IgG-mediated immune suppression in mice is epitope specific except during high epitope density conditions.

Specific IgG antibodies, passively administered together with erythrocytes, suppress antibody responses against the erythrocytes. Although used to prevent alloimmunization in Rhesus (Rh)D-negative women carrying RhD-positive fetuses, the mechanism behind is not understood. In mice, IgG suppresses efficiently in the absence of Fcγ-receptors and complement, suggesting an Fc-independent mechanism. In line with this, suppression is frequently restricted to the epitopes to which IgG binds. However, suppression of responses against epitopes not recognized by IgG has also been observed thus arguing against Fc-independence. Here, we explored the possibility that non-epitope specific suppression can be explained by steric hindrance when the suppressive IgG binds to an epitope present at high density. Mice were transfused with IgG anti-4-hydroxy-3-nitrophenylacetyl (NP) together with NP-conjugated sheep red blood cells (SRBC) with high, intermediate, or low NP-density. Antibody titers and the number of single antibody-forming cells were determined. As a rule, IgG suppressed NP- but not SRBC-specific responses (epitope specific suppression). However, there was one exception: suppression of both IgM anti-SRBC and IgM anti-NP responses occurred when high density SRBC-NP was administered (non-epitope specific suppression). These findings answer a longstanding question in antibody feedback regulation and are compatible with the hypothesis that epitope masking explains IgG-mediated immune suppression.

Indole-3-Acetic Acid Produced by Burkholderia heleia Acts as a Phenylacetic Acid Antagonist to Disrupt Tropolone Biosynthesis in Burkholderia plantarii.

Burkholderia heleia PAK1-2 is a potent biocontrol agent isolated from rice rhizosphere, as it prevents bacterial rice seedling blight disease caused by Burkholderia plantarii. Here, we isolated a non-antibacterial metabolite from the culture fluid of B. heleia PAK1-2 that was able to suppress B. plantarii virulence and subsequently identified as indole-3-acetic acid (IAA). IAA suppressed the production of tropolone in B. plantarii in a dose-dependent manner without any antibacterial and quorum quenching activity, suggesting that IAA inhibited steps of tropolone biosynthesis. Consistent with this, supplementing cultures of B. plantarii with either L-[ring-(2)H5]phenylalanine or [ring-(2)H2~5]phenylacetic acid revealed that phenylacetic acid (PAA), which is the dominant metabolite during the early growth stage, is a direct precursor of tropolone. Exposure of B. plantarii to IAA suppressed production of both PAA and tropolone. These data particularly showed that IAA produced by B. heleia PAK1-2 disrupts tropolone production during bioconversion of PAA to tropolone via the ring-rearrangement on the phenyl group of the precursor to attenuate the virulence of B. plantarii. B. heleia PAK1-2 is thus likely a microbial community coordinating bacterium in rhizosphere ecosystems, which never eliminates phytopathogens but only represses production of phytotoxins or bacteriocidal substances.

[The role of thiol antioxidants in restoring mitochondrial functions, modified by microbial metabolites].

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acethylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. The partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acethylcysteine, the full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K3). It was found that the protective effect of thiol antioxidants became prooxidant one, if the medium contained free iron and compounds with a quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.

Utilization of citrate and lactate through a lactate dehydrogenase and ATP-regulated pathway in boar spermatozoa.

Incubation of boar spermatozoa in Krebs-Ringer-Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO(2) to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH.

Inhibitory effects of green tea polyphenols on the production of a virulence factor of the periodontal-disease-causing anaerobic bacterium Porphyromonas gingivalis.

The effect of polyphenolic compounds isolated from green tea (Camellia sinensis) on the production of toxic end metabolites of Porphyromonas gingivalis was investigated. Green tea polyphenols completely inhibited the production of n-butyric acid and propionic acid at a concentration of 1.0-2.0 mg/mL in general anaerobic medium (GAM). (-)-Epigallocatechin gallate (EGCg), which is a major component of tea polyphenols also inhibited the production of phenylacetic acid at 0.5 mg/mL in GAM broth. In the experiment using resting cells of P. gingivalis, phenylacetic acid was produced from l-phenylalanine and phenylpyruvic acid, but this reaction was also inhibited by EGCg, (-)-epicatechin gallate, and (-)-gallocatechin gallate. However, (+)-catechin, (+)-gallocatechin, (-)-epicatechin, and (-)-epigallocatechin did not inhibit those reactions. These results indicate that the inhibitory effect on the production of toxic end metabolites of P. gingivalis can be attributed to the presence of the galloyl moiety, which is ester-linked with the 3-OH of the catechin moiety in the polyphenolic compounds. This study shows that continuous application of tea polyphenols on a daily basis can be considered as a useful and practical method for the prevention of periodontal diseases.

Evidence for partial agonist properties of daltroban (BM 13,505) at TP receptors in the anaesthetized open-chest rat.

We sought to determine whether the intrinsic pulmonary hypertensive activity of the purported thromboxane A2/prostanoid (TP) receptor antagonist, daltroban, was mediated by TP receptors, using the high efficacy TP receptor agonist, U-46619, and the silent TP receptor antagonist, SQ 29,548. In pentobarbitone-anesthetized, open-chest rats (n = 4-10 per group), non-cumulative injections of U-46619, dose-dependently increased mean pulmonary arterial pressure (MPAP) with an ED50 (geometric mean with 95% confidence limits in parentheses) of 1.4 (1.1-2.3) microg/kg i.v.. Daltroban increased MPAP in a bell-shaped manner, with an apparent ED50 [29 (21-35) microg/kg i.v.] being 21 fold less potent than that of U-46619. The maximal pulmonary hypertensive responses evoked by daltroban represented about half those induced by U-46619 (25.4+/-1.0 vs. 12.7+/-2 mmHg; P < 0.05 between groups). The TP receptor antagonist SQ 29,548 fully antagonized increases in MPAP evoked by equihypertensive doses of U-46619 (1.25 microg/kg) or daltroban (80 microg/kg). Further experiments were carried out to determine whether daltroban antagonized the pulmonary hypertensive responses evoked by the high efficacy agonist, U-46619, or by itself as receptor theory would predict for a partial agonist. Daltroban (10-2500 microg/kg) antagonized, although not fully, U-46619 (20 microg/kg)-evoked pulmonary hypertensive responses, since prominent intrinsic pulmonary hypertensive effects of daltroban were observed in the same range of doses. Furthermore, in contrast to U-46619 (1.25 microg/kg), daltroban (80 microg/kg) failed to evoke a second pulmonary hypertensive response following a previous injection, as would be expected for a partial agonist. Collectively, the results strongly suggest that daltroban behaves as a partial agonist at TP receptors in the pulmonary vascular bed of the rat in vivo.

The alpha-2 adrenergic agonist guanfacine improves memory in aged monkeys without sedative or hypotensive side effects: evidence for alpha-2 receptor subtypes.

The present study attempted to identify an alpha-2 agonist that could improve working memory in aged nonhuman primates without the marked hypotensive and sedative side effects produced by clonidine. Toward this end, the hypotensive, sedative, and memory-altering properties of the alpha-2 adrenergic agonists, B-HT920 and guanfacine, were compared with clonidine's effects in 9 aged rhesus monkeys. Memory capacity was assessed by a variable delay, spatial delayed response paradigm that requires the animal to remember information over short temporal intervals and to update this information on every trial. B-HT920 was found to produce a dose-response profile qualitatively similar to, but weaker than, clonidine: low doses impaired memory and began to lower blood pressure and produce sedation, while high doses improved memory. In contrast, guanfacine produced a dose-response profile opposite to that seen with clonidine: low doses improved memory without inducing hypotension or sedation, while the memory-impairing, hypotensive, and sedating properties of the drug were observed at higher doses. The potency of the 3 agonists to lower blood pressure was clonidine = B-HT920 greater than guanfacine; sedation was affected in the order clonidine greater than B-HT920 greater than guanfacine; for memory impairment, as measured by performance on the delayed response task, the rank order potency was clonidine greater than B-HT920 greater than guanfacine, while for memory improvement it was guanfacine greater than clonidine greater than B-HT920. These differences in rank order potency are consistent with the recent proposal of alpha-2 receptor subtypes, a rauwolscine-sensitive site (Rs) that binds clonidine greater than B-HT920 greater than guanfacine and a rauwolscine-insensitive site (Ri) that binds guanfacine greater than clonidine greater than B-HT920 (Boyajian and Leslie, 1987). The data suggest that the hypotensive, sedating, and memory-impairing effects of alpha-2 agonists may be due to actions at one subtype of receptor (Rs), while the memory-enhancing effects of these drugs may result from actions at another alpha-2 receptor subtype, the Ri site. The ability of low doses of guanfacine to improve memory without inducing hypotension or sedation indicates that this agonist may be an excellent candidate for treating memory disorders in man.

[Effects of guanfacine on the levels of cyclic nucleotides in anesthetized rat brain regions].

Effects of an antihypertensive drug, guanfacine, on brain regional cyclic AMP and cyclic GMP levels were studied in anesthetized rats. Cyclic nucleotides were analyzed in seven brain regions. Guanfacine decreased blood pressure and heart rate 20 min after administration. Yohimbine inhibited these hemodynamic effects of guanfacine. Guanfacine reduced cyclic AMP levels in the hypothalamus. The reducing effect of guanfacine on cyclic AMP was antagonized by yohimbine in the hypothalamus. Guanfacine lowered cyclic GMP in the cerebellum, medulla oblongata and hypothalamus. Yohimbine inhibited the effect of guanfacine on cyclic GMP in the cerebellum, medulla oblongata and hypothalamus. Prazosin showed no effect on guanfacine induced change of cyclic nucleotides in any brain region. From these results, it is concluded that guanfacine decreased cyclic AMP and cyclic GMP in the hypothalamus. In addition, it is suggested that alpha-2 adrenoceptors mainly modulate these changes of cyclic nucleotides.

Antagonism of hypotensive and bradycardic effects of centrally administered guanfacine by metiamide in anesthetized rabbits.

In anesthetized rabbits, intracerebroventricular (ICV) administration of the alpha-adrenoceptor agonist, guanfacine, like clonidine, elicited dose-related falls in arterial blood pressure and heart rate. ICV pretreatment with the histamine H2-receptor antagonist, metiamide, significantly reduced the hypotensive and bradycardic responses caused by ICV injection of either guanfacine or clonidine. The present results seem to indicate that the antagonistic effect of metiamide against either guanfacine or clonidine is probably due to its alpha-adrenoceptor blocking action rather than its histamine H2-receptor blocking action, suggesting that these interactions might be of central origin.

Effects of yohimbine stereoisomers on contractions of rat aortic strips produced by agonists with different selectivity for alpha 1- and alpha 2-adrenoceptors.

Three stereoisomers of yohimbine (corynanthine, rauwolscine and yohimbine) have been used to characterize alpha-adrenoceptors in rat aortic strips. pA2 values for each antagonist were calculated using 3 different agonists ((-)-noradrenaline, (-)-phenylephrine and guanfacine) which possess varying affinities for alpha 1 and alpha 2 receptors. Mean pA2 values were not significantly different irrespective of the agonist used and the order of the potency was corynanthine greater than yohimbine greater than rauwolscine. The results are consistent with the presence of alpha 1-adrenoceptors in rat aorta.

[Effects of guanfacine on pre- and postsynaptic alpha-adrenoceptors studied in comparison with those of clonidine].

Intravenous injections of guanfacine (1 microgram-3 mg/kg) elevated dose-dependently blood pressure in adrenalectomized, pitched rats. The vasopressor activity of guanfacine was weaker than those of clonidine, an alpha 2-agonist and 1-phenylephrine, an alpha 1-agonist. However, the maximal response produced by guanfacine was the same as that produced by clonidine, while it was much smaller than that produced by 1-phenylephrine. The dose-response curve to guanfacine for increase in blood pressure was shifted in a parallel fashion to the right by 1 mg/kg of yohimbine, an alpha 2-antagonist and by 1 mg/kg of phentolamine, a nonselective alpha 1- and alpha 2-antagonist. However, 0.1 mg/kg of prazosin, a selective alpha 1-antagonist, produced an inhibition of the blood pressure rise induced by higher doses of guanfacine, but not that induced by lower doses. Guanfacine (1 microgram-1 mg/kg) inhibited dose-dependently the tachycardia induced by electrical stimulation of spinal nerves at C7-Th1 in adrenalectomized, pithed rats. The maximal inhibition by guanfacine of heart rate increase induced by electrical stimulation was about 60%. These inhibitory effects were antagonized by yohimbine and phentolamine, but not by prazosin. These results indicate that guanfacine is an alpha 2-agonist with approximately equal potency towards the pre- and postsynaptic alpha 2-adrenoceptors, just as clonidine is, but is weaker as an alpha 2-agonist than clonidine.

[Central hypotensive effects of guanfacine in anaesthetised rabbits (author's transl)].

The effects of intracerebroventricular (i.c.v.) injection of guanfacine, a new antihypertensive agent, on blood pressure and heart rate were investigated and compared with those of clonidine in anaesthetized rabbits. The i.c.v. injection of guanfacine or clonidine induced a decrease in blood pressure and heart rate in doses which were ineffective by an intravenous route. The depressive effect of guanfacine was less than that of clonidine, but the duration of this action was obviously longer than that of clonidine. A fall in heart rate caused by guanfacine was always less conspicuous than that by clonidine in equipotent hypertensive doses. Decrease in blood pressure and heart rate caused by the two drugs were inhibited by i.c.v. pretreatment with phentolamine and 6-hydroxydopamine, respectively. These findings suggest that presynaptic and/or postsynaptic alpha-adrenoceptors in the brain may play an important role in the hypotension and bradycardia produced by guanfacine as well as clonidine. Furthermore, the uptake mechanism at the presynaptic sites of central noradrenergic neurons may be involved in the mode of hypotensive and bradycardic actions of these two drugs since these effects were abolished by pretreatment with desipramine, an inhibitor of noradrenaline uptake into the presynaptic nerve terminals.