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PubMed2006

Inhibition of transcription factor NF-kappaB activation by kappaB-Ras.

kappaB-Ras1 and kappaB-Ras2 are two small proteins that display similarity at the amino acid level to Ras-like small GTPases. Although little is known about the function of the kappaB-Ras proteins, they have been shown to interfere with activation of transcription factor NF-kappaB. They accomplish this by binding to IkappaB proteins, natural inhibitors of NF-kappaB, and delaying their stimulus-dependent degradation. In this chapter, we consider the kappaB-Ras proteins in light of their NF-kappaB regulatory properties. Three fundamental questions about kappaB-Ras function are addressed: (1) Does kappaB-Ras regulate NF-kappaB in vivo? (2) Does kappaB-Ras selectively regulate specific NF-kappaB/IkappaB complexes? (3) Does kappaB-Ras function as a true GTPase, that is, with molecular switching properties that correlate with the phosphorylation state of bound guanine nucleotide? Finally, we detail the methods currently used to study the kappaB-Ras proteins as regulators of NF-kappaB activation.

Animals↗

PubMed2004

Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells.

Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

Amino Acid Sequence↗

PubMed2004

Initiation of cellular organization in lymph nodes is regulated by non-B cell-derived signals and is not dependent on CXC chemokine ligand 13.

The molecular and cellular events that initiate the formation of T and B cell areas in developing lymph nodes are poorly understood. In this study we show that formation of the lymphoid architecture in murine neonatal lymph nodes evolves through a series of distinct stages. The initial segregation of T and B cells is regulated in a CXCL13-independent manner, characterized by the localization of B cells in a ring-like pattern in the outer cortex on day 4. However, during this CXCL13-independent phase of lymph node modeling, CXCL13 is expressed and regulated in a lymphotoxin-alpha1beta2 (LTalpha1beta2)-dependent manner. Surprisingly, neonatal B cells are unable to respond to this chemokine and also lack surface LTalpha1beta2 expression. At this time, CD45+CD4+CD3- cells are the predominant LTalpha1beta2-expressing cells and are also capable of responding to CXCL13. From day 4 on, architectural changes become CXCL13 dependent, and B cells become fully CXCL13 responsive, express LTalpha1beta2, and cluster in anatomically distinct follicles. Because the initial induction of CXCL13 is dependent on LTalpha1beta2, a role for CD45+CD4+CD3- cells in inducing chemokine expression in the developing lymph nodes is proposed and, as such, a role in initiation of the shaping of the microenvironment.

Animals↗

PubMed2005

Description and critical appraisal of principal components analysis (PCA) methodology applied to pulsed-field gel electrophoresis profiles of methicillin-resistant Staphylococcus aureus isolates.

Principal components analysis (PCA) has been described for over 50 years; however, it is rarely applied to the analysis of epidemiological data. In this study PCA was critically appraised in its ability to reveal relationships between pulsed-field gel electrophoresis (PFGE) profiles of methicillin-resistant Staphylococcus aureus (MRSA) in comparison to the more commonly employed cluster analysis and representation by dendrograms. The PFGE type following SmaI chromosomal digest was determined for 44 multidrug-resistant hospital-acquired methicillin-resistant S. aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK). Strain relatedness was determined using Dice band-matching with UPGMA clustering and PCA. The results indicated that PCA revealed relationships between MRSA strains, which were more strongly correlated with known epidemiology, most likely because, unlike cluster analysis, PCA does not have the constraint of generating a hierarchic classification. In addition, PCA provides the opportunity for further analysis to identify key polymorphic bands within complex genotypic profiles, which is not always possible with dendrograms. Here we provide a detailed description of a PCA method for the analysis of PFGE profiles to complement further the epidemiological study of infectious disease.

Cluster Analysis↗

PubMed2002

Mathematical models of protein kinase signal transduction.

We have developed a mathematical theory that describes the regulation of signaling pathways as a function of a limited number of key parameters. Our analysis includes linear kinase-phosphatase cascades, as well as systems containing feedback interactions, crosstalk with other signaling pathways, and/or scaffolding and G proteins. We find that phosphatases have a more pronounced effect than kinases on the rate and duration of signaling, whereas signal amplitude is controlled primarily by kinases. The simplest model pathways allow amplified signaling only at the expense of slow signal propagation. More complex and realistic pathways can combine high amplification and signaling rates with maintenance of a stable off-state. Our models also explain how different agonists can evoke transient or sustained signaling of the same pathway and provide a rationale for signaling pathway design.

GTP-Binding Proteins↗

PubMed2005

Chelating diamide based rate enhancement of intramolecular alkene hydroaminations catalyzed by a neutral Sc(III) complex.

[reaction: see text] Neutral scandium amido complexes are viable catalysts for intramolecular alkene hydroamination. Catalytic activity is strongly coupled to the electronic character of the Sc(III) ligand environment with chelating diamide coordination providing a precatalyst possessing substantially improved activity and superb distereoselectivity in the synthesis of trans-2,5-disubstituted pyrrolidines.

Alkenes↗

PubMed2004

Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract.

Crystallin genes are selectively expressed during lens development. Maf and Sox family proteins synergistically enhanced gammaF-crystallin promoter activity in a lens cell line. Mutational analysis of the gammaF-crystallin promoter identified a composite regulatory element containing nonconsensus Maf and Sox recognition sequences. Mutations in these recognition sequences or changes in their spacing eliminated synergistic transcription activation. The transcriptional synergy was also affected by changes in the orientation of the Maf recognition sequence that had no detectable effect on binding affinity. The interaction between Maf and Sox proteins was visualized in living cells by bimolecular fluorescence complementation analysis. The N-terminal region of Maf mediated the interaction with Sox proteins in cells. Synergistic transcription activation required the N-terminal region of Maf as well as the ancillary DNA binding domain and the unique portion of the basic region that mediate specific recognition of the gammaF-crystallin promoter element. A mutation in the ancillary DNA binding domain of Maf (R288P) that has been shown to cause cataract eliminated the transcriptional activity of Maf but had no detectable effect on DNA binding in vitro. Whereas wild-type Maf was uniformly distributed in the nucleoplasm, R288P Maf was enriched in nuclear foci. Cajal bodies and gemini of coiled bodies were closely associated with the foci occupied by R288P Maf. Wild-type Maf formed complexes with Sox proteins in the nucleoplasm, whereas R288P Maf recruited Sox proteins as well as other interaction partners to the nuclear foci. The mislocalization of normal cellular proteins to these foci provides a potential explanation for the dominant disease phenotype of the R288P mutation in Maf.

Animals↗

PubMed2006

Balloon atrial septostomy is associated with preoperative stroke in neonates with transposition of the great arteries.

BACKGROUND: Preoperative brain injury is common in neonates with transposition of the great arteries (TGA). The objective of this study is to determine risk factors for preoperative brain injury in neonates with TGA. METHODS AND RESULTS: Twenty-nine term neonates with TGA were studied with MRI before cardiac surgery in a prospective cohort study. Twelve patients (41%) had brain injury on preoperative MRI, and all injuries were focal or multifocal. None of the patients had birth asphyxia. Nineteen patients (66%) required preoperative balloon atrial septostomy (BAS). All patients with brain injury had BAS (12 of 19; risk difference, 63%; 95% confidence interval, 41 to 85; P=0.001). As expected on the basis of the need for BAS, these neonates had lower systemic arterial hemoglobin saturation (Sao2) (P=0.05). The risk of injury was not modified by the cannulation site for septostomy (umbilical versus femoral, P=0.8) or by the presence of a central venous catheter (P=0.4). CONCLUSIONS: BAS is a major identifiable risk factor for preoperative focal brain injury in neonates with TGA. Imaging characteristics of identified brain injuries were consistent with embolism; however, the mechanism is more complex than site of vascular access for BAS or exposure to central venous catheters. These findings have implications for the indications for BAS, timing of surgical repair, and use of anticoagulation in TGA.

Catheterization↗

PubMed2002

Complex splicing pattern generates great diversity in human NF1 transcripts.

BACKGROUND: Mutation analysis of the neurofibromatosis type 1 (NF1) gene has shown that about 30% of NF1 patients carry a splice mutation resulting in the production of one or several shortened transcripts. Some of these transcripts were also found in fresh lymphocytes of healthy individuals, albeit typically at a very low level. Starting from this initial observation, we were interested to gain further insight into the complex nature of NF1 mRNA processing. RESULTS: We have used a RT-PCR plasmid library based method to identify novel NF1 splice variants. Several transcripts were observed with specific insertions/deletions and a survey was made. This large group of variants detected in one single gene allows to perform a comparative analysis of the factors involved in splice regulation. Exons that are prone to skipping were systematically analysed for 5' and 3' splice site strength, branch point strength and secondary structure. CONCLUSION: Our study revealed a complex splicing pattern, generating a great diversity in NF1 transcripts. We found that, on average, exons that are spliced out in part of the mRNA have significantly weaker acceptor sites. Some variants identified in this study could have distinct roles and might expand our knowledge of neurofibromin.

Journal Article↗

PubMed2026

Germline determinants of risk and molecular subtype in young-onset lung cancer.

Young-onset lung cancer is enriched for never-smoking and oncogene-driven tumors, yet its inherited genetic basis remains poorly defined. We performed germline whole-genome sequencing in 251 young-onset lung cancer cases (median age 37), which we jointly analyzed with never-smoking cases (n=196; median age 68) and cancer-free controls (n=1,883). We identified enrichments of rare deleterious coding variants across 55 cancer-related gene sets, including EGFR/ERBB2 signaling and genes implicated by prior lung cancer GWAS. Exome-wide analyses of rare coding variants affirmed TP53 as a penetrant lung cancer predisposition gene (odds ratio [OR]=36.1, p=1.02x10-7) and discovered two novel exome-wide significant tumor subtype-dependent associations: IREB2 in cases with fusion-driven tumors (p=1.39x10-6) and SMAD6 in fusion-negative tumors (p=2.05x10-6). Structural variants contributed distinct risk, with enrichment in constrained, lung-expressed genes (OR=5.79, p=5.8x10-5) and very large germline deletions being markedly enriched in cases with fusion-driven tumors. Polygenic risk scores for lung cancer were inversely correlated with rare variant burden, consistent with additive risk from rare and common variants. Collectively, these findings delineate a complex germline architecture underlying susceptibility and molecular subtype in young-onset lung cancer.

Journal Article↗

PubMed2006

AMP-dependent protein kinase alpha 2 isoform promotes hypoxia-induced VEGF expression in human glioblastoma.

Tumor cells respond to hypoxic stress by upregulating a variety of genes involved in glucose uptake, glycolysis, and angiogenesis, all essential to maintaining nutrient availability and intracellular ATP levels. Adenosine monophosphate-dependent kinase (AMPK) is a key sensor for cellular homeostasis and is highly sensitive to changes in AMP:ATP ratios. The two catalytic AMPK alpha isoforms (AMPKalpha1, AMPKalpha2) were investigated with respect to their expression, cellular distribution, and contribution to VEGF expression under hypoxic stress in human U373 glioblastoma cells. Quantitative real-time PCR analysis showed AMPKalpha1 mRNA to be constitutively expressed in normoxia and hypoxia, whereas AMPKalpha2 mRNA levels were low in normoxia and significantly induced in hypoxia. Fluorescent immunohistochemistry showed that AMPKalpha2 protein redistributed to the nucleus under hypoxia, whereas AMPKalpha1 remained distributed throughout the cell. The AMPK chemical inhibitor, 5-iodotubericidin, effectively repressed the hypoxic induction of VEGF mRNA levels and hypoxia inducible factor-1 dependent transcription. AMPKalpha2 repression with RNA interference reduced hypoxia-induced VEGF mRNA and HIF-1 transcription, whereas AMPKalpha1 repression did not. Human glioblastoma cell lines U118 and U138 also showed hypoxia-induction of AMPKalpha2 as well as VEGF. Immunohistochemistry analysis of human astrocytoma/glioma samples revealed AMPKalpha2 present in high grade gliomas within hypoxic pseudopalisading microenvironments. These data suggest that prolonged hypoxia promotes the expression and functional activation of AMPKalpha2 and VEGF production in glioma cell lines and glioblastoma multiform tumors, thus contributing to tumor survival and angiogenesis in high grade human gliomas.

AMP-Activated Protein Kinases↗

PubMed2006

Horseshoe lung and facio-auriculo-vertebral sequence: a previously unreported association.

We describe a case of horseshoe lung in an infant with facio-auriculo-vertebral (FAV) sequence that included mild hemifacial microsomia, ear anomalies, a missing left rib, left hemivertebrae (T2-T4), and complex congenital heart disease. Of the approximately 40 cases of horseshoe lung described since 1962, most are reported in association with scimitar syndrome, and only four reported cases were associated with left lung hypoplasia. None of these cases included malformations consistent with a diagnosis of FAV sequence.

Abnormalities, Multiple↗

PubMed2002

An extensive network of coupling among gene expression machines.

Gene expression in eukaryotes requires several multi-component cellular machines. Each machine carries out a separate step in the gene expression pathway, which includes transcription, several pre-messenger RNA processing steps and the export of mature mRNA to the cytoplasm. Recent studies lead to the view that, in contrast to a simple linear assembly line, a complex and extensively coupled network has evolved to coordinate the activities of the gene expression machines. The extensive coupling is consistent with a model in which the machines are tethered to each other to form 'gene expression factories' that maximize the efficiency and specificity of each step in gene expression.

Gene Expression↗

PubMed2001

Differential processing and presentation of the H-2D(b)-restricted epitope from two different strains of influenza virus nucleoprotein.

The influenza virus strains A/NT/60/68 and A/PR/8/34 both have an immunodominant D(b)-restricted epitope in their nucleoprotein (NP) at amino acid residues 366-374, with two amino acid differences between the epitopes. Cross-reactive cytotoxic T lymphocytes (CTLs) were generated by priming mice with the influenza virus A/NT/60/68 NP and restimulating in vitro with influenza virus A/PR/8/34. CTLs that gave high levels of specific lysis recognized target cells infected with either strain of influenza virus with similar efficiency. Surprisingly, when target cells were infected with recombinant vaccinia viruses (VV) expressing the two different NPs, presentation of the D(b)-restricted epitope from the A/NT/60/68 NP was extremely poor, whereas presentation of the equivalent epitope from the A/PR/8/34 NP was as efficient as in influenza virus-infected cells. This difference was observed in spite of the fact that the two NP sequences show 94% identity at the amino acid sequence level. Experiments with additional cross-reactive CTL cell lines which recognized target cells less efficiently revealed a similar difference in presentation between the two NP epitopes in influenza virus-infected cells and showed a difference in the efficiency of presentation of the D(b)-restricted epitope from the two NP molecules independent of VV infection. The results show that two equivalent epitopes in highly similar proteins are processed with very different efficiency, even though they are both immunodominant epitopes. They also suggest that the previously described inhibition of antigen presentation by VV is a general, non-specific effect, which is more apparent for epitopes that are processed and presented less efficiently.

Animals↗

PubMed2006

Data reduction and representation in drug discovery.

Pre-clinical drug discovery relies increasingly on huge volumes of inter-related multivariate data. To make sense of these data and enable quality decision-making based on this plethora of information they must be presented in an interpretable form. Reducing the dimensionality of the data often leaves a data set that is too complex to interpret readily, so intuitive visualization methods are needed. Bioinformatics has provided much of the impetus for visualizing complex data, the cheminformatics community has been aggressive with the data-reduction problem. The increasing appreciation of the inter-related multifactorial nature of pre-clinical drug discovery makes visualization a burgeoning and active field that spans biosciences, mathematics and visual psychology.

Computational Biology↗

PubMed2002

Structural enzymology of Li(+)-sensitive/Mg(2+)-dependent phosphatases.

Li(+)-sensitive/Mg(2+)-dependent phosphatases have attracted considerable attention since they have been proposed as targets for lithium therapy in the treatment of manic-depressive patients. The members of this enzyme superfamily display low levels of sequence identity while possessing a common fold and active site. Extensive structural and biochemical data demonstrate the direct involvement of two metal ions in catalysis, and show that lithium exerts its inhibitory action by blocking the products at the active site. By exploiting the different inhibitory properties of magnesium and calcium, we have been able to solve the X-ray structures of the Li(+)-sensitive/Mg(2+)-dependent 3'-phosphoadenosine-5'-phosphatase in complex with its substrate and with its products. The structural comparison of these complexes provides a 3D picture of the different stages of the catalytic cycle. This gives new insights into the understanding of the biological function of this group of enzymes and their lithium inhibition, and should assist in the design of improved inhibitors of therapeutic value.

Amino Acid Sequence↗

PubMed2005

Optimizing the structures of minimum and transition state on the free energy surface.

Presented here is the application of a scheme for optimizing the structures of minima and transition states on the free energy surface (FES) for a path along a fixed reaction coordinate with the aid of ab initio molecular dynamics (AIMD) simulation. In the direction of the reaction coordinate, the values corresponding to the stationary points were optimized using the quasi-Newton method, in which the gradient of the free energy along the reaction coordinate was obtained by a constraint AIMD method, and the Bofill Hessian update scheme was used. The equilibrium values for the other directions were taken as the corresponding averages in the dynamic simulation. This scheme was applied to several elementary bimolecular addition reactions: (A) BH(3) + H(2)O --> H(2)O.BH(3); (B) BF(3) + NH(3) --> FB(3).NH(3); (C) SO(3) + NH(3) --> O(3)S.NH(3); (D) C(2)H(4) + CCl(2) --> H(4)C(2).CCl(2); (E) Ni(NH(2))(2) + PH(3) --> (NH(2))(2)Ni.PH(3); (F) W(CO)(5) + CO --> W(CO)(6). For reactions A, B, C, and F, no transition state (TS) exists on the potential energy surface (PES). However there is a TS on the FES. This stems from the curvature difference of the PES and -TDeltaS as a function of the reaction coordinate. For all reactions, it is found that the TS shifts toward the complexation product with increasing temperature because of the curvature increase of -TDeltaS. The equilibrium bond distances for the inactive coordinates perpendicular to the reaction coordinate always increase with temperature, which is due to the thermal excitation and anharmonicity of the PES.

Journal Article↗

PubMed2005

Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY.

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation.

Amino Acid Substitution↗