Connecting the Libraries and Athletics through Instruction and Outreach.

This column describes the approaches taken by librarians and staff at James Madison University (JMU) Libraries & Educational Technologies (LET) to extend library support to university athletics. The model resembles that used for outreach to academic programs and was first adapted to the semi-clinical, nonacademic Strength & Conditioning Department, then to JMU Athletics as a whole. Librarians offered targeted instructional sessions, orientations, and asynchronous learning modules embedded in the learning management system. This new relationship has provided an opportunity for broader collaboration, increasing LET's presence across campus.

An evaluation of pharmacogenomic information provided by five common drug information resources.

INTRODUCTION: This study evaluated whether pharmacogenomic information contained in the Food and Drug Administration (FDA)-approved package inserts of sixty-five drugs was present in five drug information resources. METHODS: The study searched for biomarkers from the FDA package inserts in 5 drug information sources: American Hospital Formulary Service Drug Information (AHFS), Facts & Comparisons 4.0 (Facts), ePocrates Online Free (ePocrates Free), Lexicomp Online (Lexicomp), and Micromedex 2.0. Each resource had the opportunity to present biomarker information for 65 drugs, a total of 325 opportunities. A binary system was used to indicate presence or absence of the biomarker information. A sub-analysis was performed on the 13 most frequently prescribed drugs in the United States. RESULTS: Package insert biomarker information was available, on average, for 81.5% of the 65 FDA-listed drugs in 2011. Percent availability for the individual resources was: Lexicomp, 95.3%; Micromedex 2.0, 92.3%; Facts, 76.9%; AHFS, 75.3%; and ePocrates Free, 67.7%. The sub-analysis of the 13 top drugs showed Lexicomp and Micromedex 2.0 had the most mentions, 92.3%; ePocrates Free had the least, 53.8%. CONCLUSION: The strongest resource for pharmacogenomic information was Lexicomp. The gap between Lexicomp and ePocrates Free is concerning. Clinicians would miss pharmacogenomic information 6.6 times more often in ePocrates Free than in Lexicomp. IMPLICATIONS: Health sciences librarians should be aware of the variation in biomarker availability when recommending drug resources for licensing and use. Librarians can also use this study to encourage publishers to include pharmacogenomics information from the package insert as a minimum standard.

Development of the research lifecycle model for library services.

QUESTION: Can the niche services of individual librarians across multiple libraries be developed into a suite of standard services available to all scientists that support the entire research lifecycle? SETTING: Services at a large, research-intensive state university campus are described. METHOD: Initial data were collected via concept mapping by librarians. Additional data were collected at conferences and meetings through interactive poster presentations. MAIN RESULTS: Services of interest to scientists for each of the stages in the research lifecycle were developed by the team to reflect the wide range of strengths of team members in aggregate. CONCLUSION: Input from researchers was the most effective tool for developing the model. A flexible research lifecycle model can be developed to match the needs of different service groups and the skills of different librarians.

A preliminary analysis of library holdings as compared to the basic resources for pharmacy education list.

The catalogs of 11 university libraries were analyzed against the Basic Resources for Pharmaceutical Education (BRPE) to measure the percent coverage of the core total list as well as the core sublist. There is no clear trend in this data to link school age, size, or rank with percentage of coverage of the total list or the "First Purchase" core list when treated as independent variables. Approximately half of the schools have significantly higher percentages of core titles than statistically expected. Based on this data, it is difficult to predict what percentage of titles on the BRPE a library will contain.

A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function.

Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.

Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end-directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC-binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.

Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.

Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization.

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif.

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.

Development of targeted online modules for recurring reference questions.

When students are given assignments with specific information needs, they may turn to the library for help. The UNC Health Sciences Library developed three short online modules to teach first-year pharmacy students how to find early/animal studies, mechanism of action information, and specific study types in an effort to lessen demand on the reference desk. The modules filled two goals: to free up time that had been spent on three common low-level questions and to provide a pedagogically sound online tool to teach students how to find answers to these three questions. The modules were created using Adobe Captivate. Developing and promoting the modules took three hours of the pharmacy librarian's time compared with nearly 23 hours spent answering individual questions via e-mail, in consultations, and at the reference desk before the modules were introduced. After introducing the modules, only one student asked for help from the library compared to more than 60 who viewed the online modules at least once.

"Hidden treasures": librarian office hours for three health sciences schools.

The changing needs of students and faculty have prompted UNC Chapel Hill's Health Sciences Library to reconsider the delivery of library services. Several years of outreach and office hours have yielded an array of "hidden treasures," or secondary outcomes, of both online and in-person office hours. The online office hours are tailored for the Schools of Medicine, Pharmacy, and Public Health. This article examines the benefits that go beyond simple consultation statistics and encompass more qualitative aspects of success resulting from increased outreach, goodwill, and stronger library-departmental partnerships.

Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome.

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.

The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes.

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Vignettes: Diverse library staff offering diverse bioinformatics services.

OBJECTIVES: The paper gives examples of the bioinformatics services provided in a variety of different libraries by librarians with a broad range of educational background and training. METHODS: Two investigators sent an email inquiry to attendees of the "National Center for Biotechnology Information's (NCBI) Introduction to Molecular Biology Information Resources" or "NCBI Advanced Workshop for Bioinformatics Information Specialists (NAWBIS)" courses. The thirty-five-item questionnaire addressed areas such as educational background, library setting, types and numbers of users served, and bioinformatics training and support services provided. Answers were compiled into program vignettes. DISCUSSION: The bioinformatics support services addressed in the paper are based in libraries with academic and clinical settings. Services have been established through different means: in collaboration with biology faculty as part of formal courses, through teaching workshops in the library, through one-on-one consultations, and by other methods. Librarians with backgrounds from art history to doctoral degrees in genetics have worked to establish these programs. CONCLUSION: Successful bioinformatics support programs can be established in libraries in a variety of different settings and by staff with a variety of different backgrounds and approaches.

Developing an interdisciplinary collaboration center in an academic Health Sciences Library.

This article describes the evolution of the Health Sciences Library's plans for an interdisciplinary, technology-enhanced collaboration center, from a technology-driven space to one with a vision of support for peer-to-peer learning and research. The center offers an exciting opportunity to be an essential partner in collaborative and interdisciplinary programs such as the new Carolina Center for Exploratory Genetic Analysis. The Library is a centrally located and neutral place, which helps minimize geographical and territorial obstacles to effective collaboration. The collaboration center raises the Library's visibility and allows staff to demonstrate the value of knowledge resources, services, technology expertise, infrastructure, and facilities for group study and collaboration.

cDNA cloning and sequence comparisons of human and chicken muscle C-protein and 86kD protein.

Thick filaments in vertebrate striated muscles are composed of myosin heavy chain (MHC) and myosin light chains (MLCs) plus at least eight other proteins: C-protein, 86kD protein (birds) or H-protein (mammals), M-protein, myomesin, titin, MM-creatine kinase, skelemin, and AMP-deaminase. Except for CPK and AMP deaminase, none have well defined functions. Analysis of cDNA clones encoding chicken C-protein and 86kD protein has revealed a high degree of shared amino acid identity, particularly in the C-terminal 40kD. To identify functionally significant regions, the human counterpart of each protein was cloned, sequenced and analysed. Two human C-protein cDNAs were isolated with significant homology to chicken fast C-protein. Clone H75, with 69% identity to chicken fast C-protein, shows the same pattern of hybridization as the chicken fast C-protein in chicken muscles. The other clone, H8 with 60% identity, shows a pattern of hybridization in chicken muscles which is consistent with the expression of chicken slow C-protein. The human 86kD protein shares 66% DNA sequence identity with the chicken 86kD protein. Assuming that essential sequences would be conserved during evolution, we compared the chicken and human proteins using PALIGN. Chicken and human fast C-proteins possess 66% peptide identity over their deduced length plus 10% conservative substitutions. Human slow C-protein and chicken fast C-protein share 44% peptide sequence identity, plus 16% conservative substitutions. Chicken and human 86kD proteins are also very similar: 54% peptide identity plus 20% conservative substitutions. This high degree of sequence identity between chicken and human C- and 86kD proteins suggests selective pressure on the primary sequence. Recent primary sequence analyses of projectin and mini-titins from Drosophila, twitchin from C. elegans, C-protein, smMLCK, 86kD protein, and M-protein from the chicken, titin from the rabbit, and skelemin from the mouse reveals that all these proteins possess multiple internal repeats of approximately 100 amino acids. These repeating domains are of two types: one is homologous to the internal repeats which define the C-2 subset of the immunoglobulin superfamily, the other is related to the fibronectin type III repeat. Both human C-proteins possess comparable internal repeats and preliminary evidence suggests the presence of the same repeats in human 86kD. This duality of repeat structure is found in many extracellular proteins and is typified by the N-CAMs.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin.

Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.

Actin filaments mediate Dictyostelium myosin assembly in vitro.

Because myosin thick filaments form in the actin-rich cortex of nonmuscle cells, we have examined the role of Dictyostelium actin filaments in the assembly of Dictyostelium myosin (type II). Fluorescence energy transfer and light-scattering assembly assays indicate that self-association of Dictyostelium myosin into bipolar thick filaments is kinetically regulated by actin filament networks. Regulation is nucleotide dependent but does not require ATP hydrolysis. Myosin assembly is accelerated approximately 5-fold by actin filaments when either 1 mM ATP or 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-P[NH]P) is present. However, actin filaments together with 1 mM ADP abolish myosin assembly. Accelerated assembly appears to require transient binding of myosin molecules to actin filaments before incorporation into thick filaments. Fluorescence energy-transfer assays demonstrate that myosin associates with actin filaments at a rate that is equivalent to the accelerated myosin assembly rate, evidence that myosin to actin binding is a rate-limiting step in accelerated thick filament formation. Actin filament networks are also implicated in regulation of thick filament formation, since fragmentation of F-actin networks by severin causes immediate cessation of accelerated myosin assembly. Electron microscopic studies support a model of actin filament-mediated myosin assembly. In ADP, myosin monomers rapidly decorate F-actin, preventing extensive formation of thick filaments. In AMP-P[NH]P, myosin assembles along actin filaments, forming structures that resemble primitive stress fibers. Taken together, these data suggest a model in which site-directed assembly of thick filaments in Dictyostelium is mediated by the interaction of myosin monomers with cortical actin filament networks.

Molecular cloning of chicken myosin-binding protein (MyBP) H (86-kDa protein) reveals extensive homology with MyBP-C (C-protein) with conserved immunoglobulin C2 and fibronectin type III motifs.

The complete nucleotide sequence of a cDNA clone encoding the chicken skeletal muscle myosin-binding protein H (MyBP-H), formerly termed 86-kDa protein, has been established and the predicted amino acid sequence compared with other proteins entered into the GenBank data base. The full-length cDNA of 2066 base pairs contains a single open reading frame of 1611 base pairs encoding a muscle-specific protein of 58,487 Da. The predicted molecular weight differs significantly from the relative mobility of 86-kDa protein in reducing sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The full-length protein expressed in Escherichia coli also exhibits an anomalously slow mobility in SDS-PAGE; this gel retardation is a property of the N-terminal 24 kDa of the protein which contain two extended motifs of alternating alanine and proline residues, resembling the N terminus of skeletal muscle myosin light chain 1 (Nabeshima, Y. I., Fujii-Kuriyama, Y., Muramatsu, M., and Ogata, K. (1984) Nature 308, 333-338). The C-terminal 40 kDa share 49.6% sequence identity and 17% conservative substitutions with chicken skeletal muscle MyBP-C (C-protein) (Einheber, S., and Fischman, D. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2157-2161). The protein contains four internal repeats of approximately 100 amino acids each, two of which bear significant resemblance to the C2 set of the immunoglobulin superfamily, and the other two are related to the type III fibronectin repeat. The arrangement of these repeats, -III-C2-III-C2-, is identical to that seen in the C-terminal 40-kDa section of MyBP-C. This repeat structure is implicated in myosin binding for the MyBP family. Finally, genomic Southern blots indicate that a single gene encodes fast skeletal muscle MyBP-H.

Complete sequence of human fast-type and slow-type muscle myosin-binding-protein C (MyBP-C). Differential expression, conserved domain structure and chromosome assignment.

Myosin-binding-protein C (MyBP-C) is a myosin-associated protein of unknown function found in the cross-bridge-bearing zone (C region) of A bands in striated muscle. Using a cDNA clone encoding the fast-type isoform of chicken MyBP-C, we screened a human fetal muscle cDNA library and isolated clones encoding the full-length human fast-type isoform of MyBP-C. cDNA clones encoding the slow-type isoform of human MyBP-C, were also isolated and fully sequenced. Northern-blot analysis demonstrated skeletal muscle-specific expression of these gene products. Using human/hamster somatic-cell hybrids, we were able to map the slow-type MyBP-C to human chromosome 12, and the fast-type MyBP-C to chromosome 19. The cDNA for human fast-type MyBP-C encodes a polypeptide of 1142 amino acids with an expected molecular mass of 128.1 kDa. Comparison of this cDNA with other members of the MyBP family reveals extensive primary-sequence conservation. Each MyBP-C contains seven immunoglobulin C2 motifs and three fibronectin type-III repeats in the arrangement C2-C2-C2-C2-C2-III-III-C2-III-C2. Regions of high identity shared by the chicken and the two human proteins are not restricted to the immunoglobulin and fibronectin motifs. Sequence comparison of all three proteins has allowed us to map a highly conserved region between the first and second C2 motifs, the only large spacer sequence present between motifs in these proteins.

Building a bioinformatics community of practice through library education programs.

This paper addresses the following questions:What makes the community of practice concept an intriguing framework for developing library services for bioinformatics? What is the campus context and setting? What has been the Health Sciences Library's role in bioinformatics at the University of North Carolina (UNC) Chapel Hill? What are the Health Sciences Library's goals? What services are currently offered? How will these services be evaluated and developed? How can libraries demonstrate their value? Providing library services for an emerging community such as bioinformatics and computational biology presents special challenges for libraries including understanding needs, defining and communicating the library's role, building relationships within the community, preparing staff, and securing funding. Like many academic health sciences libraries, the University of North Carolina (UNC) at Chapel Hill Health Sciences Library is addressing these challenges in the context of its overall mission and goals.