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Preincubation of guinea pig tracheas with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) resulted in a significant upward shift of the histamine concentration-response curve with a concomitant inhibition of prostaglandin E2 production. Preincubation of the preparations with a 5-lipoxygenase inhibitor (AA-861, 2-(12-hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-p-benzoquinone) or a leukotriene C4,D4,E4 receptor antagonist (FPL 55712, sodium 7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxy propoxy]-4-oxo-8- propyl-4H-1-benzopyran-2-carboxylate) totally blocked the L-NAME-induced tracheal hyperresponsiveness. A shift from cyclo-oxygenase to lipoxygenase products, in particular leukotrienes, is likely to be responsible for the L-NAME-induced tracheal hyperresponsiveness.

The rotA gene of Escherichia coli encodes a peptidyl-prolyl cis/trans isomerase (PPIase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPIase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS-PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPIase of E. coli is not essential and that the protein does not play an important role in protein folding.

Live attenuated Salmonella typhimurium strains expressing foreign antigens can be used for vaccination purposes. Due to deleterious effects of constitutive, high-level expression of the heterologous antigens, there is often strong selection pressure against plasmids encoding these antigens, resulting in rapid segregation in vivo. In vivo-inducible promoters may be a good alternative for constitutive promoters. The outer membrane protein PhoE of Escherichia coli is being used as a carrier for foreign antigenic determinants. Here we studied whether its expression from a plasmid is induced in S. typhimurium upon invasion of eukaryotic cells. This appeared to be the case. Furthermore, a S. typhimurium phoE mutant was constructed and the effects of the mutation on invasion, intracellular survival and virulence were studied. Survival in HEp-2 cells or in the macrophage-like cell line J744 was not, or only slightly, affected. Furthermore, the mutant appeared to be as virulent for mice as the wild-type strain.

1. VP and OT mediate their wealth of effects via 4 receptor subtypes V1a, V1b, V2, and OT receptors. 2. We here review recent insights in the pharmacological properties, structure activity relationships, species differences in ligand specificity, expression patterns, and signal transduction of VP/OT receptor. 3. Furthermore, the existence of additional VP/OT receptor subtypes is discussed.

Rats were trained to discriminate the specific 5-HT1A receptor agonist (+)-flesinoxan (R(+)-N(-)[2[4-(2,3-dihydro-2-2-hydroxy-methyl-1,4- benzodioxin-5-yl)-1-piperazinyl]ethyl]-4-fluorobenzoamide) (1.5 mg/kg p.o.) from water in a two-lever operant procedure. Generalization tests were conducted with the enantiomers and racemate of flesinoxan and the 5-HT1A receptor antagonists (S)-UH301 ((S)-5-fluoro-8-hydroxy-2-(dipropylamino)-tetralin) and WAY-100635 ((N(-)[2(-)[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride). (S)-UH301, WAY-100635 and fentanyl were investigated for their antagonistic properties. The (+)-flesinoxan stimulus generalized to (-)-flesinoxan and the racemate. The ED50 values for generalization corresponded well with the affinities of the enantiomers and the racemate for the 5-HT1A receptor. The flesinoxan cue could not be mimicked by (S)-UH301 or WAY-100635, but (S)-UH301 reduced response rates. Antagonism tests showed that both (S)-UH301 and WAY-100635 dose dependently antagonized the flesinoxan cue, with ID50 values of 0.52 and 0.03 mg/kg s.c., respectively. Fentanyl had no significant antagonistic properties. It is concluded that rats can learn to discriminate orally administered (+)-flesinoxan from water. The generalization of flesinoxan to the (-)-enantiomer and the antagonism of flesinoxan's cue by specific 5-HT1A receptor antagonists are further evidence for the involvement of flesinoxan's 5-HT1A receptor agonistic properties in its discriminative stimulus effects.

Theories on mother-daughter relationships have long been one-sided and biologically biased. The resulting images put the mental and physical health of mothers and daughters at risk. This article examines the mothers' perspective, and highlights the mutuality and dynamics in the mother-daughter relationship. New perspectives for therapeutic activities are opened up.

In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion deficiency (BLAD) was studied by means of skin transplantation experiments. Autograft and allograft behaviour in animals with BLAD was compared with the behaviour of simultaneously transplanted autografts and allografts in healthy controls. Allograft survival time was prolonged in three BLAD cattle (28, 30, and 72 days) compared to six healthy controls (12-14 days). When transplantations were repeated on one animal with BLAD using skin grafts from the same donor, accelerated rejection was observed (allograft survival time decreased from 72 days at primary to 35 days at secondary and to 21 days at tertiary transplantation), suggesting the development of immunological memory. Graft-infiltrating lymphocytes that were obtained from allograft biopsies during the period of rejection, were shown to be from recipient origin (beta 2-integrin negative). Our findings demonstrate that, although prolonged allograft survival is observed in cattle with BLAD, skin allografts are ultimately rejected.

The pulmonary delayed-type hypersensitivity (DTH) reaction induced by picryl chloride is characterized by enhanced albumin concentration in the airway tissue in the early phase (2 h after challenge with picryl sulphonic acid). During the later phase (48 h after the challenge) enhanced tracheal reactivity to carbachol in vitro and cellular (mononuclear and polymorphonuclear leukocytes) accumulation into the airway tissue in vivo are prominent features. In this present study, the effects of a novel drug, LCB 2183, were examined in the pulmonary DTH reaction. LCB 2183 (25 mg/kg twice daily intragastric gavage) failed to inhibit the enhanced albumin accumulation in the early phase of this response. In contrast, LCB 2183 abolished the tracheal hyperreactivity and the leukocyte accumulation in the airways of picryl chloride-sensitized mice 48 h after the challenge. These results demonstrate that LCB 2183 could be effective in the treatment of airway hyperreactivity and pulmonary inflammation.

Relationships between in vitro parameters (opsonic activity and anti-pneumococcal polysaccharide [PS] antibody subclasses) and in vivo mouse protection were established by logistic regression analysis. Data were from 158 mice challenged with pneumococci after vaccination with synthetic oligosaccharide- and PS-protein conjugates in combination with the adjuvant Quil A. The hypothesis that serum opsonic activity has predictive value for protection against pneumococcal infection was tested. Serum opsonic activity was well correlated with protection (chi 2 = 35.5, P < 0.001), although a stronger correlation was observed for anti-PS IgM and IgG. The combined use of IgG and opsonic activity as predictor variables yielded the best fitting model for predicting protection (chi 2 = 74.1, P < 0.001). When opsonic activity data were added to models that included various antibody isotypes, the statistical significance of the models was enhanced. Thus, the opsonic activity of antisera induced by pneumococcal vaccines can predict mouse protection.

Surfactant proteins A and D (SP-A, SP-D) can interact with lipopolysaccharide (LPS) and stimulate alveolar macrophages. The opsonic activities of SP-A and SP-D for bacteria with different types of LPS and alveolar macrophages were investigated. In flow cytometric studies with fluorescein-labeled rough (J5) and smooth (O111) Escherichia coli and rat alveolar macrophages, SP-A enhanced binding of J5 but not O111 bacteria to macrophages. Most importantly, SP-A enhanced ingestion of J5 bacteria by alveolar macrophages and subsequent bacterial killing. Immunoelectron microscopy demonstrated that J5 bacteria, the interface between the bacterium and the outer membrane of the alveolar macrophage, and ingested bacteria were heavily labeled with SP-A. In contrast, SP-D did not mediate phagocytosis. SP-A acted as an opsonin in the phagocytosis of rough LPS-containing bacteria by alveolar macrophages, emphasizing the possible role for SP-A in the alveolar defense system.

The effects of flesinoxan, a selective 5-HT1A receptor agonist, were studied under basal non-stress conditions and in the shock-probe burying paradigm. Flesinoxan (1 and 3 mg/kg s.c.) significantly reduced burying and freezing behaviour, indicating clear anxiolytic properties. Under non-stress conditions, injection of 3 mg/kg flesinoxan significantly enhanced plasma corticosterone and glucose levels, whereas prolactin secretion was significantly enhanced after both 1 mg/kg and 3 mg/kg flesinoxan. Flesinoxan (1 and 3 mg/kg) did not suppress shock-probe stress-induced rises in plasma corticosterone and glucose levels. The enhanced plasma prolactin levels induced by flesinoxan were not further affected by shock-probe exposure. Our data show that the anxiolytic effects of flesinoxan in the shock-probe burying paradigm are not related to increases in plasma corticosterone and glucose levels.

Circulating proinflammatory mediators have not been found in studies on typhoid fever, although the patients suffer from a systemic disease with characteristic protracted fever. The 14-kDa group II extracellular phospholipase A2 (PLA2) is induced by interleukin-1 and tumor necrosis factor and may mediate some of the effects of these cytokines. Circulating PLA2 concentrations were measured in 12 typhoid fever patients on various days after admission and after recovery. On admission, mean concentrations of PLA2 were elevated (1444 +/- 1560 ng/mL) and decreased gradually and significantly to day 14 (55 +/- 48 ng/mL). patients with complicated disease had significantly higher PLA2 levels on admission. PLA2 was not produced in a lipopolysaccharide-stimulated whole blood culture, indicating that PLA2 originates from other types of cells. These data indicate that PLA2 may be a mediator of disease in protracted inflammatory diseases such as thyroid fever.

To investigate the significance of endogenous, neuroleptic-like gamma-type endorphins and their putative receptors, polyclonal and monoclonal antibodies against gamma-type endorphins, which may bio-inactivate the ligands for the receptors, and monoclonal anti-idiotype antibodies, which presumably bind to the receptors, were injected into the nucleus accumbens of the rat brain. The desenkephalin-gamma-endorphin-induced antagonism of the hypomotility response elicited by challenge with apomorphine injected into the nucleus accumbens was used as test system. Both the anti-desenkephalin-gamma-endorphin antibodies and anti-idiotype antibodies blocked the action of exogenous desenkephalin-gamma-endorphin. Thus, the anti-idiotype antibodies may serve as receptor antagonists. Chronic treatment (injection into the nucleus accumbens) with the anti-idiotype antibodies induced sustained hypermotility, decreased habituation and impaired passive avoidance behavior. In such treated animals local treatment with apomorphine did not elicit hypomotility. It is suggested that gamma-type endorphins influence the setpoint for feedback regulation in dopaminergic neurons equipped with gamma-type endorphin receptor systems.

The use of angiotensin-converting-enzyme (ACE) inhibitors has been associated with increased insulin sensitivity in diabetic patients. Although such an effect could be beneficial in the treatment of hypertension or congestive heart failure in diabetic patients, it might also precipitate severe hypoglycaemia. To test this hypothesis we carried out a nested case-control study, using data in the Dutch PHARMO system (1986-92), among diabetic patients treated with insulin or with oral antidiabetic drugs, who were admitted to hospital with hypoglycaemia. We identified 94 patients who had been admitted with hypoglycaemia and selected 654 controls from the same cohort. With adjustment for a wide range of potential confounding factors, hypoglycaemia was significantly associated with current use of ACE inhibitors (odds ratio 2.8 [95% CI 1.4-5.7]). Both among users of insulin and among users of oral antidiabetic drugs, use of ACE inhibitors was significantly associated with an increased risk of hospital admission for hypoglycaemia (2.8 [1.2-6.4] and 4.1 [1.4-12.2], respectively). Although ACE inhibitors have several advantages over other antihypertensive drugs in diabetes, the risk of hypoglycaemia should be taken into account. Further investigation of the mechanism is needed since as many as 13.8% of all hospital admissions for hypoglycaemia might be attributable to use of ACE inhibitors.

Two grazing experiments were performed to study the effect of moxidectin in a 'dose and move' system on Dictyocaulus viviparus infections in calves. In the first experiment, three groups of four calves were experimentally infected with 20 larvae of D. viviparus 7 weeks before moxidectin treatment of two of these groups. A sufficient suppression of Dictyocaulus infections was observed in a 'dose and move' group, but also in a group which stayed on contaminated pasture after treatment. In contrast, high faecal larval counts and lungworm disease were observed in July-August in a non-treated pasture control group. Development of immunity against lungworm was sufficient in all three groups, on pasture. In the second experiment, four out of 26 calves, including two groups of six calves and four tracer calves, were experimentally infected with 20 larvae of D. viviparus 7 weeks before moxidectin treatment combined with a move of one of the groups. No lungworm disease was observed in this 'dose and move' group. In a pasture control group high faecal larval counts and severe clinical disease were observed in August-September and one calf had to be euthanized. Although a mean burden of 129 immature lungworms was present at the time of treatment, development of immunity appeared to be low in the 'dose and move group'. In contrast, high levels of immunity had developed in the pasture control group.

The aim of the present investigation was to study the interexaminer reliability of orthopedic tests and palpation techniques routinely used in the clinical diagnosis of disorders of the masticatory system. The tests were performed by a dentist and a physiotherapist, who both used the tests routinely when examining patients with temporomandibular disorders. Seventy-nine patients participated in this study. In the analysis, percentage agreement, intraclass correlation, and Cohen's kappa were used. The interexaminer reliability of the tests measuring maximal active mouth opening and registration of clicking during active mouth opening was high. The interexaminer reliability was fair for the tests measuring the intensity of pain during active movements and moderate for tests recording joint sounds (kappa = 0.47 to 0.59). There was high interobserver agreement on several items of the traction and translation tests, although the kappa values were low. The interexaminer reliability of the multitest scores for compression was substantial for joint sounds (kappa = 0.66) and fair for pain (kappa = 0.40). The interexaminer reliability of the multitest scores for muscle palpation and joint palpation was moderate (kappa = 0.51) and fair (kappa = 0.33), respectively. It can be concluded that most variables determined during active movements can be measured with satisfactory reliability, whereas variables for other tests are not measured with the same reliability on the basis of the kappa scores. The main symptoms of temporomandibular disorders can be evaluated reliably with multitest scores. It is recommended that clinicians calibrate their techniques regularly to improve the reliability of results in daily practice.

We have previously shown that the calcitonin (CT)-encoding exon 4 of the human calcitonin/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is surrounded by suboptimal processing sites. At the 5' end of exon 4 a weak 3' splice site is present because of an unusual branch acceptor nucleotide (U) and a weak poly(A) site is present at the 3' end of exon 4. For CT-specific RNA processing two different exon enhancer elements, A and B, located within exon 4 are required. In this study we have investigated the cooperation of these elements in CT exon recognition and inclusion by transient transfection into 293 cells of CALC-I minigene constructs. Improvement of the strength of the 3' splice site in front of exon 4 by the branchpoint mutation U-->A reduces the requirement for the presence of exon enhancer elements within exon 4 for CT-specific RNA processing, irrespective of the length of exon 4. Replacement of the exon 4 poly(A) site with a 5' splice site does not result in CT exon recognition, unless also one or more exon enhancer elements and/or the branchpoint mutation U-->A in front of exon 4 are present. This indicates that terminal and internal exons are recognised in a similar fashion. The number of additional enhancing elements that are required for CT exon recognition depends on the strength of the 5' splice site. Deletion of a large part of intron 4 also leads to partial exon 4 skipping. All these different elements contribute to CT exon recognition and inclusion. The CT exon is recognised as a whole entity and the sum of the strengths of the different elements determines recognition as an exon. Curiously, in one of our constructs a 5' splice site at the end of exon 4 is either ignored by the splicing machinery of the cell or recognised as a splice donor or as a splice acceptor site.

We tried to antagonize the endocrine and behavioural changes induced by the selective 5-HT1A receptor agonist, flesinoxan, with the putative 5-HT1A receptor antagonist, (S)-UH301 ((S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin). The interaction of (S)-UH301 (3 and 10 mg/kg s.c.) with flesinoxan (3 mg/kg s.c.) showed no antagonistic effects of (S)-UH301 on flesinoxan-induced corticosterone secretion. In fact, like flesinoxan (1 and 3 mg/kg s.c.), (S)-UH301 (3 and 10 mg/kg s.c.) itself dose dependently increased plasma corticosterone levels. Unlike flesinoxan, (S)-UH301 did not induce hyperglycemia, lower lip retraction and flat body posture. Moreover, flesinoxan-induced hyperglycemia and behavioural changes were effectively antagonized by (S)-UH301, showing potent 5-HT1A receptor antagonistic effects of (S)-UH301. Therefore we conclude that (S)-UH301 is a potent 5-HT1A receptor antagonist and that the (S)-UH301-induced corticosterone secretion is mediated by a non-5-HT1A receptor mechanism.

The Salivette was evaluated with a range of racemic beta-adrenoceptor blocking drugs with different lipophilicity. Recovery from the Salivette appeared to be independent of the stereochemical configuration of the drugs but a significant loss of drug due to the Salivette was observed for all tested drugs. The performance of the method, in terms of accuracy and precision, fitted well within the generally accepted criteria for validation, except near the limit of quantification. The Salivette is successfully used for quantitating salivary beta-blocking drugs.

To clarify the influence of opioids on social play, the effects of morphine on playful and non-playful social behavior in juvenile rats was investigated under different conditions. Environmental variables employed were different (dim and intense) levels of illumination during testing, familiarity to the test cage, and different periods of social isolation prior to testing. Under dim light conditions, morphine markedly increased playful social behavior, such as pinning, boxing/wrestling and following/chasing, whereas non-playful social behavior such as social exploration and contact behavior was hardly affected. This effect of morphine was independent of duration of previous isolation and dose-dependent, with a maximal effect at 1.0 mg/kg. The mechanism of this effect is interpreted as an action on the rewarding aspects of play. A dose of 0.1 mg/kg of morphine abolished the initial suppression of play induced by unfamiliarity to the test cage, without influencing total levels of play. This may be an effect of morphine on the integration of sensory stimuli. Under intense light conditions, whereas playful behavior was completely suppressed, morphine itself hardly affected such behavior, but decreased some aspects of non-playful social behavior. These results suggest that in juvenile rats playful and non-playful forms of social behavior are differentially regulated. In addition, opioid systems may be involved at different levels in the regulation of social play.

The prevalence of oocysts of Eimeria species in calves (n = 334), yearlings (n = 254) and cows (n = 1314) was determined on 38 Dutch dairy farms. Twelve species of Eimeria were identified in faecal specimens by sucrose-flotation. The prevalences of Eimeria spp. differed markedly in the different age classes on individual farms as well as between farms. The overall prevalence of Eimeria oocysts in faecal specimens was 46% for calves, 43% for yearlings and 16% for cows. The number of oocysts excreted was generally low in cows and yearlings, whereas high numbers of oocysts per gram of faeces (OPG) were exclusively observed in calves. No cases of clinical coccidiosis were observed in this survey. Linear regression analysis showed that there is significant reduction in the OPG levels (P < 0.05) in calves infected with Eimeria, aged between 7 and 38 weeks. Finally, the data are discussed in relation to management practices and the acquisition of immunity.

A number of Escherichia coli strains was isolated during a study of clinical mastitis on seven farms in the Netherlands. From these E. coli strains, 30 were characterised with regard to their serotype and their DNA polymorphism pattern with REP and ERIC primers. Special attention was given to recurrent E. coli mastitis in cows. The combination of serotype and DNA pattern observed, was used to study the epidemiology of clinical E. coli mastitis. The results demonstrated that the PCR reaction with the ERIC primers can be used for differentiation of E. coli strains. The DNA polymorphism patterns showed that E. coli strains isolated from cases of clinical mastitis have a great variability in genotype. More 3 than one case of clinical mastitis associated with E. coli during the same lactation period occurred infrequently. However when it took place, E. coli strains isolated from the separate episodes of inflammation, were in most instances of the same serotype and had the same DNA amplification pattern.

Unlabelled: In this study, a method was developed for the efficient entrapment of active tissue-type Plasminogen Activator (t-PA) into liposomes. Experimental conditions were varied to optimize t-PA entrapment: different buffer solutions were used (pH 4 and 7.5), the effect of the incubation concentrations of phospholipid (PL) and t-PA was monitored and the influence of liposome-size was examined. Furthermore, the effect of ultracentrifugation on t-PA containing liposomes was determined in the presence and absence of Tween 80. t-PA entrapment strongly depended on experimental conditions and ranged from 30 up to 90%. Almost quantitative+ (90%) entrapment (entrapment percentage defined as absolute entrapment (IU t-PA/mumol PL) divided by total incubation ratio (IU t-PA/mumol PL), times 100%) was obtained in Hepes buffer pH 7.5, devoid of arginine, with low ionic strength. Ultracentrifugation, used for removal of non-entrapped t-PA, was shown to have a damaging effect on the liposomes (especially in the presence of 0.05% Tween 80), leading to t-PA loss. However, because acceptable alternatives were not available, ultracentrifugation was used during this study. Therefore, the encapsulation-percentage values shown in this study are in fact underestimates for the true entrapment of t-PA., In Conclusion: almost quantitative t-PA entrapment in liposomes can be achieved by selecting the proper milieu and inducing a strong interaction between t-PA and bilayer.

Eighteen males performed two vigilance tasks with static and dynamic stimuli under the influence of oxazepam (20 and 40 mg) in a placebo-controlled, double blind, crossover design. Oxazepam (40 mg) caused impaired performance in the early part of a task with stimuli inducing frequent saccadic eye movements (dynamic task), relative to a task in which the stimuli remained at the same location (static task). This could not be explained by effects of the drug on oculomotor behavior. A larger diameter of the pupil in the dynamic task indicated that performance on this task may have required more effort. Stimulus processing requirements were higher in the dynamic task, as suggested by event-related brain potentials (ERPs), in particular the P3 wave; i.e., more resources had to be allocated in this task. This (additional) investment of resources appeared impossible after administration of oxazepam (40 mg). The conclusion was that tasks eliciting frequent eye movements require more effort and processing resources.

Resistance of microorganisms to antimicrobial agents is an increasing problem in the treatment of infectious diseases. In mixed infections, an interesting development can arise when one organism protects another from being killed by an antibiotic. Unfortunately, in the case of respiratory tract infections, experimental evidence of this development is poor. In this study, mice intranasally infected with a lethal number of pneumococci and treated with a curative dose of penicillin or amoxicillin died from pneumococcal pneumonia when they were coinoculated with beta-lactamase-producing Moraxella catarrhalis. beta-lactamase-negative M. catarrhalis did not show a similar indirect pathogenic effect. Treatment with a combination of amoxicillin and the beta-lactamase inhibitor clavulanic acid was not affected by beta-lactamase-producing M. catarrhalis. These findings help explain antibiotic failure in respiratory tract infections, even though the causative microorganism is sensitive to the antibiotic in vitro.

Antagonists for the melanocortin receptor family were identified by analysis of the effects of four melanocortin analogues on alpha-MSH(alpha-melanocyte-stimulating hormone)-induced cAMP accumulation in 293 human embryonal kidney (HEK) cells that expressed either the rat melanocortin MC3 receptor, the human melanocortin MC4 receptor or the ovine melanocortin MC5 receptor. Two peptides, [D-Arg8]ACTH(adrenocorticotrope hormone)-(4-10) and [Pro8,10,Gly9]ACTH-(4-10), antagonized the action of alpha-MSH on the melanocortin MC4 and MC5 receptors, but not the melanocortin MC3 receptor. [Ala6]ACTH-(4-10) inhibited the alpha-MSH activation of the melanocortin MC3 and MC5, but only weakly antagonized the activation of the melanocortin MC4 receptor. [Phe-I7]ACTH-(4-10) antagonized the melanocortin MC3, MC4 and MC5 receptors equally well. These antagonists were also tested to block a behavioral response induced by alpha-MSH. alpha-MSH-induced excessive grooming behavior in rats was inhibited by [Phe-I7]ACTH-(4-10), [D-Arg8]ACTH-(4-10) and [Pro8,10,Gly9]ACTH-(4-10), but not by [Ala6]ACTH-(4-10). This suggests that alpha-MSH-induced excessive grooming behavior is mediated by melanocortin MC4 receptors.

The synthesis is reported of methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-O-(3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-beta-D-glucopyranoside (4) and methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-4-O- (3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-6- O-alpha-L-fucopyranosyl-beta-D-glucopyranoside (5), which represent the invariant hexasaccharide core structure of the xylose-containing glycans of N-glycoproteins and its 6-O- fucosylated derivative. Ethyl 4-O-[3-O-allyl-4-O-benzoyl-6-O-tert-butyldimethylsilyl-2-O- (2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1- thio-beta-D-glucopyranoside (9) was coupled with methyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranoside (11). Desilylation of the resulting tetrasaccharide derivative, followed by condensation with 2,3,4,6- tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7), gave methyl 4-O-(4-O-[3-O-allyl-4- O-benzoyl-6-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-2-O-(2,3,4 -tri-O- acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)- 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside (14). Deallylation of 14, followed by condensation with 7 and deprotection, gave hexasaccharide 4. Ethyl 3,6-di-O- benzyl-2-deoxy-4-O- [4,6-di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl) - beta-D-mannopyranosyl]-2- phthalimido-1-thio-beta-D-glucopyranoside (17) was coupled with methyl 3-O- benzyl-2-deoxy-6-O- (4-methoxybenzyl)-2-phthalimido-beta-D-glucopyranoside. Demethoxybenzylation of the tetrasaccharide derivative thus obtained, followed by fucosylation using ethyl 2,3,4-tri-O- benzyl-1-thio- beta-L-fucopyranoside, gave methyl 3-O-benzyl-2-deoxy-4-O-[3,6-di-O-benzyl-2- deoxy-4-O-[4,6- di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranosyl]-2-phthalimido- beta-D-glucopyranosyl)-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L- fucopyranosyl)-beta-D-glucopyranoside (23). O-Deacetylation followed by tert-butyldimethylsilylation, benzoylation, and desilylation gave methyl 4-O-(4-O-[3-O-allyl-4-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-beta-D- xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta- D-glucopyranosyl)-3- O-benzyl-2-deoxy-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L-fucopy ran osyl)- beta-D-glucopyranoside (24). Mannosylation of 24 using 7, followed by deallylation, further mannosylation with 7, and deprotection, gave the heptasaccharide 5.

Planning future policy for medicines poses difficult problems. The main players in the drug business have their own views as to how the world around them functions and how the future of medicines should be shaped. In this paper we show how a scenario analysis can provide a powerful teaching device to readjust peoples' preconceptions. Scenarios are plausible, not probable or preferable, portraits of alternative futures. A series of four of alternative scenarios were constructed: "sobriety in sufficiency," "risk avoidance," "technology on demand," and "free market unfettered." Each scenario was drawn as a narrative, documented quantitatively wherever possible, that described the world as it might be if particular trends were to dominate development. The medical community and health policy markers may use scenarios to take a long term view in order to be prepared adequately for the future.

Bovine isolated mesenteric arterial rings were preincubated for 20 h with interferon-gamma (100 U ml-1) and relaxation in response to bradykinin (10(-12) to 3 x 10(-8) M) was then measured isometrically in an organ bath. Interferon-gamma pretreatment for 20 h markedly attenuated the endothelium-dependent bradykinin relaxation in arteries precontracted with 9,11-dideoxy-11 alpha,9 alpha-epoxymethano prostaglandin F2 alpha (U46619), and the relaxation was reversed to contraction at the highest bradykinin concentrations (-72 +/- 5% for control vs. + 6 +/- 10% for interferon-gamma). Cycloheximide (20 micrograms ml-1) present during the 20-h preincubation completely prevented the interferon-gamma effect. Methyl-L-arginine (1 mM) treatment during the 20-h preincubation also inhibited the interferon-gamma effect on bradykinin relaxation (-47 +/- 18% for interferon-gamma and methyl-L-arginine), which suggests involvement of nitric oxide during the 20-h preincubation with interferon-gamma. In control arteries, des-Arg9-bradykinin, a bradykinin B1 receptor agonist, evoked contractions, which were augmented in rings preincubated for 20 h with interferon-gamma. The bradykinin B1 receptor antagonist, des-Arg9-Leu8-bradykinin (2 microM), present in the organ bath in combination with methyl-L-arginine (1 mM) only present during the 20-h preincubation with interferon-gamma completely restored the bradykinin relaxation (-79 +/- 12%). We suggest two mechanisms. Firstly, prolonged nitric oxide release induced by interferon-gamma during the 20-h preincubation may inhibit bradykinin stimulated endothelium-derived nitric oxide release and action. Secondly, interferon-gamma caused upregulation of the bradykinin B1 receptor-mediated contraction, which may contribute to the decrease in bradykinin-induced vasodilation and cause a reversal to contraction.

Evolution of RNA secondary structure is studied using simulation techniques and statistical analysis of fitness landscapes. The transition from RNA sequence to RNA secondary structure leads to fitness landscapes that have local variations in their "ruggedness." Evolution exploits these variations. In stable environments it moves the quasispecies toward relatively "flat" peaks, where not only the master sequence but also its mutants have a high fitness. In a rapidly changing environment, the situation is reversed; evolution moves the quasispecies to a region where the correlation between secondary structures of "neighboring" RNA sequences is relatively low. In selection for simple secondary structures the movement toward flat peaks leads to pattern generation in the RNA sequences. Patterns are generated at the level of polynucleotide frequencies and the distribution of purines and pyrimidines. The patterns increase the modularity of the sequence. They thereby prevent the formation of alternative secondary structures after mutations. The movement of the quasispecies toward relatively rugged parts of the landscape results in pattern generation at the level of the RNA secondary structure. The base-pairing frequency of the sequences increases. The patterns that are generated in the RNA sequences and the RNA secondary structures are not directly selected for and can be regarded as a side effect of the evolutionary dynamics of the system.

The causes of outbreaks of lungworm disease were determined from the herd histories of 25 affected herds of young cattle. The results indicate that light pasture contamination by carriers and subsequent auto-infections are the most prominent cause of lungworm disease, followed by heavy pasture contamination by carriers. Overwintered pasture infections seem to play a minor role. Adult dairy cows seem to be the predominant carrier animal when light pasture infections are concerned. Heavy pasture infections are almost always caused by calves and yearlings.

The results of a survey on the prevalence of patent lungworm infections in herds of dairy cows in the Netherlands are presented. Low patent infections were recorded in February-March on six out of 40 farms in at least one out of 40 cows. Between mid-April and mid-June low patent infections were detected on 28 out of 39 of these farms in one to four of 40 cows. Two farms on which cows were positive in the first round were negative in the second round. One to three positive cows were found on six out of a total of 15 farms revisited in July-August. These results show that lungworm infections are cycled within herds of dairy cows in the Netherlands at a low level. This indicates that dairy cows are important as carriers for lungworm, particularly in spring. The increased patency of lungworm in cows from winter to spring may be explained by maturation of inhibited larvae.

Using a two-lever operant drug discrimination procedure, rats were trained to discriminate the 5-HT1A receptor agonist, flesinoxan (0.5 mg/kg i.p.), from saline. Hereafter, several non-serotonergic drugs were tested in generalization and antagonism tests. The flesinoxan stimulus did not generalize to the stimuli of either the alpha 1-adrenoceptor antagonist, prazosin, the alpha 2-adrenoceptor agonist, clonidine, the dopamine receptor agonist, apomorphine, the dopamine receptor antagonists, haloperidol and pimozide, the benzodiazepine receptor agonist, chlordiazepoxide, nor to the peripherally acting vasodilator, hydralazine. In antagonism studies, prazosin, haloperidol, pimozide and the alpha 2-adrenoceptor antagonist, idazoxan, failed to block the flesinoxan stimulus. In substitution tests, however, flesinoxan partially generalized to idazoxan and completely to the alpha 2-adrenoceptor antagonist, yohimbine. The affinities of yohimbine and idazoxan for the 5-HT1A receptor may explain the latter result. The present findings suggest that the central mechanism through which flesinoxan exerts its discriminative stimulus effects does not involve alpha 1- and alpha 2-adrenoceptors, dopamine and benzodiazepine receptors. Finally, the results with the blood pressure lowering agents, hydralazine, clonidine and prazosin do not support the suggestion that the centrally mediated blood pressure lowering effects of flesinoxan contribute to its internal stimulus effect.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112-6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with 31P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC50 values of AZTTP-DSG were 0.33 and 0.79 microM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 microM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.

The synthesis is reported of 4-methoxyphenyl O-(beta-D-glucopyranosyluronic acid)-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (1), 4-methoxyphenyl O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1-->4)-O-(beta-D- glucopyranosyluronic acid)-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (5), and 4-methoxyphenyl O-(beta-D-glucopyranosyluronic acid)-(1-->3)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1-->4)-O-(b eta-D- glucopyranosyluronic acid)-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (10), which are structural elements of the extracellular polysaccharide hyaluronic acid. 6-O-Levulinoyl-2,3,4-tri-O-p-toluoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) was condensed with 4-methoxyphenyl 2-deoxy-4,6-O-isopropylidene-2-phthalimido-beta-D-glucopyranoside (4). De-isopropylidenation and acetylation of the obtained disaccharide derivative yielded 4-methoxyphenyl O-(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-4 ,6-di-O - acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside, and subsequent delevulinoylation, oxidation, complete deprotection, and N-acetylation gave 1. Coupling of 4-O-allyloxycarbonyl-6-O-levulinoyl-2,3-di-O-p-toluoyl-alpha-D-glu copyranosyl trichloroacetamidiate with 4 followed by de-isopropylidenation, acetylation, and deallyloxycarbonylation of the obtained disaccharide derivative gave 8. Condensation of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate with 8 afforded trisaccharide derivative 4-methoxyphenyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--> 4)-O-(6 - O-levulinoyl-2,3-di-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-4,6-di-O- acetyl- 2-deoxy-2-phthalimido-beta-D-glucopyranoside, and subsequent delevulinoylation, oxidation, complete deprotection, and N-acetylation gave 5. 3-O-Allyloxycarbonyl-2-deoxy-4,6-O-isopropylidene-2-phthalimido-be ta-D- glucopyranosyl trichloroacetimidate was coupled with disaccharide acceptor 8, and deallyloxycarbonylation of the obtained trisaccharide derivative yielded 12. Condensation of 3 with 12 followed by de-isopropylidenation and acetylation of the obtained tetrasaccharide derivative gave 4-methoxyphenyl O-(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-O -(4,6- di-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1-->4)-O-(6-O- levulinoyl-2,3-di-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-4,6-di-O-ac etyl-2- deoxy-2-phthalimido-beta-D-glucopyranoside, and delevulinoylation, oxidation, complete deprotection, and N-acetylation yielded 10.

The synthesis is reported of methyl 3,6-di-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-beta-D-xylopyranosyl-be ta-D- mannopyranoside (2), methyl 6-O-alpha-D-mannopyranosyl-3-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (3), and methyl 3-O-alpha-D-mannopyranosyl-6-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (4). The various methyl beta-D-Manp acceptor derivatives were prepared from the corresponding methyl beta-D-Glcp derivatives via oxidation-reduction. All glycosyl donors were coupled using the trichloroacetimidate method at -40 degrees C in dichloromethane with trimethylsilyl triflate as a catalyst. Methyl-3-O-benzyl-4,6-O-benzylidene-beta-D-mannopyranoside (7) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-benzylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and hydrogenation gave methyl 4-O-acetyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta-D-mannopyr anoside (12). Coupling of 12 with 2,4,6-tri-O-acetyl-3-O-methyl-alpha-D-mannopyranosyl trichloroacetimidate (18) afforded tetrasaccharide derivative 19, and subsequent O-deacetylation gave 2. Methyl 3-O-benzyl-4,6-O-prop-2-enylidene-beta-D-mannopyranoside (22) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-prop-2-enylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and deallylation at O-6 gave methyl 4-O-acetyl-3-O-benzyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranoside (26-a), which was either condensed with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (27) or 18, to give trisaccharide derivatives 28 or 31, respectively. Debenzylation of 28 followed by condensation with 18 gave, after O-deacetylation, 3, whereas debenzylation of 31 followed by condensation with 27 gave, after O-deacetylation, 4.

Over the last 20 years several observations at the peptide level have indicated the possible existence of additional members of the vasopressin (VP)-oxytocin (OT) gene family in mammals. In this study, the human genome was analyzed for the existence of genes structurally related to the VP and OT genes. Human genomic blots probed under low stringency conditions with exon B of the human OT gene, that codes for the conserved constant region of neurophysin, revealed the presence of two distinct bands in addition to the known VP and OT gene fragments. Five clones were obtained from a library of genomic EcoRI fragments ranging from 4-8 kb, that comprised both low stringency signals, by low stringency hybridization with the OT exon B probe. One clone of 7 kb hybridized at high stringency conditions to bands of the same size as previously detected with OT exon B on a human genomic blot. However, no similarity was observed between the open reading frames of this clone and the neurophysin portion of the OT gene. Another clone of 4.8 kb was identical to a fragment of the gene for the human bone morphogenetic factor hBMP-6, a member of the TGF-beta family. The hBMP-6 gene was not detected by low stringency hybridization of the human genomic blot with the OT exon B probe. No significant similarity was found between the amino acid sequences of human OT neurophysin and hBMP-6. Therefore, no evidence can be provided that the human genome contains additional neurophysin-related genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequencing of a subtilisin-type bacterial protease and a bio-engineered variant was carried out by investigating various enzymatic digests using HPLC-frit fast atom bombardment MS methods. The fast atom bombardment mass spectral data allowed rapid identification of the enzymatically generated peptides and differentiation between both proteins. The feasibility of determining the positions and nature of mutations in the amino acid sequence depends mainly on the size of the peptides containing the modifications.

Interleukin-5-producing CV-1 cells were encapsulated in alginate and injected i.p. in guinea pigs (4 x 10(6)/animal). These cells produced approximately 8 ng interleukin-5 per 4 x 10(6) cells per day. Airway hyperresponsiveness to histamine in vivo was observed 3 and 7 days after administration. The increase in lung resistance after intravenous administration of histamine to guinea pigs was significantly potentiated, by approximately 70 to 90% in interleukin-5-treated animals. In animals treated with antibody to interleukin-5, the administration of interleukin-5-producing CV-1 cells did not induce hyperresponsiveness. The percentage of eosinophils in broncho-alveolar lavage fluid was increased by 100% at 7 days but not at 3 days after administration of interleukin-5-producing CV-1 cells. Antibody to interleukin-5 prevented the broncho-alveolar lavage eosinophilia at 7 days after interleukin-5 administration. It can be concluded that interleukin-5 induces broncho-alveolar lavage eosinophilia and airway hyperresponsiveness and that these phenomena do not occur simultaneously. These data suggest a role for interleukin-5 in the development of airway hyperresponsiveness in bronchial asthma.

A derivatization high-performance liquid chromatographic method with ultraviolet detection to monitor the plasma concentration of chloroacetaldehyde, a neurotoxic metabolite of oxazaphosphorine drugs, is presented. To prevent the rapid degradation of chloroacetaldehyde, the plasma samples are stabilized with formaldehyde. The method is linear in the concentration range 1-250 nmol/ml. Blood samples from a patient who was treated with a ten-day continuous infusion of ifosfamide were assayed. The chloroacetaldehyde concentrations did not exceed 10 nmol/ml.

The role of substance P-containing sensory nerves in the development of tracheal hyperreactivity to histamine and arecoline was investigated in an allergic model of asthma. Male Hartley-strain guinea pigs were sensitized to ovalbumin (20 mg/kg i.p.) and 14 days later exposed to either saline or ovalbumin (2%) aerosols for 8 consecutive days. One day after the last aerosol exposure the animals were killed and the tracheas were removed. Isotonic muscle shortening in response to increasing doses of histamine and arecoline was measured. Capsaicin (50 mg/kg s.c.) or vehicle pretreatment was performed 7 days prior to sensitization. Tracheas from vehicle-pretreated sensitized and ovalbumin-aerosol exposed animals had increased reactivity to both histamine and arecoline compared to saline-aerosol exposed animals. Capsaicin pretreatment did not alter tracheal reactivity in the saline-aerosol exposed group. Capsaicin pretreatment prevented the increase in tracheal reactivity caused by both agonists in sensitized and ovalbumin-aerosol exposed guinea pigs. These results suggest that capsaicin-sensitive sensory nerves are important for the development of increased tracheal reactivity in an allergic model of asthma.

Taxol is a novel and promising oncolytic agent the use of which is hampered by its neurotoxicity. We now describe a taxol-induced neuropathy in rats and its prevention by the adrenocorticotropic hormone-(4-9) (ACTH-(4-9)) analog, ORG 2766. A decrease in sensory nerve conduction velocity was seen in taxol-treated rats, both with daily injections of small amounts (6 mg/kg per week) and with weekly injections of higher amounts (9 mg/kg per week) of taxol. Concomitant administration of ORG 2766 completely prevented the occurrence of a neuropathy.

In conscious rats, i.v. administered adrenocorticotropic hormone (ACTH-(4-10)) and gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) induced a dose-dependent increase in blood pressure (BP), heart rate (HR) and pulse pressure (PP). No circadian influence on these effects was observed. The structurally related peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), only caused an increase in HR, which was not dose-dependent, whereas the stable ACTH-(4-9) analog, Org 2766, was without effect on these hemodynamic parameters. In rats under light urethane-induced anesthesia, which is known to maintain reflexes and sufficient sympathetic tone, gamma 2-MSH caused hemodynamic responses similar to those observed in conscious rats. In contrast, gamma 2-MSH had an opposite effect in rats under deep pentobarbital-induced anesthesia: a depressor effect combined with a slight bradycardia. A comparative study with rats of a more arousable Wistar rat substrain (Riv:TOX) and of a less excitable rat substrain (U:WU) showed that the dose-pressor response curves for ACTH-(4-10) and gamma 2-MSH were shifted to the left in the more excitable rats as compared to the in the less excitable rats. We conclude that a restricted amino acid sequence in the N-terminal part of the pro-opiomelanocortin (POMC)-molecule (gamma 2-MSH/ACTH-(4-10)-like) is responsible for the stimulating effects on the cardiovascular system and that those effects are strongly dependent on the state of arousal, i.e. sympathetic tone, of the rat. These stimulatory effects override a depressor phenomenon which can only be detected during central depression.

A simple and rapid high-performance liquid chromatographic method with UV detection for the determination in serum of the antimalarial artelinic acid is described. Artelinic acid is a water-soluble semi-synthetic derivative of artemisinin (or Qinghaosu), which is the parent compound of a completely new class of antimalarials with great potential value. Calibration curves of artelinic acid in serum from 0.5 to 50 micrograms/ml were found to be linear, and the extraction recovery was ca. 70% from rabbit serum and 45% from bovine and human serum. The detection limit of the method was 50 ng/ml when 250 microliters of serum were processed. Spiked samples (rabbit serum) proved to be stable for at least 43 days on storage at -20 degrees C. The method has been used for the analysis of ca. 400 samples in a pharmacokinetic study. About 50 samples can be processed in half a day.

The effects of sabeluzole, a drug that protects rat hippocampal neurones from glutamate- and N-methyl-D-aspartate (NMDA)-induced toxicity, were investigated in rat cerebellar granule cells in vitro with the whole-cell voltage clamp technique. Acute exposure of 0.1 microM sabeluzole for 20 min prior to experiments did not significantly affect glutamate receptor-mediated inward currents. Conversely, exposure of cultured granule cells to sabeluzole for 7 days reduced the NMDA-induced inward current and did not affect the non-NMDA responses evoked by kainic acid. The results suggest that chronic treatment with sabeluzole selectively reduces the functional NMDA response.

The exopolysaccharide of Lactobacillus delbrückii subsp. bulgaricus rr, isolated from skimmed milk, is a heteropolymer of D-galactopyranosyl, D-glucopyranosyl, and L-rhamnopyranosyl residues in the molar ratio 5:1:1. The structure was established by linkage analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by Smith degradation and partial acid hydrolysis, using methylation analysis, EIMS, and 1D and 2D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: -->2)-[beta-D-Galp-(1-->3)]-alpha-D-Galp-(1-->3)- beta-D-Glcp-(1-->3)-[beta-D-Galp-(1-->4)]-beta-D-Galp-(1-->4)-[alpha-L- Rhap-(1-->3)]-alpha-D-Galp-(1-->.

A sensitive and reproducible flow cytometric assay was developed for the analysis of Fc gamma and C3b(i) receptors on HSV-infected cells. The method is based on a rosette technique using fluorochrome-labeled erythrocytes sensitized with IgG or C3b(i). A comparison of flow cytometric and microscopic quantitation demonstrated that the binding of EIgG, EC3b(i) to HSV-infected cells were correlated. Flow cytometric analysis provides the opportunity to study simultaneously the distribution of E per HSV-infected cell and the total binding of E to the whole population of HSV-infected cells. Receptor activity and HSV glycoprotein cell surface expression were shown to be correlated in a linear fashion. The assay could be applied to other Fc gamma R- and C3b(i)R-bearing cells.

The non-specific lipid-transfer protein (nsL-TP), also known as sterol carrier protein 2 (SCP2), is a small (M(r) 13,000) basic protein which catalyzes in vitro the transfer of a great variety of lipids, including cholesterol, between membranes. Inherent to this transfer activity, the protein stimulates in vitro various aspects of cholesterol metabolism. nsL-TP is synthesized as a precursor (pre-nsL-TP) with a leader sequence of 20 amino acid residues. It appears that the peroxisomes play an important role in the conversion of pre-nsL-TP into the mature form. In fact, nsL-TP appears to be mainly present in peroxisomes as shown by immunogold labeling of rat liver, adrenals and testes using the anti-nsL-TP antibody. However, interpretation of the data is complicated by the fact that the antibody raised against nsL-TP also reacts with a protein with a M(r) of 58,000. From cDNA analysis it became apparent that the cross-reactive 58-kDa protein contains the complete sequence of pre-nsL-TP at its C-terminus. However, pre-nsL-TP and the 58-kDa protein are synthesized from different mRNAs. Interestingly, the N-terminal part of the 58-kDa protein was found to have significant sequence similarity with 3-oxoacyl-CoA thiolase. Both pre-nsL-TP and the 58-kDa protein contain the C-terminal peroxisomal targeting tripeptide Ala-Lys-Leu. However, as shown by subcellular fractionation studies the 58-kDa protein is exclusively localized in the peroxisomes whilst nsL-TP is not only detected in the peroxisomes but also in other subcellular fractions. Moreover, a membrane-bound form of nsL-TP was detected. This membrane-bound form is present at the cytosolic side of the membranes. The physiological function of nsL-TP is still unclear; some recent developments are discussed briefly in the last part of this review.

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.