Showing papers by "David Eisenberg published in 2006"

Toll-Like Receptor Triggering of a Vitamin D-Mediated Human Antimicrobial Response

Abstract: In innate immune responses, activation of Toll-like receptors (TLRs) triggers direct antimicrobial activity against intracellular bacteria, which in murine, but not human, monocytes and macrophages is mediated principally by nitric oxide. We report here that TLR activation of human macrophages up-regulated expression of the vitamin D receptor and the vitamin D-1-hydroxylase genes, leading to induction of the antimicrobial peptide cathelicidin and killing of intracellular Mycobacterium tuberculosis. We also observed that sera from African-American individuals, known to have increased susceptibility to tuberculosis, had low 25-hydroxyvitamin D and were inefficient in supporting cathelicidin messenger RNA induction. These data support a link between TLRs and vitamin D-mediated innate immunity and suggest that differences in ability of human populations to produce vitamin D may contribute to susceptibility to microbial infection.

Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis

Abstract: The developing science called structural genomics has focused to date mainly on high-throughput expression of individual proteins, followed by their purification and structure determination. In contrast, the term structural biology is used to denote the determination of structures, often complexes of several macromolecules, that illuminate aspects of biological function. Here we bridge structural genomics to structural biology with a procedure for determining protein complexes of previously unknown function from any organism with a sequenced genome. From computational genomic analysis, we identify functionally linked proteins and verify their interaction in vitro by coexpression/copurification. We illustrate this procedure by the structural determination of a previously unknown complex between a PE and PPE protein from the Mycobacterium tuberculosis genome, members of protein families that constitute ≈10% of the coding capacity of this genome. The predicted complex was readily expressed, purified, and crystallized, although we had previously failed in expressing individual PE and PPE proteins on their own. The reason for the failure is clear from the structure, which shows that the PE and PPE proteins mate along an extended apolar interface to form a four-α-helical bundle, where two of the α-helices are contributed by the PE protein and two by the PPE protein. Our entire procedure for the identification, characterization, and structural determination of protein complexes can be scaled to a genome-wide level.

The 3D profile method for identifying fibril-forming segments of proteins

Abstract: Based on the crystal structure of the cross-β spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed β-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using rosettadesign, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of ≈80% with a P value of ≈10−12 when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.

Unique Transcriptome Signature of Mycobacterium tuberculosis in Pulmonary Tuberculosis

Abstract: Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.

Abstract: Mycobacterial genomes contain two unique gene families, the so-called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT-6-like secretion system. This cluster, designated ESX-5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX-5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX-5 mutant of M. marinum was affected in spreading to uninfected macrophages.

Structural models of amyloid-like fibrils.

Abstract: Amyloid fibrils are elongated, insoluble protein aggregates deposited in vivo in amyloid diseases, and amyloid-like fibrils are formed in vitro from soluble proteins. Both of these groups of fibrils, despite differences in the sequence and native structure of their component proteins, share common properties, including their core structure. Multiple models have been proposed for the common core structure, but in most cases, atomic-level structural details have yet to be determined. Here we review several structural models proposed for amyloid and amyloid-like fibrils and relate features of these models to the common fibril properties. We divide models into three classes: Refolding, Gain-of-Interaction, and Natively Disordered. The Refolding models propose structurally distinct native and fibrillar states and suggest that backbone interactions drive fibril formation. In contrast, the Gain-of-Interaction models propose a largely native-like structure for the protein in the fibril and highlight the importance of specific sequences in fibril formation. The Natively Disordered models have aspects in common with both Refolding and Gain-of-Interaction models. While each class of model suggests explanations for some of the common fibril properties, and some models, such as Gain-of-Interaction models with a cross-beta spine, fit a wider range of properties than others, no one class provides a complete explanation for all amyloid fibril behavior.

The Structural Biology of Protein Aggregation Diseases: Fundamental Questions and Some Answers

Abstract: Amyloid fibrils are found in association with at least two dozen fatal diseases. The tendency of numerous proteins to convert into amyloid-like fibrils poses fundamental questions for structural biology and for protein science in general. Among these are the following: What is the structure of the cross-beta spine, common to amyloid-like fibrils? Is there a sequence signature for proteins that form amyloid-like fibrils? What is the nature of the structural conversion from native to amyloid states, and do fibril-forming proteins have two distinct stable states, the native state and the amyloid state? What is the basis of protein complementarity, in which a protein chain can bind to itself? We offer tentative answers here, based on our own recent structural studies.

Factors associated with dietary supplement use among prescription medication users

Abstract: Background: We examined the patterns of nonvitamin dietary supplement (NVDS) use among adult prescription medication users in the United States. Methods:Usingthe2002NationalHealthInterviewSurvey, we analyzed factors associated with NVDS use and prescription medication use in the prior 12 months with descriptive, 2 , and logistic regression analysis. Results: In the United States, 21% of adult prescription medication users reported using NVDSs in the prior 12 months. Of the respondents who used both prescription medications and NVDSs in the prior 12 months, 69% did not discuss this use with a conventional medical practitioner. Among adults who used prescription medications in the prior 12 months, the most commonly used supplementsincludedechinacea,ginseng,ginkgo,garlic,andglucosaminechondroitin.Prescriptionmedicationuserswith menopauseandchronicgastrointestinaldisordershadthe highest rates of NVDS use (33% and 28%, respectively), andprescriptionmedicationuserswithcoronaryheartdisease and history of myocardial infarction had the lowest rates of use (12% each). In the adjusted analysis, factors associated with increased use of NVDSs by prescription medication users included being female, being Hispanic, having more years of education, living in the West, lackingmedicalinsurance,andhavingchronicconditions.Elderly respondents were less likely to use NVDSs. Conclusion:Onein4prescriptionmedicationuserstook an NVDS in the prior 12 months, yet the majority did not share this with a conventional medical professional. Arch Intern Med. 2006;166:1968-1974

A systematic screen of β2-microglobulin and insulin for amyloid-like segments

Abstract: Identifying sequence determinants of fibril-forming proteins is crucial for understanding the processes causing >20 proteins to form pathological amyloid depositions. Our approach to identifying which sequences form amyloid-like fibrils is to screen the amyloid-forming proteins human insulin and β2-microglobulin for segments that form fibrils. Our screen is of 60 sequentially overlapping peptides, 59 being six residues in length and 1 being five residues, covering every noncysteine-containing segment in these two proteins. Each peptide was characterized as amyloid-like or nonfibril-forming. Amyloid-like peptides formed fibrils visible in electron micrographs or needle-like microcrystals showing a cross-β diffraction pattern. Eight of the 60 peptides (three from insulin and five from β2-microglobulin) were identified as amyloid-like. The results of the screen were used to assess the computational method, and good agreement between prediction and experiments was found. This agreement suggests that the pair-of-sheets, zipper spine model on which the computational method is based is at least approximately correct for the structure of the fibrils and suggests the nature of the sequence signal for formation of amyloid-like fibrils.

Correlates of use of different types of complementary and alternative medicine by breast cancer survivors in the nurses' health study.

Abstract: Purpose Among breast cancer survivors, we identified the prevalence and correlates of use of different types of complementary and alternative medicine (CAM).

Runaway domain swapping in amyloid-like fibrils of T7 endonuclease I.

Abstract: Amyloid fibrils are associated with >20 fatal human disorders, including Alzheimer's, Parkinson's, and prion diseases. Knowledge of how soluble proteins assemble into amyloid fibrils remains elusive despite its potential usefulness for developing diagnostics and therapeutics. In at least some fibrils, runaway domain swapping has been proposed as a possible mechanism for fibril formation. In runaway domain swapping, each protein molecule swaps a domain into the complementary domain of the adjacent molecule along the fibril. Here we show that T7 endonuclease I, a naturally domain-swapped dimeric protein, can form amyloid-like fibrils. Using protein engineering, we designed a double-cysteine mutant that forms amyloid-like fibrils in which molecules of T7 endonuclease I are linked by intermolecular disulfide bonds. Because the disulfide bonds are designed to form only at the domain-swapped dimer interface, the resulting covalently linked fibrils show that T7 endonuclease I forms fibrils by a runaway domain swap. In addition, we show that the disulfide mutant exists in two conformations, only one of which is able to form fibrils. We also find that domain-swapped dimers, if locked in a close-ended dimeric form, are unable to form fibrils. Our study provides strong evidence for runaway domain swapping in the formation of an amyloid-like fibril and, consequently, a molecular explanation for specificity and stability of fibrils. In addition, our results suggest that inhibition of fibril formation for domain-swapped proteins may be achieved by stabilizing domain-swapped dimers.

Crystal Structure of the Pantothenate Synthetase from Mycobacterium tuberculosis, Snapshots of the Enzyme in Action.

Abstract: Pantothenate synthetase (PS) from Mycobacterium tuberculosis represents a potential target for antituberculosis drugs. PS catalyzes the ATP-dependent condensation of pantoate and β-alanine to form pantothenate. Previously, we determined the crystal structure of PS from M. tuberculosis and its complexes with AMPCPP, pantoate, and pantoyl adenylate. Here, we describe the crystal structure of this enzyme complexed with AMP and its last substrate, β-alanine, and show that the phosphate group of AMP serves as an anchor for the binding of β-alanine. This structure confirms that binding of β-alanine in the active site cavity can occur only after formation of the pantoyl adenylate intermediate. A new crystal form was also obtained; it displays the flexible wall of the active site cavity in a conformation incapable of binding pantoate. Soaking of this crystal form with ATP and pantoate gives a fully occupied complex of PS with ATP. Crystal structures of these complexes with substrates, the reaction intermediate, a...

The diagnosis and treatment of chronic back pain by acupuncturists, chiropractors, and massage therapists.

Abstract: Objectives To describe the diagnostic and therapeutic content of visits for chronic back pain to acupuncturists, chiropractors, and massage therapists. Methods Randomly selected acupuncturists, chiropractors, and massage therapists in two states were surveyed, and then eligible providers collected data on consecutive patient visits. The authors analyzed information on diagnosis, treatment, and self-care recommendations for chronic back pain patients collected during consecutive patient visits to these complementary and alternative medicine (CAM) providers. Results Back pain was the most common reason for visits to each of these providers, with chronic back pain representing about 10% of visits to acupuncturists, 20% of visits to chiropractors, and 12% of visits to massage therapists. Diagnosis by acupuncturists included traditional questioning and inspecting the patient as well as pulse and tongue assessment and palpation of the acupuncture meridians. Treatments usually included acupuncture needling, heat of some sort, and other modalities, such as East Asian massage, herbs, and/or cupping (application of suction cups to the skin). Lifestyle recommendations were common, particularly exercise and dietary counseling. Visits to chiropractors usually included spinal and muscle/soft tissue examinations and spinal manipulation. Soft tissue techniques (eg, "active release"), stretch or strength training, and home exercise recommendations were much less common. Massage therapists usually performed a tissue assessment and commonly assessed range of motion. They emphasized Swedish, deep tissue, and trigger point massage techniques and usually made self-care recommendations, particularly increased water intake, hot/cold therapy, exercise, and body awareness. Conclusion Information on the care patients routinely receive from CAM providers will help physicians better understand these increasingly popular forms of care.

Methods and materials for characterizing and modulating interaction between heregulin and HER3

Abstract: The disclosure provided herein identifies and characterizes the domain in HER3 receptor that interacts with heregulin ligand. Typical embodiments of the invention disclosed herein include HER3 variant polypeptides having amino acid sequences which differ from the native HER3 polypeptide sequence and which have altered affinities for heregulin. Also disclosed herein are methods and materials for identifying compounds that bind to the heregulin binding domain in HER3 as well as methods and materials for modulating the interaction between HER3 and heregulin.

Detection of errors in protein models

Abstract: The detection of errors in protein models is discussed. Programs used in this field, including PROCHECK, WHAT IF, VERIFY3D and ERRAT, are described and the type of errors that they detect are outlined. Examples of the detection of errors in structures are provided with a focus on the programs VERIFY3D and ERRAT. Keywords: ERRAT; PROCHECK; VERIFY3D; WHAT IF; errors; structure validation

Granulysin peptides and methods of use thereof

Abstract: Granulysin peptides are small antimicrobial agents with potent activity against bacteria and inflammation. A pharmaceutical composition comprising granulysin peptides as an active agent is administered therapeutically to a patient suffering from a microbial infection.

Guided Imagery for Smoking Cessation in Adults: A Randomized Pilot Trial

Abstract: This pilot study describes a randomized controlled trial of an audio CD—based interactive guided imagery program for smoking cessation for adults versus a wait-listed control Feasibility, process measures, and biochemically validated abstinence were assessed at end of treatment (6 weeks) and 12 weeks, as well as at 52 weeks for intervention participants Fifty-nine percent of intervention participants attended four of six guided imagery sessions, and 94% found the technique helpful for smoking cessation Intervention participants had greater readiness to quit (Readiness to Quit Ladder, 83 vs 72, p < 05) and lower state anxiety (Spielberger Index, 32 vs 38, p < 05) at end of treatment than the control group Abstinence rates in the intervention versus control groups were 36% versus 18% (p = 43) at 6 weeks and 30% versus 12% (p = 40) at 12 weeks, respectively At 1 year, 24% of intervention participants remained abstinent A guided imagery program for smoking cessation was feasible, perceived to be

Bioinformatic challenges for the next decade(s)

Abstract: The science of bioinformatics has developed in the wake of methods to determine the sequences of the informational macromolecules—DNAs, RNAs and proteins. But in a wider sense, the biological world depends in its every process on the transmission of information, and hence bioinformatics is the fundamental core of biology. We here give a consideration of some of the key problems of bioinformatics in the coming decade, and perhaps longer.

Debating the Worth of NCCAM Research.

Abstract: Marcus and Grollman miss the mark in their critique of the National Center for Complementary and Alternative Medicine (NCCAM) (“Review for NCCAM is overdue,” D. M. Marcus and A. P. Grollman, Policy Forum, 21 July, p. 301). We believe that NCCAM, under the leadership of Stephen Straus and Margaret Chesney, has made remarkable progress in laying the groundwork and advancing rigorous research in complementary and alternative medicine (CAM). They have brought definition, a conceptual framework, strategic plans and goals, and scientific standards to the field of CAM research. The processes through which proposals are submitted, reviewed, funded, and managed are all consistent with standard NIH practice. The disciplinary diversity of the NCCAM study section members and NCCAM councils is in keeping with the breadth of CAM research. The broad representation is also consistent with current practice in other centers and institutes. NIH advisory bodies regularly include members who are grantees, and well-tested procedures are in place for managing conflicts of interest. The same procedures are used for the study sections and advisory councils for NCCAM. Marcus and Grollman's comment that the NCCAM research agenda is shaped more by politics than by science is gratuitous, as is their suggestion that the Institute of Medicine (IOM) report, Complementary and Alternative Medicine in the United States ([1][1]), was flawed because some of the members of the panel were NCCAM grantees. In fact, like NIH, the IOM has procedures for recognizing and managing conflicts of interest. Those of us who participated in it were very mindful of any potential conflicts of interest and guarded against them in our deliberations. Further, the report was carefully reviewed by external, independent reviewers before publication. Because CAM is already in the public domain, used by millions of people at a cost of billions of dollars each year and with health effects that largely have not yet been scientifically evaluated, it is appropriate that a significant focus be on clinical research. As is true in clinical trials with new conventional drugs, we should expect that many trials of CAM treatments will not show definitive efficacy, and as with most research on understudied agents, multiple studies are often needed to develop a research base adequate for mature judgment concerning efficacy or the lack thereof. We need to be patient and use our best tool, that is, science, to understand and evaluate these widely used health practices. We believe that NCCAM has established a standard not for advocacy, but rather for rigorous objectivity. 1. 1.[↵][2]Committee on the Use of Complementary and Alternative Medicine by the American Public, Complementary and Alternative Medicine in the United States (Institute of Medicine, National Academy Press, Washington, DC, 2005). # Response {#article-title-3} The main point of the letter from Folkman et al. and the response to our Policy Forum by S. E. Straus and M. A. Chesney (“In defense of NCCAM,” Policy Forum, 21 July, p. [303][3]) is that NCCAM uses standard NIH procedures for review of proposals, appointments to advisory and review groups, and management of conflict of interest. That is formally true but misleading. Because of its charter, NCCAM advisory and review groups include many individuals whose scientific credentials would not qualify them for appointment to other NIH institutes. Of greater importance, the continued funding of poor-quality proposals refutes Straus and Chesney's claim that NCCAM applies the same review standards as other NIH institutes. Except for Bondurant, the signatories of the Folkman et al. letter all hold leadership positions in CAM or integrative medicine centers supported by NCCAM. Bondurant is a senior academic officer at Georgetown University Medical Center, which has a CAM center, and he was chairman of the IOM Committee that issued the report on CAM. Berman, Eisenberg, and Folkman also served on the IOM committee. The NCCAM appropriation for 2005, $123 million, understates NIH expenditures on CAM research. In 2004, the NCCAM budget was $117.8 million, and the total NIH expenditure on CAM research was estimated at $305 million, much of which represented projects that were co-funded by NCCAM and other institutes. NCCAM recently announced the creation of five new centers that will conduct research on multicomponent traditional African and Chinese herbal medicines. Each center will receive approximately $1 million per year, which is the equivalent of 20 RO-1 research grants. Some of this research is meritorious, but much of it is not. An independent review is urgently needed to bring the evaluation of proposals by NCCAM into line with the rest of NIH and to ensure that the limited funds available for biomedical research support the best science. [1]: #ref-1 [2]: #xref-ref-1-1 "View reference 1. in text" [3]: /lookup/doi/10.1126/science.1131608

The Structural Biology of Protein Aggregation Diseases: Fundamental Questions and Some Answers

Abstract: Amyloid fibrils are found in association with at least two dozen fatal diseases. The tendency of numerous proteins to convert into amyloid-like fibrils poses fundamental questions for structural biology and for protein science in general. Among these are the following: What is the structure of the cross-beta spine, common to amyloid-like fibrils? Is there a sequence signature for proteins that form amyloid-like fibrils? What is the nature of the structural conversion from native to amyloid states, and do fibril-forming proteins have two distinct stable states, the native state and the amyloid state? What is the basis of protein complementarity, in which a protein chain can bind to itself? We offer tentative answers here, based on our own recent structural studies.