bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in Survival, Length of Stay, and Discharge Disposition of Older Trauma Patients Admitted to Trauma Centers and Nontrauma Center Hospitals
Authors: Scheetz Linda J.
Published: November 2005

Purpose: To examine the relationship of level of care (trauma center [TC], nontrauma center [NTC] hospitals) on three outcomes: survival, length of stay, and discharge disposition.

Design: Retrospective secondary analysis of a subset of data (1,418 patients age 65 to 99 years) from a large statewide study in which the purpose was to compare admission patterns (TCs and NTCs) of motor vehicle (MV) trauma patients according to age and sex. The New Jersey UB‐92 Patient Discharge Data for 2000 were used in this analysis.

Methods: Demographic and clinical variables were compared using descriptive data, independent samples t tests, Pearson chi square, and Mann‐Whitney U analyses. Logistic regression and multiple regression analyses were performed to examine relationships between level of care and three outcome variables, survival, length of stay, and discharge disposition, while controlling for age and severity of injury.

Results: NTC admission was the only predictor of survival and discharge to home, but injury severity was the strongest predictor of length of stay, followed by NTC care. The odds of survival and discharge home decreased slightly as age and injury severity increased.

Conclusions: This analysis indicated preliminary evidence that level of care influences survival, length of stay, and discharge disposition. Studies are warranted for researchers to examine the influence of postinjury variables, including complications, stress reaction, and depression on outcomes.
Linking the Practice Environment to Nurses' Job Satisfaction Through Nurse‐Physician Communication
Authors: Manojlovich Milisa
Published: November 2005

Purpose: To investigate direct and indirect relationships among the practice environment, nurse‐physician (RN‐MD) communication, and job satisfaction, as is posited in the nursing role effectiveness model (NREM).

Design: Survey.

Methods: Surveys were sent to a random sample of 500 hospital nurses throughout Michigan, and 332 (66%) responded. Main study instruments were the Conditions for Work Effectiveness Questionnaire‐II (CWEQ‐II), the Practice Environment Scale of the Nursing Work Index (PES‐NWI), the ICU Nurse‐Physician Questionnaire, and the Index of Work Satisfaction (IWS), Part B. Inferential statistical tests included multiple regression, t tests, and one‐way analysis of variance.

Findings: Practice environment (PES‐NWI) and empowerment (CWEQ‐II) scales explained 20% of the variance in RN‐MD communication. The combination of both environment scales (PES‐NWI and CWEQ‐II) and RN‐MD communication explained 61% of the variance in nursing job satisfaction scores. RN‐MD communication was also a significant mediating variable in the relationship between structure (practice environment and empowerment scales) and outcome (nursing job satisfaction).

Conclusions: Factors in the practice environment contributed both directly to nursing job satisfaction and also indirectly through RN‐MD communication. Study findings showed that a practice environment favorable to nurses improved both nurses' perceptions of their communications with physicians and their job satisfaction.
Leadership Styles and Nursing Faculty Job Satisfaction in Taiwan
Authors: Chen Hsiu‐Chin , Beck Susan L. , Amos Linda K.
Published: November 2005

Purpose: To examine nursing faculty job satisfaction and their perceptions of nursing deans' and directors' leadership styles, and to explore how the perceptions of leadership styles relate to faculty job satisfaction in Taiwan.

Methods: Descriptive, correlational, and cross‐sectional study with self‐administered questionnaires. The sample was recruited from 18 nursing programs, and 286 questionnaires were returned.

Results: Faculty perceived that Taiwan's nursing deans and directors showed more transformational than transactional leadership. Taiwan's nursing faculty were moderately satisfied in their jobs, and they were more satisfied with deans or directors who practiced the transactional leadership style of contingent reward and the transformational style of individualized consideration. A style with negative effect was passive management by exception.

Conclusions: Three types of leadership behaviors explained significant variance (21.2%) in faculty job satisfaction in Taiwan, indicating the need for further attention to training and development for effective leadership behaviors.
Increased population of CD4+CD25high regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients
Authors: Wang Xingbing , Zheng Jine , Liu Jun , Yao Junxia , He Yanli , Li Xiaoqing , Yu Jingming , Yang Jing , Liu Zhongping , Huang Shiang
Published: November 2005

Abstract:  Purpose: Regulatory T cells (T‐reg) that control harmful autoimmune T cells in the periphery may also suppress the immune response against cancer. In this study we investigated the possible involvement of CD4+CD25high T‐reg in the immune impairment of patients with acute myeloid leukemia (AML). Experimental design: The frequencies and phenotypes of CD4+CD25high T cells in the peripheral blood of AML patients were determined by flow cytometry. To assess the functional activity of CD4+CD25high T cells, CD4+CD25high, and CD4+CD25− T cells were sorted from peripheral blood mononuclear cells with FACS Vantage. The immunoregulatory properties of CD4+CD25high and CD4+CD25− T cells were characterized by proliferation assays and cytokine production assays. In addition, the frequency of apoptotic and proliferating cells in CD4+CD25high T cells were respectively evaluated by 7AAD and ki67 binding cells using flow cytometry. Results: Compared with healthy controls, AML patients had a higher proportion of CD4+CD25high T cells in peripheral blood. These cells were CD45‐RA(−), CD69(−), CD45‐RO(+), CD95(+), and intercellular CTLA‐4(+), and secreted low levels of TNF‐α and IL‐10, but no IL‐2, IL‐4, IL‐5, and IFN‐γ. They inhibited the proliferation and cytokine production (IL‐2, IFN‐γ) of CD4+CD25− T cells, but improved IL‐10 production under the co‐culture of both subsets with stimulation, thus behaving as T‐reg. Notably, CD4+CD25high T cells in AML patients presented significantly higher apoptosis and proliferation than that of healthy individuals. Conclusions: The frequency of CD4+CD25high T‐reg in peripheral blood in AML patients is significantly higher when compared with healthy individuals, likely due to the increasing proliferation of CD4+CD25high T cells.
Alterations in electrolyte equilibrium in patients with acute leukemia
Authors: Filippatos Theodosios D. , Milionis Haralampos J. , Elisaf Moses S.
Published: November 2005

Abstract:  Background and aim: A wide array of disturbances in electrolyte equilibrium is commonly seen in patients with acute leukemia (AL). These abnormalities present a potential hazard in these patients, as that of enhancing the cardiotoxic effects of certain chemotherapeutic regimens. The literature dealing with AL‐related electrolyte abnormalities and their interactions in leukemic patients was reviewed. Data synthesis: Sources included MEDLINE and EMBASE. The search strategy was based on the combination of ‘acute leukemia’, ‘electrolyte abnormalities’, ‘acid‐base disorders’, ‘potassium’, ‘sodium’, ‘magnesium’, ‘calcium’, and ‘phosphorus’. References of retrieved articles were also screened. A decrease in serum potassium, mainly owing to lysozyme‐induced tubular damage, appears to be one of the most frequent and potentially hazardous abnormalities. Other clinically significant metabolic perturbations include hyponatremia and hypercalcemia. Conclusion: A broad spectrum of electrolyte abnormalities is encountered in the clinical setting of AL, which are related to the disease process per se and/or to the therapeutic interventions. Clinicians should be vigilant for early detection and appropriate management of these disorders before the initiation of chemotherapy regimens as well as during treatment.
A case of advanced Hodgkin lymphoma with breast involvement treated with a combination of gemcitabine and pegylated liposomal doxorubicin
Authors: Fraulini Chiara , Mauro Endri , Rigolin Gian Matteo , Bissoli Sergio , Chierichetti Franca , Panareo Stefano , Feggi Luciano , Cavazzini Luigi , Castoldi Gianluigi
Published: November 2005

Abstract:  Gemcitabine in combination with pegylated liposomal doxorubicin has been recognized as an effective treatment in patients with refractory solid tumors. To our knowledge, this is the first case of relapsed Hodgkin lymphoma with breast involvement successfully treated with this association.
Glutamate receptors on human melanocytes regulate the expression of MiTF
Authors: Hoogduijn M. J. , Hitchcock I. S. , Smit N. P. M. , Gillbro J. M. , Schallreuter K. U. , Genever P. G.
Published: November 2005

Glutamate is the major excitatory neurotransmitter in the central nervous system but has also important functions in the epidermis. It is involved in keratinocyte barrier function and in re‐epithelialization processes after wounding. Recently, glutamate signalling has been suggested to be implicated in the development of melanoma. The present study examined the expression and functionality of metabotropic and ionotropic glutamate receptors on normal human melanocytes. We found that cultured melanocytes expressed the ionotropic glutamate receptors GluR2 and 4 [α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxsazolepropionic acid (AMPA) receptors] andN‐methyl‐d‐aspartate (NMDA) receptors 2A and 2C and possibly the metabotropic glutamate receptor 1. Melanocytes were also found to express specific glutamate transporters and decarboxylases, but appeared neither to produce nor to releasel‐glutamate. Stimulation with 10 or 100 μM AMPA or NMDA elevated intracellular calcium concentrations in melanocytes, and thus demonstrated the functionality of the glutamate receptors. Millimolar concentrations ofl‐glutamate did not induce melanocyte toxicity and had no stimulating effect on melanin production. However, blockage of AMPA and NMDA receptors with CFM‐2, memantine or MK801 caused a rapid and reversible change in melanocyte morphology, which was associated with disorganisation of actin and tubulin microfilaments. After 24 h of treatment with the AMPA receptor inhibitor CFM‐2, there was a sharp reduction in the expression of the crucial melanocyte differentiation and proliferation factor MiTF. The results of this study demonstrate a role for glutamate in melanocyte regulation that may have implications in melanocyte associated disorders.
Intrabodies against the EVH1 domain of Wiskott–Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells
Authors: Sato Mitsuru , Iwaya Ryo , Ogihara Kazumasa , Sawahata Ryoko , Kitani Hiroshi , Chiba Joe , Kurosawa Yoshikazu , Sekikawa Kenji
Published: November 2005

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single‐chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (VH) leader signal sequence at the N‐terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott–Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)‐2 synthesis in the T‐cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N‐terminal region of WASP may participate in IL‐2 synthesis. In transgenic mice expressing anti‐EVH1 scFvs derived from hybridoma cells producing WASP‐EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti‐EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL‐2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T‐cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL‐2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.
Complexes of Thermoactinomyces vulgaris R‐47 α‐amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with α‐(1,6) glycosidic linkages
Authors: Abe Akemi , Yoshida Hiromi , Tonozuka Takashi , Sakano Yoshiyuki , Kamitori Shigehiro
Published: November 2005

Thermoactinomyces vulgaris R‐47 α‐amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of α‐(1,4), α‐(1,4), and α‐(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes α‐(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes α‐(1,6) glycosidic linkages with a lesser efficiency. X‐ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]2 or P5; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaeα‐amylase showed that the (–) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the α‐(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites −1 and −2 at the nonreducing end of TVAI are effective in the hydrolysis of α‐(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch‐binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan‐binding domain.
Functional analysis of disease‐causing mutations in human UDP‐galactose 4‐epimerase
Authors: Timson David J.
Published: November 2005

UDP‐galactose 4‐epimerase (GALE, EC 5.1.3.2) catalyses the interconversion of UDP‐glucose and UDP‐galactose. Point mutations in this enzyme are associated with the genetic disease, type III galactosemia, which exists in two forms – a milder, or peripheral, form and a more severe, or generalized, form. Recombinant wild‐type GALE, and nine disease‐causing mutations, have all been expressed in, and purified from, Escherichia coli in soluble, active forms. Two of the mutations (N34S and G319E) display essentially wild‐type kinetics. The remainder (G90E, V94M, D103G, L183P, K257R, L313M and R335H) are all impaired in turnover number (kcat) and specificity constant (kcat/Km), with G90E and V94M (which is associated with the generalized form of galactosemia) being the most affected. None of the mutations results in a greater than threefold change in the Michaelis constant (Km). Protein–protein crosslinking suggests that none of the mutants are impaired in homodimer formation. The L183P mutation suffers from severe proteolytic degradation during expression and purification. N34S, G90E and D103G all show increased susceptibility to digestion in limited proteolysis experiments. Therefore, it is suggested that reduced catalytic efficiency and increased proteolytic susceptibility of GALE are causative factors in type III galactosemia. Furthermore, there is an approximate correlation between the severity of these defects in the protein structure and function, and the symptoms observed in patients.
Studies on the interaction of Nile red with horseradish peroxidase in solution
Authors: Hungerford Graham , Rei Ana , Ferreira M. Isabel C.
Published: November 2005

The interaction of an extrinsic probe (Nile red) with an enzyme (horseradish peroxidase) in solution was investigated using fluorescence techniques. Nile red fluorescence is very environmentally sensitive and the presence of domains of differing polarity within the enzyme was ascertained by the decomposition of the Nile red emission spectrum. Further evidence for the position of the probe inside the enzyme was obtained from a molecular modeling study. A decrease in the emission intensity of the dye during incubation with horseradish peroxidase was explained by the occurrence of resonance energy transfer between the Nile red and the heme group in the enzyme. This was supported by a calculation of the critical transfer distance and a comparison of the fluorescence intensity of the dye in both the holo‐ and apo‐enzyme. These data were then applied to the study of the effect of temperature on the structure of the enzyme, where changes in conformation were elucidated.
Mechanisms of amyloid fibril self‐assembly and inhibition
Authors: Gazit Ehud
Published: November 2005

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self‐assembled amyloid fibrils are also involved in physiological processes in several micro‐organisms. Yet, the molecular basis for the recognition and self‐assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self‐assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid‐associated disorders.
ANTECEDENTS AND CONSEQUENCES OF PSYCHOLOGICAL WORKPLACE STRAIN DURING EXPATRIATION: A CROSS‐SECTIONAL AND LONGITUDINAL INVESTIGATION
Authors: TAKEUCHI RIKI , WANG MO , MARINOVA SOPHIA V.
Published: November 2005

In this study, we examined nonlinear/interaction effects associated with the antecedents and consequences of psychological workplace strain, using cross‐sectional (N= 165) and longitudinal (N= 133) data collected from Western expatriates in China. The results of this study indicate that family characteristics interact to affect the level of psychological workplace strain experienced by expatriates. In addition, we find an inverse u‐curve relationship between psychological workplace strain and supervisory rated job performance for both cross‐sectional and longitudinal analyses. Finally, the empirical results lend support to the hypothesized positive relationship between work adjustment measured at Time 1 and job performance measured at Time 2. Implications for expatriate adjustment research and practice are discussed.
TEAMS' FEMALE REPRESENTATION AND PERCEIVED POTENCY AS INPUTS TO TEAM OUTCOMES IN A PREDOMINANTLY MALE FIELD SETTING
Authors: HIRSCHFELD ROBERT R. , JORDAN MARK H. , FEILD HUBERT S. , GILES WILLIAM F. , ARMENAKIS ACHILLES A.
Published: November 2005

We developed a team‐level temporal model and tested it with data from 92 newly formed teams in a naturalistic military setting where females were a distinct minority in all teams. Contrary to what is posited by theory on customary perceptions of females' capabilities and gender‐role congruence, a higher female proportion within teams did not adversely influence teams' early perceived potency, later social cohesion, or observers' assessments of teamwork. Consistent with theory regarding gender and abilities, however, a higher female proportion within teams contributed to better team problem solving but led to slightly worse team results on physically demanding tasks. In addition, teams' early perceived potency contributed positively to early task proficiency and later social cohesion. Furthermore, teams' social cohesion contributed positively to externally observed teamwork while controlling for task proficiency. We discuss implications of these findings for theory and practice.
DIFFERENCES IN LIPID PROFILE OF NEW ZEALAND MARINE SPECIES OVER FOUR SEASONS
Authors: MCLEAN CARLENE H. , BULLING KIM R.
Published: November 2005

New Zealand Greenshell mussels and hoki are caught throughout the year and oil is extracted from the processing discards. However, there is little information on the variability in the lipid profile during the annual cycle. The livers of male and female hoki (Macruronus novaezelandiae), Greenshell mussels (Perna canaliculus), blue mussels (Mytilus edulis aoteanus) and rock oysters (Saccostrea cucullata) were therefore collected four times during a 12‐month period, and their lipid and lipid‐soluble components were analyzed. The yield of oil obtained from hoki livers ranged from 21 ± 1 to 70 ± 2% (w/w), with a low yield occurring during their winter spawning season. Female livers generally contained a higher level of lipid than male livers. The yield of oil from whole shellfish was relatively small, ranging from 0.5 to 2% (w/w), with a higher lipid content in the summer season than in winter. Levels of omega‐3 fatty acids in hoki liver oil, 18–22% (w/w) of oil, were consistent throughout the year. Omega‐3 fatty acids in the three shellfish species were present at 9–20% (w/w) of oil, and showed large variations in quantity at different sampling dates. Blue mussels and rock oysters both produced lower levels of oil and omega‐3 fatty acids, and showed more variability compared with Greenshell mussels.
DETERMINATION OF SPHINGOLIPIDS IN NUTS AND SEEDS BY A SINGLE QUADRUPOLE LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY METHOD
Authors: FANG FANG , HO CHI‐TANG , SANG SHENGMIN , ROSEN ROBERT T.
Published: November 2005

Studies with experimental animals have shown that dietary sphingolipids significantly reduce the early stages of colon carcinogenesis and tumor formation, but knowledge about the amount and structure of sphingolipids in foods, especially foods of plant origin, is sparse because of lack of effective identification and quantification methods. Also, it is critical to know the structure of sphingolipids in plants in order to understand the potential link between diet and cancer. In this study, we developed a liquid chromatography‐mass spectrometry method with atmospheric pressure chemical ionization positive ion mode to separate, identify and quantify ceramide and cerebroside species simultaneously in 10 plant samples. The ceramides found in the samples contained mainly trihydroxy bases, especially 4‐hydroxy‐8‐sphingenine (t18:1). Both nonhydroxy fatty acids and hydroxy fatty acids were found, and these were mostly very long chain fatty acids (C > 20). Cerebrosides of the plant samples had both dihydroxy and trihydroxy bases, with dihydroxy base 4,8‐sphingadienine (d18:2) dominating. The fatty acids were composed mainly of α‐hydroxy fatty acids. Using an external calibration method, the major cerebroside found in the plants examined was quantified. This is the first report of sphingolipid content in most of the species studied.
Small-Molecule Inhibition of TNF-α
Authors: He Molly M. , Smith Annemarie Stroustrup , Oslob Johan D. , Flanagan William M. , Braisted Andrew C. , Whitty Adrian , Cancilla Mark T. , Wang Jun , Lugovskoy Alexey A. , Yoburn Josh C. , Fung Amy D. , Farrington Graham , Eldredge John K. , Day Eric S. , Cruz Leslie A. , Cachero Teresa G. , Miller Stephan K. , Friedman Jessica E. , Choong Ingrid C. , Cunningham Brian C.
Published: November 2005

We have identified a small-molecule inhibitor of tumor necrosis factor α (TNF-α) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-α activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-α subunits.
Pattern-Oriented Modeling of Agent-Based Complex Systems: Lessons from Ecology
Authors: Grimm Volker , Revilla Eloy , Berger Uta , Jeltsch Florian , Mooij Wolf M. , Railsback Steven F. , Thulke Hans-Hermann , Weiner Jacob , Wiegand Thorsten , DeAngelis Donald L.
Published: November 2005

Agent-based complex systems are dynamic networks of many interacting agents; examples include ecosystems, financial markets, and cities. The search for general principles underlying the internal organization of such systems often uses bottom-up simulation models such as cellular automata and agent-based models. No general framework for designing, testing, and analyzing bottom-up models has yet been established, but recent advances in ecological modeling have come together in a general strategy we call pattern-oriented modeling. This strategy provides a unifying framework for decoding the internal organization of agent-based complex systems and may lead toward unifying algorithmic theories of the relation between adaptive behavior and system complexity.
The Pseudo-Response Regulator
Ppd-H1
Provides Adaptation to Photoperiod in Barley
Authors: Turner Adrian , Beales James , Faure Sébastien , Dunford Roy P. , Laurie David A.
Published: November 2005

Plants commonly use photoperiod (day length) to control the timing of flowering during the year, and variation in photoperiod response has been selected in many crops to provide adaptation to different environments and farming practices. Positional cloning identified
Ppd-H1
, the major determinant of barley photoperiod response, as a pseudo-response regulator, a class of genes involved in circadian clock function. Reduced photoperiod responsiveness of the
ppd-H1
mutant, which is highly advantageous in spring-sown varieties, is explained by altered circadian expression of the photoperiod pathway gene
CONSTANS
and reduced expression of its downstream target,
FT
, a key regulator of flowering.
Direct Visualization of the Formation of Single-Molecule Conjugated Copolymers
Authors: Sakaguchi Hiroshi , Matsumura Hisashi , Gong Hui , Abouelwafa Abdelkarim M.
Published: November 2005

Electrochemical polymerization of two different kinds of thiophene monomers on an iodine-covered gold surface created highly assembled conjugated copolymers with different electronic structures. A scanning tunneling microscope revealed images of several linkage types: diblock, triblock, and multiblock. The single strand of conjugated copolymers exhibited an anomalous swinging motion on the surface. This technique presents the possibility of understanding the copolymerization process from the different monomers on the single-molecular scale and of building single-molecule superlattices on a surface through controlled electropolymerization.
Structure of a V3-Containing HIV-1 gp120 Core
Authors: Huang Chih-chin , Tang Min , Zhang Mei-Yun , Majeed Shahzad , Montabana Elizabeth , Stanfield Robyn L. , Dimitrov Dimiter S. , Korber Bette , Sodroski Joseph , Wilson Ian A. , Wyatt Richard , Kwong Peter D.
Published: November 2005

The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is immunodominant and contains features essential for coreceptor binding. We determined the structure of V3 in the context of an HIV-1 gp120 core complexed to the CD4 receptor and to the X5 antibody at 3.5 angstrom resolution. Binding of gp120 to cell-surface CD4 would position V3 so that its coreceptor-binding tip protrudes 30 angstroms from the core toward the target cell membrane. The extended nature and antibody accessibility of V3 explain its immunodominance. Together, the results provide a structural rationale for the role of V3 in HIV entry and neutralization.
Human T-Cell Leukemia Virus Type 1 Expressing Nonoverlapping Tax and Rex Genes Replicates and Immortalizes Primary Human T Lymphocytes but Fails To Replicate and Persist In Vivo
Authors: Younis Ihab , Yamamoto Brenda , Phipps Andrew , Green Patrick L.
Published: November 2005

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus associated primarily with adult T-cell leukemia and neurological disease. HTLV-1 encodes the positive
trans
-regulatory proteins Tax and Rex, both of which are essential for viral replication. Tax activates transcription initiation from the viral long terminal repeat and modulates the transcription or activity of a number of cellular genes. Rex regulates gene expression posttranscriptionally by facilitating the cytoplasmic expression of incompletely spliced viral mRNAs. Tax and Rex mutants have been identified that have defective activities or impaired biochemical properties associated with their function. To ultimately determine the contribution of specific protein activities on viral replication and cellular transformation of primary T cells, mutants need to be characterized in the context of an infectious molecular clone. Since the
tax
and
rex
genes are in partially overlapping reading frames, mutation in one gene frequently disrupts the other, confounding interpretation of mutational analyses in the context of the virus. Here we generated and characterized a unique proviral clone (H1IT) in which the
tax
and
rex
genes were separated by expressing Tax from an internal ribosome entry site. We showed that H1IT expresses both functional Tax and Rex. In short- and long-term coculture assays, H1IT was competent to infect and immortalize primary human T cells similar to wild-type HTLV-1. In contrast, H1IT failed to efficiently replicate and persist in inoculated rabbits, thus emphasizing the importance of temporal and quantitative regulation of specific mRNA for viral survival in vivo.
Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly
Authors: Alfadhli Ayna , Dhenub Tenzin Choesang , Still Amelia , Barklis Eric
Published: November 2005

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.
Herpes Simplex Virus 1 Immediate-Early and Early Gene Expression during Reactivation from Latency under Conditions That Prevent Infectious Virus Production
Authors: Pesola Jean M. , Zhu Jia , Knipe David M. , Coen Donald M.
Published: November 2005

The program of gene expression exhibited by herpes simplex virus during productive infection of cultured cells is well established; however, less is known about the regulatory controls governing reactivation from latency in neurons. One difficulty in examining gene regulation during reactivation lies in distinguishing between events occurring in initial reactivating cells versus events occurring in secondarily infected cells. Thus, two inhibitors were employed to block production of infectious virus: acyclovir, which inhibits viral DNA synthesis, and WAY-150138, which permits viral DNA synthesis but inhibits viral DNA encapsidation. Latently infected murine ganglia were explanted in the presence of either inhibitor, and then amounts of RNA, DNA, or infectious virus were quantified. In ganglia explanted for 48 h, the levels of five immediate-early and early RNAs did not exhibit meaningful differences between acyclovir and WAY-150138 treatments when analyzed by in situ hybridization or quantitative reverse transcription-PCR. However, comparative increases in viral DNA and RNA content in untreated ganglia suggested that virus was produced before 48 h postexplant. This was confirmed by the detection of infectious virus as early as 14 h postexplant. Together, these results suggest that high levels of viral gene expression at 48 h postexplant are due largely to the production of infectious virus and subsequent spread through the tissue. These results lead to a reinterpretation of previous results indicating a role for DNA replication in immediate-early and early viral gene expression; however, it remains possible that viral gene expression is regulated differently in neurons than in cultured cells.
The AAUAAA Motif of
Bamboo Mosaic Virus
RNA Is Involved in Minus-Strand RNA Synthesis and Plus-Strand RNA Polyadenylation
Authors: Chen I-Hsuan , Chou Wen-Jen , Lee Pei-Yu , Hsu Yau-Heiu , Tsai Ching-Hsiu
Published: November 2005

Bamboo mosaic virus
(BaMV) has a single-stranded positive-sense RNA genome with a 5′-cap structure and a 3′ poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3′ untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of
Nicotiana benthamiana
protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect
N. benthamiana
plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3′ UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.
Resistance to Friend Virus-Induced Erythroleukemia in
W
/
W

v

Mice Is Caused by a Spleen-Specific Defect Which Results in a Severe Reduction in Target Cells and a Lack of
Sf-Stk
Expression
Authors: Subramanian Aparna , Teal Hami E. , Correll Pamela H. , Paulson Robert F.
Published: November 2005

The characteristic progression and specificity of Friend virus for the erythroid lineage have allowed for the identification of a number of host-encoded loci that are required for disease progression. Several of these loci, including the Friend virus susceptibility gene 2 (
Fv2
), dominant white spotting gene (
W
), and Steel gene (
Sl
), regulate the initial polyclonal expansion of infected erythroid progenitor cells.
W
and
Sl
encode the Kit receptor tyrosine kinase and its ligand, stem cell factor, respectively.
W
mutant mice are severely anemic, and earlier work suggested that this defect in erythroid differentiation is the cause for the resistance to Friend virus-induced erythroleukemia. Here we show that in bone marrow,
W
/
W

v

mice have near normal numbers of target cells and the initial infection of bone marrow occurs normally in vivo. In contrast, spleen cells from
W
/
W

v

mice infected both in vitro and in vivo with Friend virus failed to give rise to erythropoietin-independent colonies at any time following Friend virus infection, suggesting that mutation of the Kit receptor specifically affects target cells in the spleen, rendering the mutant mice resistant to the development of Friend virus-induced erythroleukemia. In addition, we show that the Kit
+
pathogenic targets of Friend virus in the spleen are distinct from the pathogenic targets in bone marrow and this population of spleen target cells is markedly decreased in
W
/
W

v

mice and these cells fail to express
Sf-Stk
. These results also underscore the unique nature of the spleen microenvironment in its role in supporting the progression of acute leukemia in Friend virus-infected mice.
Protective and Therapeutic Capacity of Human Single-Chain Fv-Fc Fusion Proteins against West Nile Virus
Authors: Gould L. Hannah , Sui Jianhua , Foellmer Harald , Oliphant Theodore , Wang Tian , Ledizet Michel , Murakami Akikazu , Noonan Kristin , Lambeth Cassandra , Kar Kalipada , Anderson John F. , de Silva Aravinda M. , Diamond Michael S. , Koski Raymond A. , Marasco Wayne A. , Fikrig Erol
Published: November 2005

West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.
ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia
Authors: Jia Hong Peng , Look Dwight C. , Shi Lei , Hickey Melissa , Pewe Lecia , Netland Jason , Farzan Michael , Wohlford-Lenane Christine , Perlman Stanley , McCray Paul B.
Published: November 2005

Studies
of patients with severe acute respiratory syndrome (SARS) demonstrate
that the respiratory tract is a major site of SARS-coronavirus (CoV)
infection and disease morbidity. We studied host-pathogen interactions
using native lung tissue and a model of well-differentiated cultures of
primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2),
the receptor for both the SARS-CoV and the related human respiratory
coronavirus NL63, was expressed in human airway epithelia as well as
lung parenchyma. As assessed by immunofluorescence staining and
membrane biotinylation, ACE2 protein was more abundantly expressed on
the apical than the basolateral surface of polarized airway epithelia.
Interestingly, ACE2 expression positively correlated with the
differentiation state of epithelia. Undifferentiated cells expressing
little ACE2 were poorly infected with SARS-CoV, while
well-differentiated cells expressing more ACE2 were readily infected.
Expression of ACE2 in poorly differentiated epithelia facilitated SARS
spike (S) protein-pseudotyped virus entry. Consistent with the
expression pattern of ACE2, the entry of SARS-CoV or a lentivirus
pseudotyped with SARS-CoV S protein in differentiated epithelia was
more efficient when applied to the apical surface. Furthermore,
SARS-CoV replicated in polarized epithelia and preferentially exited
via the apical surface. The results indicate that infection of human
airway epithelia by SARS coronavirus correlates with the state of cell
differentiation and ACE2 expression and localization. These findings
have implications for understanding disease pathogenesis associated
with SARS-CoV and NL63
infections.
Neutrophils Interact with Adenovirus Vectors via Fc Receptors and Complement Receptor 1
Authors: Cotter Matthew J. , Zaiss Anne K. , Muruve Daniel A.
Published: November 2005

Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors.
Functional Divergence of Kaposi's Sarcoma-Associated Herpesvirus and Related Gamma-2 Herpesvirus Thymidine Kinases: Novel Cytoplasmic Phosphoproteins That Alter Cellular Morphology and Disrupt Adhesion
Authors: Gill Michael B. , Murphy Jo-Ellen , Fingeroth Joyce D.
Published: November 2005

The
nucleoside kinase encoded by Kaposi's sarcoma-associated herpesvirus
(KSHV) is a relatively inefficient enzyme with substrate specificity
for thymidine alone, unlike alphaherpesvirus thymidine kinases (TKs).
Similar to all gammaherpesvirus TKs, KSHV TK is composed of
two distinct domains, a conserved C-terminal kinase and a novel and
uncharacterized N terminus. Ectopic expression of KSHV TK in adherent
cells induced striking morphological changes and anchorage independence
although cells survived, a property shared with the related
rhadinovirus TKs of rhesus monkey rhadinovirus and herpesvirus saimiri.
To determine whether KSHV TK served alternate functions relevant to the
rhadinovirus life cycle and to reveal the contribution of the N
terminus, an enhanced green fluorescent protein-tagged fusion protein
and serial mutants were generated for investigation of intracellular
localization and cell biology. Analysis of truncation mutants showed
that a proline-rich region located within the N terminus cooperated
with the conserved C-terminal kinase to tether KSHV TK to a reticular
network in the cytoplasm and to induce morphological change. Fusion of
the KSHV N terminus to herpes simplex virus type 1 TK, a
nucleus-localized enzyme, similarly resulted in cytoplasmic
redistribution of the chimeric protein but did not alter cell shape or
adhesion. Unlike other human herpesvirus TKs, KSHV TKs and related
rhadinovirus TKs are constitutively tyrosine phosphorylated; a KSHV TK
mutant that was hypophosphorylated failed to detach and grow in
suspension. Loss of adhesion may enhance terminal differentiation,
viral replication, and egress at the cellular level and at the organism
level may facilitate detachment and distant migration of
KSHV-replicating cells within body fluids—promoting
oropharyngeal transmission and perhaps contributing to the multifocal
lesions that characterize KS.
Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-
d
-Ribonucleosides
Authors: Dittmer Alexandra , Drach John C. , Townsend Leroy B. , Fischer Anke , Bogner Elke
Published: November 2005

Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-
d
-nucleosides BDCRB or Cl
4
RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl
4
RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.