Structural relatedness between the 18S rRNA genes and the formyl peptide receptor genes: new insights into the phylogenesis of immune receptors.

In this study the authors examined the sequences of the ribosomal 18S rRNA of Drosophila and man and 16 mRNA sequences coding for different members of the family of the mammalian formyl peptide receptors (FPRs). The positions in the sequences of all >or=7-base oligonucleotide identities occurring in at least one of the 18S rRNAs and one of the FPR mRNAs were recorded. On the basis of the positional data, the Drosophila 18S-FPR and human 18S-FPR distances (in nucleotides) were determined for each identity. Then the actual frequency distribution of the distances (grouped into 200-unit classes) was derived. The theoretical frequency distribution of distances was also calculated under the assumption of non-relatedness between the 18S and FPR sequences. Comparison between the theoretical and the actual distributions showed that at class -500 (range from - 400 to - 600) of the 18S-FPR values the actual frequency was significantly (p < 0.01) higher than the theoretical frequency, in both Drosophila and man, suggesting that the second section of the FPR genes (approximately from nucleotide 400 to the end of sequence) may be structurally related to the first section of the ribosomal 18S genes (approximately nucelotides 1-650). The authors advance the hypothesis that the two families of genes may have used common ancestral raw genetic materials in the building of the extant sequences.

Mutation, selection, and functional repair in formyl peptide receptor genes: a view on the selection processes occurring in this gene subfamily.

Formyl peptides (FPs) released by some bacteria are powerful chemoattractants and activators of granulocytes, monocytes, and macrophages, acting through the members of a subfamily of specific seven-transmembrane G-protein-coupled formyl peptide receptors (FPRs), which are expressed only in mammals. Upon stimulation, granulocytes chemotactically move towards sites of maximal FP concentration, and release different bactericidal lytic enzymes and reactive oxygen species (ROI). In some instances, such as ischemia/reperfusion, the proinflammatory mediators released by the injured tissues and the intestinal bacteria and endotoxins, which may permeate across the damaged mucosal barrier, prime the inflowing granulocytes for an enhanced ROI production, resulting in severe damage to the host tissues. In this investigation 16 representative FPR and FPR-like mRNAs were selected to study the pattern of mutation/conservation of the individual nucleotides (nt) in the coding sequences. Mutations occur in 56.7%, 46.4%, and 87.5 % of cases in the first, second, and third nt, respectively, of the coding triplets. A probabilistic analysis demonstrated a significant nonrandom linkage between mutations in the first and second nt. Furthermore, the triplets that are variously double-mutated in the first two nt code, on average, for more hydrophobic amino acids (AA) in the transmembrane segments and more hydrophilic AA in the external and intracytoplasmic segments, thus preserving the general structure of the receptor. The authors hypothesize that when in one of the first two nt a mutation leading to a nonfunctioning protein product occurred, the mutated gene was eventually eliminated; however, a second mutation occurring in the other previously unmutated nt may have led to a protein product that is compatible with functional activity, although mutated in one (noncritical) AA. Such double mutations effecting a "functional repair" have thus survived and are retained among the extant sequences. Moreover, the combined mutation of all three nt in coding triplets occurs with a significantly higher than random frequency and this finding may be interpreted in a similar way.

The Structure of the 18S rRNA, a molecule that might be evolutionarily related to some receptors of innate immunity.

Comparisons between the sequences of insect and vertebrate 18S rRNAs and the sequences of mammalian formyl peptide and some vertebrate chemokine receptor mRNAs demonstrated non-random structural similarities between these two groups of RNAs. It has been proposed that sections of the more ancient and conserved rRNA genes could have participated in the building of these more recent genes involved in immune responses. Here we analyze the sequence architecture of the 18S rRNA in insects (Drosophila simulans) and vertebrates (man), in terms of similarities between selected segments within the individual molecules. The insect and vertebrate 18S rRNAs are basically similar, but show specific insertions/deletions and base changes. In spite of these differences, in both sequences a significantly higher-than-expected (by random occurrence) number of 7-or-more-base oligonucleotide repeats was observed between segments roughly corresponding to nt 350-1050 and nt 1150-1850, with mutual between-repeats distances comprised in the range 700-900 nt. Based on this result we performed a multialignment of segments 317-1035 of Drosophila, 360-1005 of man, 1096-1864 of Drosophila, and 1066-1736 of man, the first two segments covering the region of first occurrence of the repeats and the last two the region of recurrences. At both ends of these segments the four sequences could be aligned with relatively minor gaps and the number of base identities in all four sequences was significantly higher than expected by random coincidences. These results support the hypothesis that an ancestral gene structure, composed of a chain of about 700 nt, duplicated to form a two-unit tandem repeat which still represents the most substantial part of the 18S rRNA molecule in extant insects and vertebrates.

Modeling of granulocyte cytoskeletal responses following fMLP challenging.

Formyl peptides released from Gram-negative bacteria ligate a group of specific mammalian receptors, expressed mainly on granulocytes, monocytes, and macrophages. Receptor ligation activates different transduction cascades, eventually leading to the release of reactive oxygen species and other bactericidal chemical species, and the activation of the actin cytoskeleton with extension of lamellipodia and migration toward the sites of maximal formyl peptide concentration. In vitro, under conditions of nongradient formyl peptide concentrations, lamellipodia form all around the cell contour (chemokinesis). In granulocytes challenged under these conditions with N-formyl-methionyl-leucyl-phenylalanine, (i) the power spectrum of the contour of activated cells shows a peak at a specific periodicity, indicating that the lamellipodial extension is not completely random but stochastically conforms to a deterministic scheme, and (ii) the morphological response (percent of cells exhibiting chemokinesis) tends to reach a maximum at certain drug concentrations, then declining at higher concentrations. Accordingly, the logarithm of the drug concentration-polarizing effect curve is bell-shaped. Herein we illustrate theoretical models for the simulation of these two components of the chemokinetic responses. We show that the main traits of the general morphology and arrangement of lamellipodia may be simulated by an algorithm that starting from a situation of random distribution of active receptors on the cell membrane, encompasses in the successive calculation cycles both a local autocatalytic enhancement of the actin polymerization and a relative inhibition of the actin polymerization at some distance from the more active polymerization foci. In addition, a drug log concentration-polarizing effect bell-shaped curve may be simulated by assuming that the N-formyl-methionyl-leucyl-phenylalanine, while binding with high affinity to the specific receptor, is also able to bind to another lower affinity receptor that may effect depolarizing actions or, more generally, metabolic blocking effects. Under these conditions, at low drug concentrations the polarizing effect brought about by the ligation of the specific receptor is largely predominant. However, as the drug concentration increases and the specific receptors approach saturation, the inhibitory effects become more and more powerful and the net polarizing effect is reduced.

Formyl peptide receptors on immune and nonimmune cells: analysis of sequence conservation in FPR genes.

Formyl peptides are oligopeptides released by Gram-negative bacteria. So far, specific formyl peptide receptors (FPRs) have been described in mammals only. FPRs are seven-transmembrane G-coupled molecules and make up a relatively homogeneous group, although exhibiting different levels of affinity for the ligands. We examined the patterns of conservation/mutation within the FPR group of genes, as studied in 16 mRNAs from different species. Following alignment of the coding sections, those nucleotides identical in at least 15 sequences were assigned a "conservation index" 2; those with 8-14 identities an index 1; those with less than 8 identities an index zero. The cumulative average conservation index was 1.36. The autocorrelation function and the power spectrum of the whole series of indexes demonstrated a 3-unit periodicity. This periodicity is explained by the fact that the average conservation indexes of the first, second and third nucleotides of the coding triplets were 1.46, 1.55 (both above the mean), and 1.06 (below the mean), respectively, so that correlations at lag 3 tend to be all positive. In mRNAs, regardless of the position in the coding triplets, T is significantly more frequently conserved (average index = 1.60) than A, C, and G (1.21 - 1.38). In the nucleotides with conservation index 1 or zero, we recorded the two more frequently represented bases. In 35% of mRNA nucleotides the two more frequently represented bases were C and T; in 28% of cases the two more frequently represented bases were A and G; other couples occurred with lower frequencies. Both mutations may arise following C methylation with subsequent transformation into T (by deamination), either in the template or the coding DNA strand. Thus, we hypothesized that in FPR mRNAs there is an evolutionary trend of transformation from G to A and from C to T, the latter being the more stable of the bases.

Indomethacin-induced ileitis is associated with tensiometric, vascular and oxidative changes in the experimental rat model.

BACKGROUND: Indomethacin-induced enteritis is a model of inflammatory bowel disease. MATERIALS AND METHODS: To further characterize this model, rats received two injections of indomethacin (7.5 mg kg(-1)) 24 h apart and histological damage of intestinal mucosa, oxidative stress, alterations of intestinal motility and mesenteric vascular bed (MVB) reactivity were investigated after 5 days. RESULTS: The results show that indomethacin caused several histological and functional changes at the ileal level. In particular, response to carbachol as well as the nonadrenergic-noncholinergic inhibitory response to electrical field stimulation (EFS) was lower in the treated than control rats. Moreover, nitric oxide (NO)-component of the inhibitory response was higher in the treated than control rats. Mesenteric vessels preparations from the treated rats showed increased noradrenaline (NA)-induced perfusion pressure, whereas relaxant responses to acetylcholine, although not significantly reduced in the treated rats, had a higher nitrergic component. This finding suggests that vascular dysfunction may contribute to chronic inflammation. Indomethacin injection also determined acute and severe oxidative stress in ileum mucosa. CONCLUSIONS: In conclusion, our study contributes to further characterize the rat model of indomethacin-induced enteritis and suggests that it is suitable for drug screening in rats, as this model can be obtained in a very short period and is simple and reproducible.

Functional alterations of mesenteric vascular bed, vas deferens and intestinal tracts in a rat hindlimb unloading model of microgravity.

1. Prolonged bed rest or exposure to microgravity may cause several alterations in autonomic nervous system response (ANSR). 2. Hindlimb unloading (HU) rats were used as an animal model of simulated microgravity to investigate ANSR changes. The experiments were carried out to investigate the effects of simulated microgravity on the autonomic nervous response of the perfused mesenteric vascular bed (MVB), vas deferens and the colon and duodenum from 2-week HU rats. 3. In MVB preparations of HU rats, the frequency-dependent increases in perfusion pressure with perivascular nerve stimulation (PNS; 8-40 Hz) were inhibited, whereas the noradrenaline (NA) concentration-dependent (1-100 microM) perfusion pressure increases were potentiated. The latter most probably reflected up-regulation of alpha-adrenergic receptor function. Relaxant responses of NA-precontracted MVB to PNS (4-30 Hz) or isoprenaline were not different between control and HU preparations, while vasodilation induced by the endothelial agonist ACh was reduced. 4. Transmural stimulation (2-40 Hz) induced frequency-dependent twitches of the vas deferens which were reduced in vas deferens of HU rats, while the sensitivity to NA-induced contraction was significantly increased. 5. In the gastroenteric system of HU rat, direct contractile responses to carbachol or tachykinin as well as relaxant or contractile responses to nervous stimulation appeared unchanged both in the proximal colon rings and in duodenal longitudinal strips. 6. In conclusion, HU treatment affects peripheral tissues in which the main contractile mediators are the adrenergic ones such as resistance vessels and vas deferens, probably by reducing the release of neuromediator. This study validates NA signalling impairment as a widespread process in microgravity, which may most dramatically result in the clinical phenotype of orthostatic intolerance.

Effects of in vivo treatment with interleukins 1beta and 6 on rat mesenteric vascular bed reactivity.

1. Inflammatory bowel disease (IBD) is a condition that involves proinflammatory cytokines such as interleukins 1beta and 6 (ILs). In this disease, it has been shown that an abnormal microcirculatory system is implicated. 2. Therefore, the effects of in vivo treatment for three days with interleukins 1beta and 6 were investigated on rat isolated mesenteric vascular bed (MVB). 3. A significant concentration-dependent increase in vascular response to noradrenaline (NA) was found, with a significant difference in Emax between control (93.01 +/- 16.78 mmHg) and treated preparations (137.91 +/- 5.20 mmHg). Endothelin-1(ET-1) induced a significantly greater increase of perfusion pressure in treated rats in comparison with control rats at the highest concentration used (0.1 microm). 4. The concentration-dependent decrease of perfusion pressure induced by acetylcholine (ACh) in MVB precontracted with NA was significantly reduced in specimens from treated rats in comparison with control rats, with a significant difference in Emax between control and treated preparations. 5. Perivascular nerve stimulation (PNS) evoked contractions with no difference between treatments. Similarly, no difference in relaxant effect was found after PNS in specimens precontracted with NA, in the presence of guanethidine. 6. These findings indicate that the precocious inflammation acts only at postsynaptic level, facilitating vascular contraction. These data seem to support the hypothesis that vascular dysfunction caused by overproduction of ILs may contribute, among other immunological factors, to vasculitis in IBD that leads to intestinal ischaemia through vasoconstriction.

Interleukins 1 beta and 6 induce functional alteration of rat colonic motility: an in vitro study.

BACKGROUND: In rodents, interleukins administration induces intestinal changes similar to those found in inflammatory bowel disease. We investigated the effects of in vivo subchronic treatment with IL-1 beta and IL-6 on rat colonic mucosa and circular smooth muscle. MATERIALS AND METHOD: We evaluated transmucosal electrical parameters (Ussing chambers) and early changes of in vitro direct contractility induced by carbachol and tachykinins. Alterations in excitatory and inhibitory neurotransmission were studied with electrical field stimulation (EFS). RESULTS: Treatment with interleukins induces inflammation proved by fever, early signs of colonic histological damage and changes in mucosal ion transport. Concentration response-curve to carbachol was significantly lower in treated rats (P<0.02) with significant difference in Emax between control (1.67+/-0.17 g) and treated preparations (1.20+/-0.13 g) (P<0.05). Concentration response-curve to NK2 agonist was significantly lower in the treated rats (P<0.005) with a significant difference in Emax between the control (0.26+/-0.04 g) and treated preparations (0.12+/-0.02 g) (P<0.02). None of the drugs used induces changes in EC50. The contractile reflex response to electrically induced distension was significantly higher in the treated rats and more reduced after administration of atropine. Adding NK2 receptor antagonist resulted in a further reduction being observed in the treated and control rats (P=NS). Relaxation by EFS on cholinergic tone was not different between treatments, although pretreatment with L-NNA resulted in greater relaxation in the treated (-21.7%) than in the control rats (-14.8%). CONCLUSION: Early inflammation induced by a subchronic treatment with ILs causes changes in mucosal ionic transport parameters, a reduction in the direct contractile response, and an alteration in the neurotransmission (by an enhancing cholinergic component) that may affect the physiological pattern of colonic motility and the sensory reflex.

Involvement of kappa-opioid receptors in peripheral response to nerve stimulation in kappa-opioid receptor knockout mice.

1 The present study aimed to evaluate the role of kappa-opioid receptors at two peripheral sites, the vas deferens and the proximal colon, in kappa-opioid receptor knockout mice. We investigated the role of the kappa-opioid receptor in the vas deferens twitch response and in the colonic "off-contraction", a rebound contractile response which follows the inhibitory response to low frequencies stimulation (10, 20, 30 Hz) and which has been suggested to "locally" reproduce the contractile component of the peristaltic reflex. 2 Transmural stimulation of the vas deferens at lower frequencies (10 Hz, 10 V, 1 ms pulse trains lasting 0.5 s) evoked a contractile response that was significantly higher in the preparations from knockout mice because of lack of kappa-opioid receptors than in wild type mice. A selective kappa-opioid receptor agonist, U-50,488H, induced a dose-dependent inhibition of the electrically stimulated contraction in vas deferens. The percentages of reduction of the twitch response were significantly lower in knockout mice than in wild type mice after treatment with U-50,488H. The reduction of twitch response caused by U-50,488H was not reversed by administration of nor-binaltorphimine (nor-BNI) (5 x 10-6 m), a selective kappa-opioid receptor antagonist, in preparations from both knockout mice and wild type mice. U-50,488H has no effect on postsynaptic adrenergic receptors, as its administration did not affect the direct contractile response to noradrenaline. 3 Transmural stimulation (5 Hz, 20 V, 2 ms pulse trains lasting 30 s) induced inhibition of spontaneous activity of colonic strips during the period of stimulation, followed by an "off-contraction" after the cessation of stimulation. The statistical evaluation of the "off-contraction" responses between the two strains showed no significant difference. The off-contraction, measured in specimens from knockout mice, was inhibited concentration-dependently by U-50,488H (P < 0.01) and significantly less than from wild type mice. 4 The effect of U-50,488H was not reversed by administration of nor-BNI (5 x 10-6 m), either in preparations from knockout mice or from wild type mice. 5 Our data may suggest that kappa-opioid receptors are involved in some peripheral responses to the nerve stimulation, as indicated by the effect of U-50,488H, a selective kappa-opioid receptor agonist. However, the involvement of kappa-opioid receptor was also present, although less apparent, in kappa -opioid receptor knockout mice, suggesting either that this drug acts not only on kappa-opioid receptors but also on other receptor sites, such as kappa-like receptors. An alternative interpretation can be related to a sodium channel blocking action of U-50,488H, which could explain the inhibitory effects of twitch response still present but less evident in knockout strain and the lack of effect of the antagonist nor-BNI.

Idiopathic chronic constipation: tachykinins as cotransmitters in colonic contraction.

BACKGROUND: Tachykinins (TKs) have been shown to be involved in the excitatory enteric motor pathway. This study aimed to examine the direct and nerve-mediated effect of specific NK1, NK2 and NK3 receptor agonists and antagonists in colonic preparations from control subjects and patients with idiopathic chronic constipation (ICC). MATERIALS AND METHODS: Cumulative concentrations of Sar9Met(O2)11 substance P (selective NK1 receptor agonist), [Ala5,beta-Ala8]-neurokinin A (4-10) (selective NK2 receptor agonist) and [MePhe7]-neurokinin B (selective NK3 receptor agonist) were tested on colonic circular muscle strips to evaluate the direct drug effects. In addition, in the presence of atropine, the role of TKs in the off-contraction that follows the typical inhibitory response evoked by low frequencies of electrical field stimulation (0.5--10 Hz, 20 V, 1 ms pulse trains lasting 1 min) was investigated. RESULTS: In control preparations, the rank order of potency was: NK2 receptor-selective agonist > NK3 receptor-selective agonist > NK1 receptor-selective agonist. The off-contraction was found to be reduced by about 30--40% in colonic circular muscle from ICC patients with respect to controls. Incubation with the NK1 receptor agonist did not modify the off-contraction measurements in either control or ICC preparations. Conversely, both NK2 and NK3 receptor agonists significantly (P < 0.01) increased the off-contraction in ICC preparations only. This increased response was fully antagonized by MEN-10627, a NK2 and NK3 receptor antagonist depending on the dose. CONCLUSIONS: We may conclude that ICC is hyporesponsive to TKs and that the contractile reflex to distension is greatly reduced in ICC disease, but can be restored by incubation with NK2 and NK3 receptor agonists.

Effects of erythromycin on human colonic circular muscle in idiopathic chronic constipation.

BACKGROUND: Erythromycin has been shown to have profound prokinetic effects on the gastrointestinal tract of humans and animals, probably through its action on endogenous motilin receptors. The purpose of this study was to determine both the direct and indirect effects ('off contraction') of erythromycin and motilin on ex vivo circular muscle strips of the distal colon from patients with or without idiopathic chronic constipation (ICC). MATERIALS AND METHODS: Cumulative concentrations of erythromycin (1-20 microM) and motilin (0.05-1 microM) were tested in both control and ICC preparations in order to evaluate the direct drugs effect. A range doses of both erythromycin (0.5-10 microM) and motilin (0.05-0.5 microM) were tested on their ability to affect the off-contraction that follows the typical inhibitory response evoked by low frequencies of Electrical Field Stimulation (EFS) (1-5 Hz, 20 V, 1 msec pulse trains lasting 1 min). RESULTS: The direct effect of both erythromycin and motilin was a slight increase (less than 10% of the maximal ACh-induced contraction) in the basal tension, with no dose-response relationship. The off-contraction, evoked by EFS, was not affected by drugs pretreament in control preparations. Conversely, in ICC preparations both drugs significantly increased the off-contraction (about 30%). CONCLUSIONS: Erythromycin causes mainly an indirect contractile effect in circular muscle strips from ICC patients. This effect may be related to the activation of inhibitory neuronal motilin receptors. This activation might potentiate NANC relaxation, proportionally increasing the circumferential reflex contraction that follows the EFS-induced relaxation.

Cholinergic stimulation and nonadrenergic, noncholinergic relaxation of human colonic circular muscle in idiopathic chronic constipation.

The aim of our study was to further investigate the pathophysiological mechanism underlying idiopathic chronic constipation (ICC), a disorder of colonic motility. A possible alteration of excitatory and inhibitory neurotransmission and also the role of inhibitory neurotransmitters such as nitric oxide (NO), 5'-adenosine triphosphate (ATP), and vasoactive intestinal peptide (VIP) has been evaluated on preparations of distal colon from patients with or without ICC. The isometric tension was recorded from isolated circular muscle strips of both experimental groups during pharmacological and electrical field stimulation (EFS). The contractile response obtained by acetylcholine (ACh 20 microM), EFS (20 Hz, 20 V, 1 msec, pulse trains lasting 1 min) and substance P (SP 1 microM) was significantly lower in ICC than in control preparations. The effect of inhibitory nonadrenergic, noncholinergic innervation was evaluated using EFS at low frequencies (0.5-8 Hz), after cholinergic and sympathetic blockade with atropine (3 microM) and guanethidine (3 microM). The maximum relaxation value expressed as percentage of inhibition of SP-induced contraction was significantly higher in ICC than in control preparations (87+/-2.4 and 67+/-6.3, respectively; P<0.05). Experiments with substances that antagonize or reduce the effect of putative inhibitory mediators (VIP 6-28, apamin and N(G)-nitro-L-arginine) suggest that an alteration in NO and ATP release is present in ICC preparations. In particular at a higher inhibitory frequency NO-mediated relaxation is enhanced in ICC vs control, supporting the hypothesis that excessive NO production may be involved in pathophysiological mechanism of constipation.

Postoperative changes in serum interleukin-2 concentrations.

OBJECTIVE: To evaluate the postoperative changes in circulating interleukin-2 (IL-2) concentration according to the severity of the surgical injury and other postoperative variables that could influence IL-2 production. DESIGN: Prospective observational study. SETTING: University hospital, Italy. SUBJECTS: 43 patients about to undergo major operations (gastric and colo-rectal resection for cancer), intermediate operations (open cholecystectomy or mastectomy for cancer), and minor operations (hernia repair or breast lump); 24 healthy age and sex matched controls. MAIN OUTCOME MEASURES: Postoperative changes in serum concentrations of IL-2 after different degrees of surgery on the 1st, 3rd and 8th postoperative days correlated with changes in in vivo cellular mediated immunity (skin tests), duration of operation, blood transfusion or postoperative H2-blockers and antiprostaglandins. RESULTS: There were no significant variations in IL-2 serum concentrations postoperatively on ANOVA, and when the data were normalised, there were no significant changes in the median postoperative values after minor and intermediate operations. There was a slight but not significant increase in IL-2 concentrations after major operations. Neither blood transfusion nor duration of operation correlated with postoperative changes in IL-2, while postoperative antiprostaglandins and H2-blockers seemed to provide slight but not significant protection against a reduction in IL-2 concentrations. CONCLUSIONS: Circulating IL-2 does not necessarily correlate with reported in vitro postoperative production of IL-2 and therefore seems to be of little use in monitoring immunosuppression in surgical patients.