Effect of aluminium ion on bioavailability of levofloxacin following oral administration of cilexetil ester of levofloxacin as prodrug in rats.

A prodrug of levofloxacin (LVFX), cilexetil ester of LVFX (LVFX-CLX), was synthesized to examine whether the prodrug can avoid chelate formation with metal cations in the gastrointestinal tract. LVFX-CLX exhibited a 10-times higher partition coefficient than LVFX. In vitro, LVFX was precipitated by 76.1% in the presence of a 10-times higher concentration of aluminium chloride (Al3+), but LVFX-CLX was not. LVFX-CLX was rapidly hydrolyzed enzymatically by rat plasma, intestinal mucosal and liver homogenates at 37 °C, but not by pancreatic enzymes and luminal fluid. The minimum inhibitory concentration values of LVFX-CLX against S. aureus, E. coli and P. aeruginosa were far higher than that of LVFX. In rats, area under the plasma concentration-time curve from zero to 4 h (AUC0-4h) of LVFX after oral administration of LVFX-CLX was 1.34-fold higher than that after LVFX, though it did not reach significance level. Co-administration of Al3+ with LVFX and LVFX-CLX in rats decreased AUC0-4h of plasma LVFX by 75% and 60%, respectively, however, the AUC0-4h of plasma LVFX after co-administration of LVFX-CLX and Al3+ was 2.2-times higher than that after co-administration of LVFX and Al3+. These results suggested that the use of LVFX-CLX may reduce the modulation of intestinal microflora caused by LVFX and the suppressive effect of Al3+ on intestinal absorption of LVFX.

Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes.

Hemidesmosomes (HDs) mediate cell adhesion to the extracellular matrix and have morphological association with intermediate-sized filaments (IFs) through cytoplasmic plaques. Though several proteins have been located in HDs, most of them have not been well characterized, with the exception of the 230-kD antigen of bullous pemphigoid (BP), an autoimmune skin blistering disease. Only recently we have succeeded in isolating HDs from bovine corneal epithelial cells and in identifying five major components on SDS-PAGE (Owaribe K., Y. Nishizawa, and W. W. Franke. 1991. Exp. Cell Res. 192:622-630). In this study we report on immunological characterization of one of the major components, termed HD1, with an apparent molecular mass of 500 kD. Immunofluorescence microscopy showed colocalization of HD1 with BP antigen at the basement membrane zone of those tissues that have typical HDs, including skin epidermis, corneal and tracheal epithelia, and myoepithelium. In cultured keratinocytes, HD1 demonstrated colocalization with BP antigen in the precise way, while being absent from focal adhesions. Immunoelectron microscopy revealed that an epitope of HD1 was located on the cytoplasmic side of HDs. Taking all these results together, we conclude that HD1 is a new hemidesmosomal component. Interestingly, HD1 also exists in endothelial and glial cells, which lack typical HDs.

A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization.

An 82-kD protein has been purified from the undercoat of the adherens junction isolated from the rat liver. The purification scheme includes low salt extraction followed by DEAE-cellulose ion exchange, DNase I-actin affinity, and carboxyl methyl-cellulose ion exchange chromatographies. The purified 82-kD protein was essentially free of contaminants as judged by SDS-PAGE combined with silver staining. The substoichiometric 82-kD protein largely inhibited the actin filament assembly; when the molar ratio of the 82-kD protein to G-actin was 1:1,000, the viscosity was reduced to 28% of the control value. Direct electron microscopic studies revealed that the 82-kD protein selectively inhibited monomer addition at the barbed ends of actin filaments. By use of the antibody raised against the 82-kD protein, this protein was shown by immunofluorescence microscopy to be localized at the cell-to-cell adherens junction in various types of cells. In contrast, the 82-kD protein was not concentrated at the cell-to-substrate adherens junctions (focal contacts). These findings have led us to conclude that the 82-kD protein is a barbed end-capping protein which is associated with the undercoat of the cell-to-cell adherens junction. Hence, we have tentatively designated the 82-kD protein as radixin (from the Latin word radix meaning root).

A new high molecular mass protein showing unique localization in desmosomal plaque.

A high molecular mass protein of 680 kD was identified and purified from the isolated desmosomes in bovine muzzle epidermal cells. This protein, called "desmoyokin" (from the English, yoke) here, showed no binding ability with keratin filaments in vitro, and its molecule had a characteristic dumbell shape approximately 170 nm in length. We have succeeded in obtaining one monoclonal antibody specific to desmoyokin. By the use of this monoclonal antibody and antidesmoplakin monoclonal antibody, desmoyokin was shown to be colocalized with desmoplakin at the immunofluorescence microscopic level; desmoyokin occurred only in the stratified epithelium, not in the simple epithelium nor in the other tissues. At the electron microscopic level, these two proteins were clearly seen to be sorted out in the plaque of desmosomes with desmoyokin at the periphery and desmoplakin at the center of the disk-shaped desmosomal plaque, suggesting that these two plaque proteins play distinct roles in forming and maintaining the desmosomes in stratified epithelium.

Remodeling of desmosomal and hemidesmosomal adhesion systems during early morphogenesis of mouse pelage hair follicles.

Early hair follicle morphogenesis proceeds with the formation of a hair placode, the downgrowth of the hair plug into the mesenchyme, and the development of an elongated hair follicle - processes that involve a series of exchange of messages between epithelium and mesenchyme. Regulation of epithelial cell adhesion during hair morphogenesis has been demonstrated in terms of the changing expression patterns of E- and P-cadherins. In this study, distribution patterns of several major components of desmosomes and hemidesmosomes, which are the most prominent cell adhesion systems in epidermal tissues, were examined during early morphogenesis of mouse pelage hair follicles. We found that both desmosomal and hemidesmosomal adhesion systems became downregulated in hair placodes and were much reduced or almost lost in hair plugs, which persisted in the region containing hair matrix. Downregulation of the adhesion systems in hair plugs was confirmed by electron microscopy. Similar distribution patterns of these molecules were obtained in the developing follicles in cultured skin. It may be that epidermal cells at the initial stages of hair development respond to the first mesenchymal message by grossly changing their cell adhesion systems and that the resultant changes in cell adhesivity underlie early hair follicle morphogenesis.

High-level secretion of two antibody single chain Fv fragments by Pichia pastoris.

The diagnostic and therapeutic applications of antibody single-chain Fv (sFv) fragments often require large amounts of protein that can be problematic and expensive to obtain. Here we report the secretion of two sFv fragments by the yeast Pichia pastoris at levels up to 250 mg/l. Soluble sFv fragments were purified from culture supernatants in one step by affinity or metal-chelating chromatography, and were indistinguishable from their bacterially expressed counterparts in terms of affinity. Secretion of functional sFv fragments by Pichia pastoris provides a low cost, high yield alternative to current sFv expression systems.

Immunization of rats reduces nicotine distribution to brain.

The effect of active immunization against nicotine on the initial distribution of nicotine to brain was studied in anesthetized rats. Animals received nicotine 0.03 mg/kg nicotine (equivalent to the nicotine dose absorbed by a human smoking two cigarettes) as a rapid injection in the jugular vein. In control animals, the arterial serum nicotine concentration initially exceeded the venous concentration 4.6-fold, similar to the initial arteriovenous difference produced by cigarette smoking in humans. Animals immunized with the nicotine analog CMUNic maintained this arteriovenous gradient, but with both arterial and venous nicotine concentrations several times higher than in controls. The arterial nicotine concentration was higher in immunized animals even at the first (7.5 s) sampling time. The brain nicotine concentration at 3 min was 36% lower in the immunized animals. The time course of nicotine distribution to brain was studied in a second group of animals. Brain nicotine concentration was reduced in rats immunized with CMUNic over the entire 6-min sampling period immediately following nicotine dosing (mean reduction 38%). A reduction was found at the earliest sampling time (30 s) and was maximal at 1 min (48%). Nicotine protein binding in serum was markedly increased in animals immunized with CMUNic compared to controls (91.2 versus 10.9%), and the unbound nicotine concentration in serum was lower (10.0 versus 13.4 ng/ml). The reduction in brain nicotine concentration correlated with antibody affinity for nicotine, and the percentage of nicotine bound in serum. These data demonstrate that nicotine-specific antibodies produced by active immunization rapidly bind nicotine in arterial blood, reduce the unbound nicotine concentration, and reduce the early distribution of nicotine to brain. Effects were observed using a clinically relevant nicotine dose and route of administration. These data suggest that the use of immunization to modify the behavioral effects of nicotine may be possible.

A nicotine conjugate vaccine reduces nicotine distribution to brain and attenuates its behavioral and cardiovascular effects in rats.

Vaccination of animals to elicit drug-specific antibodies, or the passive transfer of such antibodies from other animals, can reduce the behavioral effects of drugs such as cocaine and heroin. To study the potential application of this approach to treating nicotine dependence, IgG was isolated from rabbits immunized with a nicotine-protein conjugate vaccine. Anesthetized rats received immune IgG containing nicotine-specific antibodies (Nic-IgG) or control-IgG i.v.. Thirty minutes later, rats received nicotine at 0.03 mg/kg i.v., equivalent on an mg/kg basis to the nicotine intake from two cigarettes by a smoker. Compared to control-IgG, Nic-IgG reduced the brain nicotine concentration in a dose-related manner (65% reduction at the highest IgG dose). Pretreatment with Nic-IgG also reduced the distribution to brain of five repeated doses of nicotine (equivalent to the nicotine intake from 10 cigarettes) administered over 80 min. To study blood pressure effects, rats received control-IgG or Nic-IgG 1 day prior to administering nicotine. Nicotine-induced systolic blood pressure increases were attenuated by Nic-IgG in a dose-related manner, and were almost completely blocked by the highest Nic-IgG dose. Pretreatment with Nic-IgG also completely prevented the nicotine-induced stimulation of locomotor activity observed in rats receiving control-IgG. Nic-IgG did not prevent locomotor activation from cocaine, demonstrating its specificity for nicotine. These data demonstrate that the administration of nicotine-specific antibodies can reduce or prevent some of the pharmacokinetic, cardiovascular, and behavioral consequences of nicotine in rats. Effects were observed at nicotine doses and nicotine serum concentrations equal to or exceeding those typically associated with nicotine exposure in cigarette smokers. A potential role for immunization in the treatment of nicotine dependence is suggested.

Passive immunization against nicotine prevents nicotine alleviation of nicotine abstinence syndrome.

Passive immunization against nicotine interferes with its locomotor and pressor effects. The current study determined whether immunization could prevent another nicotine action: the reversal of nicotine abstinence syndrome. IgG containing 4.4-5.6% nicotine-specific antibody was isolated from rabbits immunized with 3'-amino-methyl-nicotine conjugated to a carrier protein. Twenty rats were rendered dependent by 7 days of subcutaneous infusion of 3.15 mg/kg/day nicotine (expressed as the base). Upon termination of nicotine infusion, each rat was injected intraperitoneally with 150 mg of IgG from normal serum (n=13) or from nicotine antiserum (n=7). Twenty-two and one-half hours later, all rats were observed over 15 min for baseline nicotine abstinence signs. Two and one-half hours after baseline observations, seven of the 13 rats pretreated with control IgG and all seven rats pretreated with nicotine-specific IgG were then challenged by 0.12 mg/kg (sc) nicotine. The remaining six rats pretreated with control IgG were challenged with saline alone. All rats were then observed again for abstinence signs. Nicotine injection caused significantly less reduction of abstinence signs in the immunized rats. The nicotine effect in immunized rats was comparable to the saline effect in nonimmunized rats. Immunization also significantly reduced free serum nicotine concentration and nicotine distribution to the brain. These results raise the possibility that immunization might prevent nicotine consumption from relieving the discomforts of smoking cessation.

Identification of human hair stained with oxidation hair dyes by gas chromatographic-mass spectrometric analysis.

This paper describes the gas chromatographic-mass spectrometric (GCMS) analysis of oxidation hair dyes from human hair. Diamines from the dyes were directly extracted from the hair in basic solution and aminophenols were extracted after neutralization. Both extracts were derivatised with trifluoroacetic anhydride and analysed by GCMS. Five components of oxidation hair dyes namely, p-phenylenediamine, toluene-2,5-diamine, o-aminophenol, m-aminophenol and p-aminophenol were clearly identified, whilst no other compounds originating from the hair dyes were detected. The presence and relative amounts of these dye components from hair extracts may assist in the discrimination of human hair especially in cases involving forensic science.

An experimental model of death from anaphylactic shock with compound 48/80 and postmortem changes in levels of histamine in blood.

The present study was made on an experimental animal model of a death from anaphylaxis, in which postmortem changes in levels of histamine and 1-methylhistamine, in whole blood were measured. Instead of the usual immunological method administering compound 48/80, a degranulating agent of mast cell and the effect closely resembling the immuno-reaction, resulted in reliable death in a short time. The animals that died rapidly after the injection of compound 48/80, were found to have large increases in levels of histamine and 1-methylhistamine soon after the administration. These results were similar to the results of injecting histamine exogenously. On the other hand, the animals that died after a longer time showed no increases in levels of those amines within about 24 h, but 24 hours after death histamine levels were only increased tremendously without rise in 1-MHA levels. These phenomena closely resembled those in the control animals that were treated with overdoses of Nembutal.

Demonstration of oxidation dyes on human hair.

This paper describes a method of selected ion monitoring (SIM) analysis which can demonstrate the staining of human hair with oxidation hair dyes. Hair samples were decomposed with NaOH-Na2S2O4 solution by heating (100 degrees C, 30 min) in a stream of nitrogen. Basic and neutral ether extracts from the reaction mixture were trifluoroacetylated with trifluoroacetic anhydride in ethyl acetate and were then analyzed by SIM. The minimum lengths of a single hair for the detection of the 5 components of oxidation hair dyes acting as indicators were 1 mm for toluene-2,5-diamine, 2 mm for p-phenylenediamine, 20 mm for p-aminophenol, 50 mm for m-aminophenol and 100 mm for o-aminophenol. This method was applied to practical cases and the results were good.

Inhibition of nicotine-induced seizures in rats by combining vaccination against nicotine with chronic nicotine infusion.

The ability of a nicotine vaccine to protect against nicotine-induced seizures was studied in rats. Groups of 10 rats were vaccinated with 3 doses of either a nicotine conjugate vaccine over 6 weeks to elicit high titers of nicotine-specific antibodies or with a control vaccine. Rats were then pretreated with a 1-week subcutaneous infusion of either nicotine 1 mg/kg/day or saline and then received a single 2 mg/kg ip dose of nicotine to provoke seizures. Vaccination reduced the incidence of seizures. The combination of vaccination and pretreatment with nicotine infusion was more effective than either treatment alone. These data suggest that vaccination is protective against this toxic effect of nicotine and that combining vaccination and chronic nicotine administration may provide a novel strategy for blocking some effects of nicotine.

Simple extractive derivatization of methamphetamine and its metabolites in biological materials with Extrelut columns for their GC-MS determination.

A simple, extractive heptafluoro-n-butyrylation with Extrelut columns was devised to simultaneously measure methamphetamine (MAMP), amphetamine (AMP), 4-hydroxymethamphetamine (HMAMP), and 4-hydroxyamphetamine (HAMP) in biological materials by gas chromatography-mass spectrometry (GC-MS) using 4-methoxymethamphetamine-d5 as the internal standard. Human urine, human whole blood, and porcine skeletal muscle spiked with the stimulant standards were used for evaluating the method. After deproteinization and adjustment of the pH to 12.6, the sample was applied to an Extrelut column. Using the present method, AMP, MAMP, and HMAMP could be determined in an actual forensic case study.

[Development of the method for analysis of theophylline and its related compounds using capillary high-performance liquid chromatography/fast atom bombardment-mass spectrometry].

A convenient, reliable and sensitive method for analysis of theophylline (TH), theobromine (TB) and caffeine (CA) in biological samples by capillary high performance liquid chromatography (HPLC)/fast atom bombardment (FAB)-mass spectrometry (MS) was developed using 7-ethyltheophylline (7-ETH) as an internal standard (IS). The capillary column (0.3 mm i.d.) enabled the introduction of entire effluent to the frit interface for FAB-MS; and a special column switching device was utilized for sample concentration and gradient formation. These conditions realized simultaneous determination of the compounds of as low as 50-500 ng/ml concentrations. Actually, the compounds in blood and urine could be identified and quantified for healthy subjects having TH and/or CA spontaneously. In addition, TH in human hair obtained from the patients with bronchial asthma, who had taken 400 mg TH daily for more than 2 years, could be detected by the capillary HPLC/FAB-MS. This method seems very useful for the analysis of clinical or forensic samples, because of its ability of final drug identification.

[Screening of drugs and chemicals by wide-bore capillary gas chromatography. II. Detection of drugs and chemicals in the blood].

This paper describes the detection limit for 23 drugs and chemicals in the blood by means of a screening method that uses a gas chromatographic system equipped with a wide-bore capillary column and a nitrogen phosphorus detector. The detection limit by this method was determined as being 1 mm of peak height at the detector's range of 100 and 8 of attenuation. Using this scale, the absolute detection limit was in the range of 1 pg for malathion and sumithion to 1 ng for meprobamate. The detection limit of drugs and chemicals in the blood was 5 ng/ml for sumithion to 8 micrograms/ml for meprobamate. Therefore, this screening method is able to detect the presence of drugs even a therapeutic-level dosages, with the exception of compounds such as haloperidol, which have extremely low therapeutic dosage levels.

[Screening of drugs and chemicals by wide-bore capillary gas chromatography with flame ionization and nitrogen phosphorus detectors].

A method is presented for forensic toxicological screening of drugs and chemicals in blood and urine by wide-bore capillary gas chromatography with flame ionization detectors (FID) and nitrogen phosphorus detectors (NPD). The presence of drugs and chemicals in blood and urine specimens was confirmed by comparing these gas chromatograms with those of typical drug-free specimens. Peak components of drug-free specimens were piperidone, p-cresol, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, n-butylphthalate, bis (2-ethylhexyl) phthalate, squalene, cholesterol and two alcohols (unidentified) on FID chromatograms and were piperidone, indole, nicotine, cotinine, hydroxycotinine, caffeine and several unknown urine constituents on NPD chromatograms. In practical cases, the presence of drugs and chemicals in postmortem specimens was easily ascertained by the present method.