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PubMedDate not supplied

Mutations in the reverse transcriptase component of telomerase (TERT) in patients with bone marrow failure.

Human telomerase has two core components, the RNA molecule (TERC) that provides the template for telomere repeat elongation and a reverse transcriptase (TERT) that is responsible for the addition of telomere repeats at the ends of each chromosome. Mutations in TERC have been found in the autosomal-dominant form of the inherited bone marrow failure syndrome dyskeratosis congenita and in a subset of patients with aplastic anemia and myelodysplasia. These patients have short telomeres compared to age-matched controls. These observations suggest that uncharacterised cases of dyskeratosis congenita/aplastic anemia may have mutations in TERT or other molecules that associate with TERC in the telomerase complex. We have therefore screened the TERT gene for mutation by denaturing HPLC in 80 patients with inherited and acquired bone marrow failure (24 with dyskeratosis congenita, 36 with constitutional aplastic anemia, 13 with idiopathic aplastic anemia and 7 with other forms of bone marrow failure). 15 different TERT mutations have been identified. Of these, 5 are in flanking intron sequences, 6 are synonymous and 4 are non-synonymous (missense) substitutions in the coding sequence. These are the first natural mutations of TERT to be described and we highlight their possible pathogenic role in the development of bone marrow failure.

Adult↗

PubMed2005

DPP10 modulates Kv4-mediated A-type potassium channels.

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase (DPP) IV known as DPP10 was recently identified and linked to asthma susceptibility; however, the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels (Nadal, M. S., Ozaita, A., Amarillo, Y., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) Neuron. 37, 449-461). We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to DPPX activity and significantly different from DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude, and modified the voltage dependence and kinetic properties of the current such that they resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization, we found DPP10 to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming the association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10, and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity.

Amino Acid Sequence↗

PubMed2003

Vaccine immunity to pathogenic fungi overcomes the requirement for CD4 help in exogenous antigen presentation to CD8+ T cells: implications for vaccine development in immune-deficient hosts.

Systemic fungal infections with primary and opportunistic pathogens have become increasingly common and represent a growing health menace in patients with AIDS and other immune deficiencies. T lymphocyte immunity, in particular the CD4+ Th 1 cells, is considered the main defense against these pathogens, and their absence is associated with increased susceptibility. It would seem illogical then to propose vaccinating these vulnerable patients against fungal infections. We report here that CD4+ T cells are dispensable for vaccine-induced resistance against experimental fungal pulmonary infections with two agents, Blastomyces dermatitidis an extracellular pathogen, and Histoplasma capsulatum a facultative intracellular pathogen. In the absence of T helper cells, exogenous fungal antigens activated memory CD8+ cells in a major histocompatibility complex class I-restricted manner and CD8+ T cell-derived cytokines tumor necrosis factor alpha, interferon gamma, and granulocyte/macrophage colony-stimulating factor-mediated durable vaccine immunity. CD8+ T cells could also rely on alternate mechanisms for robust vaccine immunity, in the absence of some of these factors. Our results demonstrate an unexpected plasticity of immunity in compromised hosts at both the cellular and molecular level and point to the feasibility of developing vaccines against invasive fungal infections in patients with severe immune deficiencies, including those with few or no CD4+ T cells.

Animals↗

PubMed2004

Generating and dimerizing the transient 16-electron phosphinidene complex [Cp*Ir=PAr]: a theoretical and experimental study.

The properties of the 16-electron phosphinidene complex [CpRIr=PR] were investigated experimentally and theoretically. Density functional theory calculations show a preferred bent geometry for the model complex [CpIr=PH], in contrast to the linear structure of [CpIr=NH]. Dimerization to give [[CpIr=PH]2] and ligand addition to afford [Cp(L)Ir=PH] (L=PH3, CO) were calculated to give compounds that were energetically highly favorable, but which differed from the related imido complexes. Transient 16-electron phosphinidene complex [Cp*Ir=PAr] could not be detected experimentally. Dehydrohalogenation of [Cp*IrCl2(PH2Ar)] in CH2Cl2 at low temperatures resulted in the novel fused-ring systems 17 (Ar=Mes*) and 20 (Ar=Mes), with dimeric [[Cp*Ir=PAr]2] being the likely intermediate. Intramolecular C-H bond activation induced by steric factors is considered to be the driving force for the irreversible formation of 17 and 20. ONIOM calculations suggest this arises because of the large steric congestion in [[Cp*Ir=PAr]2], which forces it toward a more reactive planar structure that is apt to rearrange.

Computer Simulation↗

PubMedDate not supplied

Angiogenesis in glioma: molecular mechanisms and roadblocks to translation.

Gliomas are characterized by very high levels of neo-vascularization holding out the hope that therapies aimed at angiogenesis will have a significant impact on this intractable family of tumors. Intense research into the molecular mechanisms that drive the formation of new blood vessels in response to tumor growth has revealed a great deal of complexity, at the heart of which are competing pro- and anti-angiogenic influences. The relevant signaling pathways, and how they might be manipulated to interfere in the promotion of vessel growth are discussed. Several types of anti-angiogenic lead compounds are already in clinical trials, but assessing their impact on brain tumors is not straightforward. We discuss in depth some of the practical aspects of using imaging to more meaningfully follow tumor progression and response to treatment, which is particularly relevant to the use of therapies that target blood flow directly, which is fundamental to modern imaging modalities.

Angiogenesis Inhibitors↗

PubMed2006

Local activation of the tissue factor-factor VIIa pathway in patients with pneumonia and the effect of inhibition of this pathway in murine pneumococcal pneumonia.

OBJECTIVE: The tissue factor (TF)-factor VIIa (FVIIa) complex not only is essential for activation of blood coagulation but also affect the inflammatory response during sepsis. The objective of this study was to determine the role of TF-FVIIa in pneumonia caused by Streptococcus pneumoniae, the most important causative organism in community-acquired pneumonia and a major cause of sepsis. DESIGN: A controlled, in vivo laboratory study. SETTING: Research laboratory of a health sciences university. PATIENTS AND SUBJECTS: Patients with unilateral community-acquired pneumonia and female BALB/c mice. INTERVENTIONS: Bilateral bronchoalveolar lavage was performed in patients with community-acquired pneumonia. In mice, pneumonia was induced by intranasal inoculation with S. pneumoniae with or without concurrent inhibition of TF-FVIIa by subcutaneous injections of recombinant nematode anticoagulant protein (rNAPc2). MEASUREMENTS AND MAIN RESULTS: Patients with unilateral community-acquired pneumonia demonstrated elevated concentrations of FVIIa, soluble TF, and thrombin-antithrombin complexes in bronchoalveolar lavage fluid obtained from the infected site compared with the uninfected site. Mice with S. pneumoniae pneumonia displayed increased TF expression and fibrin deposits in lungs together with elevated thrombin-antithrombin complex levels in bronchoalveolar lavage fluid; inhibition of TF-FVIIa by rNAPc2 attenuated the procoagulant response in the lung but did not affect host defense, as reflected by an unaltered outgrowth of pneumococci and an unchanged survival. CONCLUSIONS: These data suggest that TF-FVIIa activity contributes to activation of coagulation in the lung during pneumococcal pneumonia but does not play an important role in the antibacterial host defense in this murine model.

Adult↗

PubMed2006

Kinases and G proteins join the Wnt receptor complex.

Wnt proteins form a family of secreted signaling proteins that play a key role in various developmental events such as cell differentiation, cell migration, cell polarity and cell proliferation. It is currently thought that Wnt proteins activate at least three different signaling pathways by binding to seven transmembrane receptors of the Frizzled family and the co-receptor LRP6. Despite our growing knowledge of intracellular components that mediate a Wnt signal, the molecular events at the membrane have remained rather unclear. Now several publications(1-4) indicate that Frizzled receptors are G-protein coupled and kinases were identified that phosphorylate the co-receptor LRP6. These data deepen our understanding of Wnt-mediated signal transduction and provide more insight into how specificity may be achieved.

Animals↗

PubMed2005

Activation of coagulation and inhibition of fibrinolysis in the lung after inhalation of lipopolysaccharide by healthy volunteers.

Pneumonia is frequently associated with changes in coagulation and fibrinolysis in the bronchoalveolar space. To determine the effect of lipopolysaccharide (LPS) on the hemostatic balance in the human lung, six healthy subjects inhaled nebulized LPS or saline in a randomized cross-over study and bronchoalveolar lavage fluid was obtained six hours thereafter. LPS induced soluble tissue factor and thrombin-antithrombin complexes and inhibited plasminogen activator activity in BALF. Additionally plasminogen activator inhibitor type 1 production was upregulated after LPS inhalation. LPS also elicited local activation of neutrophils (release of elastase, myeloperoxidase and bactericidal/permeability increasing protein) and secretion of interleukin (IL)-6 and IL-8. Inhalation of LPS by healthy humans reproduces major features of the procoagulant response to inflammatory and infectious lung diseases and may be used as a novel model to evaluate pathogenetic mechanisms and new interventions.

Administration, Inhalation↗

PubMed2003

Hospitals in a globalized world: a view from Canada.

Globalization is a complex, multidimensional phenomenon that has already influenced the way hospitals operate and will increasingly impact the healthcare landscape and patients' experience worldwide. This paper briefly analyzes the direct and indirect effects of globalization on healthcare systems and services, mainly focusing on the experience of academic health sciences centres. Building their analysis on the belief that globalization is neither negative nor positive in itself, the authors compare alternative definitions of globalization, suggest possible ways in which it could impact health systems, examine how the role of large teaching and research institutions could evolve over the next decade or so, and put forward some fundamental questions faced by healthcare institutions. In the first part of the paper, the complex and multidimensional nature of globalization is analyzed and the highly polarized debate on the nature of this phenomenon briefly summarized. The second part focuses on the effects of globalization on health and healthcare. A pre-existing conceptual framework is used to analyze the complex linkages between globalization and health, and alternative scenarios are presented to illustrate the current and potential effects of international trade policies and regulations on health systems. In the third part, changes in hospitals' structure, organization and functions triggered by globalization and the introduction of new information and communication technologies are examined. The analysis is built around five main elements: patients, human resources, capital, information and funding. Finally, the paper highlights some of the most fundamental challenges, both practical and ethical, that healthcare institutions have to face in the transition to a new era of globalized health services.

Academic Medical Centers↗

PubMed1998

Characterization of the initial steps of precursor import into rat liver mitoplasts.

Mitochondria have two independent protein-import machineries, one in the outer membrane (the Tom system) and the other in the inner membrane (the Tim system). Here, we have characterized the initial steps of precursor import into rat liver mitoplasts. The import reaction was separated into two stages, consisting of precursor binding to the mitoplasts at 0-10 degreesC, and a subsequent chase reaction at 30 degreesC. This assay revealed four distinct precursor-import steps: DeltaPsi-dependent initial binding of the precursor, precursor transfer to the Tim23-Tim17 stage, DeltaPsi-dependent translocation of the presequence across the inner membrane, and the complete translocation of the mature portion of the precursor. Antibodies against the intermembrane space domain of Tim23 inhibited neither the precursor binding nor the subsequent translocation of the presequence across the inner membrane. In contrast, the antibodies inhibited the complete translocation of the mature domain of the precursor across the inner membrane. Immunoprecipitation with anti-Tim23 IgGs revealed that the precursor-Tim23 complex increased with time and temperature after the initial targeting of the precursor to the mitoplasts. These results suggest that the precursor is first targeted to the inner membrane component DeltaPsi-dependently, then transferred to the Tim system consisting of Tim23-Tim17, and finally imported into the matrix.

Amino Acid Sequence↗

PubMed1999

Cloning of a novel cytochrome P450 (CYP4C15) differentially expressed in the steroidogenic glands of an arthropod.

Biosynthesis of ecdysteroids, arthropod steroid molting hormones, proceeds from dietary cholesterol through a complex and still incompletely elucidated pathway. Most of the known steps are catalyzed by cytochrome P450 enzymes (CYPs) but none of their genes has yet been identified. We have established a cDNA library of crayfish steroidogenic glands (Y organs). A full length CYP-cDNA was characterized containing a 1539 bp open reading frame encoding a predicted protein of 513 amino acid residues. This novel CYP was assigned to the CYP4 family and designated CYP4C15. Northern blots demonstrated predominant expression of this gene in the active molting glands, suggesting a role in ecdysteroid biosynthesis rather than detoxification.

Amino Acid Sequence↗

PubMed2005

Assembly and mechanosensory function of focal adhesions: experiments and models.

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.

Actin Cytoskeleton↗

PubMed2005

Mycobacterium tuberculosis protein tyrosine phosphatase PtpB structure reveals a diverged fold and a buried active site.

Intracellular pathogenic bacteria manipulate host signal transduction pathways to facilitate infection. Mycobacterium tuberculosis protein tyrosine phosphatases (PTPs) PtpA and PtpB are thought to be secreted into host cells and interfere with unidentified signals. To illuminate the mechanisms of regulation and substrate recognition, we determined the 1.7 A resolution crystal structure of PtpB in complex with the product phosphate. The protein adopts a simplified PTP fold, which combines features of the conventional PTPs and dual-specificity phosphatases. PtpB shows two unusual elaborations--a disordered, acidic loop and a flexible, two-helix lid that covers the active site--that are specific to mycobacterial orthologs. Biochemical studies suggest that substrate mimicry in the lid may protect the phosphatase from oxidative inactivation. The insertion and deletion of large structural elements in PtpB suggest that, outside the active site module, the PTP family is under unusual selective pressure that promotes changes in overall structure.

Binding Sites↗

PubMed2004

Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta enhancer.

Transcriptional activation of the interferon-beta (IFN-beta) gene requires assembly of an enhanceosome containing the transcription factors ATF-2/c-Jun, IRF-3/IRF-7, NF-kappaB and HMGI(Y). These factors cooperatively bind a composite DNA site and activate expression of the IFN-beta gene. The 3.0 A crystal structure of the DNA-binding domains of ATF-2/c-Jun and two IRF-3 molecules in a complex with 31 base pairs (bp) of the PRDIV-PRDIII region of the IFN-beta enhancer shows that association of the four proteins with DNA creates a continuous surface for the recognition of 24 bp. The structure, together with in vitro binding studies and protein mutagenesis, shows that protein-protein interactions are not critical for cooperative binding. Instead, cooperativity arises mainly through nucleotide sequence-dependent structural changes in the DNA that allow formation of complementary DNA conformations. Because the binding sites overlap on the enhancer, the unit of recognition is the entire nucleotide sequence, not the individual subsites.

Activating Transcription Factor 2↗

PubMed2004

Fast, but error-prone, responses during acute alcohol intoxication: effects of stimulus-response mapping complexity.

BACKGROUND: Although moderate doses of alcohol can impair performance on tasks that require information processing, little is known about the locus of the alcohol effects within the processing stream. This study used a psychological refractory period paradigm to investigate the effect of alcohol on the central, cognitive stage of information processing when task complexity is manipulated by altering stimulus-response compatibility. METHODS: Thirty-four healthy male social drinkers were assigned to one of two groups (n = 17) that performed two tasks. Each trial consisted of a task 1 stimulus (tone) followed by a task 2 stimulus (letter) that was presented after one of four stimulus onset asynchronies (50, 200, 500, or 1100 msec). A baseline test of performance was obtained before the groups received a beverage containing either 0.0 g/kg (placebo) or 0.65 g/kg alcohol. Both groups were retested when blood alcohol concentration (BAC) was increasing and was decreasing. RESULTS: The alcohol group made significantly more errors in task 1 compared with their drug-free baseline measure during the ascending phase of the BAC curve, and error rates increased to a greater extent for the more complex arbitrary stimulus-response mapping condition. Moreover, this increase in errors continued unabated during the descending phase of the BAC curve. Increasing BACs also slowed performance (longer reaction time), but unlike errors, reaction time returned to drug-free baseline levels when BAC was decreasing. CONCLUSIONS: The results provide evidence that an acute dose of alcohol can impair one aspect of the central, cognitive stages of information processing. The possibility that errors in information processing remain during decreasing BACs even after processing speed has returned to drug-free levels has important practical implications relating to the detrimental consequences of acute alcohol intoxication.

Adult↗

PubMed2002

Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients.

The opportunistic human pathogen Achromobacter (Alcaligenes) xylosoxidans has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF). However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF. Misidentification of A. xylosoxidans as Burkholderia cepacia complex is especially problematic and presents a challenge to effective infection control in CF. To address the problem of accurate identification of A. xylosoxidans, we developed a PCR assay based on a 16S ribosomal DNA sequence. In an analysis of 149 isolates that included 47 A. xylosoxidans and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively. The availability of this assay will enhance identification of A. xylosoxidans, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.

Alcaligenes↗

PubMed2004

Lipid/peptide/nucleotide separation with MALDI-ion mobility-TOF MS.

Matrix-assisted laser desorption/ionization when combined with ion mobility-orthogonal time-of-flight mass spectrometry is a viable technique for fast separation and analysis of biomolecules in complex mixtures. Isobaric lipid, peptide, and oligonucleotide ions are preseparated before mass analysis by differences of up to 30% in mobility drift time. Ions of similar chemical type fall along well-defined "trend lines" (with deviations of approximately 3%) when plotted in two-dimensional representations of ion mobility as a function of m/z. Discussion of fundamental and technical limitations of the technique point to its potential for being most useful when applied to systems such as bodily fluids and intact tissue, where an alternative chemical or chromatographic preseparation step prior to mass analysis is either impractical or undesirable.

Analgesics, Opioid↗

PubMed1972

The dissociation of avidin-biotin complexes by guanidinium chloride.

Avidin molecules in which a fraction of the four binding sites were occupied by biotin did not dissociate completely in 6.4m-guanidinium chloride. Only unoccupied subunits dissociated. The remainder recombined to form the tetrameric avidin-biotin complex. The rate at which unoccupied subunits were unfolded and dissociated was only decreased by one-half in species in which three of the four binding sites were occupied by biotin. These results can be explained by assuming that unfolding of unoccupied subunits followed by dissociation from the tetramer is initiated by penetration of guanidinium ions into the binding site and disorganization of this region of the subunit. When a site is occupied by biotin this pathway is blocked and the subunit does not unfold. Each subunit behaves independently and is not markedly stabilized when neighbouring subunits are occupied.

Avidin↗