Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T2 family and the mechanism of S-RNase action.

The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

The T cell repertoire is shaped by positive and negative selection of thymocytes that express low levels of T cell receptor (TCR) and both CD4 and CD8. TCR-mediated signals that determine these selection processes are only partly understood. Vav, a GDP-GTP exchange factor for Rho-family proteins, is tyrosine phosphorylated following TCR stimulation, suggesting that it may transduce TCR signals. We now demonstrate that mice lacking Vav are viable and display a profound defect in the positive selection of both class I- and class II-restricted T cells. In contrast, Vav is not essential for negative selection, though in its absence negative selection is much less effective. Vav may influence the efficiency of TCR-induced selection events by regulating the intracellular calcium flux of thymocytes.

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.

Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking. Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors.

Sheep learn to recognize the odours of their lambs within two hours of giving birth, and this learning involves synaptic changes within the olfactory bulb. Specifically, mitral cells become increasingly responsive to the learned odour, which stimulates release of both glutamate and GABA (gamma-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. Here we investigate the possible role of NO in olfactory learning. We find that the neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odour. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP concentrations. We propose that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.

Activation of the high affinity IgE receptor (Fc epsilon RI) of mast cells, a member of the antigen receptor family, leads to the release of allergic mediators, a critical event in the onset of immediate hypersensitivity. Stimulation of Fc epsilon RI results in the rapid association and activation of the Syk tyrosine kinase. Using Syk-deficient mast cells we show that they fail to degranulate, synthesize leukotrienes and secrete cytokines when stimulated through Fc epsilon RI, conclusively demonstrating an essential role for Syk in Fc epsilon RI signalling. Furthermore, our data strongly supports a model of Fc epsilon RI engagement leading to the sequential activation of the tyrosine kinases Lyn and then Syk. A similar mechanism is likely to apply to signal transduction through all members of the antigen receptor family.

Many flowering plants have developed a self-incompatibility mechanism, which is controlled by a single polyallelic locus (the S-locus), to prevent inbreeding. The products of the S-locus in the styles of solanaceous plants are an allelic series of glycoproteins with RNase activity [McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F. & Clarke, A. E. (1989) Nature 342, 955-957]. These S-RNases show some amino-acid-sequence similarity with two fungal RNases (T2 and Rh), including the presence of two active-site His residues, which suggests a common three-dimensional structure. Disulphide bonding is important in the maintenance of the three-dimensional structure of the fungal RNases [Kurihara, H., Mitsui, Y., Ohgi, K., Irie, M., Mizuno, H. & Nakamura, T. (1992) FEBS Lett. 306, 189-192] and the S-RNases [Tsai, D. S., Lee, H.-S., Post, L. C., Kreiling, K. M. & Kao, T.-H. (1992) Sex. Plant Reprod. 5, 256-263]. We have used the S2-allele RNase of Nicotiana alata, which has nine Cys residues, to establish the pattern of disulphide bonding. The disulphide bonds Cys16-Cys21, Cys45-Cys94, Cys153-Cys182 and Cys165-Cys176 are consistent with the S2-RNase having a similar three-dimensional structure to RNase Rh. A free Cys residue (Cys95) adjacent to Cys45-Cys94 promotes a rapid specific disulphide migration when the protein is exposed to denaturing conditions.

Self-incompatibility is a mechanism developed by many plants to prevent inbreeding. The products of the self-incompatibility (S)-locus in the styles of solanaceous plants are a series of glycoproteins with ribonuclease activity. In this study, we report on the N-glycans from the stylar self-incompatibility S3- and S6-ribonucleases of Nicotiana alata, which were enzymically released and fractionated by high-pH anion-exchange HPLC. A total of 14 N-glycans were identified and characterized by a combination of electrospray-ionization mass-spectrometry, 1H-NMR spectros-copy, chemical degradation, and methylation analyses. This patterns of N-glycosylation is much more complex than that previously found on the N.alata S1- and S2-RNases, each of which contained only four N-glycans.

Phacomatoses are hereditary disease caused by germinative matrix disorder. Apart from known proliferative and tumor processes on peripheral nerves and their roots which make up a familiar picture of this disease to all neurologist, other tissue and organ malformations of octo and mesodermal origin may occur. This is a case report of a girl with neurofibromatosis type I after Riccardi with occlusive hydrocephalus complication. We pointed to a great number of neurofibromatosis complications, their prompt detection and treatment.

Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimer's brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimer's brain.

Gametophytic self-incompatibility, a mechanism that prevents inbreeding in some families of flowering plants, is mediated by the products of a single genetic locus, the S-locus. The products of the S-gene in the female sexual tissues of Nicotiana alata are an allelic series of glycoproteins with RNase activity. In this study, we report on the microheterogeneity of N-linked glycosylation at the four potential N-glycosylation sites of the S2-glycoprotein. The S-glycoproteins from N.alata contain from one to five potential N-glycosylation sites based on the consensus sequence Asn-Xaa-Ser/Thr. The S2-glycoprotein contains four potential N-glycosylation sites at Asn27, Asn37, Asn38 and Asn 150, designated sites I, II, IV and V, respectively. Site III is absent from the S2-glycoprotein. Analysis of glycopeptides generated from the S2-glycoprotein by trypsin and chymotrypsin digestions revealed the types of glycans and the degree of microheterogeneity present at each site. Sites I (Asn27) and IV (Asn138) display microheterogeneity, site II (Asn37) contains only a single type of N-glycan, and site V (Asn150) is not glycosylated. The microheterogeneity observed at site I on the S2-glycoprotein is the same as that observed at the only site, site I, on the S1-glycoprotein (Woodward et al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylation consensus sequence at site I is conserved in all S-glycoproteins from other species of self-incompatible solanaceous plants, glycosylation at this site may be important to their function. No other post-translational modifications (e.g. O-glycosylation, phosphorylation) were detected on the S2-glycoprotein.

Acetylcholine (ACh) and noradrenaline (NA) release in the olfactory bulb (OB) of ewes was monitored using microdialysis. Both ACh and NA release increased at parturition in multiparous but not in primiparous ewes. However, vaginocervical stimulation performed 6 hr postpartum induced an increase of ACh and NA release in both primiparous and multiparous ewes, indicating that a maturation process had occurred. Finally, pharmacological challenges to the ACh and NA inputs revealed differential responsiveness between nulliparous and multiparous nongestant ewes. These results suggest that the first parturition induces changes in neural circuitry involving ACh and NA inputs to the OB.

Two soluble tumour-necrosis-factor-alpha(TNF)-binding proteins are derived from the extracellular domains of the p55 and p75 TNF receptors. They are considered to play a pivotal regulatory role in TNF-mediated inflammatory processes, including diseases such as rheumatoid arthritis, by competing with the cell surface receptors for TNF and lymphotoxin (LT, tumour-necrosis factor beta). The extracellular domains of the two receptors each contain four similar cysteine-rich repeats of about 40 amino acids, in common with several other cell surface proteins including the p75 nerve-growth-factor receptor and the CD40 and Fas antigens. The aim of this study was to characterize the involvement of the four cysteine-rich repeats of the human p55 TNF receptor in TNF and LT binding by both membrane-bound and soluble forms of the receptor. Individual repeats were systematically deleted by PCR mutagenesis and the variants transiently expressed in COS cells. Immunoprecipitated receptor variants exhibited the expected sizes on SDS/PAGE gels, and bound a panel of conformation-dependent anti-(TNF receptor) antibodies. Binding of TNF by the four soluble derivatives was compared with binding by the wild-type soluble receptor using a TNF-affinity column and a BIAcore Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and 125I-TNF binding to U937 cells. delta 4, which lacks the fourth cysteine-rich repeat, bound TNF comparably with the full-length soluble receptor. TNF-binding affinity was unaltered by deletion of the fourth membrane-proximal cysteine-rich repeat, as determined by Scatchard analysis of the transmembrane derivatives. We conclude that the fourth cysteine-rich repeat is not required for TNF binding. In contrast, both the soluble and the transmembrane derivatives lacking any one of the first, second or third repeats failed to bind TNF. Although we cannot entirely exclude the possibility that this may be due to indirect conformational change, rather than the removal of essential epitopes, our results suggest that the first three repeats are each required for TNF binding by both the soluble and the cell-surface receptor.

Oestrus behaviour and the luteinizing hormone (LH) surge are induced in ovariectomized ewes by oestradiol (E2) after a period of progesterone priming with a low level of E2 (Pge2) and we have previously shown that these effects are primarily mediated through their action on the mediobasal hypothalamus (MBH). The aim of the present study was to assess what neurochemical changes in the MBH are induced by these steroids that might mediate their action on oestrus behaviour and LH release. Eight ovariectomized ewes were implanted with microdialysis probes in the MBH and submitted to three artificial cycles, so that they exhibited either both oestrus behaviour and an LH surge (Pge2 + E2), an LH surge alone (E2 alone) or neither oestrus behaviour nor an LH surge (Pge2 alone). Microdialysis and blood samples were collected every 30 min from 4 h before the end of Pge2 treatment until the end of oestrus. Behavioural tests with a ram were made to assess receptivity. Dopamine (DA) levels were found to increase significantly at the termination of Pge2 treatment after both Pge2 + E2 and Pge2 treatments. When the ewes received E2 after a Pge2 + low estradiol priming (Pge2 + E2), DA levels decreased 16 h later (4 h after E2) whereas they did not change after E2 or Pge2 alone. By contrast, serotonin (5HT) levels did not change significantly during the first 24 h but then increased when ewes received E2 alone and decreased when they were treated with Pge2 + E2. gamma-Aminobutyric acid (GABA) concentrations decreased significantly at the beginning of the sampling period after all treatments but this decrease lasted longer after Pge2 + E2 and was most pronounced at the beginning of receptivity. No significant long term effects of these steroid treatments were found on noradrenaline (NA), aspartate, glutamate, glycine and taurine levels. However, E2 administration was followed during the next few hours by a significant increase in glycine and to a smaller extent in glutamate and GABA. More importantly, when ewes were treated with Pge2 + E2, NA levels increased significantly following the behavioural interactions with a ram when the ewes were sexually receptive. In contrast to this, DA levels only increased during interactions with the ram when the ewes were not receptive. 5HT levels increased after tests where the ewe was either receptive or unreceptive to the male. GABA, aspartate and glycine levels increased in the sample just preceding the test and then decreased during it.(ABSTRACT TRUNCATED AT 400 WORDS)

Crosslinking of B- or T-cell antigen receptors results in the rapid tyrosine phosphorylation of a number of proteins, including Vav, a protein expressed in cells of the haematopoietic system. Vav contains an array of structural motifs that include Src-homology domains SH2/SH3 and regions of homology to the guanine-nucleotide-exchange protein Dbl, pleckstrin and protein kinase C (refs 5-9). Using the RAG-complementation approach, we have analysed in vivo differentiation and in vitro responses of B- and T-lineage cells generated by injection of embryonic stem cells homozygous for a null mutation in the vav gene into blastocysts of RAG-1- or RAG-2-deficient mice. Here we report that antigen receptor-mediated proliferative responses of B and T cells in vitro are severely reduced in the absence of Vav. We also suggest a direct link between the low proliferative response of Vav-deficient B and T cells and the reduced number of these cells in peripheral lymphoid organs of chimaeric mice.

The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans (S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).

Tridacnin, a glycoprotein lectin, was isolated from the symbiotic marine clam Hippopus hippopus and the structure of its major N-glycan chains determined. Tridacnin contains only N-linked glycans which were quantitatively cleaved by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Following purification by anion-exchange HPLC, the structures of the oligosaccharides were established using a combination of electrospray ionisation mass spectrometry, 1H-NMR spectroscopy and linkage analysis. The N-glycans are primarily of the oligomannose type but, in addition, some contain a novel 6-O-Me group on the terminal mannose residue of the chain. The N-glycan chains had the following structures. [formula: see text]

The tyrosine kinase Syk (relative molecular mass 72,000), which is widely expressed in haematopoietic cells, becomes associated with and activated by engagement of the B-cell antigen receptor. Furthermore, it has been implicated in signalling through the receptors for interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF) and Fc, the T cell receptor, as well as through receptors for several platelet agonists. A homologous kinase, ZAP-70, is crucial in signalling through the T-cell receptor and in T-cell development. Using homologous recombination in embryonic stem cells, we created mice null for the syk gene which showed petechiae in utero and died shortly after birth. Irradiated mice reconstituted with Syk-deficient fetal liver showed a block in B-cell development at the pro-B to pre-B cell transition, consistent with a key role for Syk in pre-B-cell receptor signalling. Despite the production of small numbers of immature B cells, Syk-deficient radiation chimaeras failed to accumulate mature B cells, indicating a possible role for this protein in the production or maintenance of mature B cells. In addition, whereas the development of alpha beta T cells proceeded normally, Syk-deficient mice showed impaired development of thymocytes using the V gamma 3 variable region gene (V gamma 3+ thymocytes). Finally, we show that Syk is not required for signalling through the IL-2 and G-CSF receptors.

A polymeric fraction containing the putative O-antigen has been isolated from the lipopolysaccharide of the reference strain (CDC 4534-60) for serogroup O9 of Serratia marcescens. The major component of the fraction was a polymer with a disaccharide repeating-unit of L-rhamnose (Rha) and 2-acetamido-2-deoxy-D-galactose (GalNAc) with the following structure:----3)D-GalpNAc(beta 1----3)L-Rhap(alpha 1----. Evidence for the presence in the fraction of a similar, minor polymer containing 4-substituted rhamnose residues was provided by the NMR spectra, methylation analysis, and Smith degradation.

Partially acetylated glucorhamnans have been isolated from the lipopolysaccharides of three strains of Serratia marcescens. The polymer from the reference strain (C.D.C. 864-57) for serogroup O4 has the disaccharide repeating-unit shown below, in which acetylation at position 2 of the rhamnosyl residue is approximately 90% complete. Similar glucorhamnans from the reference strain (C.D.C. 843-57) for serogroup O7 and from a pigmented strain (NM) of serogroup O14 differ only in the configuration of the L-rhamnopyranosyl residue (beta) and the extent of O-acetylation (O7, almost stoichiometric; NM, 80-90%). Glucorhamnans of the second type have been isolated previously from the lipopolysaccharides of other strains of S. marcescens, including the reference strain for serogroup O6 and another pigmented O14 strain (N.C.T.C. 1377). In all cases, the lipopolysaccharide extracts also contained acidic glycans, but the glucorhamnans are believed to constitute the integral side-chains. (Formula: see text).