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The global transcriptional activator of Saccharomyces cerevisiae, Gcr1p, mediates the response to glucose by stimulating protein synthesis and CLN-dependent cell cycle progression.

Growth of Saccharomyces cerevisiae requires coordination of cell cycle events (e.g., new cell wall deposition) with constitutive functions like energy generation and duplication of protein mass. The latter processes are stimulated by the phosphoprotein Gcr1p, a transcriptional activator that operates through two different Rap1p-mediated mechanisms to boost expression of glycolytic and ribosomal protein genes, respectively. Simultaneous disruption of both mechanisms results in a loss of glucose responsiveness and a dramatic drop in translation rate. Since a critical rate of protein synthesis (CRPS) is known to mediate passage through Start and determine cell size by modulating levels of Cln3p, we hypothesized that GCR1 regulates cell cycle progression by coordinating it with growth. We therefore constructed and analyzed gcr1delta cln3delta and gcr1delta cln1delta cln2delta strains. Both strains are temperature and cold sensitive; interestingly, they exhibit different arrest phenotypes. The gcr1delta cln3delta strain becomes predominantly unbudded with 1N DNA content (G1 arrest), whereas gcr1delta cln1delta cln2delta cells exhibit severe elongation and apparent M phase arrest. Further analysis demonstrated that the Rap1p/Gcr1p complex mediates rapid growth in glucose by stimulating both cellular metabolism and CLN transcription.

Base Sequence↗

Distinct mechanisms control the stability of the related S-phase cyclins Clb5 and Clb6.

The yeast S-phase cyclins Clb5 and Clb6 are closely related proteins that are synthesized late in G1. Although often grouped together with respect to function, Clb5 and Clb6 exhibit differences in their ability to promote S-phase progression. DNA replication is significantly slowed in clb5Delta mutants but not in clb6Delta mutants. We have examined the basis for the differential functions of Clb5 and Clb6 and determined that unlike Clb5, which is stable until mitosis, Clb6 is degraded rapidly at the G1/S border. N-terminal deletions of CLB6 were hyperstabilized, suggesting that the sequences responsible for directing the destruction of Clb6 reside in the N terminus. Clb6 lacks the destruction box motif responsible for the anaphase promoting complex-mediated destruction of Clb5 but contains putative Cdc4 degron motifs in the N terminus. Clb6 was hyperstabilized in cdc34-3 and cdc4-3 mutants at restrictive temperatures and when S/T-P phosphorylation sites in the N terminus were mutated to nonphosphorylatable residues. Efficient degradation of Clb6 requires the activities of both Cdc28 and Pho85. Finally, hyperstabilized Clb6 expressed from the CLB6 promoter rescued the slow S-phase defect exhibited by clb5Delta cells. Taken together, these findings suggest that the SCF(Cdc4) ubiquitin ligase complex regulates Clb6 turnover and that the functional differences exhibited by Clb5 and Clb6 arise from the distinct mechanisms controlling their stability.

Amino Acid Motifs↗

De novo development of membranous nephropathy in cadaver renal allografts.

Glomerulonephritis in transplant recipients often reflects recurrence of the immunopathogenetic mechanism causing the original renal disease. Membranous nephropathy (MN), a progressive immune complex mediated glomerular disease and the commonest cause of idiopathic nephrotic syndrome in adults, has been virtually unreported in transplant recipients. Two cases are reported here of typical MN (by clinical, light, immunofluorescent and electron microscopic criteria) developing de novo in the transplants of patients whose original diseases were anti-GBM nephritis and focal glomerular sclerosis. NM developed following episodes of viral hepatitis and renal infarction respectively. Possible mechanisms by which this lesion might develop in these patients are investigated and discussed. Chronic immune complex nephropathy (MN) can develop de novo in immunosuppressed transplant recipients apparently initiated by events in the post-transplant period.

Adolescent↗

Effects of noncovalently bound quinones on the ground and triplet states of zinc chlorins in solution and bound to de novo synthesized peptides.

The Qy absorption band of two chlorophyll derivatives, zinc chlorin e6 (ZnCe6) and zinc pheophorbide a (ZnPheida), in aqueous solution is bathochromically shifted on addition of quinones, e.g., 1,4-benzoquinone (BQ), with a corresponding shift of the fluorescence band. This is due to a complex formation of zinc chlorins induced by BQs and subsequent rearrangement. The time-resolved absorption spectra after laser pulse excitation show triplet quenching of the pigments by BQ and other quinones via electron transfer. The effects of electron transfer to noncovalently bound BQs were also studied with de novo synthesized peptides, into which ZnCe6 and ZnPheida were incorporated as model systems for the primary steps of photosynthetic reaction centers. Whereas the photophysical properties are similar to those of the unbound zinc chlorins, no BQ-mediated complex formation was observed.

Benzoquinones↗

Midkine enhances fibrinolytic activity of bovine endothelial cells.

A hitherto unknown function of midkine (MK) was found in the regulation of fibrinolytic activity of vascular endothelial cells. Recombinant murine MK enhanced plasminogen activator (PA)/plasmin levels in bovine aortic endothelial cells (BAECs) in a dose- and time-dependent manner. After incubation with 10 ng/ml MK for 18 h, PA and plasmin levels increased 6- and 4-fold, respectively. This effect was attributed to a moderate upregulation of urokinase-type PA expression as well as to a significant down-regulation of PA inhibitor-1 (PAI-1) expression. BAECs constitutively synthesized and secreted MK and its production was enhanced 2-fold with 1 microM retinoic acid or 10 microM retinol. It was found that MK served as a substrate for tissue transglutaminase. In the culture medium, MK existed as a transglutaminase-mediated complex of 36 kDa. Addition of anti-MK antibody to BAEC cultures resulted in a decrease of basal PA activity and an increase of basal PAI-1 levels and attenuated the ability of retinol to enhance PA activity 50% and potentiated the ability to increase PAI-1 levels 4-fold. Furthermore, MK and basic fibroblast growth factor (bFGF) acted more than additively in enhancing PA levels. We conclude that in BAECs MK is a novel autocrine factor sustaining the fibrinolytic property. MK functions as a mediator of retinoid and cooperates with bFGF to enhance fibrinolytic activity of BAECs.

Animals↗

Modeling and functional analysis of the interaction between von Willebrand factor A1 domain and glycoprotein Ibalpha.

Binding of the von Willebrand factor (vWF) A1 domain to the glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to reactive substrates under high shear stress conditions, a key event in hemostasis and thrombosis. We have now used the known three-dimensional structure of the A1 domain to model the interaction with the GP Ibalpha sequence 271-279, which has previously been implicated in ligand binding. Docking procedures suggested that A1 domain residues in strand beta3 and preceding loop (residues 559-566) as well as in helix alpha3 (residues 594-603) interact with Asp residues 272, 274, 277 and sulfated Tyr residues 278 and 279 in GP Ibalpha. To verify this model, 14 mutant A1 domain fragments containing single or multiple side chain substitutions were tested for their ability to mediate platelet adhesion under flow. Each of the vWF residues Tyr(565), Glu(596), and Lys(599) proved to be strictly required for A1 domain function, which, in agreement with previous findings, was also dependent on Gly(561). Moreover, an accessory functional role was apparent for a group of positively charged residues, including Arg at positions 629, 632, 636 and Lys at positions 643 and 645, possibly acting in concert. There was, however, no evidence from the model that these residues directly participate in forming the complex with GP Ibalpha. These results provide a partial model of the vWF-GP Ibalpha interaction linked to the manifestation of functional activity in platelet adhesion.

Amino Acids↗

SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V: localization of SHIP-2 to the activated platelet actin cytoskeleton.

The platelet receptor for the von Willebrand factor (VWF) glycoprotein Ib-IX-V (GPIb-IX-V) complex mediates platelet adhesion at sites of vascular injury. The cytoplasmic tail of the GPIbalpha subunit interacts with the actin-binding protein, filamin, anchoring the receptor in the cytoskeleton. In motile cells, the second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3) induces submembraneous actin remodeling. The inositol polyphosphate 5-phosphatase, Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2), hydrolyzes PtdIns(3,4,5)P3 forming phosphatidylinositol 3,4 bisphosphate (PtdIns(3,4)P2) and regulates membrane ruffling via complex formation with filamin. In this study we investigate the intracellular location and association of SHIP-2 with filamin, actin, and the GPIb-IX-V complex in platelets. Immunoprecipitation of SHIP-2 from the Triton-soluble fraction of unstimulated platelets demonstrated association between SHIP-2, filamin, actin, and GPIb-IX-V. SHIP-2 associated with filamin or GPIb-IX-V was active and demonstrated PtdIns(3,4,5)P3 5-phosphatase activity. Following thrombin or VWF-induced platelet activation, detection of the SHIP-2, filamin, and receptor complex decreased in the Triton-soluble fraction, although in control studies the level of SHIP-2, filamin, or GPIb-IX-V immunoprecipitated by their respective antibodies did not change following platelet activation. In activated platelets spreading on a VWF matrix, SHIP-2 localized intensely with actin at the central actin ring and colocalized with actin and filamin at filopodia and lamellipodia. In spread platelets, GPIb-IX-V localized to the center of the platelet and showed little colocalization with filamin at the plasma membrane. These studies demonstrate a functionally active complex between SHIP-2, filamin, actin, and GPIb-IX-V that may orchestrate the localized hydrolysis of PtdIns(3,4,5)P3 and thereby regulate cortical and submembraneous actin.

Actins↗

Human recombinant soluble decay accelerating factor inhibits complement activation in vitro and in vivo.

Complement plays a role in activating the inflammatory response and has been implicated in the pathogenesis of some inflammatory diseases. With a view toward controlling unwanted C activation, we evaluated the C regulator, human decay accelerating factor (DAF). Three forms of recombinant DAF were purified from transfected Chinese hamster ovary cells: glycophosphatidylinositol (GPI)-linked membrane DAF (mDAF) extracted from cell membranes; spontaneously shed soluble DAF (sDAF) derived from mDAF; and a novel secreted protein (seDAF), generated by deletion of the signal for GPI attachment. We show that all three molecules inhibit both the classical and alternative pathways of C activation. The following observations indicate that mDAF extracted from Chinese hamster ovary cells reincorporates into RBC membranes via its GPI anchor: 1) cells that are preincubated with mDAF and then washed remain fully protected from C-mediated hemolysis; 2) incubation with phosphatidylinositol-specific phospholipase C abolishes this protection; and 3) sDAF and seDAF, which lack a GPI anchor, do not associate with cell membranes. mDAF is a more potent inhibitor of C-mediated hemolysis than either sDAF or seDAF, suggesting that incorporation into cell membranes greatly enhances the efficiency with which DAF inhibits C activation on the cell surface. In contrast, C activation in the fluid phase is inhibited by sDAF and seDAF, but not by mDAF, possibly due to interference by serum lipoproteins. A reversed passive Arthus reaction in guinea pigs was used to evaluate the ability of recombinant seDAF to inhibit C activation in vivo. When administered at dermal sites, seDAF reduced the severity of immune complex-mediated inflammatory reactions induced by a reversed passive Arthus reaction, as judged by both gross and histologic examination. These data indicate that seDAF may be useful as an anti-inflammatory therapeutic.

Animals↗

Immunoregulatory effects of covalent antigen-antibody complexes. III. Enhancement or suppression depending on the time of administration of complex relative to a T-independent antigen.

The photosensitive affinity label NAP (4-azido-2-nitrophenyl) was used to make a stable covalent-bonded monomeric immune complex (Ag2Ab) between rabbit anti-NAP antibody and a bihaptenic compound containing NAP linked to fluorescein (NAP-aminocaproyl-lysyl-Fl). This complex injected into mice had marked effects on their subsequent response to fluorescein coupled to a thymus-independent carrier (Fl-ficoll). Depending on the time at which the complex was administered relative to challenge, it was possible to obtain either enhancing or suppressive effects. The enhancing but not the suppressive effect of complex was dependent on immune recognition of the rabbit IgG carrier. While the suppressive effect probably results from complex-mediated inactivation of T-independent B cells, it is suggested that the enhancing effect results from priming of the T-dependent B cells by Fl-Ficoll followed by their triggering into antibody production by rabbit IgG-specific helper cells.

Animals↗

Rheumatoid factor in rheumatoid arthritis associated renal disease and in lupus nephritis.

To test the hypothesis that rheumatoid factor (RF) protects against (immune complex mediated) renal disease, patients with rheumatoid arthritis (RA; 48 with nephropathy of various types, 35 without renal disease) and systemic lupus erythematosus (SLE; 35 with and 17 without nephritis) were evaluated for the presence and titre of RF. There was no correlation between RF and nephropathy in RA, whereas in SLE RFs were almost exclusively seen in patients without nephropathy. This result supports the above hypothesis for lupus nephropathy but not for RA associated renal disease, and it may be explained by a more pronounced role for immune complexes in SLE and interference of RFs with the complexes.

Arthritis, Rheumatoid↗

Immunopathology of scleritis.

Conjunctival and scleral biopsies from 25 patients with necrotizing scleritis and 5 patients with recurrent nonnecrotizing scleritis were studied by histopathologic, immunofluorescence, and immunoperoxidase techniques. Vasculitis with fibrinoid necrosis and neutrophil invasion of the vessel wall was present in 75% of the scleral and 52% of the conjunctival specimens. Vascular immunodeposits were found in 93% of the scleral and 79% of the conjunctival tissue tested by immunofluorescence techniques. A dramatic increase in the number of inflammatory cells over normal controls was detected in both tissues by immunoperoxidase techniques. In the conjunctival epithelium, there were significantly more T-helpers, macrophages, and B cells. In the conjunctival substantia propria, there were significantly more T cells of all types, macrophages, and B cells. Likewise, scleral specimens showed an increase over controls of T cells of all types and macrophages. HLA-DR expression was dramatically increased in both tissues. Immune-complex-mediated vasculitis plays a pivotal role in the pathogenesis of necrotizing scleritis and recurrent nonnecrotizing scleritis. Induced HLA-DR expression on ocular nonimmune cells and T cell controlled responses also may participate.

Adult↗

Regulation of Notch signalling by non-visual beta-arrestin.

Signalling activity of the Notch receptor, which plays a fundamental role in metazoan cell fate determination, is controlled at multiple levels. We uncovered a Notch signal-controlling mechanism that depends on the ability of the non-visual beta-arrestin, Kurtz (Krz), to influence the degradation and, consequently, the function of the Notch receptor. We identified Krz as a binding partner of a known Notch-pathway modulator, Deltex (Dx), and demonstrated the existence of a trimeric Notch-Dx-Krz protein complex. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway. Our results establish a novel mode of regulation of Notch signalling and define a new function for non-visual beta-arrestins.

Animals↗

Gag order.

The Polycomb group (PcG) complex mediates heritable silencing of numerous genes during development. In a recent issue of Molecular Cell, Wang et al. (2004) characterize the order of PcG protein recruitment to specific regions of Ubx and substantiate a binding-in trans looping model of repression that may interfere with transcription initiation or elongation.

Animals↗

Waldenström's macroglobulinaemia presenting as reticulate purpura and bullae in a patient with hepatitis B virus infection.

Cutaneous manifestations of Waldenström's macroglobulinaemia (WM) include purpura, ulcers, urticaria, leukocytoclastic vasculitis, and immunobullous dermatoses. No association has been reported previously of WM and hepatitis B virus (HBV) infection. A 40-year-old female HBV carrier was admitted to hospital because of generalized oedema, oliguria, haematuria, hypertension, fever and blood-tinged sputum. Cutaneous manifestations included generalized petechiae, palpable purpura mainly on the legs, multiple necrotic ulcerations and gangrenous changes on the toes, and necrotic, giant confluent reticulate purpura on the trunk surmounted by several tense bullae. Laboratory investigations revealed monoclonal gammopathy of IgM kappa type (6.7 g/L), membranoproliferative glomerulonephritis associated with HBV infection, Bence Jones proteinuria, and an increased number of abnormal plasmacytoid cells in the bone marrow. Pathologic examination demonstrated immune complex-mediated vasculitis with deposits of IgM in the walls of dermal vessels and secondary subepidermal bulla formation. HBV infection may have caused WM or modified the clinical course in this fatal case.

Adult↗

An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter.

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pull down is lost by mutation of the activation domain.

Amino Acid Sequence↗

Effect of dexamethasone on T-cell receptor/CD3 expression.

Glucocorticoid hormones (GCH) are anti-inflammatory and immunosuppressive agents that inhibit T-cell growth and activation. Since the T-cell receptor (TCR)/CD3 complex mediates T-lymphocyte activation, we studied the effect of in vitro dexamethasone (DEX), a synthetic GCH, on TCR/CD3 expression. DEX-treatment of a hybridoma T-cell line and normal un-transformed T-cell clones induced a decrease of the TCR/ CD3 membrane expression after 4 days. After 4 weeks, TCR/CD3 was undetectable. However, the amount of mRNAs coding TCR/CD3 chains, including TCR alpha, TCR beta, CD3 gamma, CD3 theta and CD3 epsilon, as well as the amount of CD3 epsilon protein, a major component of the complex, were unaltered. By contrast, a decrease of the mRNAs deriving from the TCR zeta gene locus, as well as of the TCR zeta protein which is responsible for the membrane expression of the TCR/CD3 complex, was induced. These data suggest that the down-modulation of TCR expression is due to the diminution of TCR zeta gene products in DEX-treated cells.

Animals↗

Reversible depletion of synovial lining cells after intra-articular treatment with liposome-encapsulated dichloromethylene diphosphonate.

We studied the depletion and repopulation of synovial lining cells in mice. A single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP) in the mouse knee joint caused selective elimination of synovial lining cells. Depletion of cells occurred within a few days as evidenced by light microscopic, electronmicroscopic and immunohistochemical studies. Maximal depletion was seen on day 7. Repopulation was observed in the following weeks, starting at the bone side of the joint. Until day 30, full recovery (60% recovery) was not observed in the lining lying adjacent to the dermis. Side effects on cartilage metabolism, such as inhibition of proteoglycan synthesis or degradation of proteoglycans from the matrix was minor but significant, 1 and 2 days after liposome treatment but thereafter full recovery was observed. Selective elimination of lining cells from the joint enabled us to study the in vivo role of these cells in the onset and subsequent pathology of experimental arthritis. An immune-complex-mediated experimental arthritis elicited in lining cell depleted joints that had received CL2MDP-liposomes 7 days earlier prevented inflammation as compared to controls.

Animals↗

Studies on the mechanisms involved in the inflammatory response in a reversed passive Arthus reaction in guinea-pig skin: contribution of neutrophils and endogenous mediators.

1. Mediators of inflammation can increase vascular permeability in at least two different ways: by acting directly on endothelial cells or, indirectly, through an incompletely understood mechanism, dependent on circulating neutrophils. Neutrophil-dependent oedema formation has been described in the skin of rabbits, rats, hamsters, mice and man. In contrast, we presented evidence in a previous study that local oedema formation induced by i.d. injection of chemoattractants in guinea-pig skin was neutrophil-independent. In the present study, we sought evidence of neutrophil-dependent oedema formation in immune-complex-mediated vasculitis, the reversed passive Arthus (RPA) reaction, in guinea-pig skin. We also investigated whether haemorrhage in the RPA reaction was neutrophil-dependent (as it is in other species) and the role of endogenous mediators of inflammation (prostaglandins, nitric oxide, histamine, PAF and leukotrienes) in contributing to the local inflammatory response. 2. In the RPA reaction, most oedema formation occurred over the first 60 min whereas 111In-neutrophil accumulation was still increasing from 60 to 240 min. The different kinetics of these two events suggested that they may be dissociated. 3. Oedema formation was partially inhibited by a long-acting PAF antagonist (UK-74,505) and an H1 histamine receptor antagonist (mepyramine) but not by a 5-lipoxygenase inhibitor (ZM 230487). A nitric oxide synthesis inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) suppressed oedema formation by 68% whereas a cyclo-oxygenase inhibitor suppressed oedema by 27%. 4. 111In-neutrophil accumulation in the RPA reaction was partially suppressed by UK-74,505. In contrast, ZM 230487 was without effect at doses which abrogated arachidonic acid-induced 111In-neutrophil accumulation. 5. The anti-CD18 monoclonal antibody, (mAb) 6.5E F(ab')2, effectively inhibited 111In-neutrophil accumulation induced by PAF, zymosan-activated plasma (ZAP) and in the RPA reaction. However, oedema formation measured in the same sites was not altered. In contrast, oedema formation in the RPA reaction was partially suppressed by 6.5E whole mAb which was 2.5 times more potent than 6.5EF(ab')2 at inhibiting guinea-pig neutrophil adhesion to protein-coated plastic. Haemorrhage induced by PAF and in the RPA reaction was significantly inhibited by 6.5E F(ab')2 pretreatment.6. We conclude that in the RPA reaction in guinea-pig skin, oedema formation is partially neutrophil dependent as assessed by using an anti-CD18 mAb, whereas ZAP-induced oedema formation is neutrophil-independent. Haemorrhage was also dependent on neutrophil accumulation. In addition, our studies support a role for PAF in mediating both oedema formation and "'In-neutrophil accumulation in the RPA reaction. Endogenous release of histamine also appears to be important in mediating oedema formation suggesting that mast cells play a critical role in increases of vascular permeability in inflammatory reactions in guinea-pig skin. Moreover, our results confirm previous findings which suggest a dominant role for nitric oxide in maintaining cutaneous blood flow in the guinea-pig.

Animals↗