Evidence for arginine residues in the immunoglobulin-binding sites of human Clq.

The immune complex binding activity of human Clq was lost following treatment of the protein with the arginine-selective reagents cyclohexane 1,2-dione and phenylglyoxal. Both inactivations followed pseudo-first-order kinetics. The affinity of Clq for immune complexes was reduced 7-fold following cyclohexane-1,2-dione treatment, and could be substantially restored by treatment of the modified protein with hydroxylamine. Heat-aggregated IgG protected Clq against inactivation by both reagents. Incorporation of 25 molecules of [7-14C]phenylglyoxal per Clq molecule completely inactivated the protein. These data are consistent with the presence of arginyl residues in the immunoglobulin recognition sites of human Clq.

Binding of complement component C1q by spectrin.

125I-labelled human C1q was found to bind to human spectrin. Scatchard plots for the binding process were non-linear, indicating the possible presence of multiple classes of binding sites for C1q on spectrin. The binding was ionic-strength-dependent; the extent of binding decreased with increasing ionic strength. Chemical modification of arginine and histidine residues on C1q as well as pretreatment of C1q at pH 4.45 or at 56 degrees C reduced its spectrin binding activity. The amount of 125I-labelled C1q bound to immune complexes was reduced by the presence of spectrin. Spectrin was also able to deplete the complement haemolytic activity of human serum in a dose-dependent manner.

Binding of complement component C1q by rat adipocyte membranes.

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.

C1q binding to mitochondria: a possible artefact?

Mitochondrial preparations, obtained from human tonsils and from rat spleen, liver, heart, and kidney tissues, bound [125I]C1q with affinities of 10(7)-10(8)M-1. The binding of C1q was not affected by treatment of the mitochondrial preparations with pronase, trypsin, or phospholipase D, but it was lowered 5-6-fold following treatment of the mitochondria with DNase and RNase. Binding of C1q to mitochondrial preparations was also greatly diminished by limited chemical modification of C1q with cyclohexane-1,2-dione. It is suggested that the reported binding of C1q to mitochondria may have arisen from the protein binding to DNA and/or RNA contaminating the mitochondrial preparations.

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine.

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.

Direct immunization with synthetic peptidyl-polyamide resin. Comparison with antibody production from free peptide and conjugates with carrier proteins.

An 11-amino acid residue peptidyl-linkage agent-polyamide resin complex was synthesized by the fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid-phase system. Mice were immunized with the free peptide, peptidyl-resin and peptide coupled to the carrier proteins ovalbumin (Ova) and keyhole limpet haemocyanin (KLH). The immunogenicity of these materials was assessed by measurement of the capacity of the various antisera to bind the peptide in an enzyme-linked immunosorbent assay (ELISA). The peptidyl-resin exhibited enhanced immunogenicity compared to the free peptide. It is suggested that the time needed for screening for immunogenicity of large numbers of synthetic peptides thus be greatly shortened by using peptidyl-resins for immunization. This method eliminates laborious cleavage of peptide from resin, purification, coupling to carrier and the difficulties of handling peptides of low solubility.

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon.

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (K(D) 0.47+/-0.05 nM), of high capacity (Bmax 2.1+/-0.1 fmol mg(-1) wet weight tissue) and reversible (kinetically derived K(D) 0.36+/-0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide gamma (NPgamma) > or = NKA > or = [Lys5, MeLeu9,Nle10]NKA (4-10) (NK2 agonist) >> substance P (SP) > neurokinin B (NKB) > or = [Pro9]SP (NK1 agonist) >> senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968 > or = MEN11420 > GR94800 > or = MEN10627 > MEN10376 > or = R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 microM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear.

Structure-activity relationships of neurokinin A (4-10) at the human tachykinin NK(2) receptor: the role of natural residues and their chirality.

A structure-activity study of neurokinin A (NKA) (4-10) was performed to investigate the importance of residue and chirality for affinity and efficacy at the NK(2) receptor in human colon circular muscle. Two series of NKA(4-10) analogues were produced with either L-alanine or the D-enantiomer substituted. Their activities were determined in vitro by means of radioligand binding and isolated smooth muscle pharmacology. NKA was more potent than NKA(4-10) at the human, unlike the rabbit, NK(2) receptor. The contractile response of NKA(4-10) was unaffected by N-terminal acetylation. L-Ala substitution of Asp(4), Val(7), Leu(9), and Met(10) caused an 8- to 80-fold decrease, and substitution of Phe(6) caused a 5000-fold decrease in binding affinity (P < 0.01). Positions Ser(5) and Gly(8) were not significantly affected. In functional studies, a similar pattern was observed. The replacement of residues with their respective D-enantiomer drastically reduced binding affinity and functional potency, particularly at positions 6 and 7 (P < 0.05). NKA(4-10) analogues L-Ala(6), L-Ala(8), D-Phe(6), D-Val(7), and D-Met(10) were partial agonists. An excellent correlation was observed between binding and functional data (r = 0.95). A retro-inverso analogue of NKA(4-10) was inactive. In conclusion, the side chains of Asp(4), Phe(6), Val(7), Leu(9), and Met(10) are structurally important features of NKA(4-10) for agonist activity, and changes in amino acid chirality are detrimental to binding affinity and functional activity. Overall, our data are broadly similar to those of previous studies in the rat. However, at the human NK(2) receptor, unlike the rat, [Ala(8)]NKA(4-10) was an antagonist.

Bufokinin: immunoreactivity, receptor localization and actions in toad intestine and mesenteric circulation.

In this study, we have mapped the immunoreactivity and the binding sites for bufokinin, a tachykinin peptide from the toad intestine. Dense bufokinin-immunoreactive fibers were present at the myenteric plexus, but no cell bodies were stained, suggesting an extrinsic origin. Bufokinin nerve fibers were also associated with submucosal blood vessels and mesenteric arteries. Autoradiographic binding sites for [(125)I]Bolton-Hunter-bufokinin were densely localized over the intestinal circular and longitudinal muscle, submucosal blood vessels and the endothelium of mesenteric arteries. Mesenteric veins had minimal immunoreactivity and binding sites. In the anesthetized toad, topical application of bufokinin onto the mesentery caused a 2.7-fold increase in arterial blood flow, observed using intravital microscopy. This study supports a role for bufokinin as an endogenous spasmogen and hemodynamic regulator in the toad intestine.

Stabilization of lethal and hemolytic activities of box jellyfish (Chironex fleckeri) venom.

The stability of both the lethal and hemolytic activities of box jellyfish (Chironex fleckeri) tentacle extract was assessed after various extraction procedures. Both activities were higher when no buffers or water were used during the initial extraction. Also, when the extract was first filtered through a Sep-pak C18 cartridge, the residual lethal titre, after incubation for 24 hr at room temperature, was increased 16-fold and hemolysis was increased 2.6-fold. Evidence for proteolytic activity in the extract was also obtained and monitored by size exclusion HPLC.

Protection of monkeys against the lethal effects of male funnel-web spider (Atrax robustus) venom by immunization with a toxoid.

A stable toxoid was prepared from robustoxin (the lethal polypeptide neurotoxin in the venom of the male funnel-web spider, Atrax robustus) by polymerization with glutaraldehyde. This material was non-toxic in new-born mice. Administration of the toxoid to three Macaca fascicularis monkeys (50-80 micrograms/kg s.c. at 14-day intervals for 8-12 weeks) produced no toxic effects; anti-robustoxin antibodies were detected in serum samples by immunodiffusion tests within 13-27 days. In vivo evidence of successful protection with the toxoid was obtained by challenging the monkeys with male A. robustus venom (50 micrograms/kg i.v.) under anaesthesia with pentobarbitone (one monkey), or with ketamine, halothane and nitrous oxide, 1-26 weeks after the last injection of the toxoid. Only minor respiratory, cardiovascular and skeletal motor disturbances were produced, and all monkeys recovered fully and uneventfully. Challenge with the same dose of venom in non-immunized or robustoxin N-terminal decapeptide ovalbumin conjugate-treated monkeys resulted in typical lethal neurotoxic effects, culminating in severe hypotension or death from circulatory and respiratory failure within 280 min.

Benzyl-benzoate foam: effects on mite allergens in mattress, serum and nasal secretory IgE to Dermatophagoides pteronyssinus, and bronchial hyperreactivity in children with allergic asthma.

Home mattresses of 24 asthmatic children with house dust mite allergy were sprayed with either benzyl-benzoate foam or placebo in a double blind fashion, 10 days before the children left the residential house for asthmatic children Istituto Pio XII (located in the Italian Alps in an environment free of mites) and went back to their own home for the Christmas and Easter holidays. A further group of 8 children, whose mattresses received no treatment, was kept as an absolute control. Two days after spraying, benzyl-benzoate or placebo were vacuumed from the mattresses. Acarex test was performed immediately before spraying and at the end of each holiday period of 20 and 10 days, respectively. Bronchial hyperreactivity as well as serum and nasal secretory specific IgE for Dermatophagoides pteronyssinus were assessed in all children immediately before leaving and within 48 hr after returning to the residential house. The results of the study show that sprayed benzyl-benzoate foam was no more effective than placebo in reducing the level of house dust mite recovered from patients' mattresses, or in reducing bronchial hyperreactivity and IgE concentration in serum and nasal secretions.

Evidence for a high molecular weight pre-robustoxin molecule in the venom of the male Sydney funnel-web spider (Atrax robustus).

Robustoxin is the lethal polypeptide toxin in Atrax robustus venom. A monoclonal antibody was produced using synthetic, unfolded robustoxin conjugated to keyhole limpet haemocyanin as the immunogen. This monoclonal antibody did not protect newborn mice against challenge with the crude venom of the male Sydney funnel-web spider, but did slightly prolong their survival time. Western blotted crude venom of the male Sydney funnel-web spider showed two monoclonal antibody binding bands. One band at low M(r) corresponded to robustoxin (M(r) 4854), while the other higher M(r) band (approximately 37,000) may be due to a pre-robustoxin molecule.

Structure-activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A.

A series of analogues of neurokinin A(4-10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser5 were assessed at rat fundus NK2 receptors. In membrane binding studies with [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), all compounds except [Asp5]NKA(4-10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4-10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe5]NKA(4-10) is in fact neurokinin B(4-10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn5]NKA(4-10) was equally as weak in contractile studies as the acidic [Asp5]NKA(4-10). However, [Glu5]NKA(4-10) and [Gln5]NKA(4-10) were no different from NKA(4-10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.

Carbonic anhydrase activity in the blood of adult sheep, fetal sheep and lambs.

The activity of carbonic anhydrase (E.C.4.2.1.1) (CA) has been measured in the blood of adult and fetal sheep and lambs. The mean activity in adult sheep was 0.89 enzyme units (EU) per 100 micrograms of Hb. The activity in fetal sheep aged 90 days was just below 20% of this and in fetuses near full term was just under 40% of the mean adult level. The regression line gave an increase of CA activity (per 100 micrograms Hb) of 0.004 EU/day. The appearance of CA in fetal blood normally occurred before any detectable production of adult Hb. One aberrant fetus showed early development of the adult pattern in the red cells, having adult type Hb and adult levels of CA during the period of 116-128 days of fetal age. In the period after birth the CA level in the blood rose rapidly, reaching the adult level 30 days after birth. During this period activity per 100 micrograms HB increased by 0.014 EU/day, significantly faster than during fetal life.

Bufokinin: actions and distribution in the toad cardiovascular system.

1. Bufokinin is a substance P-like neuropeptide and potent spasmogen isolated from the intestine of the cane toad Bufo marinus. In the present study, we investigated the effects of bufokinin on systemic blood pressure and heart rate in the anaesthetized toad and the distribution of bufokinin-like immunoreactivity in the toad vasculature. 2. Intravenous bufokinin caused a dose-dependent fall in systemic blood pressure (maximum fall 20 mmHg) with an ED50 of 2.9 pmol. At higher doses, the effect was prolonged and blood pressure did not return to baseline within 60 min. There was no significant change in heart rate associated with hypotension. 3. Bufokinin-like immunoreactivity was mapped in whole mounts of toad blood vessels and organs using a mouse polyclonal antibody BK3 (at 1:5000) and the avidin-biotin method. Bufokinin-immunoreactive fibres were associated with most blood vessels examined: a moderately dense perivascular network of varicose fibres was present around renal arteries, with sparser immunoreactive fibres in the ventral aorta, sciatic artery, anterior abdominal vein and hepatic portal vein. 4. Bufokinin-immunoreactive fibres, mainly following blood vessels, were seen in whole mounts of the urinary/bladder and tongue, but not in the air sac. In the heart ventricle, varicose fibres were found in the valve cusps, intracardiac ganglia, epicardium and myocardium close to the endocardium, but not in the rest of the myocardium. 5. The vasodepressor action of bufokinin and the presence of bufokinin-like immunoreactivity in varicose fibres in various vessels suggest a role for bufokinin in haemodynamic regulation and/or sensory nerve function in the toad. The lack of any reflex tachycardia in response to the falls in blood pressure was of note.

Binding and functional potency of neurokinin A analogues in the rat fundus: A structure-activity study.

Structure-activity relationships of neurokinin A (NKA) and the two analogues NKA(4-10) and [Nle10]NKA(4-10) were investigated at the rat fundus NK-2 receptor, using selected amino acid substitutions. Both radioligand binding with [125I][Lys5,Tyr(I2)7,MeLeu9, Nle10] NKA(4-10) and functional studies were performed and correlated. In membrane binding experiments loss of His1 and Lys2, or replacement of Lys2 with Ala did not substantially alter binding affinity of NKA. NKA(4-10) free acid was unable to compete with the radioligand. [Nle10]NKA(4-10) binding affinity to rat fundus membrane preparations was decreased when substituting Asp4 with Gln or Asn, or Val7 with either Tyr or Ile. Replacement of Ser5 with the negatively charged Glu also decreased the binding affinity, but substitution with the positively charged Lys substantially increased the affinity of [Nle10] NKA(4-10) for the NK-2 receptor. Lengthening NKA(4-10) or [Nle10]NKA(4-10) with Ala11 or Nle11, respectively, decreased the binding affinity of the peptide. In both binding and functional studies, replacement of any of the residues of NKA(4-10), except for Ser5, with alanine decreased the affinity of the peptide for the NK-2 receptor. Ala substitutions at positions 4, 6, and very obviously at 8, 9 and 10 of NKA(4-10) yielded peptides unable to achieve a maximum contractile response, although they did not demonstrate antagonist activity. These data confirm the importance of the NKA carboxyl terminus, and the requirement for Phe6, Val7, Gly8, Leu9 and Met10 integrity for interaction with the NK-2 receptor. They also suggest that Ser5 is a good site to target modifications leading to the design of new potential drugs.

Oxygen carriage and carbonic anhydrase activity in the blood of a marsupial, the Tammar wallaby (Macropus eugenii), during early development.

Blood O2 transport and Hb type have been studied in pouch young and adult of a marsupial, the Tammar Wallaby. The O2-Hb equilibrium curves (at 35.5 degrees C and PCO2 = 34 Torr) had a high P50 in the first few days of life, up to 49 Torr. This fell to 32 Torr by 2 weeks of age. Also (delta log P50/delta PCO2) was low but it rose to adult levels by 2 weeks of age. The curves in these early pouch young showed a change in Hill coefficient (nH) at between 32 and 62% saturation, nH rising to more than 4.0 at higher O2 saturations. This indicates interaction between more than 4 Hb subunits. Model calculations showed that such curves could be produced by a mixture of 2 Hb components; one with a low P50 and low nH, and one with a high P50 and high nH. In this model the nH values were different from the nH values of either component. The temperature effect on P50 in early pouch young was higher than in adult Tammars and similar to that reported for adult eutherians. In the first 4 days all red cells were nucleated and four Hb types were present. Carbonic anhydrase activity in the blood before birth was about 30% of the adult levels. These levels remained until 2 days after birth, when a rapid rise in activity began, near-adult levels being reached at 5 days despite the animals being still very immature.

Comparison of nedocromil sodium and sodium cromoglycate administered by pressurized aerosol, with and without a spacer device in exercise-induced asthma in children.

To compare the effectiveness of nedocromil sodium (NS) and sodium cromoglycate (SCG) administered by metered dose inhaler (MDI) in preventing exercise-induced asthma (EIA), 12 asthmatic children with EIA were studied in a randomized, double-blind, cross-over, placebo-controlled study. NS and SCG were given by MDI alone, and by MDI with a 700 ml spacer device (Fisonair, Fisons, UK), in order to assess the benefit of using such a device. Following a baseline exercise challenge, the protective effect of NS, SCG or placebo was evaluated in each subject. The percentage fall in forced expiratory volume in one second, and percentage protection were measured. NS and SCG provided a significant and comparable protection from EIA, and both were better than placebo. No further improvement was observed after drug administration via the spacer. Both NS and SCG are effective in preventing EIA in children, when administered at the recommended clinical dose, and the use of a spacer for administering the drug provides no advantage if the technique of inhalation is good.

Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts.

1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.