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PubMed2006

The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261.

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

Binding Sites↗
PubMed1996

Translocation arrest of an intramitochondrial sorting signal next to Tim11 at the inner-membrane import site.

The import of proteins from the cytosol into the mitochondrial matrix involves the concerted action of two separate import systems: the TOM system in the outer membrane, and the TIM system in the inner membrane. Here we report that the inner-membrane system also sorts proteins to the intermembrane space. Some intermembrane-space proteins, such as cytochromes b2 and c1, are synthesized with a complex pre-sequence consisting of a positively charged matrix targeting signal followed by an uncharged sequence that acts as sorting signal for the intermembrane space. We show that this sorting signal can be efficiently crosslinked to an inner-membrane protein of relative molecular mass 11K after the mature part of the precursor has been sorted to the intermembrane space. The 11K protein, which we term Tim11, is a component of the protein import system in the inner membrane.

Adenosine Triphosphate↗
PubMed2005

A structural basis for allosteric control of DNA recombination by lambda integrase.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage lambda to integrate or excise its genome into and out of the host chromosome. lambda recombination is carried out by the bacteriophage-encoded integrase protein (lambda-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of lambda-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of lambda-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.

Allosteric Regulation↗
PubMed2004

The FHA domain protein SNIP1 is a regulator of the cell cycle and cyclin D1 expression.

Smad nuclear interacting protein 1 (SNIP1) is an evolutionarily conserved protein containing a forkhead-associated (FHA) domain that regulates gene expression through interactions with multiple transcriptional regulators. Here, we have used short interfering RNAs (siRNAs) to knockdown SNIP1 expression in human cell lines. Surprisingly, we found that reduction in SNIP1 levels resulted in significantly reduced cell proliferation and accumulation of cells in the G1 phase of the cell cycle. Consistent with this result, we observed that cyclin D1 protein and mRNA levels were reduced. Moreover, SNIP1 depletion results in inhibition of cyclin D1 promoter activity in a manner dependent upon a previously characterized binding site for the AP-1 transcription factor family. SNIP1 itself is induced upon serum stimulation immediately prior to cyclin D1 expression. These effects were independent of the tumour suppressors p53 and retinoblastoma (Rb), but were consistent with an interaction with BRG1, a component of the ATP-dependent chromatin remodelling complex, Swi/Snf. These results define both a new function for SNIP1 and identify a previously unrecognized regulator of the cell cycle and cyclin D1 expression.

Base Sequence↗
PubMed2005

An outbreak of serotype 1 Streptococcus pneumoniae meningitis in northern Ghana with features that are characteristic of Neisseria meningitidis meningitis epidemics.

BACKGROUND: The Kassena-Nankana District (KND) of northern Ghana lies in the African meningitis belt, where epidemics of bacterial meningitis have been reoccurring every 8-12 years. These epidemics are generally caused by Neisseria meningitidis, an organism that is considered to be uniquely capable of causing meningitis epidemics. METHODS: We recruited all patients with suspected meningitis in the KND between 1998 and 2003. Cerebrospinal fluid samples were collected and analyzed by standard microbiological techniques. Bacterial isolates were subjected to serotyping, multilocus sequence typing (MLST), and antibiotic-resistance testing. RESULTS: A continual increase in the incidence of pneumococcal meningitis was observed from 2000 to 2003. This outbreak exhibited strong seasonality, a broad host age range, and clonal dominance, all of which are characteristic of meningococcal meningitis epidemics in the African meningitis belt. The case-fatality rate for pneumococcal meningitis was 44.4%; the majority of pneumococcal isolates were antibiotic sensitive and expressed the serotype 1 capsule. MLST revealed that these isolates belonged to a clonal complex dominated by sequence type (ST) 217 and its 2 single-locus variants, ST303 and ST612. CONCLUSIONS: The S. pneumoniae ST217 clonal complex represents a hypervirulent lineage with a high propensity to cause meningitis, and our results suggest that this lineage might have the potential to cause an epidemic. Serotype 1 is not included in the currently licensed pediatric heptavalent pneumococcal vaccine. Mass vaccination with a less complex conjugate vaccine that targets hypervirulent serotypes should, therefore, be considered.

Drug Resistance, Bacterial↗
PubMed2006

Effect of a novel molybdenum ascorbate complex on ex vivo myocardial performance in chemical diabetes mellitus.

BACKGROUND: The insulin-like action of metal complexes on target tissues, including the heart, has been reported in experimental diabetes mellitus. Since streptozotocin-induced diabetes is associated with insulin deficiency and left ventricular dysfunction, this study was designed to determine whether the novel metal complex molybdenum ascorbate [MoO(2)(aa)(2)] would improve cardiac function in this model of diabetes. METHODS: Diabetes was induced in Sprague-Dawley rats (n = 6) following an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks of diabetes, cardiac function was determined in isolated working hearts perfused with 11 mmol/L glucose, 1.2 mmol/L palmitate and 3% albumin. MoO(2)(aa)(2 )was added directly into the perfusate of working hearts at a concentration of 200 micromol/L for a period of 30 minutes. Age-matched control rats served as controls (n = 6). RESULTS: Cardiac function, expressed as heart rate (HR) and aortic flow, was significantly decreased in diabetic hearts compared with control hearts. The diabetic state was associated with 23% and 60% reductions in HR and aortic flow, respectively. Short-term addition of MoO(2)(aa)(2) was beneficial and partially prevented the attenuation in diabetic rat heart function. MoO(2)(aa)(2 )increased HR by 15%, while aortic flow was increased by 85%. In control hearts, MoO(2)(aa)(2) had no effect on HR and increased aortic flow by 12%. CONCLUSION: This study extends previous observations on the benefit of metal complexes in experimental diabetes. Our results indicate that short-term treatment with MoO(2)(aa)(2) partially reversed the left ventricular dysfunction associated with the streptozotocin model of diabetes.

Animals↗
PubMed2006

Tim17p regulates the twin pore structure and voltage gating of the mitochondrial protein import complex TIM23.

The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.

Membrane Transport Proteins↗
PubMed2002

Human cell DNA replication is mediated by a discrete multiprotein complex.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.

Cell Line↗
PubMed2002

Increased CCR5 affinity and reduced CCR5/CD4 dependence of a neurovirulent primary human immunodeficiency virus type 1 isolate.

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.

AIDS Dementia Complex↗
PubMed2005

Exposure and binding of selected immunodominant La/SSB epitopes on human apoptotic cells.

OBJECTIVE: Opsonization of apoptotic cells by autoantibodies bound to surface membrane-translocated La/SSB antigens may initiate tissue damage in the setting of congenital heart block. By injecting pregnant mice with human anti-La antibodies, we previously demonstrated the formation of IgG-apoptotic cell complexes in the developing mouse fetus; however, the binding of anti-La antibodies to human-specific epitopes could not be addressed. Accordingly, the objective of the current study was to delineate the epitope specificity of human La antibodies that are exposed on the surface of apoptotic cells. METHODS: We used fluorescence microscopy and flow cytometry to assess the binding of human anti-La antibodies affinity purified against immunodominant epitopes of La to human cells undergoing spontaneous apoptosis, in a murine xenograft model in vivo and in cultured human fetal cardiocytes rendered apoptotic in vitro, respectively. RESULTS: Anti-La antibodies bound to immunodominant epitopes of La within the NH(2)-terminus and the RNA recognition motif (RRM) region of apoptotic human cells, in both xenografts and fetal cardiocytes. In contrast, human antibodies affinity purified against the COOH-terminal La epitope did not bind apoptotic cells in either model. This defines the topology of redistributed La during apoptosis, with surface exposure of the NH(2)-terminus and RRM regions. The potential importance of anti-La NH(2)-terminal and anti-La RRM specificity was confirmed by detection of this reactivity in mothers of children with congenital heart block. CONCLUSION: These findings provide insight into both the molecular modification of the La autoantigen during apoptosis and the specificity of antibodies capable of binding to surface-exposed La. Subsequent formation of surface immune complexes may lead to tissue injury in patients with autoimmune diseases such as congenital heart block.

Animals↗
PubMed2006

Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells.

IL-23 is regarded as a major pro-inflammatory mediator in autoimmune disease, a role which until recently was ascribed to its related cytokine IL-12. IL-23, an IL-12p40/p19 heterodimeric protein, binds to IL-12Rbeta1/IL-23R receptor complexes. Mice deficient for p19, p40 or IL-12Rbeta1 are resistant to experimental autoimmune encephalomyelitis or collagen-induced arthritis. Paradoxically, however, IL-12Rbeta2- and IL-12p35-deficient mice show remarkable increases in disease susceptibility, suggesting divergent roles of IL-23 and IL-12 in modulating inflammatory processes. IL-23 induces IL-17, which mediates inflammation and tissue remodeling, but the role of IL-12 in this respect remains unidentified. We investigated the roles of exogenous (recombinant) and endogenous (macrophage-derived) IL-12 and IL-23, on IL-17-induction in human T-cells. IL-23 enhanced IL-17 secretion, as did IL-2, IL-15, IL-18 and IL-21. In contrast, IL-12 mediated specific inhibition of IL-17 production. These data support the role of IL-23 in inflammation through stimulating IL-17 production by T lymphocytes, and importantly indicate a novel regulatory function for IL-12 by specifically suppressing IL-17 secretion. These data therefore extend previous reports that had indicated unique functions for IL-23 and IL-12 due to distinct receptor expression and signal transduction complexes, and provide novel insights into the regulation of immunity, inflammation and immunopathology.

Animals↗
PubMed2005

Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution.

The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange. This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways. We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects. The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution.

Alleles↗
PubMed2004

Restriction enzyme-generated siRNA (REGS) vectors and libraries.

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme-generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 x 10(5) clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.

Animals↗
PubMed2005

Using surveys in public participation processes for risk decision making: the case of the 2003 British GM Nation? Public debate.

This article takes as its case study the "GM Nation?" public debate, a major participation process on the commercialization of agricultural biotechnology, which occurred in Britain during the summer of 2003. We investigate possible self-selection biases in over 36,000 open questionnaire responses on the risks and benefits of genetically modified crops and food obtained during GM Nation? A comparison sample of equivalent responses from a statistically representative sample (n = 1,363) of the British general public obtained shortly after the conclusion of the debate is reported. This comparison shows that the GM Nation? open responses were indeed not fully representative of British "public opinion" regarding agricultural biotechnology. Rather, such opinion is not a unitary whole, but fragmented, with considerable ambivalence coexisting alongside outright opposition to GM agriculture. The methodological implications for multistage participation processes are discussed: in particular, the need to anticipate outcomes of complex design decisions, and to include representative public surveys as standard where measures of broader public attitudes to risk are an important objective.

Agriculture↗
PubMed2003

Medical errors, incidents, accidents and violations.

Atul Gawande: 'The real problem isn't how to stop bad doctors from harming, even killing their patients. It's how to prevent good doctors from doing so.' A. Gawande: When doctors make mistakes. The New Yorker, 1st February 1999; 40-55.Errors are an integral part of human behaviour and performance, and in this respect, medical/ surgical practice is no exception. Undoubtedly medical errors account for substantial patient morbidity and mortality. The subject is complicated due to complex and at times confusing taxonomy and the lack of agreed definitions and classification of medical/ surgical errors. It is not possible to eliminate errors from clinical practice but we can improve the quality of medical care by adopting error-tolerant operating medical systems (E-TOMS) based on progress in cognitive psychology, human factors, and human reliability assessment made during the past 30 years. E-TOMS should enable detection, reporting and targeted reduction of errors, and together with effective team dynamics, good clinical governance incorporating root-cause analysis of adverse events during the delivery of health care should improve the quality of care that we can provide for our patients.

Journal Article↗
PubMed2006

Comparison of in vitro and in vivo complement activation by metal and bioabsorbable screws used in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of the study was to evaluate the biocompatibility of polylactide (PLLA) screws in comparison with standard metal screws for fixation of the patellar tendon graft in human anterior cruciate ligament (ACL) reconstruction. METHODS: A total of 41 patients (22 women and 19 men) were prospectively randomized for the use of metal interference screws (20 patients) or biologically resorbable PLLA screws from Linvatec, Largo, FL (21 patients). Average age at the time of surgery was 26 years (15 to 51 y). Synovial fluid and plasma were collected preoperatively and after 6 weeks in both groups. Plasma was analyzed for C5a and synovial fluid, as well as for terminal SC5b-9 complement complex (TCC) and interleukin (IL)-8. At 1 year after surgery, serum was incubated with metal, PLLA, and no screws; this was followed by analysis of C5a after 1 and 6 hours of incubation. Inflammatory mediators were measured through enzyme-linked immunosorbent assay (ELISA). RESULTS: In the BioScrew group, 4 patient samples showed high C5a concentration in synovial fluid after 6 weeks, but no statistically significant difference was observed between the 2 groups (P = .11). One patient in the BioScrew group had a high TCC value after 6 weeks, but no statistically significant difference was seen between the 2 groups (P = .20). In the in vitro study, no increased C5a generation was observed in sera incubated with a BioScrew or a metal screw compared with controls. CONCLUSIONS: No statistically significant difference was observed between the BioScrew and metal screw groups concerning C5a, TCC, and IL-8 formation. However, some patients in the BioScrew group showed elevated values. LEVEL OF EVIDENCE: Level II, prospective randomized trial.

Absorbable Implants↗
PubMed2003

Longitudinal study of tuberculosis outcomes among immunologically naive Aché natives of Paraguay.

This study documents the course of a tuberculosis epidemic in an immunologically naive group of South American Indians within fewer than 20 years after first sustained contact with outsiders. Groups of Northern Aché (ah-CHAY) of eastern Paraguay were contacted and settled on reservations between 1971-1979. Not surprisingly, the Aché are very susceptible to tuberculosis, and the epidemiological characteristics of the disease are quite different from those of populations that have had tuberculosis for centuries. Within 6 years of the first detected case of tuberculosis among the Aché, the prevalence rate of active tuberculosis cases reached 18.2%, and of infected cases among adults, 64.6%, some of the highest rates ever reported for any human group. Remarkably, males and females are equally likely to have been diagnosed with active tuberculosis, Aché children between birth and 5 years of age are least vulnerable to tuberculosis, high nutritional and socioeconomic status do not decrease the risk of disease or infection, and children immunized with BCG are less responsive to tuberculin challenge than are other children. Moreover, similar to the Yanomamö, but unlike populations of European or African descent, a high percentage of Aché with active disease test negative on tuberculin challenge tests (purified protein derivative; PPD). These differences may be due to a high prevalence of diminished cell-mediated immunity, and T-helper 2 dominance. We also hypothesize that these immunological characteristics, low genetic diversity, hostile intergroup interactions, and behavioral noncompliance to treatment protocols together contribute to the high rates of active disease observed. Existing tuberculosis control programs are poorly equipped to handle the impact of these causal complexities on the course of recent tuberculosis epidemics that have quickly spread throughout native communities of Latin America during the last decade.

Adolescent↗
PubMed2002

Stabilization and first direct spectroscopic evidence of the o-quinone methide derived from vitamin E.

[reaction: see text] The o-quinone methide (2) derived from vitamin E (1) can be stabilized at low temperatures in a complex with the highly polar N-methylmorpholine-N-oxide (5). The lifetime of 2 can thus be prolonged from less than 10 s to several minutes. In the complex formed, 2 adopts a zwitterionic, aromatic structure with the exocyclic methylene group in perpendicular arrangement to the ring plane, stabilized by the negatively charged oxygen in 5.

Drug Stability↗