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PubMed2005

Mediator and TFIIH govern carboxyl-terminal domain-dependent transcription in yeast extracts.

In Saccharomyces cerevisiae, the RNA polymerase II (RNA Pol II) carboxyl-terminal domain (CTD) is required for viability, and truncation of the CTD results in promoter dependent transcriptional defects. A CTD-less RNA Pol II is unable to support transcription in yeast extracts, but basal transcription reactions reconstituted from highly purified general transcription factors are CTD-independent. To reconcile these two findings, we have taken a biochemical approach using yeast extracts and asked whether there is a factor in the cell that confers CTD-dependence upon transcription. By placing a cleavage site for the tobacco etch virus protease prior to the CTD, we have created a highly specific method for removing the CTD from RNA Pol II in yeast whole cell extracts. Using derivatives of this strain, we have analyzed the role of the Srb8-11 complex, Mediator, and TFIIH, in CTD-dependent basal transcription by either mutation or immunodepletion of their function. We have found that Mediator is a direct intermediary of CTD-dependent basal transcription in extracts and that the requirement for Mediator and the CTD in basal transcription originates from their ability to compensate for a limiting amount of TFIIH activity in extracts.

Endopeptidases↗
PubMed2002

Levels of phospho-Smad2/3 are sensors of the interplay between effects of TGF-beta and retinoic acid on monocytic and granulocytic differentiation of HL-60 cells.

We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor-beta (TGF-beta) receptor serine/threonine kinases, in TGF-beta-dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-beta-dependent differentiation of these cells to monocytes, but not retinoic acid-dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D(3) also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-beta1. Simultaneous treatment of these cells with TGF-beta1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho-Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-beta1 alone. TGF-beta1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-beta-induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor-alpha (RAR-alpha), ATRA is unable to reduce levels of TGF-beta-induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-beta, whereby a putative RAR-alpha-dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-beta, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-beta-dependent differentiation of the cells to monocytic end points.

Cell Differentiation↗
PubMed1980

A micromethod for the analysis of cryoglobulins via laser nephelometry: evaluation and comparison to C1q binding activity in autoimmune diseases in pediatrics.

Quantitative determinations of cryoglobulins (IgG, IgA, IgM, and C3) were performed by a laser nephelometry microtechnique on 250 serum samples from a group of pediatric patients suspected of having immune complex-mediated disorders. Approximately 50% of these samples were cryoglobulin positive. Patients with cryoglobulins were examined as three separate groups: systemic lupus erythematosus, presumptive autoimmune disorders, and chronic bacterial or viral infections. Nearly all of these patients have mixed cryoglobulins. The relation of cryoglobulinemia with serum hypocomplementemia and renal involvement was examined in a group of systemic lupus erythematosus patients. High levels of cryoglobulins were found in patients with hypocomplementemia and anti-DNA antibodies without clinical evidence of nephritis. Highly significant correlations were oberved between C1q binding activity, presence of cryoglobulins, and serum hypocomplementemia in systemic lupus erythematosus patients. Only 60% of the cryoglobulin positive samples had immune complex demonstrable by C1q binding. Cryoglobulin analysis using the laser nephelometry microtechnique permits screening of pediatric patients for the presence of immune complex, permits detection of low levels of cryoglobulins, allows quantitative determination of the specific imunoglobulin classes in the precipitate, and requires only a very small amount of blood suitable for the pediatric population.

Autoimmune Diseases↗
PubMed1991

Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death.

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.

Animals↗
PubMed2006

Optic nerve dynein motor protein distribution changes with intraocular pressure elevation in a rat model of glaucoma.

Acute intraocular pressure (IOP) elevation causes accumulation of retrogradely-transported brain derived neurotrophic factor and its receptor at the optic nerve head (ONH) in rats and monkeys. Obstruction of axonal transport may therefore be involved in glaucoma pathogenesis, but it is unknown if obstruction is specific to certain transported factors or represents a generalized failure of retrograde axonal transport. The dynein motor complex mediates retrograde axonal transport in retinal ganglion cells (RGC). Our hypothesis was that elevated IOP interferes with dynein-mediated axonal transport. We studied the distribution of dynein subunits in the retina and optic nerve after acute and chronic experimental IOP elevation in the rat. IOP was elevated unilaterally in 54 rats. Dynein subunit distribution was compared in treated and control eyes by immunohistochemistry and Western blotting at 1 day (n=12), 3 days (n=4), 1 week (n=15), 2 weeks (n=12) and 4 weeks (n=11). For immunohistochemistry, sections through the ONH were probed with an anti-dynein heavy chain (HC) antibody and graded semi-quantitatively by masked observers. Other freshly enucleated eyes were microdissected for separate Western blot quantification of dynein intermediate complex (IC) in myelinated and unmyelinated optic nerve, ONH and retina. Immunohistochemistry showed accumulation of dynein HC at the ONH in IOP elevation eyes compared to controls (P<0.001, Wilcoxon paired sign-rank test, n=29). ONH dynein IC was elevated by 46.5% in chronic IOP elevation eyes compared to controls by Western blotting (P<0.001, 95% CI=25.9% to 67.8%, n=17). The maximum increase in ONH dynein IC was 78.7% after 1 week (P<0.05, n=5), but significant increases were also detected after 4 h and 4 weeks of IOP elevation (P<0.05, n=4 rats per group). Total retinal dynein IC was increased by 8.7% in chronic IOP elevation eyes compared to controls (P<0.03, 95% CI 1.4% to 16.1%, n=24). In the retina, IOP elevation particularly affected the 72 kD subunit of dynein IC, which was 100.7% higher in chronic IOP elevation eyes compared to controls (P<0.00001, 95% CI 71.0% to 130.4%, n=21). Dynein IC changes in myelinated and unmyelinated optic nerve were not significant (P>0.05). We conclude that dynein accumulates at the ONH with experimental IOP elevation in the rat, supporting the hypothesis that disrupted axonal transport in RGC may be involved in the pathogenesis of glaucoma. The effect of IOP elevation on other motor proteins deserves further investigation in the future.

Animals↗
PubMed1989

Immune mechanisms in the primary and secondary vasculitides.

Vasculitis, or angiitis, affects both the central and peripheral nervous systems and is increasingly recognized as a cause of neurologic dysfunction. The vascular inflammation may be systemic or organ restricted. Recent work has expanded our knowledge on the pathogeneses of vasculitis. Historically, immune complex mediated vasculitis is best defined but studies now delineate cell mediated immune mechanisms including T cell and endothelial cell interactions. Additional factors in the genesis of vascular inflammatory diseases are also being explored. There are numerous underlying etiologies ranging from primary immunologic disturbances to infections, malignancies, and toxins. In order to plan rational therapies, clinicians need to be aware of current work on the pathogeneses of vascular inflammation as well as exploring specific etiologies in their individual patients.

Humans↗
PubMed1996

New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking reagent.

BACKGROUND: To study very large macromolecular complexes, it would be useful to be able to incorporate probe molecules, such as fluorescent tags or photoactivatable crosslinkers, into specific sites on proteins. Current methods for doing this use relatively large amounts of highly purified protein, limiting the general utility of these approaches. The need for covalent posttranslational chemistry also makes it extremely difficult to use modified proteins in studies of native complexes in crude lysates or in living cells. We set out to develop a protein tag that would circumvent these problems. RESULTS: A very simple type of molecular recognition, metal-ligand complexation, can be used to deliver a nickel-based crosslinking reagent to proteins containing a six-histidine (His6) tag. When activated with a peracid, the His6-Ni complex mediates oxidative crosslinking of nearby proteins. The crosslinking reaction does not involve freely diffusible intermediates, and thus only those proteins in close proximity to the His6-tagged polypeptide are crosslinked. CONCLUSIONS: The His6 tag, commonly used as an affinity handle for the purification of recombinant proteins, can also be used as an internal receptor for an oxidative protein-crosslinking reagent. No covalent protein modifications are necessary, since the His6 tag is introduced at the DNA level. The crosslinking reaction is fast, efficient in most cases, and provides products that are easily separated from most other proteins present. This methodology should find widespread use in the study of multiprotein complexes.

Binding Sites↗
PubMed2003

Activity of the Caenorhabditis elegans UNC-86 POU transcription factor modulates olfactory sensitivity.

The activity of transcription factors modulates several neural pathways that mediate complex behaviors. We describe here the role of the POU transcription factor UNC-86 in the olfactory behavior of Caenorhabditis elegans. unc-86-null mutants are defective in response to odor attractants but avoid odor repellents normally. Continuous UNC-86 activity is necessary for maintenance of odortaxis behavior; hyperactivation of UNC-86 by fusion to a VP16 activation domain dramatically enhances sensitivity to odor attractants and promotes odor-attractant adaptation. UNC-86 is not expressed in olfactory sensory neurons but is expressed throughout the life of the animal in the AIZ interneurons of the odorsensory pathway. We suggest that UNC-86 transcriptional activity regulates the expression of genes that mediate synaptic properties of AIZ and that hyperactive UNC-86::VP16 may enhance the expression of synaptic components to affect the capacity to analyze and process sensory information.

Animals↗
PubMed2006

Heterotrimeric G-proteins: a short history.

Some 865 genes in man encode G-protein-coupled receptors (GPCRs). The heterotrimeric guanine nucleotide-binding proteins (G-proteins) function to transduce signals from this vast panoply of receptors to effector systems including ion channels and enzymes that alter the rate of production, release or degradation of intracellular second messengers. However, it was not until the 1970s that the existence of such transducing proteins was even seriously suggested. Combinations of bacterial toxins that mediate their effects via covalent modification of the alpha-subunit of certain G-proteins and mutant cell lines that fail to generate cyclic AMP in response to agonists because they either fail to express or express a malfunctional G-protein allowed their identification and purification. Subsequent to initial cloning efforts, cloning by homology has defined the human G-proteins to derive from 35 genes, 16 encoding alpha-subunits, five beta and 14 gamma. All function as guanine nucleotide exchange on-off switches and are mechanistically similar to other proteins that are enzymic GTPases. Although not readily accepted initially, it is now well established that beta/gamma complexes mediate as least as many functions as the alpha-subunits. The generation of chimeras between different alpha-subunits defined the role of different sections of the primary/secondary sequence and crystal structures and cocrystals with interacting proteins have given detailed understanding of their molecular structure and basis of function. Finally, further modifications of such chimeras have generated a range of G-protein alpha-subunits with greater promiscuity to interact across GPCR classes and initiated the use of such modified G-proteins in drug discovery programmes.

Amino Acid Sequence↗
PubMedDate not supplied

Inhibition of tetrahydrobiopterin synthesis reduces nitric oxide production by isolated glomeruli in immune complex glomerulonephritis.

We studied the effect of modulators of tetrahydrobiopterin (BH4) synthesis on the production of nitrite by glomeruli from rats with immune complex-mediated glomerular inflammation. Glomeruli were isolated 24 h after induction of accelerated nephrotoxic nephritis and cultured at 2,000/ml for 48 h. Lipopolysaccharide dose dependently increased ex vivo nitrite production. 2,4-Diamino-6-hydroxypyrimidine (DAHP), an inhibitor of the de novo synthetic pathway of BH4, inhibited nitrite production by up to 50% and this effect was reversed by sepiapterin, a substrate for the salvage pathway of BH4 synthesis. Sepiapterin alone increased nitrite generation indicating that BH4 is rate limiting. Inhibition of both pathways with DAHP and methotrexate led to 80% inhibition of nitrite. Pharmacological inhibition of BH4 synthesis may be a useful mechanism to reduce nitric oxide synthesis in immune complex inflammation.

Animals↗
PubMedDate not supplied

A simple method for purification of human monocytes and evaluation of IgG Fc-receptor-mediated phagocytosis.

A simple and reproducible, although not completely original method for purification of human monocytes and evaluation of IgG Fc-receptor-mediated phagocytosis has been set up and is described here. The purity of monocyte preparations obtained by the use of this method is quite elevated (over 95%), but the yield, although satisfactory for this purpose, is rather poor. As to the test of phagocytosis, a time-response curve using opsonized sheep erythrocytes showed that 10-min incubation gives the best and most reproducible results. A peroxidase reaction is suggested in order to obtain a better evaluation of erythrocytes within monocytes. The test has been proved suitable for evaluation of Fc-receptor function of monocytes in primary biliary cirrhosis, an immunological liver disease, and might be also useful in immune complexes-mediated conditions.

Adult↗
PubMed1990

PAF and immune complex-induced injury.

Acute immune complex-mediated dermal vasculitis and pulmonary alveolitis have been induced in rats by the intradermal injection or intrapulmonary instillation of rabbit polyclonal antibody to bovine serum albumin (BSA), followed by intravenous injection of antigen. In the dermis, using reconstitution experiments in neutrophil-depleted rats and platelet-activating factor (PAF) receptor antagonists, we have shown that accessibility of PAF receptors on neutrophils is required for the full expression of dermal vascular injury. In the lung, intratracheal instillation of PAF receptor antagonists (with anti-BSA) results in a 54% suppression of pulmonary vascular leakage, suggesting that PAF receptors are necessary for the full expression of injury. These data suggest that in immune complex-induced tissue damage in which neutrophils and their oxygen radicals and proteases play a key role, there is an interplay of PAF and neutrophils required for the full expression of injury. The possible mechanisms involved will be discussed.

Animals↗
PubMed1995

RNA polymerase II C-terminal domain required for enhancer-driven transcription.

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.

Amanitins↗
PubMed1996

Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice.

Despite its widespread distribution on both lymphoid and myeloid cells, the biological role of the low-affinity immunoglobulin-G receptor, Fc gamma RII, is not fully understood. Defects in this receptor or its signalling pathway in B cells result in perturbations in immune-complex-mediated feedback inhibition of antibody production. We now report that Fc gamma RII-deficient animals display elevated immunoglobulin levels in response to both thymus-dependent and thymus-independent antigens. Additionally, the effector arm of the allergic response is perturbed in these mice. Mast cells from Fc gamma RII-/- are highly sensitive to IgG-triggered degranulation, in contrast to their wild-type counterparts. Fc gamma RII-deficient mice demonstrate an enhanced passive cutaneous analphylaxis reaction, the result of a decreased threshold for mast-cell activation by Fc gamma RIII cross-linking. These results demonstrate that Fc gamma RII acts as a general negative regulator of immune-complex-triggered activation in vivo for both the afferent and efferent limbs of the immune response. Exploiting this property offers new therapeutic opportunities for the treatment of allergic and autoimmune disorders.

Animals↗
PubMedDate not supplied

HCV-related cryoglobulinemic vasculitis: an update on its etiopathogenesis and therapeutic strategies.

Cryoglobulinemic vasculitis (CV) is an immune-complex-mediated systemic vasculitis involving small-medium sized vessels. A causative role of hepatitis C virus (HCV) in over 4/5 patients has been definitely established on the basis of epidemiological, pathological, and laboratory studies. There is great geographical heterogeneity in the prevalence of CV as well as other HCV-related immuno-lymphoproliferative disorders. Thus, unknown environmental and/or genetic co-factors should contribute to the pathogenesis of these conditions. Due to the biological properties, HCV genomic sequences cannot be integrated into the host genome; the virus could trigger the immunological alterations only indirectly by exerting a chronic stimulus to the immune system. Recent laboratory observations gave us new important insights on the complex pathogenetic mechanism(s) of HCV-related CV. Firstly, the HCV envelop protein E2, able to bind CD81 molecule expressed on B-lymphocytes, might be involved in the first steps of HCV-driven autoimmune and lymphoproliferative phenomena. The interaction between HCV-E2 and CD81 may increase the frequency of VDJ rearrangement in antigen-reactive B-cell. One possible consequence may be the activation of anti-apoptotic Bcl-2 protoncogene that leads to extended B-cell survival. Interestingly, t(14, 18) translocation along with Bcl-2 activation have been demonstrated in B-lymphocytes of 80% HCV-related CV. The B-lymphocyte expansion is responsible for a wide autoantibody and immune-complex production, including mixed cryoglobulins. CV shows a relatively benign clinical course; however, its cumulative survival is significantly worse if compared to general population. For a correct therapeutic approach to HCV-related CV we must deal with conflicting conditions: HCV infection, autoimmune, and lymphoproliferative alterations. Therapeutic strategy of CV includes etiologic, pathogenetic, and/or symptomatic therapies, which should be tailored for the single patient according to the severity of clinical symptoms. A careful clinical monitoring of patients with HCV-related CV is mandatory in all cases, with particular attention to neoplastic complications.

Cryoglobulinemia↗
PubMed2004

Translators and mediators: bilingual young people's accounts of their interpreting work in health care.

The interpreting work bilingual young people do in health care settings has largely been seen as a social problem, indicating deficiencies either in parents' language skills or in the provision of professional interpreting services. Little research has addressed this contribution young people make to health care work from their perspectives. This study explored the accounts of bilingual young people from four linguistic groups in London, including those from established minority groups and those more recently arrived. Young people reported extensive experience of interpreting in a number of settings, and identified a range of benefits to themselves and their families arising from their contributions, as well as some problems faced in achieving successful encounters. Focusing on young people's own accounts enabled their work to be conceptualised not as merely 'inappropriate and inadequate interpreting' but as a varied contribution to the informal economy of health care that ranged from simple translation to complex mediation between families, the wider community and the health care system.

Adolescent↗
PubMed2003

SNARE-protein-mediated disease resistance at the plant cell wall.

Failure of pathogenic fungi to breach the plant cell wall constitutes a major component of immunity of non-host plant species--species outside the pathogen host range--and accounts for a proportion of aborted infection attempts on 'susceptible' host plants (basal resistance). Neither form of penetration resistance is understood at the molecular level. We developed a screen for penetration (pen) mutants of Arabidopsis, which are disabled in non-host penetration resistance against barley powdery mildew, Blumeria graminis f. sp. hordei, and we isolated the PEN1 gene. We also isolated barley ROR2 (ref. 2), which is required for basal penetration resistance against B. g. hordei. The genes encode functionally homologous syntaxins, demonstrating a mechanistic link between non-host resistance and basal penetration resistance in monocotyledons and dicotyledons. We show that resistance in barley requires a SNAP-25 (synaptosome-associated protein, molecular mass 25 kDa) homologue capable of forming a binary SNAP receptor (SNARE) complex with ROR2. Genetic control of vesicle behaviour at penetration sites, and plasma membrane location of PEN1/ROR2, is consistent with a proposed involvement of SNARE-complex-mediated exocytosis and/or homotypic vesicle fusion events in resistance. Functions associated with SNARE-dependent penetration resistance are dispensable for immunity mediated by race-specific resistance (R) genes, highlighting fundamental differences between these two resistance forms.

Arabidopsis↗
PubMed1997

Pre-TCR signaling components trigger transcriptional activation of a rearranged TCR alpha gene locus and silencing of the pre-TCR alpha locus: implications for intrathymic differentiation.

A rearranged TCR alpha transgene remains transcriptionally inactive in rag-2-/- thymocytes but can be induced by CD3-mediated signals with concomitant maturation of double-negative (DN) thymocytes to the CD4+CD8+ double-positive (DP) stage. Reciprocally, the same signals silence pre-TCR alpha (pT alpha) expression. In normal C57BL/6 thymocytes, TCR alpha expression is not detected in DN thymocytes while, in contrast, TCR beta expression is initiated at the most immature c-kit+CD44+CD25- stage and continues throughout thymocyte development. pT alpha expression is first detected at the intermediate c-kit +/- CD44+CD25+ DN stage, increases during transition to the more mature c-kit-CD44-CD25+ stage and is lost at the DP stage. Thus, although TCR beta and pT alpha expression are independent, the pre-TCR complex mediates signals controlling the appearance of alpha beta TCR through selective regulation of TCR alpha and pT alpha genes.

Animals↗