Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: beta-elimination (or desulfuration), which results on the loss of an H(2)S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH(-) anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electric field. Alkylation appears to substantially reduce both beta-elimination and the subsequent amido bond lysis.

Despite the importance of the Vav family proteins for B cell receptor (BCR) signaling, their activation mechanisms remain poorly understood. We demonstrate here that adaptor molecules Grb2 and BLNK, in addition to Vav, are required for efficient Rac1 activation in response to BCR stimulation. Loss of either Grb2 or BLNK results in decreased translocation of Vav3 to membrane rafts. By expression of Vav3 as a raft-targeted construct, the defective Rac1 activation in Grb2- or BLNK-deficient B cells is restored. Hence, our findings suggest that Grb2 and BLNK cooperate to localize Vav into membrane rafts, thereby contributing to optimal activation of Vav in B cells.

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

It is well known that microbial pathogens are able to subvert the host immune system in order to increase microbial replication and propagation. Recent research indicates that another arm of the immune response, that of the chemokine system, is also subject to this sabotage, and is undermined by a range of microbial pathogens, including viruses, bacteria, and parasites. Currently, it is known that the chemokine system is being challenged by a number of mechanisms, and still more are likely to be discovered with further research. Here we first review the general mechanisms by which microbial pathogens bypass mammalian chemokine defences. Broadly, these can be grouped as viral chemokine interacting proteins, microbial manipulation of host chemokine and chemokine receptor expression, microbial blockade of host chemokine receptor signalling, and the largely hypothetical mechanisms of microbial enhancement of host anti-chemokine networks (including digestion, antagonism, and neutralisation of host chemokines and chemokine receptors). We then discuss the potential results of these interactions in terms of outcome of infection.

The agonist-specific coupling properties of the three cloned human alpha(1)-adrenoceptor subtypes have been compared, when expressed at similar levels in Chinese hamster ovary (CHO) cell lines, using noradrenaline and the (+/-)- meta- and (+/-)- para- structural isomers of octopamine as agonists. The alpha(1A)- and the alpha(1B)-adrenoceptor subtypes coupled to both the release of arachidonic acid and to the accumulation of inositol phosphates, whereas the alpha(1D)-adrenoceptor subtype only coupled effectively to the accumulation of inositol phosphates. The rank order of potencies of the three agonists tested was the same for all the three receptor subtypes when coupled to either signalling pathway: noradrenaline > meta-octopamine > para-octopamine. Meta-octopamine was a partial agonist of the alpha(1A)-adrenoceptor subtype when coupled to arachidonic acid release, whereas para-octopamine was a full agonist of this pathway. In contrast, meta-octopamine was a full agonist at the alpha(1B)-adrenoceptor subtype when coupled to arachidonic acid release, whereas para-octopamine was a partial agonist of this pathway. Neither meta-octopamine, nor para-octopamine acted as full agonists when coupling any of the three alpha(1)-adrenoceptor subtypes to the accumulation of inositol phosphates. Para-octopamine was only a weak partial agonist of this pathway for all three receptor subtypes. The results show that the modulation of arachidonic acid release and inositol 1,4,5-trisphosphate production occurs in both a subtype- and agonist-specific manner for the alpha(1A)-, alpha(1B)- and alpha(1D)-adrenoceptor subtypes. In addition, the alpha(1A)-adrenoceptor exhibits agonist-specific coupling (agonist trafficking) to the different second messenger pathways.

Phosphoinositide 3-kinases (PI3Ks) regulate numerous biological processes, including cell growth, differentiation, survival, proliferation, migration and metabolism. In the immune system, impaired PI3K signalling leads to immunodeficiency, whereas unrestrained PI3K signalling contributes to autoimmunity and leukaemia. New insights into the role of PI3Ks in lymphocyte biology have been derived from gene-targeting studies, which have identified the PI3K subunits that are involved in B-cell and T-cell signalling. In particular, the catalytic subunit p110delta seems to be adapted to transmit antigen-receptor signalling in B and T cells. Additional recent work has provided new insights into the molecular interactions that lead to PI3K activation and the signalling pathways that are regulated by PI3K.

Autoimmune polyendocrinopathy syndrome type 1 is a recessive Mendelian disorder resulting from mutations in a novel gene, AIRE, and is characterized by a spectrum of organ-specific autoimmune diseases. It is not known what tolerance mechanisms are defective as a result of AIRE mutation. By tracing the fate of autoreactive CD4+ T cells with high affinity for a pancreatic antigen in transgenic mice with an Aire mutation, we show here that Aire deficiency causes almost complete failure to delete the organ-specific cells in the thymus. These results indicate that autoimmune polyendocrinopathy syndrome 1 is caused by failure of a specialized mechanism for deleting forbidden T cell clones, establishing a central role for this tolerance mechanism.

Dopamine has been implicated in the control of sexual behaviour, but its role seems quite complex and controversial. The aim of the present experiments was to investigate the effects of dopamine (DA) acting on D2 receptors in the mediobasal hypothalamus (MBH) on sexual behaviour in female sheep. To achieve this, the D2 agonist, quinpirole, was administered bilaterally via microdialysis probes into the MBH of ovariectomized ewes either before or after oestradiol (E2) administration. Quinpirole (100 ng/ml) infused for 6 h just before E2 hastened the onset of oestrus behaviour and the luteinizing hormone surge, whereas the same treatment given 6-12 h or 18-21 h after E2 decreased the intensity of sexual receptivity without affecting LH or prolactin secretion. We then tested the hypothesis that E2 stimulates the onset of oestrus partly by decreasing DA activation of D2 receptors. In this case the D2 antagonists pimozide or spiperone (100 ng/ml) were infused into the MBH via microdialysis probes for 11 h in the absence of E2 administration. A significant number of ewes showed induction of receptivity with both antagonists, although its intensity was significantly lower than that induced by E2. These treatments generally did not significantly alter extracellular concentrations of monoamines or aminoacids although quinpirole modulated the ability of sexual interactions to increase noradrenaline release. These experiments show that DA acts via D2 receptors in the MBH to control female sexual behaviour in a biphasic manner: the onset of sexual motivation and receptivity requiring an initial increase in activation followed by a decrease. This dual action could explain some of the controversies concerning DA action on sexual behaviour.

The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca(2+) concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca(2+)-binding subunit of troponin, troponin C (TnC), with a single Ca(2+)-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca(2+)-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca(2+)-binding sites. Ca(2+) binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca(2+)-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca(2+) binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.

A novel receptor, named 1C7 or NKp30 and involved in natural cytotoxicity, was recently identified. This receptor is encoded by the 1C7 gene, which is located within the class III region of the human MHC, HLA. It is a member of the immunoglobulin gene superfamily (IgSF) and, remarkably, is expressed at the mRNA level as six different splice variants in human. Recent investigations have indicated that the 1c7 gene of the mouse is silenced by in-frame stop codons. In this study, the molecular characterization of the rat 1c7 gene is described. cDNA derived from this gene encode a protein of 192 amino acid residues predicted to contain a single IgV-set domain in the extracellular region and a positively charged residue in the transmembrane region. Expression of the gene was detected in freshly isolated rat Natural Killer (NK) and T splenocytes. Transfection of rat 1C7 into the NK cell line RNK-16 induced cytolytic activity against glioma as well as lymphoma tumor cells. In addition, binding of a r1C7-Fc fusion protein by a panel of target cells correlated with susceptibility to killing by RNK-16-1C7 effector cells. These results indicate that the r1C7 molecule could function as an NK activating receptor as previously reported for the human NKp30 receptor molecule.

RhoG, a member of the Rho family of GTPases, has been implicated as a regulator of the actin cytoskeleton. In this study, we show a novel function for the small GTPase RhoG on the regulation of the interferon-gamma promoter and nuclear factor of activated T cells (NFAT) gene transcription in lymphocytes. Optimal function of RhoG for the expression of these genes requires a calcium signal, normally provided by the antigen receptor. In addition, RhoG potentiation of NFAT requires the indirect activity of Rac and Cdc42; however, pathways distinct from those activated by Rac and Cdc42 mediate RhoG activation of NFAT-dependent transcription. Using effector domain mutants of RhoG we found that its ability to potentiate NFAT-dependent transcription correlates with its capacity to increase actin polymerization, supporting the suggestion that NFAT-dependent transcription is an actin-dependent process. RhoG also promotes T-cell spreading on fibronectin, a property that is independent of its ability to enhance NFAT-dependent transcription. Hence, these results implicate RhoG in leukocyte trafficking and the control of gene expression induced in response to antigen encounter.

The cloned Drosophila octopamine/tyramine receptor can be coupled to second messenger pathways in an agonist-specific fashion by the endogenously occurring biogenic amines, octopamine and tyramine, when expressed in Chinese hamster ovary cells. We have mutated to alanine a range of receptor amino acids that could potentially form hydrogen bonds with the beta-hydroxyl group of octopamine based on homologies with alpha- and beta-adrenergic receptor subtypes. After stable expression of the mutant receptors in CHO cells we have compared the ability of octopamine and tyramine to displace [(3)H]yohimbine binding to membrane fractions from the mutant cell lines with their ability to modulate adenylyl cyclase activity in intact cells. The results suggest that none of the mutated amino acids residues, at least in isolation, are likely to be involved in interactions with the beta-hydroxyl group of the octopamine side chain. It is possible that amino acids not mutated in the present study are somehow involved in this interaction. Alternatively, it is also possible that the beta-hydroxyl group of the octopamine side chain is capable of interacting with more than one of the amino acids mutated in the present study.

Antigenic peptides are loaded onto class I MHC molecules in the endoplasmic reticulum (ER) by a complex consisting of the MHC class I heavy chain, beta(2)-microglobulin, calreticulin, tapasin, Erp57 (ER60) and the transporter associated with antigen processing (TAP). While most mammalian species transport these peptides into the ER via a single allele of TAP, rats have evolved different TAPs, TAP-A and TAP-B, that are present in different inbred strains. Each TAP delivers a different spectrum of peptides and is associated genetically with distinct subsets of MHC class Ia alleles, but the molecular basis for the conservation (or co-evolution) of the two transporter alleles is unknown. We have determined the crystal structures of a representative of each MHC subset, viz RT1-A(a) and RT1-A1(c), in association with high-affinity nonamer peptides. The structures reveal how the chemical properties of the two different rat MHC F-pockets match those of the corresponding C termini of the peptides, corroborating biochemical data on the rates of peptide-MHC complex assembly. An unusual sequence in RT1-A1(c) leads to a major deviation from the highly conserved beta(3)/alpha(1) loop (residues 40-59) conformation in mouse and human MHC class I structures. This loop change contributes to profound changes in the shape of the A-pocket in the peptide-binding groove and may explain the function of RT1-A1(c) as an inhibitory natural killer cell ligand.

Although monosodium urate monohydrate (MSU) crystals have been recognized since the 18th century as the etiologic agent of gout, it is still unknown why certain hyperuricemic individuals remain asymptomatic, and how an acute attack of gout spontaneously resolves. We hypothesized that mononuclear phagocytes hold the key to these questions, and that the state of monocyte/macrophage differentiation is critical.

Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins.

Mice lacking the p110delta catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110delta(-/-) B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110delta function is required for BCR-mediated calcium flux, activation of phosphlipaseCgamma2, and Bruton's tyrosine kinase. Thus, p110delta plays a critical role in B cell homeostasis and function.

Sheep form an olfactory recognition memory for their lambs within 2 h of parturition and will subsequently reject the approaches of any strange lamb and protest vocally. In this study we report that following olfactory memory formation, ewes exposed to either their own or a strange lamb show c-fos mRNA expression in the medial frontal cortex, although levels of expression in the pyramidal output cell layer V were significantly higher in ewes that rejected strange lambs. Reversibly inactivating this region by the retrodialysis of the anaesthetic tetracaine before birth reduced aggressive motor responses towards lambs but not protest vocalisations. Similar treatment during the critical period for olfactory memory formation and lamb recognition (0-4 h post-partum) had no effect on ewes maternal behaviour towards their own lambs. It did, however, prevent the normal selective expression of aggressive rejection, and reduced protest vocalisation behaviours directed towards strange lambs. These rejection behaviours did appear 1 h after the termination of tetracaine infusions despite the ewes not being given the opportunity to interact with their own lambs during this time. Therefore, tetracaine blockade of the medial frontal cortex prevents animals from responding with motor aggression, but not vocal aggression, to odour cues from strange lambs, but has no effect on the formation of an olfactory recognition memory for their own lambs. Both pre- and post-partum aggressive rejection of strange lambs was associated with increased concentrations of dopamine, serotonin, glutamate and GABA. When these behaviours were inhibited by the tetracaine infusions, extracellular concentrations of these neurotransmitters were all increased by the anaesthetic but did not change in response to lambs. These findings suggest that a functional medial frontal cortex is not required for the formation of an olfactory recognition memory or for mediating pro-active maternal behaviours. It is however required for the mediation of motor but not vocal aspects of aggressive rejection responses directed towards aversive odour cues from strange lambs.

Metalloproteinases (MMP) produced by both cancer and normal stromal fibroblast cells play a critical role in the metastatic spread of tumours, however little is known of the regulation of their release. In this report we demonstrate that breast cancer cells in culture release apparently full length soluble EMMPRIN that promotes the release of pro-MMP2 from fibroblasts. The generation of MMP2 is mediated by activation of phospholipase A(2) and 5-lipoxygenase. These results suggest that the production of soluble EMMPRIN, phospholipase A(2) and 5-lipoxygenase activities are sites for potential therapeutic intervention.

In recent years, substantial progress has been made towards the identification of intracellular signalling molecules that couple multi-subunit immune-recognition receptors (MIRRs) to their various effector functions. Among these, the VAV proteins have been observed to have a crucial role in regulating some of the earliest events in receptor signalling. VAV proteins function, in part, as guanine-nucleotide exchange factors (GEFs) for the RHO/RAC family of GTPases. This review focuses on the role of VAV proteins in the regulation of lymphocyte development and function, and emphasizes the regulatory roles that these proteins have through both GEF-dependent and -independent mechanisms.

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270 kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200 kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Le(a). After the release of oligosaccharides by reductive beta-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex(0-->2)NeuAc(1-->)(x)Hex(x)HexNAc(x)(-ol), x=1-6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Le(a) epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.