstim in bone marrow of rats.

To clarify the role of the granulocyte colony-stimulating factor (G-CSF) receptor in the nonlinear elimination of a recombinant human G-CSF derivative, nartograstim (NTG), the accompanying changes in the in vivo NTG total body clearance at steady state (CLss) or the early-phase tissue uptake clearance (CLuptake) in rats were compared with the change in the number of G-CSF receptors in bone marrow. The infusion rate-dependent decrease in CLss in control rats confirmed the existence of a saturable elimination mechanism for NTG. The Michaelis-Menten constant (Km) and maximal velocity for this saturable process were estimated to be 107 pM and 15.5 pmol.h-1.kg-1, respectively. The Km for this saturable process was comparable with the dissociation constant (Kd) for the specific binding of NTG to bone marrow cells. After administration of excess NTG, the CLuptake of tracer amounts of 1251-NTG by bone marrow and spleen, which corresponds to the receptor density in the tissues, was reduced at 2 h but gradually recovered. This change in CLuptake corresponds well to the change in the in vitro NTG-binding capacity in each isolated cell. This reduction in CLuptake might be due to the downregulation of G-CSF receptors on the cell surface. On the other hand, the saturable CLss in cyclophosphamide-treated rats was 17% of that in control rats, whereas the saturable CLss in rats given NTG repeatedly was twofold greater than in controls, which is associated with the upregulation of G-CSF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturable uptake of a recombinant human granulocyte colony-stimulating factor derivative, nartograstim, by the bone marrow and spleen of rats in vivo.

Kinetic analysis of the in vivo tissue distribution of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivative, nartograstim (NTG; nartograstim is an international nonproprietary name of KW-2228, Kyowa Hakko Kogyo, Tokyo, Japan) was studied in rats to determine the mechanism of the growth factor's nonlinear pharmacokinetic properties. The early-phase tissue uptake clearance (CL(uptake)) by each tissue was determined within 5 min after the i.v. administration of 125I-NTG. Coadministration of various amounts (0.6-150 microgram/kg) of unlabeled NTG did not produce any significant change in CL(uptake) by kidneys and liver. The hepatic and renal extraction ratios of NTG were low, 0.10 to 0.15, which suggests that there is no saturable uptake system in either of these organs. CL(uptake) by the kidneys was comparable to the glomerular filtration rate of unbound NTG. By contrast, a dose-dependent reduction in CL(uptake) by bone marrow and spleen was clearly observed with increasing doses of unlabeled NTG, i.e., at the maximal dose, these values were 14% and 20%, respectively, of those after a tracer dose. A saturable process was, therefore, involved in the tissue uptake of 125I-NTG by bone marrow and spleen. The intrinsic clearance (Vmax/Km) of this saturable uptake by bone marrow, 1.4 ml min-1 kg-1, was greater than that by the spleen, 0.09 ml min-1 kg-1. The sum of the intrinsic clearances of the saturable process was comparable with the value for the clearance of the saturable elimination process of NTG obtained previously by nonlinear pharmacokinetic analysis.(ABSTRACT TRUNCATED AT 250 WORDS)