Expression of inflammatory cytokines in pigs experimentally infected with Mycoplasma hyopneumoniae.

The expression of interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and IL-6 were studied over a period of 35 days in the lungs of pigs experimentally infected with Mycoplasma hyopneumoniae, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), morphometric analysis and in-situ hybridization. Fifteen colostrum-deprived pigs aged 14 days were inoculated intranasally with M. hyopneumoniae. IL-1, TNF-alpha and IL-6 were detected by RT-PCR in the lungs of the infected pigs from 7 days post-inoculation (dpi) onwards, but not in the uninfected control pigs. Concurrent expression of all three cytokines was always observed, in association with lung lesions. Inflammatory cytokine-positive cells were detected in the lungs at 7 dpi, their number increasing at 21dpi, and decreasing thereafter. The results suggest that such cytokines play a role in mediating and regulating inflammation in M. hyopneumoniae infection.

Synthetic peptide-derived antibody-based immunohistochemistry for the detection of porcine circovirus 2 in pigs with postweaning multisystemic wasting syndrome.

Porcine circovirus 2 (PCV2) was detected consistently in formalin-fixed paraffin wax-embedded lymph node and spleen from experimentally and naturally infected pigs by synthetic peptide-derived polyclonal antibody-based immunohistochemistry. Synthetic peptides were generated from open reading frame 2 of PCV2 by solid-phase peptide synthesis, purified by high performance liquid chromatography, and injected into rabbits to produce polyclonal antibody. Positive cells had large nuclei with abundant cytoplasm, and resembled macrophages. In serial sections, a similar distribution of PCV2 antigen and DNA was confirmed in virus-infected cells by immunohistochemistry and in-situ hybridization, respectively. The immunohistochemical method described was successfully applied to formalin-fixed, paraffin wax-embedded tissues and should prove helpful in diagnosing postweaning multisystemic wasting syndrome.

Classical swine fever virus induces tumor necrosis factor-alpha and lymphocyte apoptosis.

The expression of tumor necrosis factor (TNF)-alpha and apoptosis was studied in lymph nodes from pigs infected with Classical swine fever virus (CSFV). Pigs were inoculated with CSFV and euthanized at 3, 5, 7, and 10 days postinoculation. An increase in TNF-alpha expression was detected in CSFV-infected lymph nodes using a reverse transcription-polymerase chain reaction, and TNF-alpha protein was detected in lymph nodes by immunohistochemistry. The majority of TNF-alpha-positive cells also expressed the SWC3a antigen, a specific marker for porcine leukocytes. By combined use of in situ hybridization and immunohistochemistry, CSFV infection was detected in lymph nodes macrophage. Lymphocytes death occurred by apoptosis that was characterized by condensed shrunken cells and the formation of apoptotic bodies, some of them contained pyknotic nuclear remnants. Apoptosis was detected in situ by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) reaction. A double-labeling experiment using immunohistochemistry and TUNEL reaction for the detection of CSFV and apoptosis demonstrated that the majority of labeled cells were positive for CSFV or apoptosis. This suggests that CSFV can induce apoptosis directly and indirectly. Apoptotic cells induced by viral infection were more abundant than CSFV-infected cells in all lymph nodes tested. A double-labeling experiment using immunohistochemistry and TUNEL reaction for the detection of TNF-alpha and apoptosis demonstrated that labeled cells were positive for either TNF-alpha or apoptosis, and both. The present study addressed two important issues regarding CSFV-induced apoptosis: (i) viral infection and apoptosis colocalize at the cell level; and (ii) one or more factors (e.g., TNF-alpha) released from macrophages may induce apoptosis in uninfected bystander cells.

Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin.

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against beta(2) glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.

Transcriptional repression of euchromatic genes by Drosophila heterochromatin protein 1 and histone modifiers.

In Drosophila, heterochromatin protein 1 (HP1) suppresses the expression of euchromatic genes that are artificially translocated adjacent to heterochromatin by expanding heterochromatin structure into neighboring euchromatin. The purpose of this study was to determine whether HP1 functions as a transcriptional repressor in the absence of chromosome rearrangements. Here, we show that Drosophila HP1 normally represses the expression of four euchromatic genes in a dosage-dependent manner. Three genes regulated by HP1 map to cytological region 31 of chromosome 2, which is immunostained by anti-HP1 antibodies in the salivary gland. The repressive effect of HP1 is decreased by mutation in Su(var)3-9, whose mammalian orthologue encodes a histone H3 methyltransferase and mutation in Su(var)2-1, which is correlated with histone H4 deacetylation. These data provide genetic evidence that an HP1-family protein represses the expression of euchromatic genes in a metazoan, and that histone modifiers cooperate with HP1 in euchromatic gene repression.

FK506 promotes adenosine release from endothelial cells via inhibition of adenosine kinase.

The immunosuppressants, cyclosporin A and tacrolimus (FK506) induce an increase in plasma levels of adenosine and mimic ischemic preconditioning. However, the mechanism of action of the two drugs on adenosine metabolism is not clear. Since inhibition of adenosine kinase promotes an increase in endogenous adenosine release, we tested a hypothesis that FK506 induces adenosine release via inhibition of adenosine kinase activity. In cultured endothelial cells, FK506 enhanced release of tracer adenosine and inhibited uptake of tracer adenosine. It also reduced adenosine kinase activity of the cell membrane fraction. In addition, FK506 does not inhibit membrane transport of tracer adenosine. These observations indicate that FK506 inhibits in situ adenosine kinase activity in endothelial cells. Other cell signaling inhibitors were found to inhibit adenosine uptake via inhibition of adenosine transport. In conclusion, FK506 promotes adenosine release from endothelial cells by a novel mechanism involving inhibition of adenosine kinase activity associated with the membrane.

Expression and functional analysis of three isoforms of human heterochromatin-associated protein HP1 in Drosophila.

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein associated with pericentromeric heterochromatin in Drosophila. HP1-like proteins have also been found associated with heterochromatin in human cells. The goal of this study was to determine whether proteins of the structurally conserved human HP1 family exhibit conserved heterochromatin targeting and silencing properties in Drosophila. We established transgenic lines of Drosophila melanogaster expressing each of the three human HP1 proteins, HP1Hsalpha, HP1HSbeta, and HP1Hsgamma, under the Hsp70 heat shock promoter. We show that all three isoforms of human HP1 are stably expressed in Drosophila and are associated with heterochromatin in Drosophila chromosomes. Like Drosophila HP1, all three human HP1 proteins are delocalized by an HP1-POLYCOMB chimeric protein, implying that both human HP1 and Drosophila HP1 interact in a common protein complex, and that at least some aspects of heterochromatin structure are highly conserved throughout the evolution of eukaryotes. Ectopic expression of two of the three human HP1 family proteins significantly enhances heterochromatic silencing in Drosophila.

Microalbuminuria in urban Zimbabwean women.

The prevalence of microalbuminuria (MAU) in African populations has not been reported, nor has the relationship between MAU and hypertension been reported for these populations. We collected spot urine samples from 370 women, 25 years and older as a part of a population-based, cross-sectional blood pressure survey in an urban community in Zimbabwe and analysed the samples for albumin and beta2-microglobulin. The age-adjusted prevalence of hypertension was 30% for women 25 years and older in this community. After excluding the samples with hematuria (11%), the prevalence of MAU (3.0 < or = albumin-to-creatinine ratio (ACR, mg/mmol) <25.0) in the study population was 9%. When age-adjusted to the population in the community, the prevalence was 8% among women 25 years and older. The prevalence of MAU was substantially higher in hypertensive (HT) than in normotensive (NT) women (16% vs 4%, P<0.001). A significantly higher level of log ACR in HT was found in each age group except the youngest age group (age 25-34). In age-adjusted multiple regression, percent fat mass was negatively associated with log ACR (beta = -1. 18, 95% CI (-0.23, -2.21), P = 0.02). In a similar regression analysis, higher log beta8-microglobulin-to-creatinine ratio was very strongly associated with higher log ACR (beta = 0.34, 95% CI (0.25, 0.43), P<0.0001) and significantly associated with lower percent fat mass (beta = -1.02, 95% CI (-0.25, -1.8), P = 0.01). These results suggest that MAU is frequently caused by hypertension, but that other diseases may contribute to its presence.

Apoptosis and regulation of Bax and Bcl-X proteins during human neonatal vascular remodeling.

To verify that apoptosis is one of the possible mechanisms of neonatal vascular remodeling during the transition from fetal to neonatal circulation, we assayed for apoptosis and evaluated the expression of apoptosis-regulatory proteins in umbilical vessel versus ascending aorta, ductus arteriosus (DA) versus adjacent pulmonary artery and aorta, or aorta versus its branching arteries. Twenty-two umbilical cords (UCs), 6 DAs with adjacent aortas and pulmonary arteries, and 4 aortic arches with their branching great arteries were obtained from neonates. Smooth muscle cell (SMC) apoptosis in umbilical vessels was identified in all UCs. The expressions of Bax and Bcl-X were stronger in umbilical artery than in the neonatal aorta, but Bcl-2 was weak in both arteries in immunohistochemistry. In the immunoblot analysis of UCs, the expression of the proapoptotic short isoform of Bcl-X was stronger than in other tissue, and caspase-3 was selectively activated, whereas it was not in the other components of the cardiovascular system. In contrast, the expression patterns of the FasAg and Fas ligand were similar in umbilical artery and aorta. Regulation of Bcl-2 family proteins was also observed in other vascular sites at which SMCs undergo apoptosis on hemodynamic changes during birth, such as the DA and the branching points of the great arteries from the aortic arch. Apoptosis is involved in the regression of human umbilical vessels and the DA and in the remodeling of the branching great arteries during the neonatal period, when Bcl-2 family proteins are likely to play a key role.

Hydroxyoxazolidines as alpha-aminoacetaldehye equivalents: novel inhibitors of calpain.

The synthesis of [1-[(5-hydroxy-4-(phenylmethyl)-3-oxazolidinyl)carbonyl]-2-ethylpropy lcarbamic acid phenylmethyl ester (2; MDL 104,903), a potent inhibitor of calpain, is described. Synthesis of related compounds, which offer insights into the mechanism of action for 2, are also described, as is an O-acetyl prodrug derivative of 2.

Cross-cultural measurement of experience: Taiwanese and Americans' peak performance, peak experience, misery, failure, sport, and average events.

To compare Taiwanese and Americans on selected experiential personality dimensions, the Experience Questionnaire was translated and tested with 27 Taiwanese in an American university. Descriptions by 129 Taiwanese of peak performance, peak experience, misery, failure, sport, and average events were compared with those made by 123 Americans. Analysis of variance with repeated measures of factors indicated that both samples uniformly characterized processes of peak performance as full focus with clarity of self in process. The Taiwanese considered failure more significant than the Americans who denied clarity of self in misery and failure and more generally endorsed peak experience than the Taiwanese. The study extends the credibility of experience: experiential events can simultaneously have cross-cultural generality and inner processes that are culturally sensitive.

Comparative absorption of bismuth in Sprague-Dawley rats following oral administration of preparations containing bismuth sucrose octasulfate, bismuth subsalicylate, and bismuth subcitrate.

The absorption of bismuth from De-Nol (bismuth subcitrate, DN), Pepto-Bismol (bismuth subsalicylate, PB) and bismuth sucrose octasulfate (BISOS) was examined in male Sprague-Dawley rats after a single oral dose of each compound (60mg bismuth). Bismuth was analysed in blood, urine, kidney, brain, liver, and lung using graphite furnace atomic absorption spectrophotometry. Bismuth Cmax averaged 18.4 +/- 11.6 ng mL(-1) for BISOS, 292 +/- 130 ng mL(-1) for DN, and 21.5 +/- 9.63 ng mL(-1 ) for PB. Cmax was significantly lower for BISOS compared to DN (p<0.05) but not significantly different for BISOS compared to PB (p > 0.05). Bismuth AUC was 1356 +/- 474 ng h(-1) mL (-1) for BISOS, 2129 +/- 452 ng h(-l) mL(-1) for DN, and 1824 +/- 919 ng h(-1) for PB, which indicated a lower extent of absorption from BISOS compared to DN. Kidney, liver, and lung levels of bismuth were also significantly lower for BISOS compared to DN (p < 0.05). Bismuth urinary excretion was significantly lower for BISOS (0.04 +/- 0.02%) compared to DN (0.27 +/- 0.15%) but not significantly different compared to PB (0.07 +/- 0.03%). These data suggest that the absorption of bismuth following oral administration of bismuth sucrose octasulfate is significantly lower than that from De-Nol and similar to that from Pepto-Bismo.

Determination of bioavailability and systemically available fractions of drugs undergoing reversible metabolism: application to 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethylamino)ethyl]benzamide and its sulfide and sulfone metabolites in rats.

Methods are discussed which permit the calculation of the bioavailability (F) and fraction of an oral dose entering the central circulation (f) of a drug and its interconversion metabolite. The interrelationships between the F and f and between the F and systemically available fractions afforded by reversible metabolism are also derived and described. The application of these principles is illustrated by the pharmacokinetic analysis of 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethylamino)ethyl]benzamide (ML-1035, 1) and its sulfide (2) and sulfone (3) metabolites in rats. Like intravenous ML-1035, ML-1035 administered orally underwent metabolic interconversion with 2 but not with 3 in this species. Both ML-1035 and 2 were absorbed rapidly and are pharmacologically active. On average, 8.3 and 13% of an oral dose (152.4 mumol/kg) of ML-1035 were bioavailable as ML-1035 and its sulfide metabolite, respectively, while 23 and 65% of a molar equivalent dose of the sulfide metabolite were bioavailable as either compound, respectively. Thus, the sulfide metabolite is better absorbed than ML-1035 in rats. Following oral administration of either ML-1035 or 2, the systemically available fractions of both compounds were weakly to moderately influenced by the reversible metabolism process in rats. Moreover, the bioavailability of the sulfone metabolite was very poor (2.5-4%) following separate oral administration of ML-1035, 2, and 3.

Pharmacokinetics and metabolic interconversion of intravenous 4-amino-5-chloro-2-[(methylsulfinyl)ethoxy]-N-[2-(diethylamino)ethyl] benzamide and its sulfide and sulfone metabolites in rats.

The pharmacokinetics of a new 5-hydroxytryptamine (5HT3) receptor antagonist, 4-amino-5-chloro-2-[(methylsulfinyl)ethoxy]-N-[2-(diethylamino)ethyl] benzamide (ML-1035, 1), and its sulfone and sulfide metabolites were examined in 12 rats. Each of these compounds (25.4 mumol/kg) was administered to rats intravenously. Their plasma concentrations were measured by high-performance liquid chromatography. These plasma data revealed that 1, a sulfoxide, underwent interconversion with its sulfide metabolite. However, no interconversion was observed between 1 and its sulfone metabolite. Examination of mean times and additional properties of the 1/sulfide metabolite system revealed that total exposure times of 1 and the sulfide metabolite were moderately and weakly, respectively, influenced by the metabolic interconversion process. However, the tissue distribution process strongly influenced the total exposure times of both compounds. The disposition of the sulfone metabolite of 1 was also strongly influenced by the tissue distribution process. In addition, < 3% of the intravenous dose of 1 or the sulfide was available to the general circulation as the sulfone metabolite.

Disposition kinetics of ML-1035 sulfoxide enantiomers and the prochiral sulfide in rats.

ML-1035, 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethyl-amino)ethyl]benzamide, is a sulfoxide compound and a racemic gastroprokinetic agent with a chiral center at the sulfur atom. We have investigated the disposition kinetics of (R)-ML-1035 sulfoxide (R) and (S)-ML-1035 sulfoxide (S) after the single enantiomers and the racemic mixture were administered to rats in separate experiments. There was no noticeable chiral inversion after either enantiomer dose. Both enantiomers were rapidly absorbed. After dosing with enantiomers or with the racemate, the resulting plasma concentration-time curve of R was closely parallel to that of S in both intravenous and oral experiments, suggesting that the two enantiomers have approximately the same disposition kinetics. After intravenous enantiomer doses, only S underwent conversion to sulfide, suggesting that sulfidation in the liver is enantioselective. However, the enantioselective sulfidation after intravenous dosing did not introduce a difference in the global plasma disposition profiles between R and S, since the reduction reaction is a minor metabolic process. Other metabolic reactions such as sulfonation and mono-N-desethylations were not enantioselective. After oral administration, conversion to sulfide was observed for both enationers, implicating the existence of a nonhepatic pathway in sulfidation. Administration of a prochiral sulfide dose was associated with an enantioselective sulfoxidation, in which the R/S concentration ratios increased as a function of time. In addition, enatiomeric interaction causing changes in pharmacokinetic parameters was observed after the oral racemate dose, while the interaction is negligible after an intravenous racemate dose, indicating a route dependency in enantiomeric interaction.

Sex-differences in the disposition of substituted benzamides: pharmacokinetics of a gastroprokinetic agent (4-amino-5-chloro-2-[2- (methylsulfinyl) ethoxy]-N-[2-(diethylamino)ethyl] benzamide hydrochloride) (ML-1035) in male and female New Zealand white rabbits.

The disposition of 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N- [2-(diethylamino)ethyl] benzamide hydrochloride (ML-1035) following intravenous (10 mg kg-1) and oral (200 mg kg-1) dosing was investigated in male and female New Zealand white rabbits. After intravenous dosing ML-1035 was eliminated with a half-life of 1.45 +/- 0.49 h in males and 0.79 +/- 0.08 h in females. Volume of distribution at steady-state was 2.08 +/- 0.98 l kg-1 in males and 9.11 +/- 5.86 l kg-1 in females. Clearance averaged 2.99 +/- 1.11 l h-1 kg-1 in males and 16.73 +/- 7.29 l h-1 kg-1 in females. All pharmacokinetic parameters were significantly different between males and females (p < 0.05). Absolute bioavailability after oral administration was 7.35 per cent for males and 12.31 per cent for females, suggesting that ML-1035 undergoes significant first-pass elimination. Plasma area under the curve for the metabolites of ML-1035 after both oral and intravenous administration were also different between the two sexes. These data suggest that the disposition of ML-1035 shows significant differences between male and female rabbits.

Validation of a column-switching high-performance liquid chromatographic (HPLC) method for determination of ML-1035 and its five metabolites in plasma.

A fully automated column-switching HPLC procedure has been developed and validated for quantitation of ML-1035 and its five metabolites in plasma employing direct injection. Plasma samples were injected onto a CN extraction column (4 x 4.6 mm, 5 microns) for micellar cleanup with 0.5% sodium dodecyl sulfate (SDS) in 50 mM phosphate. The proteinaceous components were solubilized and flushed out. The extracted compounds were then eluted by forward flush onto a C8 analytical column (150 x 4.6 mm, 5 microns) for further analysis using fluorescence detection (excitation, 308 nm; emission, 350 nm). After the subsequent washing and reequilibration with a sequence of three solvent mixtures, the extraction column was ready for the next injection. The limit of quantitation for all compounds of interest was about 10 to 15 ng/ml using 100 microliters of plasma. Excellent precision, accuracy, and linearity were obtained for all compounds over a range of 10 to 1500 ng/ml. The practicality of the HPLC method was also validated with plasma samples from dogs receiving ML-1035. Longevity for both extraction and analytical columns is excellent. Micellar cleanup coupled with the column-switching technique is a promising HPLC procedure when using direct injection of biological fluids.

Pharmacokinetic evaluation of two human epidermal growth factors (hEGF51 and hEGF53) in rats.

The pharmacokinetic profiles of two iodinated human epidermal growth factors (125I-hEGF51 and 125I-hEGF53) in rats receiving a single intravenous dose have been investigated using three independent bioanalytical techniques. Based on a tetrachloroacetic acid precipitation methodology, hEGF51 and hEGF53 were found to have distribution half-lives of 0.80 +/- 0.2 and 0.80 +/- 0.18 min, and elimination half-lives of 79.8 +/- 24.1 and 77.9 +/- 21.1 min, respectively. Evaluated by immunoprecipitation, distribution half-lives were 0.59 +/- 0.09 and 0.63 +/- 0.15 min, and elimination half-lives were 117.8 +/- 22.9 and 118.7 +/- 38.8 min, respectively. Both precipitation techniques produced similar, parallel plasma concentration-time curves, and there were no significant differences in other calculated kinetic parameters, including clearance and volume of distribution evaluated by either technique. Plasma disposition profiles of both peptides were also confirmed by visualization with SDS-PAGE and autoradiography, and were found to be similar to those generated by tetrachloroacetic acid and immunoprecipitation methods. Thus, three independent methods strongly suggest that both peptides have the same disposition profile, which exhibits a very rapid disappearance rate in the distribution phase followed by a much slower elimination process. These results also indicate that the pharmacokinetic behavior of human epidermal growth factor is not altered by deletion of two amino acids from the carboxyl terminus. In addition, the incubation study suggests that about 23% of the exogenous peptides were associated with red blood cells.

Determination of ML-1035 enantiomers in plasma by chiral high-performance liquid chromatography.

ML-1035, is a gastroprokinetic agent structurally related to metoclopramide. Because ML-1035 contains an asymmetric chiral sulphoxide moiety, a chiral HPLC method has been developed to separate and quantitate its R- and S-enantiomers in plasma. The ML-1035 enantiomers present in plasma are extracted with dichloroethane under alkaline conditions, the extract evaporated to dryness and reconstituted in the mobile phase. Samples are chromatographed on a Chiralcel OD HPLC column with hexane-absolute ethanol (1% TEA) (1:1, v/v) as the mobile phase. The enantiomers of the unchanged drug are determined by fluorescence measurement (ex: 310 nm, em: 350 nm). The method provides a linear response for both enantiomers over a concentration range of 25 (limit of determination) to 2500 ng ml-1 with correlation coefficients of 0.9987 or greater. The inter-assay precision is 9.5% or less and the accuracy ranges from 93.9 to 103.4% of the theoretical value. The method is used to determine the plasma concentrations of the R- and S-enantiomers following oral and intravenous administration of R- or S-enantiomers to dogs. The method is also adapted to measure enantiomer levels from in vitro reaction mixtures so that the possibility of metabolic inversion may be assessed. The data suggest that no significant level of inversion between the enantiomers occurred either in vivo or in vitro.