Copyright (c) 2009 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu Recent documents in eScholarship@UMMS en-us Fri, 20 Nov 2009 05:53:24 PST 3600 http://escholarship.umassmed.edu/pgfe_pp/1 http://escholarship.umassmed.edu/pgfe_pp/1 Thu, 19 Nov 2009 14:56:16 PST Several forward and reverse proteomic approaches are available that can be used to identify interaction partners for a protein of interest. Here we provide methods for identifying interacting partners by the yeast two-hybrid system (a reverse proteomic method) and by tandem immuno-affinity purification of protein complexes combined with mass spectrometry (a forward proteomic method). Albertha J. M. Walhout Caenorhabditis elegans Caenorhabditis elegans Proteins Protein Interaction Mapping Two-Hybrid System Techniques Tandem Mass Spectrometry 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 http://escholarship.umassmed.edu/gsbs_diss/433 http://escholarship.umassmed.edu/gsbs_diss/433 Mon, 16 Nov 2009 05:50:54 PST ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF's p53-independent tumor suppressor activity is not understood. The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF's p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF's ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression. To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF. Ramesh C. Kovi ADP-Ribosylation Factors Apoptosis Tumor Suppressor Protein p53 Phosphoproteins DNA-Binding Proteins Tumor Suppressor Protein p14ARF Colorectal Neoplasms Genes, Tumor Suppressor Academic Dissertations Dissertations, UMMS http://escholarship.umassmed.edu/oapubs/2039 http://escholarship.umassmed.edu/oapubs/2039 Fri, 13 Nov 2009 14:50:26 PST The scientific advances made in the year leading up to the 15th Conference on Retroviruses and Opportunistic Infections were overshadowed, to some extent, by setbacks in the AIDS vaccine research arena and in particular, the failure of the Merck STEP trial. Arguably, these disappointments were offset by strong advances that were being made in basic science and pathogenesis. In particular, recent discoveries into cellular factors that influence virus-host cell interplay and new insights into the mechanisms of viral pathogenesis were highlighted at the meeting. These research discoveries paint an optimistic picture regarding the development of new strategies to combat HIV and AIDS. Mario Stevenson http://escholarship.umassmed.edu/oapubs/2038 http://escholarship.umassmed.edu/oapubs/2038 Fri, 13 Nov 2009 14:50:21 PST Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (C(H)) region genes, except Cdelta]. The recombination is initiated by the B cell-specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, gammaH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur. Janet Stavnezer Animals Cytidine Deaminase Germinal Center Humans Immunoglobulin Class Switching Immunoglobulin Isotypes Models, Genetic *Recombination, Genetic http://escholarship.umassmed.edu/oapubs/2037 http://escholarship.umassmed.edu/oapubs/2037 Fri, 13 Nov 2009 14:50:16 PST Jonathan M. Spector Africa South of the Sahara Asia Asphyxia Neonatorum Fetal Death Humans Infant, Newborn *Perinatal Mortality Resuscitation Stillbirth http://escholarship.umassmed.edu/oapubs/2036 http://escholarship.umassmed.edu/oapubs/2036 Fri, 13 Nov 2009 14:50:11 PST The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two "patches" of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion. Jennifer A. Songer Alleles Amino Acid Sequence Amino Acids Carrier Proteins Conserved Sequence Exosomes Models, Molecular Molecular Sequence Data Mutant Proteins Mutation Protein Binding Protein Subunits Protein Transport Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins http://escholarship.umassmed.edu/oapubs/2035 http://escholarship.umassmed.edu/oapubs/2035 Fri, 13 Nov 2009 14:50:06 PST Ten healthy human volunteers were subjected to progressive lower body negative pressure (LBNP) to the onset of cardiovascular collapse to compare the response of noninvasively determined skin and fat corrected deep muscle oxygen saturation (SmO2) and pH to standard hemodynamic parameters for early detection of imminent hemodynamic instability. Muscle SmO2 and pH were determined with a novel near infrared spectroscopic (NIRS) technique. Heart rate (HR) was measured continuously via ECG, and arterial blood pressure (BP) and stroke volume (SV) were obtained noninvasively via Finometer and impedance cardiography on a beat-to-beat basis. SmO2 and SV were significantly decreased during the first LBNP level (-15 mmHg), whereas HR and BP were late indicators of impending cardiovascular collapse. SmO2 declined in parallel with SV and inversely with total peripheral resistance, suggesting, in this model, that SmO2 is an early indicator of a reduction in oxygen delivery through vasoconstriction. Muscle pH decreased later, suggesting an imbalance between delivery and demand. Spectroscopic determination of SmO2 is noninvasive and continuous, providing an early indication of impending cardiovascular collapse resulting from progressive reduction in central blood volume. Babs R. Soller Adipose Tissue Adult Blood Pressure Blood Volume Electrocardiography Female Heart Rate *Hemodynamics Humans Hydrogen-Ion Concentration Hypovolemia Lower Body Negative Pressure Male Models, Cardiovascular Muscle, Skeletal Oxygen *Oxygen Consumption Skin *Spectrophotometry, Infrared Stroke Volume Time Factors Vascular Resistance http://escholarship.umassmed.edu/oapubs/2034 http://escholarship.umassmed.edu/oapubs/2034 Fri, 13 Nov 2009 14:50:01 PST The intensity of exercise above which oxygen uptake (Vo2) does not account for all of the required energy to perform work has been associated with lactate accumulation in the blood (lactate threshold, LT) and elevated carbon dioxide output (gas exchange threshold). An increase in hydrogen ion concentration ([H+]) is approximately concurrent with elevation of blood lactate and CO2 output during exercise. Near-infrared spectra (NIRS) and invasive interstitial fluid pH (pHm) were measured in the flexor digitorum profundus during handgrip exercise to produce a mathematical model relating the two measures with an estimated error of 0.035 pH units. This NIRS pHm model was subsequently applied to spectra collected from the vastus lateralis of 10 subjects performing an incremental-intensity cycle protocol. Muscle oxygen saturation (SmO2) was also calculated from spectra. We hypothesized that a H+ threshold could be identified for these subjects and that it would be different from but correlated with the LT. Lactate, gas exchange, SmO2, and H+ thresholds were determined as a function of Vo2 using bilinear regression. LT was significantly different from both the gas exchange threshold (Delta = 0.27 +/- 0.29 l/min) and H+ threshold (Delta = 0.29 +/- 0.23 l/min), but the gas exchange threshold was not significantly different from the H+ threshold (Delta = 0.00 +/- 0.38 l/min). The H+ threshold was strongly correlated with LT (R2 = 0.95) and the gas exchange threshold (R2 = 0.85). This initial study demonstrates the feasibility of noninvasive pHm estimations, the determination of H+ threshold, and the relationship between H+ and classical metabolic thresholds during incremental exercise. Babs R. Soller Adult Anaerobic Threshold Exercise Extracellular Fluid Feasibility Studies Female *Hand Strength Humans Hydrogen-Ion Concentration Lactic Acid Male Models, Biological *Muscle Contraction Muscle, Skeletal Oxygen Consumption Pulmonary Gas Exchange Quadriceps Muscle *Spectroscopy, Near-Infrared United States http://escholarship.umassmed.edu/oapubs/2033 http://escholarship.umassmed.edu/oapubs/2033 Fri, 13 Nov 2009 14:49:55 PST PURPOSE: The therapeutic importance of immune responses against single versus multiple antigens is poorly understood. There also remains insufficient understanding whether responses to one subset of antigens are more significant than another. Autoantibodies are frequent in cancer patients. They can pose no biological significance or lead to debilitating paraneoplastic syndromes. Autoreactivity has been associated with clinical benefits, but the magnitude necessary for meaningful results is unknown. Autologous tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor generate immune infiltrates in preexisting metastases with associated tumor destruction. We sought to identify targets of responses from this vaccination strategy. EXPERIMENTAL DESIGN: Postvaccination sera used in screening a cDNA expression library prepared from a densely infiltrated metastasis of a long-term surviving melanoma patient identified several autoantigens. Additional autoantigens were identified through similar screenings in non-small cell lung cancer and murine models, and proteins implicated in cancer propagation. ELISAs for several targets were established using recombinant proteins, whereas others were evaluated by petit serologies. RESULTS: Eleven gene products were identified through serologic screening from two patients showing highly favorable clinical outcomes. A subset of antigens revealed significant changes in antibody titers compared with weak responses to other proteins. Time course analyses showed coordinated enhanced titers against several targets as a function of vaccination in responding patients. CONCLUSIONS: This study shows the range of biologically significant antigens resulting from a whole-cell vaccine. Targets include autoantigens that are components of cell cycle regulation. Potent antibody responses against multiple autoantigens are associated with effective tumor destruction without clinical autoimmunity. Taylor Sittler Antibody Formation Autoantigens Autoimmunity Cancer Vaccines Cell Line, Tumor Humans Immunotherapy Lymphocytes, Tumor-Infiltrating Neoplasms Prognosis Treatment Outcome Tumor Cells, Cultured http://escholarship.umassmed.edu/oapubs/2032 http://escholarship.umassmed.edu/oapubs/2032 Fri, 13 Nov 2009 14:49:50 PST PURPOSE: In this study, we investigated whether an oncofetal protein, IMP3, can serve as a new biomarker to predict progression and metastasis of early-stage urothelial carcinoma of the bladder. EXPERIMENTAL DESIGN: The expression of IMP3 in 242 patients with primary superficial bladder urothelial carcinoma and metastatic urothelial carcinoma was evaluated by immunohistochemistry. Patients with primary superficial urothelial carcinoma of the bladder were further investigated by use of survival analysis. RESULTS: Twenty percent (42 of 214) of primary superficial urothelial carcinomas and 93% (26 of 28) of metastatic urothelial carcinomas expressed IMP3. Kaplan-Meier plots and log-rank tests showed that patients with IMP3-positive tumors had a much lower progression-free survival (P = 0.0002) and disease-free survival rate (P = 0.0067) than did those with IMP3-negative tumors. The 5-year progression-free and disease-free survival rates were 91% and 94% in IMP3-negative patients versus 64% and 76% in IMP3-positive patients, respectively. Sixty percent of IMP3-positive patients with superficial invasive urothelial carcinoma at initial diagnosis went on to develop metastases, whereas no metastasis was found in IMP3-negative patients (P = 0.0017). In the multivariable Cox analysis, patients with IMP3 expression in their superficial urothelial carcinomas subsequently developed invasive tumors or metastasis at a rate that was about five times greater than cases without expression of IMP3 adjusting for other well-known clinical variables (tumor stage and grade, etc.). CONCLUSIONS: Our findings indicate that IMP3 is an independent prognostic marker that can identify a group of patients with a high potential to develop progression and who might benefit from early aggressive therapy. Lioudmila Sitnikova Aged Aged, 80 and over Carcinoma, Papillary Disease Progression Female Humans Male Middle Aged Neoplasm Invasiveness Neoplasm Metastasis Neoplasm Proteins Prognosis RNA-Binding Proteins Survival Analysis Tumor Markers, Biological Urinary Bladder Neoplasms Urothelium http://escholarship.umassmed.edu/oapubs/2031 http://escholarship.umassmed.edu/oapubs/2031 Fri, 13 Nov 2009 14:49:45 PST Oral health care in pregnancy is often avoided and misunderstood by physicians, dentists, and patients. Evidence-based practice guidelines are still being developed. Research suggests that some prenatal oral conditions may have adverse consequences for the child. Periodontitis is associated with preterm birth and low birth weight, and high levels of cariogenic bacteria in mothers can lead to increased dental caries in the infant. Other oral lesions, such as gingivitis and pregnancy tumors, are benign and require only reassurance and monitoring. Every pregnant woman should be screened for oral risks, counseled on proper oral hygiene, and referred for dental treatment when necessary. Dental procedures such as diagnostic radiography, periodontal treatment, restorations, and extractions are safe and are best performed during the second trimester. Xylitol and chlorhexidine may be used as adjuvant therapy for high-risk mothers in the early postpartum period to reduce transmission of cariogenic bacteria to their infants. Appropriate dental care and prevention during pregnancy may reduce poor prenatal outcomes and decrease infant caries. Hugh Silk Dental Caries Female Gingivitis Granuloma, Pyogenic Humans Infant, Newborn Mouth Diseases *Oral Health Periodontitis Pregnancy Pregnancy Complications Pregnancy Outcome Prenatal Care Tooth Mobility http://escholarship.umassmed.edu/oapubs/2030 http://escholarship.umassmed.edu/oapubs/2030 Fri, 13 Nov 2009 14:49:40 PST Differentiation of naive CD4+ T cells into T helper type 1 (Th1) effector cells requires both T cell receptor (TCR) signaling and cytokines such as interleukin-12 and interferon gamma (IFN-gamma). Here, we report that a third cytokine signal, mediated by the Janus family tyrosine kinase 3 (Jak3) and signal transducer and activator of transcription 5 (STAT5) pathway, is also required for Th1 cell differentiation. In the absence of Jak3-dependent signals, naive CD4+ T cells proliferate robustly but produce little IFN-gamma after Th1 cell polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4 or to impaired upregulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 cell differentiation. Min Shi Animals CD4-Positive T-Lymphocytes Cell Differentiation *Chromatin Assembly and Disassembly Cytokines Epigenesis, Genetic Interferon-gamma Janus Kinase 3 Mice Mice, Mutant Strains Promoter Regions, Genetic STAT Transcription Factors STAT5 Transcription Factor Signal Transduction Th1 Cells http://escholarship.umassmed.edu/oapubs/2029 http://escholarship.umassmed.edu/oapubs/2029 Fri, 13 Nov 2009 14:49:35 PST Primate lentiviruses encode four "accessory proteins" including Vif, Vpu, Nef, and Vpr/Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. We present evidence that the Vpx proteins of HIV-2/SIV(SM) promote virus infection by antagonizing an antiviral restriction in macrophages. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. Our results indicate that macrophage harbor a potent antiviral restriction and that primate lentiviruses have evolved Vpx to counteract this restriction. Natalia Sharova Animals COS Cells Cercopithecus aethiops DNA-Binding Proteins Gene Expression Regulation, Viral Gene Silencing HIV-2 Leukocytes, Mononuclear Macrophages RNA Interference RNA, Small Interfering Recombinant Fusion Proteins Viral Regulatory and Accessory Proteins http://escholarship.umassmed.edu/oapubs/2028 http://escholarship.umassmed.edu/oapubs/2028 Fri, 13 Nov 2009 14:49:30 PST Two activities of human papillomavirus type 16 E6 (HPV16 E6) are proposed to contribute to the efficient immortalization of human epithelial cells: the degradation of p53 protein and the induction of telomerase. However, the requirement for p53 inactivation has been debated. Another E6 target is the hAda3 protein, a p53 coactivator and a component of histone acetyltransferase complexes. We have previously described the role of hAda3 and p53 acetylation in p14ARF-induced human mammary epithelial cell (MEC) senescence (P. Sekaric, V. A. Shamanin, J. Luo, and E. J. Androphy, Oncogene 26:6261-6268, 2007). In this study, we analyzed a set of HPV16 E6 mutants for the ability to induce hAda3 degradation. E6 mutants that degrade hAda3 but not p53 could abrogate p14ARF-induced growth arrest despite the presence of normal levels of p53 and efficiently immortalized MECs. However, two E6 mutants that previously were reported to immortalize MECs with low efficiency were found to be defective for both p53 and hAda3 degradation. We found that these immortal MECs select for reduced p53 protein levels through a proteasome-dependent mechanism. The findings strongly imply that the inactivation of the p14ARF-p53 pathway, either by the E6-mediated degradation of p53 or hAda3 or by cellular adaptation, is required for MEC immortalization. Vladimir A. Shamanin Cell Aging Cell Line *Cell Transformation, Viral Epithelial Cells Human papillomavirus 16 Humans Oncogene Proteins, Viral Repressor Proteins Transcription Factors Tumor Suppressor Protein p14ARF Tumor Suppressor Protein p53 http://escholarship.umassmed.edu/oapubs/2027 http://escholarship.umassmed.edu/oapubs/2027 Fri, 13 Nov 2009 14:49:25 PST BACKGROUND: Obesity is often comorbid with depression and individuals with this comorbidity fare worse in behavioral weight loss treatment. Treating depression directly prior to behavioral weight loss treatment might bolster weight loss outcomes in this population, but this has not yet been tested in a randomized clinical trial. METHODS AND DESIGN: This randomized clinical trial will examine whether behavior therapy for depression administered prior to standard weight loss treatment produces greater weight loss than standard weight loss treatment alone. Obese women with major depressive disorder (N = 174) will be recruited from primary care clinics and the community and randomly assigned to one of the two treatment conditions. Treatment will last 2 years, and will include a 6-month intensive treatment phase followed by an 18-month maintenance phase. Follow-up assessment will occur at 6-months and 1- and 2 years following randomization. The primary outcome is weight loss. The study was designed to provide 90% power for detecting a weight change difference between conditions of 3.1 kg (standard deviation of 5.5 kg) at 1-year assuming a 25% rate of loss to follow-up. Secondary outcomes include depression, physical activity, dietary intake, psychosocial variables and cardiovascular risk factors. Potential mediators (e.g., adherence, depression, physical activity and caloric intake) of the intervention effect on weight change will also be examined. DISCUSSION: Treating depression before administering intensive health behavior interventions could potentially boost the impact on both mental and physical health outcomes. TRIAL REGISTRATION: NCT00572520. Kristin L. Schneider Adult Aged *Behavior Therapy Comorbidity Depressive Disorder, Major Female Follow-Up Studies Humans Male Middle Aged Obesity Research Design Young Adult http://escholarship.umassmed.edu/oapubs/2026 http://escholarship.umassmed.edu/oapubs/2026 Fri, 13 Nov 2009 14:49:19 PST We have developed a top-down, rule-based mathematical model to explore the basic principles that coordinate mechanochemical events during animal cell migration, particularly the local-stimulation-global-inhibition model suggested originally for chemotaxis. Cells were modeled as a shape machine that protrudes or retracts in response to a combination of local protrusion and global retraction signals. Using an optimization algorithm to identify parameters that generate specific shapes and migration patterns, we show that the mechanism of local stimulation global inhibition can readily account for the behavior of Dictyostelium under a large collection of conditions. Within this collection, some parameters showed strong correlation, indicating that a normal phenotype may be maintained by complementation among functional modules. In addition, comparison of parameters for control and nocodazole-treated Dictyostelium identified the most prominent effect of microtubules as regulating the rates of retraction and protrusion signal decay, and the extent of global inhibition. Other changes in parameters can lead to profound transformations from amoeboid cells into cells mimicking keratocytes, neurons, or fibroblasts. Thus, a simple circuit of local stimulation-global inhibition can account for a wide range of cell behaviors. A similar top-down approach may be applied to other complex problems and combined with molecular manipulations to define specific protein functions. Javier Satulovsky Animals *Cell Movement Cell Shape Dictyostelium Feedback, Biochemical Microtubules *Models, Biological Signal Transduction http://escholarship.umassmed.edu/oapubs/2025 http://escholarship.umassmed.edu/oapubs/2025 Fri, 13 Nov 2009 14:49:14 PST A hallmark of male germ cell gene expression is the generation by alternative polyadenylation of cell-specific mRNAs, many of which utilize noncanonical A(A/U)UAAA-independent polyadenylation signals. Cleavage factor I (CFIm), a component of the pre-mRNA cleavage and polyadenylation protein complex, can direct A(A/U)UAAA-independent polyadenylation site selection of somatic cell mRNAs. Here we report that the CFIm subunits NUDT21/CPSF5 and CPSF6 are highly enriched in mouse male germ cells relative to somatic cells. Both subunits are expressed from spermatogenic cell mRNAs that are shorter than the corresponding somatic transcripts. Complementary DNA sequencing and Northern blotting revealed that the shorter Nudt21 and Cpsf6 mRNAs are generated by alternative polyadenylation in male germ cells using proximal poly(A) signals. Both sets of transcripts contain CFIm binding sites within their 3'-untranslated regions, suggesting autoregulation of CFIm subunit formation in male germ cells. CFIm subunit mRNA and protein levels exhibit distinct developmental variation during spermatogenesis, indicating stage-dependent translational and/or posttranslational regulation. CFIm binding sites were identified near the 3' ends of numerous male germ cell transcripts utilizing A(A/U)UAAA-independent sites. Together these findings suggest that CFIm complexes participate in alternative polyadenylation directed by noncanonical poly(A) signals during spermatogenesis. Becky L. Sartini Amino Acid Sequence Animals Base Sequence Cleavage And Polyadenylation Specificity Factor Gene Expression Regulation Male Mice Molecular Sequence Data Polyadenylation Protein Subunits RNA Precursors *RNA Splice Sites Sequence Homology, Amino Acid Spermatogenesis Spermatogonia Testis mRNA Cleavage and Polyadenylation Factors http://escholarship.umassmed.edu/oapubs/2024 http://escholarship.umassmed.edu/oapubs/2024 Fri, 13 Nov 2009 14:49:09 PST BACKGROUND: Extent of left ventricular dysfunction in patients with acute myocardial infarction (AMI) is an important predictor of subsequent morbidity and mortality. It is unclear, however, how often ejection fraction (EF) findings are evaluated in the setting of AMI, and the characteristics of patients who do not have their EF evaluated, particularly from the more generalizable perspective of a population-based investigation. PURPOSE: The purpose of this study was to examine nearly 3 decade long trends (1975-2003) in the evaluation of EF in patients admitted with confirmed AMI (n = 12,760) to all greater Worcester (Massachusetts) hospitals during 14 annual periods. RESULTS: The percentage of patients undergoing evaluation of EF before hospital discharge increased substantially between 1975 (4%) and 2003 (73%). Despite these encouraging trends, approximately one quarter of patients in our most recent study year did not receive an EF evaluation. In the mid-1970s through mid-1980s, radionuclide ventriculography was typically used to assess EF, whereas echocardiography was most often used to evaluate EF during more recent periods. Predictors of not undergoing an evaluation of cardiac function included older age, shorter length of hospital stay, code status limitations, dying during hospitalization, Medicare insurance, several comorbidities, and a recent non-Q-wave myocardial infarction. CONCLUSIONS: The results of this community-wide study suggest that a considerable proportion of patients with AMI fail to have their EF evaluated. Efforts remain needed to optimize the use of cardiac imaging studies and link the results of these studies to improved patient outcomes. Paul A. Santolucito Aged Aged, 80 and over Cause of Death Disease Progression Female Humans *Inpatients Length of Stay Male Massachusetts Middle Aged Myocardial Infarction Prognosis Retrospective Studies Stroke Volume Ventricular Function, Left http://escholarship.umassmed.edu/oapubs/2023 http://escholarship.umassmed.edu/oapubs/2023 Fri, 13 Nov 2009 14:49:04 PST A high-fat diet causes activation of the regulatory protein c-Jun NH2-terminal kinase 1 (JNK1) and triggers development of insulin resistance. JNK1 is therefore a potential target for therapeutic treatment of metabolic syndrome. We explored the mechanism of JNK1 signaling by engineering mice in which the Jnk1 gene was ablated selectively in adipose tissue. JNK1 deficiency in adipose tissue suppressed high-fat diet-induced insulin resistance in the liver. JNK1-dependent secretion of the inflammatory cytokine interleukin-6 by adipose tissue caused increased expression of liver SOCS3, a protein that induces hepatic insulin resistance. Thus, JNK1 activation in adipose tissue can cause insulin resistance in the liver. Guadalupe Sabio Adipocytes Adipose Tissue Animals Dietary Fats Enzyme Activation Glucose Insulin Insulin Receptor Substrate Proteins *Insulin Resistance Interleukin-6 Liver MAP Kinase Signaling System Mice Mitogen-Activated Protein Kinase 8 Phosphorylation Proto-Oncogene Proteins c-akt *Signal Transduction *Stress, Physiological Suppressor of Cytokine Signaling Proteins http://escholarship.umassmed.edu/oapubs/2022 http://escholarship.umassmed.edu/oapubs/2022 Fri, 13 Nov 2009 14:48:59 PST Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis (PFGE), that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB(Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O(6)-methylguanine (O(6)-meG) base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB(Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly. Anetta Nowosielska *Base Pair Mismatch Cisplatin *DNA Repair DNA Replication DNA, Bacterial Escherichia coli Methylnitronitrosoguanidine http://escholarship.umassmed.edu/oapubs/2021 http://escholarship.umassmed.edu/oapubs/2021 Fri, 13 Nov 2009 14:48:55 PST PURPOSE OF REVIEW: Drug resistance results when the balance between the binding of inhibitors and the turnover of substrates is perturbed in favor of the substrates. Resistance is quite widespread to the HIV-1 protease inhibitors permitting the protease to process its 10 different substrates. This processing of the substrates permits the virus HIV-1 to mature and become infectious. The design of HIV-1 protease inhibitors that closely fit within the substrate-binding region is proposed to be a strategy to avoid drug resistance. RECENT FINDINGS: Cocrystal structures of HIV-1 protease with its substrates define an overlapping substrate-binding region or substrate envelope. Novel HIV-1 protease inhibitors that were designed to fit within this substrate envelope were found to retain high binding affinity and have a flat binding profile against a panel of drug-resistant HIV-1 proteases. SUMMARY: The avoidance of drug resistance needs to be considered in the initial design of inhibitors to quickly evolving targets such as HIV-1 protease. Using a detailed knowledge of substrate binding appears to be a promising strategy for achieving this goal to obtain robust HIV-1 protease inhibitors. Madhavi N. L. Nalam http://escholarship.umassmed.edu/oapubs/2020 http://escholarship.umassmed.edu/oapubs/2020 Fri, 13 Nov 2009 14:48:49 PST Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation. Matthew R. Murawski Animals Bronchoalveolar Lavage Fluid Dendritic Cells Humans *Immunity, Innate Macrophages, Peritoneal Mice Mice, Inbred C57BL Mice, Knockout Neutrophils Respiratory Syncytial Viruses Toll-Like Receptor 2