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).

Structural studies have been carried out on the putative O-specific polysaccharide of the reference strain (C.D.C. 3607-60) for Serratia marcescens O13. Circumstantial evidence that the O13 antigen is a microcapsular, acidic polymer, rather than an integral part of the lipopolysaccharide, has been obtained. Degradative and spectroscopic studies established that the polymer is based on the repeating unit shown, in which the glucuronic acid residue of the linear pentasaccharide carries the lateral 2-acetamido-2-deoxy-beta-D-glucopyranosyl substituent in only about half of the units. The same polymer, again with non-stoichiometric substitution, is also produced by strain IP 421 (O13:H7). The latter strain also produces a neutral polymer which appears to constitute the side chain of the lipopolysaccharide. This polymer, which has a disaccharide repeating-unit of 2-substituted beta-D-ribofuranosyl and 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residues, has been isolated previously from the lipopolysaccharides of the reference strains for S. marcescens serogroups O12 and O14, and appears to be the antigen known to be shared by these strains. (Formula: see text).

In rheumatoid arthritis there is a chronic immune and inflammatory reaction which can lead to the destruction of the diseased joint. Cytokine gene expression was studied in synovial cells using cDNA probes specific for human interleukin 1 (IL-1), -alpha and IL-1 beta, tumour necrosis factor (TNF), -alpha and TNF beta (lymphotoxin); protein molecules which induce cartilage degradation and bone resorption. In all cases studied, IL-1 mRNA was present in freshly isolated synovial cells from fluid or membrane. Compared to levels of IL-1 mRNA found in optimally activated normal blood mononuclear cells, the levels of IL-1 alpha mRNA were high in seven of the nine patients studied, whereas IL-1 beta mRNA, the dominant form in blood, was relatively lower. TNF alpha and TNF beta mRNA were also detected. Rheumatoid synovial cells, cultured without any stimulus, continued to express high levels of IL-1 alpha mRNA for up to 5 days, compared to the 24 h response of activated blood cells; IL-1 beta mRNA in culture was also prolonged. Cultures of rheumatoid joint cells produced IL-1 bioactivity, with roughly equal amounts of IL-1 alpha and beta, as assessed using neutralizing antibodies. TNF bioactivity was also detected which may be of importance as TNF induces the production of IL-1. The finding of these mediators produced in large amounts in active rheumatoid synovial cells suggests that mutually stimulatory cell interactions, mediated by these molecules, may be important in the chronic inflammation and tissue destruction in rheumatoid arthritis.

The acute monocytic leukemia cell line THP-1 secretes predominantly IL-1 beta after treatment with bacterial lipopolysaccharide and tumour promoting phorbol ester (PMA). IL-1 alpha is also secreted, but represents less than 10% of the total IL-1 activity. This differential is reflected at the level of mRNA as IL-1 beta mRNA is more abundant than IL-1 alpha mRNA. Studies of transcription in isolated nuclei however indicate that each gene is transcribed at a similar rate, suggesting that post-transcriptional mechanisms regulate the relative abundance of IL-1 alpha and IL-1 beta mRNA. Measurement of RNA half life after addition of alpha-amanitin (an inhibitor of RNA polymerase II) indicate that IL-1 alpha mRNA is not as stable as IL-1 beta mRNA suggesting one mechanism for the different relative levels of RNA.

The expression of the mRNA encoding tumour necrosis factor, lymphotoxin and interleukin-6 by peripheral blood mononuclear cells was analysed. Unstimulated cells contained no detectable mRNA for these cytokines, however each mRNA was transiently expressed after stimulation with either the combination of phytohaemagglutinin and phorbol ester or the single stimulus of lipopolysaccharide. The dual stimulus yielded the stronger signal. The cytokine mRNA's had short half lives, but were stabilised following protein synthesis inhibition. Cyclosporin A completely blocked induction of lymphotoxin and partially inhibited induction of TNF and IL-6 mRNA. The features of regulation described in this paper suggest these genes belong within the "early" set of genes expressed following immune cell activation.

T cell clones derived from patients with autoimmune diseases were found to be capable of producing tumor necrosis factor (TNF). This was demonstrated by stimulating the clones, in the absence of accessory cells, with antibodies against the Ti/T3 complex and with recombinant interleukin 2 (IL2). Analysis of RNA extracted from these clones showed that TNF mRNA was more abundant than lymphotoxin (LT) mRNA. We also found that TNF protein in the supernatants of these clones was generally more abundant than LT as assessed by using the murine L929 cell assay. TNF production was not limited to T cells from autoimmune individuals, since the T cell tumor HUT78 and T cells purified from the peripheral blood of healthy individuals also made TNF. Unlike the T cell clones, HUT78 produced greater amounts of LT mRNA than TNF mRNA. Induction of TNF mRNA in T cells from healthy individuals displayed a two-signal requirement (phorbol myristate 13-acetate and phytohemagglutinin or OKT3 and phorbol myristate 13-acetate), similar to that described for the induction of the T cell lymphokines IL 2 and interferon-gamma (IFN-gamma). Additionally we found that IL2 alone was sufficient to induce TNF in these cells when they had been precultured with phytohemagglutinin for 7 days to express IL 2 receptors. The cloned T cells we have characterized also produce IFN-gamma which was detected in the supernatants of the clones using a radioimmunoassay. The evidence suggests that T cells can produce TNF and have the potential to deliver by themselves the dual and synergistic signals of TNF/LT and IFN-gamma to target cells, a process which may be of importance in the pathogenesis of human autoimmunity.

An acidic, partially acetylated galactomannan has been isolated from the lipopolysaccharide of the reference strain (C.D.C. 864-57) for Serratia marcescens serogroup O4. From the results of methylation analysis, Smith degradations, and n.m.r. spectroscopic studies of the O-deacetylated polymer, it was concluded that the repeating unit has the structure shown, in which the acetal-linked pyruvic acid has the R configuration. The polymer is believed to confer O specificity on the organism, but not to constitute the side chain of the lipopolysaccharide. (formula; see text).

The production and growth regulatory activity of transforming growth factor beta were studied in human thyroid tissue. As estimated by its mRNA expression in fresh tissue samples, transforming growth factor beta was produced in normal and in diseased thyroid glands. Transforming growth factor beta mRNA was mainly produced by thyroid follicular cells and in lesser quantities by thyroid infiltrating mononuclear cells. The concentrations of transforming growth factor beta mRNA were lower in iodine-deficient nontoxic goiter than in Graves' disease and normal thyroid tissue. Transforming growth factor beta protein secretion by cultured thyroid follicular cells was also low in nontoxic goiter, but could be increased by addition of sodium iodide (10 microM) to the culture medium. Recombinant transforming growth factor beta did not affect basal tritiated thymidine incorporation in cultured thyroid follicular cells, but inhibited, at a concentration of 10 ng/ml, the growth stimulatory influence of insulin-like growth factor I, epidermal growth factor, transforming growth factor alpha, TSH, and partly that of normal human serum on cultured thyroid follicular cells. This inhibition was greater in Graves' disease than in nontoxic goiter. These results suggest that transforming growth factor beta may act as an autocrine growth inhibitor on thyroid follicular cells. Decreased transforming growth factor beta production and decreased responsiveness to transforming growth factor beta may be cofactors in the pathogenesis of iodine-deficient nontoxic goiter.

Recombinant tumour necrosis factor (TNF) promotes survival and induces proliferation in the tumour cells from two malignancies of B lymphocytes--hairy-cell leukaemia and B-chronic lymphocytic leukaemia. Culture with TNF also induces TNF mRNA and protein, so the cytokine may act as an autocrine tumour growth factor. These growth promoting effects are antagonised by alpha but not by gamma interferon.