Copyright (c) 2009 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/oapubs Recent documents in Open Access Articles en-us Sat, 21 Nov 2009 15:05:40 PST 3600 http://escholarship.umassmed.edu/oapubs/2059 http://escholarship.umassmed.edu/oapubs/2059 Wed, 18 Nov 2009 14:50:53 PST The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions. Xiaoyu Wang Animals Bovine papillomavirus 1 Chromatin Immunoprecipitation DNA-Binding Proteins Gene Expression Regulation, Viral Hela Cells Human papillomavirus 11 Human papillomavirus 18 Humans Intracellular Signaling Peptides and Proteins Mice Neoplasm Proteins Oncogene Proteins, Viral Papillomaviridae Proteasome Endopeptidase Complex Protein Stability RNA, Small Interfering *Transcription, Genetic Two-Hybrid System Techniques Ubiquitination Viral Proteins http://escholarship.umassmed.edu/oapubs/2058 http://escholarship.umassmed.edu/oapubs/2058 Wed, 18 Nov 2009 14:50:47 PST BACKGROUND: Cell adhesion plays an important role in proliferation, metastasis, and tumor growth and may represent a potential vulnerability in treatment of prostate cancer patients. Bicalutamide (Casodex) has been used as an anti-androgen agent for prostate cancer patients during hormone ablation therapy. This study focuses on the effect of Bicalutamide on cell adhesion to fibronectin (FN) in prostate cancer cells. METHODS: Androgen-dependent LNCaP prostate cancer cells were stimulated with androgen before being irradiated with doses of 0, 5, 10, or 15 Gy. Cell adhesion to fibronectin was then measured to ascertain androgen's role in integrin mediated prostate cancer cell adhesion. Flow cytometry was used to analyze surface expression of integrin subtypes in LNCaP cells. RESULTS: LNCaP cell adhesion to FN was significantly increased by stimulation with androgen when treated with 10 or 15 Gy ionizing radiations but not at 0 or 5 Gy. This increase was inhibited by treatment with Bicalutamide. LNCaP cells exposed to high dose radiation showed an increased expression of alpha(V) and beta(1) integrins in response to androgen treatment while Bicalutamide abolished this effect. CONCLUSIONS: Our data show that Bicalutamide inhibits the effect of androgen on cell adhesion to FN through changes of integrin subtypes in cells given high dose radiation. This suggests new molecular targets and possible treatment strategies for prostate cancer patients to improve the outcome during hormone ablation therapy and radiation therapy. Tao Wang Adenocarcinoma Androgens Anilides Antineoplastic Agents Cell Adhesion Cell Line, Tumor Dose-Response Relationship, Radiation Fibronectins Humans Integrins Male Nitriles Prostatic Neoplasms Radiation, Ionizing Tosyl Compounds http://escholarship.umassmed.edu/oapubs/2057 http://escholarship.umassmed.edu/oapubs/2057 Wed, 18 Nov 2009 14:50:42 PST An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of Human Immunodeficiency Virus Type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial conducted in healthy adult volunteers of both genders. Robust cross-subtype HIV-1-specific T cell responses were detected in IFNgamma ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds. Shixia Wang AIDS Vaccines Adolescent Adult HIV Antibodies HIV Envelope Protein gp120 Human Experimentation Humans Immunoglobulin G Interferon-gamma Middle Aged Neutralization Tests T-Lymphocytes Vaccines, DNA http://escholarship.umassmed.edu/oapubs/2056 http://escholarship.umassmed.edu/oapubs/2056 Wed, 18 Nov 2009 14:50:37 PST Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine. Shixia Wang Animals Antibodies, Bacterial Antigens, Viral Bacterial Proteins Blotting, Western Enzyme-Linked Immunosorbent Assay Female Immunity, Mucosal Immunoglobulin G Mice Mice, Inbred BALB C Plague Plague Vaccine Plasminogen Activators Protein Engineering Vaccines, DNA Vaccines, Synthetic Yersinia pestis http://escholarship.umassmed.edu/oapubs/2055 http://escholarship.umassmed.edu/oapubs/2055 Wed, 18 Nov 2009 14:50:31 PST Influenza virus infection of the respiratory tract is characterized by a neutrophil infiltrate accompanied by inflammatory cytokine and chemokine production. We and others have reported that Toll-like receptor (TLR) proteins are present on human neutrophils and that granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment enhances IL-8 (CXCL8) secretion in response to stimulation with TLR ligands. We demonstrate that influenza virus can induce IL-8 and other inflammatory cytokines from GM-CSF-primed human neutrophils. Using heat inactivation of influenza virus, we show that viral entry but not replication is required for cytokine induction. Furthermore, endosomal acidification and viral uncoating are necessary. Finally, using single-cell analysis of intracellular cytokine accumulation in neutrophils from knockout mice, we prove that TLR7 is essential for influenza viral recognition and inflammatory cytokine production by murine neutrophils. These studies demonstrate neutrophil activation by influenza virus and highlight the importance of TLR7 and TLR8 in that response. Jennifer P. Wang Animals Cell Line Cytokines Granulocyte Macrophage Colony-Stimulating Factors, Recombinant Humans Immunity, Innate Influenza A Virus, H3N2 Subtype Ligands Macrolides Membrane Glycoproteins Mice Mice, Inbred C57BL Mice, Knockout Neutrophil Activation Neutrophils RNA, Viral Toll-Like Receptor 7 Toll-Like Receptor 8 Toll-Like Receptors Virus Internalization Virus Replication http://escholarship.umassmed.edu/oapubs/2054 http://escholarship.umassmed.edu/oapubs/2054 Wed, 18 Nov 2009 14:50:26 PST OBJECTIVE: Although initial research suggests that computerized physician order entry reduces pediatric medication errors, no comprehensive error surveillance studies have evaluated the effect of computerized physician order entry on children. Our objective was to evaluate comprehensively the effect of computerized physician order entry on the rate of inpatient pediatric medication errors. METHODS: Using interrupted time-series regression analysis, we reviewed all charts, orders, and incident reports for 40 admissions per month to the NICU, PICU, and inpatient pediatric wards for 7 months before and 9 months after implementation of commercial computerized physician order entry in a general hospital. Nurse data extractors, who were unaware of study objectives, used an established error surveillance method to detect possible errors. Two physicians who were unaware of when the possible error occurred rated each possible error. RESULTS: In 627 pediatric admissions, with 12,672 medication orders written over 3234 patient-days, 156 medication errors were detected, including 70 nonintercepted serious medication errors (22/1000 patient-days). Twenty-three errors resulted in patient injury (7/1000 patient-days). In time-series analysis, there was a 7% decrease in level of the rates of nonintercepted serious medication errors. There was no change in the rate of injuries as a result of error after computerized physician order entry implementation. CONCLUSIONS: The rate of nonintercepted serious medication errors in this pediatric population was reduced by 7% after the introduction of a commercial computerized physician order entry system, much less than previously reported for adults, and there was no change in the rate of injuries as a result of error. Several human-machine interface problems, particularly surrounding selection and dosing of pediatric medications, were identified. Additional refinements could lead to greater effects on error rates. Kathleen E. Walsh Attitude of Health Personnel Child Child, Preschool Drug Therapy, Computer-Assisted Female Health Services Research Hospitalization Hospitals, Pediatric Humans Infant Intensive Care Units, Neonatal Intensive Care Units, Pediatric Male *Medical Order Entry Systems Medical Records Systems, Computerized Medication Errors Pharmaceutical Preparations Physician's Practice Patterns Retrospective Studies Safety Management Total Quality Management United States http://escholarship.umassmed.edu/oapubs/2053 http://escholarship.umassmed.edu/oapubs/2053 Wed, 18 Nov 2009 14:50:20 PST OBJECTIVE: To assess the technical feasibility of using a retrievable, closed cell intracranial stent delivered through a microcatheter for safe removal of foreign bodies or clot. METHODS: In vitro and in vivo testing were performed to demonstrate the feasibility of using retrievable intracranial stents for foreign body or clot removal. In vitro testing was performed in an anatomically correct silicone vascular replica by partially deploying the stent around a coil, then retracting the stent into the microcatheter to trap the coil. Withdrawal of the stent delivery system into the guide catheter resulted in coil removal. Subsequently, the technique was evaluated in a porcine model of intracranial aneurysms, wherein both fresh clot and herniated coils were extracted from the carotid arteries. RESULTS: In these experimental procedures, both herniated coils and fresh clot were safely and easily removed from the in vitro and in vivo models. No periprocedural adverse events were observed. CONCLUSION: These in vitro and in vivo studies suggest the potential use of retrievable stents for the removal of foreign bodies or clot from the intracranial circulation. Ajay K. Wakhloo Animals *Blood Vessel Prosthesis Device Removal Equipment Failure Analysis Female Foreign Bodies Intracranial Thrombosis Prosthesis Design *Stents Swine http://escholarship.umassmed.edu/oapubs/2052 http://escholarship.umassmed.edu/oapubs/2052 Wed, 18 Nov 2009 14:50:15 PST The use of major histocompatibility complex (MHC) tetramers in the detection and analysis of antigen-specific T cells has become more widespread since its introduction 11 years ago. Early challenges in the application of tetramer staining to CD4+ T cells centred around difficulties in the expression of various class II MHC allelic variants and the detection of low-frequency T cells in mixed populations. As many of the technical obstacles to class II MHC tetramer staining have been overcome, the focus has returned to uncertainties concerning how oligomer valency and T-cell receptor/MHC affinity affect tetramer binding. Such issues have become more important with an increase in the number of studies relying on direct ex vivo analysis of antigen-specific CD4+ T cells. In this review we discuss which problems in class II MHC tetramer staining have been solved to date, and which matters remain to be considered. Sabrina S. Vollers Antibody Affinity CD4-Positive T-Lymphocytes Flow Cytometry Histocompatibility Antigens Class II Humans Staining and Labeling http://escholarship.umassmed.edu/oapubs/2051 http://escholarship.umassmed.edu/oapubs/2051 Wed, 18 Nov 2009 14:50:10 PST Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (alpha TS) from Escherichia coli, a (beta alpha)(8) TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several (15)N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of alpha TS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several beta-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact beta-barrel and a frayed alpha-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of alpha TS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure. Ramakrishna Vadrevu Amides Amino Acids Deuterium Escherichia coli Hydrogen Hydrogen Bonding Isotope Labeling Magnetic Resonance Spectroscopy Molecular Weight Nitrogen Isotopes *Protein Folding Protein Structure, Secondary Protein Subunits Thermodynamics Time Factors Tryptophan Synthase Urea http://escholarship.umassmed.edu/oapubs/2050 http://escholarship.umassmed.edu/oapubs/2050 Wed, 18 Nov 2009 14:50:04 PST To exit infected cells, human immunodeficiency virus type 1 (HIV-1) exploits the vacuolar protein-sorting pathway by engaging Tsg101 and ALIX through PTAP and LYPx(n)L late assembly (L) domains. In contrast, less-complex retroviruses often use PPxY L domains to recruit Nedd4 family ubiquitin ligases. Although HIV-1 Gag lacks PPxY motifs, we now show that the budding of various HIV-1 L-domain mutants is dramatically enhanced by ectopic Nedd4-2s, a native isoform with a truncated C2 domain. The effect of Nedd4-2s on HIV-1 budding required a catalytically active HECT domain and was specific, since other Nedd4 family proteins showed little activity and an unrelated retrovirus was not rescued. The residual C2 domain of Nedd4-2s was critical for the enhancement of HIV-1 budding and for the association of Nedd4-2s with Gag, as reflected by its incorporation into virus-like particles. Interestingly, the incorporation of Nedd4-2s also depended on its active site, indicating that the ability to form a thioester with ubiquitin was required. These data suggest a novel mechanism by which HIV-1 Gag can connect to cellular budding machinery. Yoshiko Usami Amino Acid Motifs Cell Line HIV-1 Humans Protein Binding Protein Interaction Mapping Ubiquitin Ubiquitin-Protein Ligases gag Gene Products, Human Immunodeficiency Virus http://escholarship.umassmed.edu/oapubs/2049 http://escholarship.umassmed.edu/oapubs/2049 Wed, 18 Nov 2009 14:49:58 PST Objective: To assess primary care provider (PCP) attitudes and self-reported behavior with regard to identifying and managing depression in adult patients before and after a chronic disease/collaborative care intervention.Method: A self-administered cross-sectional survey was conducted in 6 targeted practices among 39 family practice physicians, family nurse practitioners, and residents before and after implementation of a depression in primary care project. In this project, the sites received tools and training in depression screening and guideline-concordant treatment, facilitated referral services for patients to access mental health providers, psychiatric phone consultation, patient education materials, and services of a depression care manager. The project was conducted from June 2003 through June 2006.Results: Comparison of responses prior to and after the intervention showed that significantly or nearly significantly larger proportions of PCPs endorsed the importance of depression as a patient presenting problem (p = .000), increased provision of supportive counseling (p = .13), more often identified counseling or therapy as effective (p = .07), and more often referred patients to mental health services (p = .001). PCPs also reduced their perception that treating depression is time consuming (p = .000).Conclusions: After a chronic disease/collaborative care approach to depression treatment in primary care was implemented, PCP attitudes and behaviors about depression treatment were significantly modified. More guideline-concordant care, and increased collaboration with mental health services, was reported. Implications for future primary care depression intervention activities and research are discussed. Carole C. Upshur http://escholarship.umassmed.edu/oapubs/2048 http://escholarship.umassmed.edu/oapubs/2048 Wed, 18 Nov 2009 14:49:53 PST Emerging clinical guidelines recommend shared decision making to individualize drug regimens for older adults with Type 2 diabetes mellitus. While the current health education campaign for diabetes in the United States recommends physician-initiated medication-related discussions about adherence and side effects, little emphasis is placed on soliciting patient concerns. This study's aim was to explore the concerns of older adults with diabetes about the complexity of their drug regimens and to determine whether they discussed medication-related concerns with their physician. Twenty-two patients with Type 2 diabetes age 65 years and older who used five or more medications were selected from an urban academic geriatric medicine practice in the United States. In-depth semi-structured interviews were conducted to uncover participants' perceptions of multiple medication use and related discussions with providers. The predominant theme that emerged was the variability in medication-related topics that patients perceived they could discuss with their physician. While most participants described physician-initiated discussions about adherence and side effects, many did not bring up concerns about medication cost or their desire to reduce medication burden. In order to encourage greater patient involvement in medication decision making for diabetes treatment, educational messages promoting patient-physician dialogue need to take more account of patient concerns. Jennifer Tjia Age Factors Aged Diabetes Mellitus, Type 2 Drug Interactions Female Humans Interviews as Topic Male *Patient Compliance *Patient Participation Physician-Patient Relations Polypharmacy http://escholarship.umassmed.edu/oapubs/2047 http://escholarship.umassmed.edu/oapubs/2047 Wed, 18 Nov 2009 14:49:48 PST OBJECTIVES: To determine whether prescription drug benefits are associated with the use of guideline recommended medications by older persons with type 2 diabetes mellitus (DM). DESIGN: Cross-sectional study. PARTICIPANTS: A national sample of Medicare beneficiaries with DM aged 65 and older and an indication for angiotensin-converting enzyme inhibitor (ACEI) or angiotensin II-receptor blocker (ARB) use or high risk of coronary heart disease (hypertension or current smoking) who participated in the 2003 Medicare Current Beneficiary Survey. MEASUREMENTS: Prescription drug coverage was measured according to self-report and verified according to insurance claims. Outcome variables were use of an ACEI or an ARB (ACEI/ARB) or a statin or use of an ACEI/ARB and a statin. Survey-weighted multinomial logistic regression was used to identify the independent effect of drug coverage on one of two categories of recommended medication use (ACEI/ARB or statin or ACEI/ARB and statin) compared with the reference category of none after controlling for sociodemographic characteristics and health status. RESULTS: The final study sample was 1,181 (weighted N=4.0 million). Overall, 23% had no drug coverage, 16% Medicaid coverage, 43% employer coverage, 9% Medigap coverage, and 9% Department of Veterans Affairs (VA) or state-sponsored low-income coverage. Overall, 33% received a statin and an ACEI/ARB, 44% only an ACEI/ARB or a statin, and 23% neither. After adjustment, VA and state-sponsored drug benefits were most strongly associated with combined ACEI/ARB and statin use (relative risk ratio (RRR)=4.83, 95% confidence interval (CI)=2.24-10.4)), followed by employer-sponsored coverage (RRR=2.60, 95% CI=1.67-4.03)). CONCLUSIONS: Prescription drug benefits from VA and state-sponsored drug programs are strongly associated with use of recommended medications by older adults with DM. Jennifer Tjia Aged Angiotensin-Converting Enzyme Inhibitors Cardiovascular Diseases Diabetes Mellitus, Type 2 Drug Utilization Female Guideline Adherence Health Benefit Plans, Employee Humans Hydroxymethylglutaryl-CoA Reductase Inhibitors *Insurance Coverage Insurance, Medigap *Insurance, Pharmaceutical Services Male Medicaid *Medicare Practice Guidelines as Topic Receptors, Angiotensin United States United States Department of Veterans Affairs http://escholarship.umassmed.edu/oapubs/2046 http://escholarship.umassmed.edu/oapubs/2046 Wed, 18 Nov 2009 14:49:42 PST BACKGROUND: Recent data suggest that Clostridium difficile-associated diarrhea is becoming more severe and difficult to treat. Antibody responses to C. difficile toxin A are protective against symptomatic disease and recurrence. We examined the safety and pharmacokinetics (pk) of a novel neutralizing human monoclonal antibody against C. difficile toxin A (CDA1) in healthy adults. METHODS: Five cohorts with 6 subjects each received a single intravenous infusion of CDA1 at escalating doses of 0.3, 1, 5, 10, and 20 mg/kg. Safety evaluations took place on days 1, 2, 3, 7, 14, 28, and 56 post-infusion. Samples for pk analysis were obtained before and after infusion, and at each safety evaluation. Serum CDA1 antibody concentrations and human anti-human antibody (HAHA) titers were measured with enzyme-linked immunosorbent assays. A noncompartmental model was used for pk analysis. RESULTS: Thirty subjects were enrolled. The median age was 27.5 yrs. There were no serious adverse events (AE) related to CDA1. Twenty-one of the 48 reported non-serious adverse events were possibly related to CDA1, and included transient blood pressure changes requiring no treatment, nasal congestion, headache, abdominal cramps, nausea, and self-limited diarrhea. Serum CDA1 concentrations increased with escalating doses: mean C(max) ranged from 6.82 microg/ml for the 0.3 mg/kg cohort to 511 microg/ml for the 20 mg/kg cohort. The geometric mean values of the half-life of CDA1 ranged between 25.3 and 31.8 days, and the volume of distribution approximated serum. No subject formed detectable HAHA titers. CONCLUSION: Administration of CDA1 as a single intravenous infusion was safe and well tolerated. C(max) increased proportionally with increasing doses. A randomized study of CDA1 in patients with C. difficile associated diarrhea is underway. Claribel P. Taylor Adult Antibodies, Anti-Idiotypic Antibodies, Bacterial Antibodies, Monoclonal effects Antitoxins Bacterial Toxins Enterotoxins Enzyme-Linked Immunosorbent Assay Female Half-Life Humans Infusions, Intravenous Male Middle Aged http://escholarship.umassmed.edu/oapubs/2045 http://escholarship.umassmed.edu/oapubs/2045 Wed, 18 Nov 2009 14:49:36 PST The authors report herein two diagnostically challenging cases centered on the myeloid/natural killer (myeloid/NK) cells, a variant of myeloblasts, to illustrate the importance of advanced flow cytometric immunophenotyping and an updated understanding of surface markers in hematopoietic malignancies. Myeloid/NK cell acute leukemia is a very rare subtype of leukemia. Although its NK-cell nature is debatable, it represents a variant of leukemia with distinct morphological and immunophenotypical features. The first case is a de novo myeloid/NK-cell acute leukemia with a striking clinical, morphologic and immunophenotypic resemblance to acute promyelocytic leukemia (APL), but which could be distinguished by its CD11a, CD18, CD117 and CD9 expression. This case illustrates the importance of utilizing the APL surrogate surface phenotype of HLA-DR(low), CD11a(low) and CD18(low) by flow cytometric study to rule in/out APL immunophenotypically. In the second case, we show that myeloid/NK-cell blasts can present as a variant of blasts in a preleukemic disease as refractory anemia with excess blasts-1 (RAEB-1), where the blasts were negative for CD34, CD117 and HLA-DR. The recognition of such blast variant is important in appropriately classifying such preleukemic diseases by blast percentage. Guilin Tang http://escholarship.umassmed.edu/oapubs/2044 http://escholarship.umassmed.edu/oapubs/2044 Wed, 18 Nov 2009 14:49:31 PST BACKGROUND: Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or whether it must be enforced by separate mechanisms. It has been hypothesized that scaffold proteins promote graded behavior. RESULTS: Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust in that it resists perturbation by loss of several negative-feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. CONCLUSIONS: The results suggest that the MAPK cascade module is inherently ultrasensitive but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context. Satoe Takahashi Adaptor Proteins, Signal Transducing Cell Membrane Genes, Fungal *MAP Kinase Signaling System Mutation Pheromones Receptor Cross-Talk Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins http://escholarship.umassmed.edu/oapubs/2043 http://escholarship.umassmed.edu/oapubs/2043 Wed, 18 Nov 2009 14:49:26 PST Eradication of hepatitis C virus (HCV) infection requires a complex and coordinated interplay between innate and adaptive immune responses that, when it fails, leads to chronic infection. In this review, the innate immune mechanisms by which HCV is sensed and by which HCV undermines host defense are discussed. The critical role of dendritic cells in antigen presentation and T-cell activation in addition to type I interferon production and interference of HCV with innate immune cell functions are reviewed. Finally, current and emerging therapeutic approaches targeting innate immune pathways are evaluated. Gyongyi Szabo Animals Hepacivirus Hepatitis C Humans Immunity, Innate Lymphocyte Activation T-Lymphocytes http://escholarship.umassmed.edu/oapubs/2042 http://escholarship.umassmed.edu/oapubs/2042 Wed, 18 Nov 2009 14:49:21 PST Pkdrej, a member of the polycystin-1 gene family, is expressed only in the male germ line. Male mice that are homozygous for a targeted mutation in the Pkdrej allele (Pkdrej(tm/tm)) are fertile in unrestricted mating trials, but exhibit lower reproductive success when competing with wild-type males in sequential mating trials and in artificial insemination of mixed-sperm populations. Following mating, sperm from Pkdrej(tm/tm) mice require >2 h longer than those of wild-type males to be detected within the egg/cumulus complex in the oviduct. Sperm from mice of both genotypes are able to capacitate in vitro. However, one of the component processes of capacitation, the ability to undergo a zona pellucida-evoked acrosome reaction, develops more slowly in sperm from Pkdrej(tm/tm) animals than in sperm from wild-type males. In contrast, a second component process of capacitation, the transition to hyperactivated flagellar motility, develops with a similar time course in both genotypes. These two behavioral consequences of capacitation, exocytotic competence and altered motility, are therefore differentially regulated. These data suggest that Pkdrej controls the timing of fertilization in vivo through effects on sperm transport and exocytotic competence and is a factor in postcopulatory sexual selection. Keith A. Sutton Acrosome Animals Copulation Female Fertilization Genotype Male Mating Preference, Animal Mice Mice, Transgenic Receptors, Cell Surface Reproduction *Sperm Capacitation Sperm Motility TRPP Cation Channels http://escholarship.umassmed.edu/oapubs/2041 http://escholarship.umassmed.edu/oapubs/2041 Wed, 18 Nov 2009 14:49:15 PST Saccharomyces cerevisiae mating pheromones trigger dissociation of a heterotrimeric G protein (Galphabetagamma) into Galpha-guanosine triphosphate (GTP) and Gbetagamma. The Gbetagamma dimer regulates both mitogen-activated protein (MAP) kinase cascade signaling and cell polarization. Here, by independently activating the MAP kinase pathway, we studied the polarity role of Gbetagamma in isolation from its signaling role. MAP kinase signaling alone could induce cell asymmetry but not directional growth. Surprisingly, active Gbetagamma, either alone or with Galpha-GTP, could not organize a persistent polarization axis. Instead, following pheromone gradients (chemotropism) or directional growth without pheromone gradients (de novo polarization) required an intact receptor-Galphabetagamma module and GTP hydrolysis by Galpha. Our results indicate that chemoattractant-induced cell polarization requires continuous receptor-Galphabetagamma communication but not modulation of MAP kinase signaling. To explore regulation of Gbetagamma by Galpha, we mutated Gbeta residues in two structurally distinct Galpha-Gbeta binding interfaces. Polarity control was disrupted only by mutations in the N-terminal interface, and not the Switch interface. Incorporation of these mutations into a Gbeta-Galpha fusion protein, which enforces subunit proximity, revealed that Switch interface dissociation regulates signaling, whereas the N-terminal interface may govern receptor-Galphabetagamma coupling. These findings raise the possibility that the Galphabetagamma heterotrimer can function in a partially dissociated state, tethered by the N-terminal interface. Shelly Catherine Strickfaden Alleles *Cell Polarity GTP-Binding Protein alpha Subunits GTP-Binding Protein beta Subunits GTP-Binding Protein gamma Subunits Guanosine Triphosphate Heterotrimeric GTP-Binding Proteins Hydrolysis Models, Biological Mutation Phenotype Pheromones Protein Binding Receptors, Pheromone Recombinant Fusion Proteins Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction Suppression, Genetic Tropism http://escholarship.umassmed.edu/oapubs/2040 http://escholarship.umassmed.edu/oapubs/2040 Wed, 18 Nov 2009 14:49:09 PST PURPOSE: In an environment of multiple campaigns promoting judicious antibiotic use in children, identification of effective strategies is important. We assessed physician responses to a community-level intervention with respect to antibiotic prescribing, related practices, and perceived effectiveness. METHODS: This study was a mixed qualitative and quantitative evaluation of a randomized controlled community-wide educational intervention in 16 Massachusetts communities. Physicians in intervention communities received locally endorsed guidelines, group educational sessions, and biweekly newsletters. Parents simultaneously received materials in physicians' offices and by mail. After the intervention, we conducted a mailed physician survey and individual interviews to assess the impact of the intervention. We compared survey responses for intervention and control physicians, and we analyzed interview transcripts to provide in-depth information about selected topics. RESULTS: Among survey respondents (n = 168), 91% of intervention and 4% of control physicians reported receiving intervention materials. Physicians received information from multiple other sources. More intervention than control physicians reported decreased antibiotic prescribing from 2000-2003 (75% vs 58%, P = .03), but there were no differences between groups in knowledge, attitudes, or behaviors favoring judicious antibiotic use. Both groups were concerned about antibiotic resistance and reported room to reduce their own prescribing. Interviewed physicians suggested frequent repetition of messages, brief written materials on specific topics for themselves and patients, and promotion in the mass media as the most effective strategies to reduce prescribing. CONCLUSIONS: In multiple communities an intervention in physician offices to promote judicious antibiotic prescribing reached its intended audience, but physicians' self-reported attitudes and practices were similar in intervention and control communities. Campaigns that repeat brief, consistent reminders to multiple stakeholder groups may be most effective at assuring judicious antibiotic use. Christopher J. Stille Anti-Bacterial Agents *Drug Utilization Evaluation Studies as Topic Family Practice Female Health Care Surveys *Health Knowledge, Attitudes, Practice Humans Male Massachusetts Medicaid Parents Patient Education as Topic Pediatrics *Physician's Practice Patterns Qualitative Research United States