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PubMed2003

mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.

Animals↗

PubMed2002

A kinetic framework for a mammalian RNA polymerase in vivo.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

Animals↗

PubMed2006

Caspase inhibitors promote alternative cell death pathways.

The use of caspase inhibitors has revealed the existence of alternative backup cell death programs for apoptosis. The broad-spectrum caspase inhibitor zVAD-fmk modulates the three major types of cell death. Addition of zVAD-fmk blocks apoptotic cell death, sensitizes cells to necrotic cell death, and induces autophagic cell death. Several studies have shown a crucial role for the kinase RIP1 and the adenosine nucleotide translocator (ANT)-cyclophilin D (CypD) complex in necrotic cell death. The underlying mechanism of zVAD-fmk-mediated sensitization to necrotic cell death involves the inhibition of caspase-8-mediated proteolysis of RIP1 and disturbance of the ANT-CypD interaction. RIP1 is also involved in autophagic cell death. Caspase inhibitors and knockdown studies have revealed negative roles for catalase and caspase-8 in autophagic cell death. The positive role of RIP1 and the negative role of caspase-8 in both necrotic and autophagic cell death suggest that the pathways of these two types of cell death are interconnected. Necrotic cell death represents a rapid cellular response involving mitochondrial reactive oxygen species (ROS) production, decreased adenosine triphosphate concentration, and other cellular insults, whereas autophagic cell death first starts as a survival attempt by cleaning up ROS-damaged mitochondria. However, when this process occurs in excess, autophagy itself becomes cytotoxic and eventually leads to autophagic cell death. A better understanding of the molecular mechanisms of these alternative cell death pathways may provide therapeutic tools to combat cell death associated with neurodegenerative diseases, ischemia-reperfusion pathologies, and infectious diseases, and may also facilitate the development of alternative cytotoxic strategies in cancer treatment.

Adenine Nucleotide Translocator 1↗

PubMed2006

Cardiac involvement in adults with m.3243A>G MELAS gene mutation.

Cardiac data in adults with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS syndrome) or asymptomatic gene carriers with the mitochondrial deoxyribonucleic acid adenine-to-guanine point mutation at nucleotide pair 3243 are scarce. Twelve subjects (mean age 35 +/- 13 years), 8 with MELAS syndrome (patients) and 4 asymptomatic gene carriers (carriers), were enrolled in the study. Each subject underwent electrocardiography, exercise testing, Holter monitoring, echocardiography, and genetic and biochemical analysis for respiratory chain enzyme activity (complex I rest activity) in skeletal muscle. On electrocardiography and Holter monitoring, none of the subjects had evidence of preexcitation, cardiac arrhythmias, or conduction abnormalities. Patients had significantly lower (42 +/- 17% from normal vs 103 +/- 14%, p <0.02) exercise tolerance. All but 1 of the patients and none of the gene carriers had ragged red fibers on muscle biopsy. The mean percentage of gene mutation in skeletal muscle tended to be higher in patients (53 +/- 19%, range 19% to 73%) compared with carriers (33 +/- 20%, range 15% to 62%). Mean complex I rest activity in patients (36 +/- 18%, range 10% to 58%) was significantly (p <0.01) lower compared with carriers (120 +/- 60%, range 72% to 205%). Left ventricular (LV) abnormalities were confined to patients with MELAS syndrome. Two patients had LV hypertrophy, 5 had LV systolic abnormalities, and 5 had LV diastolic dysfunction. Apart from 1 patient with an isolated LV diastolic abnormality, all patients with LV abnormalities had ragged red fibers. Patients with abnormal systolic LV function had a trend toward a higher percentage of mutated skeletal muscle (59.7 +/- 10.7% vs 35.8 +/- 21.3%, p <0.10) and significantly lower complex I rest activity (26.7 +/- 14.0% vs 97.8% +/- 57.9, p <0.01). In conclusion, none of the MELAS gene carriers had cardiac abnormalities, whereas most patients with the MELAS phenotype, particularly those with ragged red fibers, had LV involvement.

Adolescent↗

PubMed2004

The yeast Rad7/Rad16/Abf1 complex generates superhelical torsion in DNA that is required for nucleotide excision repair.

Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions. The nature of the dual incision reactions on either side of the damaged base has been extensively investigated. However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined. We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway. GGR removes lesions from DNA that is not actively transcribing. We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions. Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component. The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER--a process referred to as excision. We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA.

Adenosine Triphosphatases↗

PubMed2004

Metal-catalyzed di-tert-butylsilylene transfer: synthesis and reactivity of silacyclopropanes.

Metal-catalyzed di-tert-butylsilylene transfer was developed as a mild, operationally simple, functional-group-tolerant method for silacyclopropane formation. Di-tert-butylsilylene was transferred from cyclohexene silacyclopropane 1 to an alkene through the use of a metal salt. Silacyclopropanation occurred at temperatures as low as -27 degrees C when AgOTf or AgOC(O)CF(3) were used as catalysts. Complex silacyclopropanes were formed stereospecifically and diastereoselectively from functionalized alkenes. Silacyclopropanes reacted with various carbonyl compounds, including aldehydes, ketones, formate esters, and formamides, in an overall process that efficiently converts alkenes into oxasilacyclopentanes with defined stereochemistry.

Journal Article↗

PubMed2006

Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity.

PURPOSE: The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. EXPERIMENTAL DESIGN: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. RESULTS: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. CONCLUSIONS: These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.

Antibodies, Monoclonal↗

PubMed2002

Anti-viral cytotoxic T cells inhibit the growth of cancer cells with antibody targeted HLA class I/peptide complexes in SCID mice.

A number of experimental antibody mediated cancer therapies aim to redirect cytotoxic T cells (CTLs) of non-tumour specificity to cancer cells. It has been previously demonstrated that cancer cells targeted with recombinant HLA-class I/viral peptide complexes via antibody delivery systems can be killed by virus specific CTLs. This novel therapeutic system has been developed with a simple pre-clinical model using the recombinant anti-CD20 B9E9 scFvSA fusion protein to target HLA-A2/peptide complexes to CD20 +ve Daudi lymphoma cells. In vitro data confirmed that, although binding of the B9E9 scFvSA fusion protein alone to Daudi cells had no effect on their growth, effective CTL mediated killing of Daudi cells could be achieved by targeting with B9E9 sfvScSA and recombinant HLA-A2/MI complexes at dilutions as low as 100 pg/ml. In contrast the free HLA-A2/MI complexes only significantly inhibited CTL activity at concentrations in excess of 100 ng/ml. The in vivo tumour protection assays in SCID mice demonstrated that only 1 of the 4 mice that received anti-HLA-A2/M1 CTLs and Daudi cells targeted with the B9E9 scFvSA fusion protein and HLA-A2/M1 complexes developed a tumour. In contrast in the control mice that received CTL and native Daudi cells all 4 developed tumours, as did all 4 that received targeted Daudi cells but no CTLs. Similar results were obtained in a parallel experiment using Daudi cells targeted with B9E9 scFvSA and HLA-A2/BMLF1 complexes and a CTL line to HLA-A2/BMLF1. The demonstration of in vivo activity for targeted HLA class I/peptide complexes combined with anti-viral T cells, supports the further clinical development of the system where it may be combined with autologous CTLs produced by vaccination or ex vivo expansion.

Animals↗

PubMed2005

Angiogenesis and apoptosis in glioma: two arenas for promising new therapies.

New therapies for gliomas are urgently needed in view of the very marginal increase in patient survival that has been achieved over the past two decades, which is only somewhat mitigated by improvements in quality of life. Two relatively recent fields of research that hold out great promise in this area, are angiogenesis and apoptosis. Depriving growing tumors of the blood supply they need, or tipping the balance in the cancer cell towards cell death, both provide conceptually elegant approaches to therapy, with the hope of great efficacy and little toxicity. However, attempts at successfully translating exciting laboratory findings to the clinic have been slowed by the complexity of the underlying biology. In this article we examine some of the issues that have impeded progress, and examine the potential role that integrins may play as targets, with a role in both angiogenesis and apoptosis.

Animals↗

PubMed2004

Human amygdala responses during presentation of happy and neutral faces: correlations with state anxiety.

BACKGROUND: Previous functional imaging studies demonstrating amygdala response to happy facial expressions have all included the presentation of negatively valenced primary comparison expressions within the experimental context. This study assessed amygdala response to happy and neutral facial expressions in an experimental paradigm devoid of primary negatively valenced comparison expressions. METHODS: Sixteen human subjects (eight female) viewed 16-sec blocks of alternating happy and neutral faces interleaved with a baseline fixation condition during two functional magnetic resonance imaging scans. RESULTS: Within the ventral amygdala, a negative correlation between happy versus neutral signal changes and state anxiety was observed. The majority of the variability associated with this effect was explained by a positive relationship between state anxiety and signal change to neutral faces. CONCLUSIONS: Interpretation of amygdala responses to facial expressions of emotion will be influenced by considering the contribution of each constituent condition within a greater subtractive finding, as well as 1) their spatial location within the amygdaloid complex; and 2) the experimental context in which they were observed. Here, an observed relationship between state anxiety and ventral amygdala response to happy versus neutral faces was explained by response to neutral faces.

Adult↗

PubMed2005

Architecture of the ribosome-channel complex derived from native membranes.

The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of "native" ribosome-channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.

Animals↗

PubMed2004

Envisaging the physicochemical processes during the preparation of supported catalysts: Raman microscopy on the impregnation of Mo onto Al2O3 extrudates.

Raman microscopy has been applied to study the preparation of shaped Mo/Al(2)O(3) catalysts. The speciation of different Mo complexes over gamma-Al(2)O(3) support bodies was followed in time after pore volume impregnation with aqueous solutions containing different Mo complexes. The addition of NO(3-) to the impregnation solutions allows for a quantitative Raman analysis of the distribution of different complexes over the catalyst bodies as this ion can be used as an internal standard. After impregnation with an acidic ammonium heptamolybdate (AHM) solution, the strong interaction between Mo(7)O(24)(6-) and Al(2)O(3) results in slow transport of this complex through the support and extensive formation of Al(OH)(6)Mo(6)O(18)(3-) near the outer surface of the support bodies. This may be prevented by decreasing the interaction between Mo and Al(2)O(3). In this way, transport is facilitated and a homogeneous distribution of Mo is obtained on a reasonable time scale. A decrease in interaction between Mo and Al(2)O(3) can be achieved by using alkaline impregnation solutions or by the addition of complexing agents, such as citrate and phosphate, to the impregnation solution. In general, time-resolved in situ Raman microscopy can be a valuable tool to study the physicochemical processes during the preparation of supported catalysts.

Journal Article↗

PubMed2003

Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids.

Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and alpha-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting.

Animals↗

PubMed2003

Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity.

Adiponectin is an adipocyte-specific secretory protein that circulates in serum as a hexamer of relatively low molecular weight (LMW) and a larger multimeric structure of high molecular weight (HMW). Serum levels of the protein correlate with systemic insulin sensitivity. The full-length protein affects hepatic gluconeogenesis through improved insulin sensitivity, and a proteolytic fragment of adiponectin stimulates beta oxidation in muscle. Here, we show that the ratio, and not the absolute amounts, between these two oligomeric forms (HMW to LMW) is critical in determining insulin sensitivity. We define a new index, S(A), that can be calculated as the ratio of HMW/(HMW + LMW). db/db mice, despite similar total adiponectin levels, display decreased S(A) values compared with wild type littermates, as do type II diabetic patients compared with insulin-sensitive individuals. Furthermore, S(A) improves with peroxisome proliferator-activated receptor-gamma agonist treatment (thiazolidinedione; TZD) in mice and humans. We demonstrate that changes in S(A) in a number of type 2 diabetic cohorts serve as a quantitative indicator of improvements in insulin sensitivity obtained during TZD treatment, whereas changes in total serum adiponectin levels do not correlate well at the individual level. Acute alterations in S(A) (DeltaS(A)) are strongly correlated with improvements in hepatic insulin sensitivity and are less relevant as an indicator of improved muscle insulin sensitivity in response to TZD treatment, further underscoring the conclusions from previous clamp studies that suggested that the liver is the primary site of action for the full-length protein. These observations suggest that the HMW adiponectin complex is the active form of this protein, which we directly demonstrate in vivo by its ability to depress serum glucose levels in a dose-dependent manner.

3T3-L1 Cells↗

PubMed2003

Structural diversification and neo-functionalization during floral MADS-box gene evolution by C-terminal frameshift mutations.

Frameshift mutations generally result in loss-of-function changes since they drastically alter the protein sequence downstream of the frameshift site, besides creating premature stop codons. Here we present data suggesting that frameshift mutations in the C-terminal domain of specific ancestral MADS-box genes may have contributed to the structural and functional divergence of the MADS-box gene family. We have identified putative frameshift mutations in the conserved C-terminal motifs of the B-function DEF/AP3 subfamily, the A-function SQUA/AP1 subfamily and the E-function AGL2 subfamily, which are all involved in the specification of organ identity during flower development. The newly evolved C-terminal motifs are highly conserved, suggesting a de novo generation of functionality. Interestingly, since the new C-terminal motifs in the A- and B-function subfamilies are only found in higher eudicotyledonous flowering plants, the emergence of these two C-terminal changes coincides with the origin of a highly standardized floral structure. We speculate that the frameshift mutations described here are examples of co-evolution of the different components of a single transcription factor complex. 3' terminal frameshift mutations might provide an important but so far unrecognized mechanism to generate novel functional C-terminal motifs instrumental to the functional diversification of transcription factor families.

Amino Acid Motifs↗

PubMed2006

Dobutamine does not influence inflammatory pathways during human endotoxemia.

OBJECTIVE: Catecholamines have anti-inflammatory and anticoagulant properties. Dobutamine is a synthetic catecholamine frequently used in patients with septic myocardial dysfunction. The objective was to determine whether a continuous infusion of dobutamine exerts immunomodulatory effects in healthy volunteers challenged with endotoxin. DESIGN: Prospective, open-label study. SETTING: Clinical research unit of a university hospital. PARTICIPANTS: Sixteen male healthy volunteers. INTERVENTIONS: Volunteers received a constant infusion with dobutamine (10 microg.kg.min, n = 8) or physiologic saline (n = 8). All participants were challenged with a bolus injection of endotoxin prepared from Escherichia coli (4 ng/kg). Dobutamine infusion was commenced 1 hr before endotoxin challenge and was continued until 3 hrs thereafter. MEASUREMENTS AND MAIN RESULTS: Dobutamine infusion was associated with an increase in mean arterial blood pressure (peak 122 +/- 5 mm Hg) and heart rate (peak 84 +/- 4 beats/min, both p < .05 vs. saline). Endotoxin injection induced the systemic release of cytokines (tumor necrosis factor-alpha, interleukins-6, -8, and -10) and secretory phospholipase A2, endothelial cell activation (increase in the plasma levels of soluble E-selectin and von Willebrand factor), activation of coagulation (increased plasma levels of soluble tissue factor, F1 + 2 prothrombin fragment, and thrombin-antithrombin complexes), and activation with subsequent inhibition of fibrinolysis (increased plasma concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor type I, and plasmin-alpha2-antiplasmin complexes). None of these responses were influenced by dobutamine. CONCLUSIONS: Dobutamine, infused in a clinically relevant dose, does not influence inflammatory and coagulant pathways during human endotoxemia.

Adult↗

PubMed2005

Importance of counting in biology.

In this study, analysis of biological problems through structural and quantitative approaches is described. Classical genetics on major histocompatibility complex in avian species has been evaluated. Experiments showing that antibodies from various individuals were generally distinct have led to further analysis of immunoglobulin light chain diversity by means of O'Farrell's 2D gel technique. The transition of the research towards vaccine development has been described. 'It's not the voting that's democracy, it's the counting.' - Tom Stoppard (Czech-born dramatist), Jumpers (1972) act 1.

Allergy and Immunology↗

PubMed2001

Extraintestinal Complications of Inflammatory Bowel Disease.

Extraintestinal complications of inflammatory bowel disease (IBD) are often secondary to the underlying disease. Therefore, the first priority is to get active IBD into remission with medications, since surgery for IBD is not indicated for the treatment of extraintestinal complications. Symptoms of extraintestinal complications usually can be treated with simple agents; the treatment of patients with refractory symptoms and the use of more complex drug regimens should be done in cooperation with specialists on affected organ systems. Careful consideration of prescribed drugs is necessary because they may negatively influence the course of IBD.

Journal Article↗