bluuebunny/crossref_metadata_2025 huggingface dataset:
A restraint molecular dynamics and simulated annealing approach for protein homology modeling utilizing mean angles
Authors: Möglich Andreas , Weinfurtner Daniel , Maurer Till , Gronwald Wolfram , Kalbitzer Hans Robert
Published: April 2005

Background

We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Döker et al. (1999, BBRC, 257, 348–350). The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS.

Results

To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor γ (Ppar γ). The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar γ was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms.

Conclusion

In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data.
Shortest triplet clustering: reconstructing large phylogenies using representative sets
Authors: Sy Vinh Le , von Haeseler Arndt
Published: April 2005

Background

Understanding the evolutionary relationships among species based on their genetic information is one of the primary objectives in phylogenetic analysis. Reconstructing phylogenies for large data sets is still a challenging task in Bioinformatics.

Results

We propose a new distance-based clustering method, the shortest triplet clustering algorithm (STC), to reconstruct phylogenies. The main idea is the introduction of a natural definition of so-called k-representative sets. Based on k-representative sets, shortest triplets are reconstructed and serve as building blocks for the STC algorithm to agglomerate sequences for tree reconstruction in O(n2) time for n sequences.

Simulations show that STC gives better topological accuracy than other tested methods that also build a first starting tree. STC appears as a very good method to start the tree reconstruction. However, all tested methods give similar results if balanced nearest neighbor interchange (BNNI) is applied as a post-processing step. BNNI leads to an improvement in all instances. The program is available at http://www.bi.uni-duesseldorf.de/software/stc/.

Conclusion

The results demonstrate that the new approach efficiently reconstructs phylogenies for large data sets. We found that BNNI boosts the topological accuracy of all methods including STC, therefore, one should use BNNI as a post-processing step to get better topological accuracy.
Deduction of probable events of lateral gene transfer through comparison of phylogenetic trees by recursive consolidation and rearrangement
Authors: MacLeod Dave , Charlebois Robert L , Doolittle Ford , Bapteste Eric
Published: April 2005

Background

When organismal phylogenies based on sequences of single marker genes are poorly resolved, a logical approach is to add more markers, on the assumption that weak but congruent phylogenetic signal will be reinforced in such multigene trees. Such approaches are valid only when the several markers indeed have identical phylogenies, an issue which many multigene methods (such as the use of concatenated gene sequences or the assembly of supertrees) do not directly address. Indeed, even when the true history is a mixture of vertical descent for some genes and lateral gene transfer (LGT) for others, such methods produce unique topologies.

Results

We have developed software that aims to extract evidence for vertical and lateral inheritance from a set of gene trees compared against an arbitrary reference tree. This evidence is then displayed as a synthesis showing support over the tree for vertical inheritance, overlaid with explicit lateral gene transfer (LGT) events inferred to have occurred over the history of the tree. Like splits-tree methods, one can thus identify nodes at which conflict occurs. Additionally one can make reasonable inferences about vertical and lateral signal, assigning putative donors and recipients.

Conclusion

A tool such as ours can serve to explore the reticulated dimensionality of molecular evolution, by dissecting vertical and lateral inheritance at high resolution. By this, we mean that individual nodes can be examined not only for congruence, but also for coherence in light of LGT. We assert that our tools will facilitate the comparison of phylogenetic trees, and the interpretation of conflicting data.
Abacavir, efavirenz, didanosine, with or without hydroxyurea, in HIV-infected adults failing initial nucleoside/protease inhibitor-containing regimens
Authors: Swindells Susan , Cohen Calvin J , Berger Daniel S , Tashima Karen T , Liao Qiming , Pobiner Bonnie F , Snidow Jerry W , Pakes Gary E , Hernandez Jaime E ,   the NZTA4008 Study Team
Published: April 2005

Background

Hydroxyurea (HU) is an immunomodulatory agent that has been documented to enhance the antiretroviral activity of nucleoside reverse transcriptase inhibitors, such as abacavir (ABC) and didanosine (ddI), and would be expected to improve virologic efficacy.

Methods

A 48-week, phase IV, multicenter, open-label, proof-of-concept clinical trial was conducted to evaluate second-line, protease inhibitor (PI)-sparing therapy with ABC/efavirenz (EFV)/ddI plus HU or without HU in HIV-infected subjects failing to achieve HIV-1 RNA ≤ 400 copies/mL after ≥ 16 weeks of treatment with lamivudine/zidovudine or lamivudine/stavudine, plus 1 or 2 PIs. Subjects were assigned to ABC (300 mg twice daily)/ EFV (600 mg once daily)/ ddI (400 mg once daily) plus HU (500 mg twice daily) (n = 30) or this regimen without HU (n = 24).

Results

Baseline mean HIV-1 RNA was 3.86 log10 copies/mL and CD4+ cell count was 345 cells/mm3. A similar percentage of subjects in the non-HU arm (58%) and HU arm (53%) completed the study. Intent-to-treat: missing = failure analysis showed no differences in proportions of subjects in the non-HU and HU arms achieving undetectable plasma HIV-1 RNA levels at week 24 (<400 copies/mL: 58% [14/24] vs 57% [17/30], P = 0.899; <50 copies/mL (50% [12/24] vs 47% [14/30], P = 0.780). Median change from baseline in CD4+ cell count in the non-HU and HU arms at week 48 was +114 cells/mm3 and -63 cells/mm3 (P = 0.007), respectively. Both regimens were generally well tolerated, although more subjects in the HU arm withdrew prematurely from the study due to adverse events (23% vs 4%). Four cases of possible ABC-related hypersensitivity were observed.

Conclusion

ABC/EFV/ddI was an effective and well-tolerated second-line regimen for nucleoside/PI-experienced HIV-infected subjects. The addition of HU blunted the CD4+ cell response, did not appear to enhance antiviral activity, and resulted in more treatment-limiting adverse events.
Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B
Authors: Soto José Luis , Cabrera Carmen M , Serrano Salvio , López-Nevot Miguel Ángel
Published: April 2005

Background

The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines

Methods

We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP).

Results

The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes.

Conclusion

These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.
Finite volume analysis of temperature effects induced by active MRI implants with cylindrical symmetry: 1. Properly working devices
Authors: Busch Martin HJ , Vollmann Wolfgang , Schnorr Jörg , Grönemeyer Dietrich HW
Published: April 2005

Background

Active Magnetic Resonance Imaging implants are constructed as resonators tuned to the Larmor frequency of a magnetic resonance system with a specific field strength. The resonating circuit may be embedded into or added to the normal metallic implant structure. The resonators build inductively coupled wireless transmit and receive coils and can amplify the signal, normally decreased by eddy currents, inside metallic structures without affecting the rest of the spin ensemble. During magnetic resonance imaging the resonators generate heat, which is additional to the usual one described by the specific absorption rate. This induces temperature increases of the tissue around the circuit paths and inside the lumen of an active implant and may negatively influence patient safety.

Methods

This investigation provides an overview of the supplementary power absorbed by active implants with a cylindrical geometry, corresponding to vessel implants such as stents, stent grafts or vena cava filters. The knowledge of the overall absorbed power is used in a finite volume analysis to estimate temperature maps around different implant structures inside homogeneous tissue under worst-case assumptions. The "worst-case scenario" assumes thermal heat conduction without blood perfusion inside the tissue around the implant and mostly without any cooling due to blood flow inside vessels.

Results

The additional power loss of a resonator is proportional to the volume and the quality factor, as well as the field strength of the MRI system and the specific absorption rate of the applied sequence. For properly working devices the finite volume analysis showed only tolerable heating during MRI investigations in most cases. Only resonators transforming a few hundred mW into heat may reach temperature increases over 5 K. This requires resonators with volumes of several ten cubic centimeters, short inductor circuit paths with only a few 10 cm and a quality factor above ten. Using MR sequences, for which the MRI system manufacturer declares the highest specific absorption rate of 4 W/kg, vascular implants with a realistic construction, size and quality factor do not show temperature increases over a critical value of 5 K.

Conclusion

The results show dangerous heating for the assumed "worst-case scenario" only for constructions not acceptable for vascular implants. Realistic devices are safe with respect to temperature increases. However, this investigation discusses only properly working devices. Ruptures or partial ruptures of the wires carrying the electric current of the resonance circuits or other defects can set up a power source inside an extremely small volume. The temperature maps around such possible "hot spots" should be analyzed in an additional investigation.
Tissue Doppler echocardiographic quantification. Comparison to coronary angiography results in Acute Coronary Syndrome patients
Authors: Donal Erwan , Raud-Raynier Pascale , Coisne Damien , Allal Joseph , Herpin Daniel
Published: April 2005

Background

Multiples indices have been described using tissue Doppler imaging (DTI) capabilities. The aim of this study was to assess the capability of one or several regional DTI parameters in separating control from ischemic myocardium.

Methods

Twenty-eight patients with acute myocardial infarction were imaged within 24-hour following an emergent coronary angioplasty. Seventeen controls without any coronary artery or myocardial disease were also explored. Global and regional left ventricular functions were assessed. High frame rate color DTI cineloop recordings were made in apical 4 and 2-chamber for subsequent analysis. Peak velocity during isovolumic contraction time (IVC), ejection time, isovolumic relaxation (IVR) and filling time were measured at the mitral annulus and the basal, mid and apical segments of each of the walls studied as well as peak systolic displacement and peak of strain.

Results

DTI-analysis enabled us to discriminate between the 3 populations (controls, inferior and anterior AMI). Even in non-ischemic segments, velocities and displacements were reduced in the 2 AMI populations. Peak systolic displacement was the best parameter to discriminate controls from AMI groups (wall by wall, p was systematically < 0.01). The combination IVC + and IVR< 1 discriminated ischemic from non-ischemic segments with 82% sensitivity and 85% specificity.

Conclusion

DTI-analysis appears to be valuable in ischemic heart disease assessment. Its clinical impact remains to be established. However this simple index might really help in intensive care unit routine practice.
Research: Is resection of tumours involving the pelvic ring justified? : A review of 49 consecutive cases
Authors: Yuen Alex , Ek Eugene T , Choong Peter FM
Published: April 2005

Introduction

Pelvic surgery is challenging and impacts significantly on limb and visceral function, thus, raising the question "is heroic surgery justifiable". This study assessed the functional, oncologic and surgical outcomes following pelvis tumour resections.

Methods

Between 1996–2003, 49 patients (mean age 43 years) underwent pelvic tumour resections- 38 primary malignant tumours, 5 secondary tumours and 6 benign tumours. Bone tumours comprised 5 osteosarcomas, 5 Ewings sarcomas, and 12 chondrosarcomas. Of the soft tumours, 9 were of neural origin. Tumours involved the ilium, acetabulum, pubic bones, sacrum or a combination of these. Functional assessment was performed and no patient had metastases at presentation.

Results

There were 41 limb sparing resections and 8 hindquarter amputations. Surgical margins were intralesional (1), marginal (13), wide (26), and radical (3). Of limb sparing surgery, prosthetic reconstructions were performed in 10 patients, biologic reconstructions in 6, a combination of these in 3 and no reconstruction in others. There was 1 intraoperative death, 7 local recurrences and 19 metastases. Death from disease occurred at a mean of 14.2 months with a mean followup of 27 (1–96) months. Amputation and periacetabular resections had worse functional outcomes. Emotional acceptance was surprisingly high.

Conclusion

Pelvic resections are complex. Functional outcome is significantly affected by surgery. Disease control is similar to limb tumours. Emotional acceptance of surgery in survivors was surprisingly high. Major pelvic resection for malignancy appears justified.
Monochorionic triamniotic triplet pregnancy with a co-triplet fetus discordant for congenital cystic adenomatoid malformation of the lung
Authors: Gul Ahmet , Aslan Halil , Cebeci Altan , Ceylan Yavuz , Tekirdag Ali Ismet
Published: April 2005

Background

Spontaneous monochorionic triamniotic pregnancy is rare and is at increased risk for pregnancy complications. The presence of an anomalous fetus further complicates the management.

Case presentation

We present a case of monochorionic triamniotic triplet pregnancy diagnosed at 15 weeks of gestation with one fetus having developed a multicystic lung lesion, suggestive of congenital cystic adenomatoid malformation (CCAM). At 24 weeks, the largest cyst measured 10 mm in diameter. We managed the pregnancy conservatively and delivered three live male fetuses with birth weights 1560 g, 1580 g and 1590 g at 35 weeks of gestation. Two newborns were admitted to the neonatal intensive care unit with respiratory distress, the third one died due to sepsis 7 days postpartum. One of the newborns was discharged healthy at 24 days postpartum. The newborn with CCAM developed a pneumothorax on the right side, recovered after treatment, and was discharged after one month. Computerized tomography (CT) of the infant at 3 months demonstrated two cystic lesions in the middle lobe of the right lung measuring 25 mm and 15 mm. A repeat CT of the infant at 6 months showed a 30 mm solitary cystic mass.

Conclusion

Monochorionic triamniotic triplet pregnancy with a co-triplet fetus discordant for CCAM, present rarely and can be managed conservatively. These findings may help in decision making and counselling of parents.
Study on Oxygen Quenching Processes of the Excited State Dendrimer Molecules
Authors: Momotake Atsuya , Horiuchi Hiroaki , Hayakawa Junpei , Mizutani Takuo , Nishimura Yoshinobu , Hiratsuka Hiroshi , Arai Tatsuo
Published: April 2005

Since the excited singlet state of the core stilbene in dendrimers G1–G4 has ca. 5-ns lifetime, oxygen quenched their excited singlet state producing the singlet oxygen. The efficiency of singlet oxygen production based on the quenching of the stilbene core is the highest in G4.
Copacabana's other side: Marcos Bernstein and The Other Side of the Street at the Berlin Film Festival 2004
Authors: Hermanns Ute
Published: April 2005

Marcos Bernstein, Brazilian screenwriter and director, attended the Berlin International Film Festival in 2004. His film O outro lado da rua (The Other Side of the Street) proved to be a great success due, in part, to the participation in the film of Oscar-nominated actress Fernanda
Montenegro (who starred in Central Station, for which Bernstein wrote the screenplay). This interview focuses on what led Bernstein to direct his first movie, his experience as a screenwriter, his conception of film-making and the plot and themes of this new film. As a result of his
fascination with Fernanda Montenegro, the ‘first lady’ of Brazilian film, he resolved to make a movie about the life and love affairs of older people in Copacabana, the beach-front Rio de Janeiro district where many old people live.
Report on the Munich Film Festival 2004
Authors: Fisher Jaimey
Published: April 2005

Although the Munich festival has long had to play second fiddle to Berlin's A-list event, it remains in many ways more important for Germany's media industry. This status is reflected in the character and constitution of both film festivals: while Berlin offers a higher profile venue
for international competition and independent film - it usually attracts more Hollywood stars as well as film buyers - the Munich festival's biggest prizes are intended for domestic talent. This year's festival was one of the best of the last several, not only because it offered excellent
individual films, but also because of its productive mixture of experienced directors and relative newcomers.
The Expanding Role of the Pediatrician in Improving Child Health in the 21st Century
Authors: Satcher David , Kaczorowski Jeffrey , Topa David
Published: April 2005

Children in the United States increasingly are at risk for health problems that are precipitated or exacerbated by social, community, and environmental factors. Currently, pediatricians are unable to sufficiently address these health conditions without expanding their roles beyond that of providing health care to individual patients. Thus, to improve child health, physicians must work within their communities to identify the needs of the population they serve and take appropriate action to influence the private and public policies that address these needs. Healthy People 2010 establishes a well-supported framework that confronts the social and community factors that affect children's health and serves as a resource for community-minded pediatricians. In addition to Healthy People 2010, other successful initiatives have been created by pediatricians, and they must be expanded if the pediatric community is to alleviate the social, community, and environmental factors that negatively affect child health.
Community Pediatrics: The Rochester Story
Authors: Haggerty Robert J. , Aligne C. Andrew
Published: April 2005

There are so many problems facing children today (eg, violence, poor nutrition, substance abuse, teen pregnancy) that conventional medical care can only address a small portion of these concerns. Thus, to be optimally effective, pediatrics needs to be linked to other disciplines and programs that address these issues by using different paradigms. Robert Haggerty, the originator of the term “community pediatrics,” reflects on how one can successfully practice community pediatrics in an academic setting and model it for young physicians while also improving the health of children at the community level. Here we tell the story of the years that Haggerty was chief of pediatrics at the University of Rochester and took on the challenge of fulfilling the department's responsibility to all children in the county. Because of his pioneering work, his tenure was heralded as a critical period in the development of the field of community pediatrics.
Communities as Teachers: Learning to Deliver Culturally Effective Care in Pediatrics
Authors: Sidelinger Dean E. , Meyer Dodi , Blaschke Gregory S. , Hametz Patricia , Batista Milagros , Salguero Rachel , Reznik Vivian
Published: April 2005

A patient's culture has an effect on her or his view of illness, decision to seek care, and adherence to treatment plans and follow-up visits. In this article, we describe community-academic partnerships designed to teach improved delivery of culturally effective care conducted in pediatric residency training programs in New York, New York, and San Diego, California. Columbia University–Children's Hospital of NewYork-Presbyterian focuses most of residents' cultural-training experiences within 1 community program, a home-visitation program (Best Beginnings) with which residents work in various capacities throughout residency. The University of California, San Diego and Naval Medical Center San Diego use a series of cultural “immersion experiences” as a primary method. The creation of community-academic partnerships for the purpose of service and training can be a critical asset in the development of culturally effective care training: community partners become teachers and local communities serve as classrooms.
Building Healthier Communities for Children and Families: Applying Asset-Based Community Development to Community Pediatrics
Authors: Pan Richard J. , Littlefield Diane , Valladolid Sara G. , Tapping Peggy J. , West Daniel C.
Published: April 2005

Social capital is the power of social networks and relationships, which constitute the social environment. Social capital has been associated with many measures of health and development. Asset-based community development (ABCD) provides a framework to increase social capital and build stronger, healthier communities for children. ABCD is a strength-based approach to community building that emphasizes bringing together community assets including individual community members, voluntary associations, and institutions. How pediatricians can apply ABCD to child health is described.
Integrating Pediatrics and Public Health
Authors: Goldhagen Jeffrey
Published: April 2005

The demographics of children in the United States and the determinants of child health outcomes are evolving rapidly. This evolution will require those responsible for children's health and well-being to acquire new skills and access new resources in the future to ensure the relevance of the field of pediatrics to the health of children. The objective of this article is to define a new paradigm for the practice of pediatrics that integrates the knowledge, attitudes, and skills of pediatrics with the principles, practice, and resources of public health to address the current challenges to child health in the United States. Historical and contemporary advances in the understanding of child health provide a precedent and framework to facilitate this integration.
Continuum Regression
Authors: Sundberg Rolf
Published: April 2005

When, in a multiple regression, regressors are near‐collinear, so‐called regularized or shrinkage regression methods can be highly preferable to ordinary least squares, by trading bias for variance. Continuum regression, introduced by Stone and Brooks in 1990, ties together several more classical regularized regression methods, such as principal components regression, partial least squares regression, and ridge regression.
StPetersburg School of Probability
Authors: Seneta Eugene
Published: April 2005

The term St. Petersburg School of Probability is used to describe the chief St. Petersburg mathematicians who worked on probabilistic problems, and mathematicians in other cities of the Russian Empire whose work influenced them. The St. Petersburg group is taken as beginning with the return from Paris of M. V. Ostrogradsky and V. Ya. Buniakovsky, whose subsequent probabilistic creativity was strongly influenced by Laplace'sThéorie analytique. Buniakovsky's association with Chebyshev led to the probabilistic flourishing of Chebyshev and his pupils Markov and Liapunov. Markov's creativity in Markov chains was stimulated by his antagonistic interaction with P. A. Nekrasov (Moscow), and his probabilistic oeuvre in general was initiated against a background of support by A. V. Vasiliev (Kazan).
Identifying stereoisomers of the asymmetric hydrogenation catalyst [Me‐BPE‐Rh(COD)]+BF
Authors: Fox Martin E. , McCague Raymond
Published: April 2005

The performance of a catalyst used in asymmetric synthesis is likely to be dependent upon its stereoisomeric purity. An impurity was detectable by 31P NMR in early development batches of the asymmetric hydrogenation catalyst [(S,S)‐Me‐BPE‐Rh(COD)]+BF made from the ligand bis((2S,5S)‐2,5‐dimethylphospholano)ethane [(S,S)‐Me‐BPE]. Its identity as a stereoisomer with one chiral and one meso‐phospholane ring was deduced by comparison of the 31P NMR spectra and GC traces of the ligand with a deliberately synthesized mixture of isomers. Interestingly, the impurity corresponded to a trans‐meso isomer formed by thermal (200°C) pyramidal inversion at phosphorus of the initially synthesized cis‐meso‐phospholane when the ligand was purified by distillation. Low levels of this trans‐meso/chiral impurity do not significantly impair the enantioselectivity of the rhodium complex as an asymmetric hydrogenation catalyst, but high levels of stereochemical impurities resulted in a loss of both enantioselectivity and activity. Therefore it is indeed important to establish that a catalyst used in asymmetric catalysis is sufficiently stereoisomerically pure. Owing to strict control of the stereochemical purity of the key hexane‐2,5‐diol intermediate, the impurity is not detected in production batches. Chirality 17:177–185, 2005. © 2005 Wiley‐Liss, Inc.
Configurational stability of 2‐benzoylcyclohexanone: Unexpected solvent effects on the rate of racemization
Authors: Tumambac Gilbert E. , Francis Christopher J. , Wolf Christian
Published: April 2005

The kinetics of the racemization of 2‐benzoylcyclohexanone 1 in hexanes, ethanol, and mixtures thereof have been investigated by time dependence of enantiomeric purity using enantioselective HPLC. In pure hexanes and ethanol, the racemization half‐lives were determined as 552 and 23.8 min, respectively, at 66°C. Surprisingly, racemization of 1 in mixtures of hexanes and ethanol was found to involve an induction period followed by a sigmoidal decrease of the enantiomeric excess with half‐lives varying between 11.5 and 24.0 min. This unexpected solvent influence on the rate of racemization of 1 was attributed to complex isomerization mechanisms involving three possibly interconverting enol tautomers of 1. Chirality 17:171–176, 2005. © 2005 Wiley‐Liss, Inc.
Complexes containing actin and spectrin from erythrocyte and brain
Authors: Lin Diane C. , Flanagan Michael D. , Lin Shin
Published: April 2005

A complex of proteins with properties similar to those of erythrocyte spectrinband 4.1‐actin complex has been idientified in a preparation derived from bovine brain. The complex has an apparent sedimentation coefficient of about 26S, and contains brain spectrin (also called fodrin) and actin as major components. The actin in the complex is in the oligomeric form, which nucleates assembly of actin filaments that grow from the “barbed” end. The complex cross‐links actin filaments, resulting in an increase in low‐shear viscosity. Whether the complex contains a protein analogous to erythrocyte band 4.1 is not known. However, it can be demonstrated that brain spectrin has the capability to interact with band 4.1 in a way which increases its ability to cross‐link actin filaments.
Organizational forms of actin in 13762 ascites mammary tumor cell microvilli
Authors: Carraway Coralie A. Carothers , Jung Goeh , Carraway Kermit L.
Published: April 2005

The organization of microvillus actin and its associated proteins have been investigated in sublines of mammary ascites tumors (MAT) with mobile (MAT‐B1) and immobile (MAT‐C1) cell surface receptors. Microvilli isolated from these sublines differ in morphology (branched for MAT‐C1 versus unbranched for MAT‐B1) and the presence of a 58,000‐dalton polypeptide (58K). 58K is found associated with MAT‐C1 microvilli, microvillar cytoskeletons obtained by nonionic detergent extractions, and microvillar membranes prepared under conditions which depolymerize actin microfilaments. By extraction with actin‐stabilizing buffers (isotonic Triton‐Mg‐ATP) microvillar actin can be fractionated into four forms. About 40% of the actin is sedimented at low speed (7,500g, 15 min). The pellets contain microfilaments; actin and α‐actinin are the predominant proteins. High‐speed pellets from these low‐speed supernates contain about 10% of the actin as a transmembrane complex with a cell surface glycoprotein (cytoskeleton‐associated glycoprotein, [CAG] 75–80,000 daltons) in MAT‐B1 cells or with CAG and 58K in MAT‐C1 cells. Transmembrane complexes can be purified from MAT‐B1 and MAT‐C1 microvillar membranes in Triton‐containing buffer by gel filtration or sucrose density gradient centrifugation. The presence of only CAG and actin in the MAT‐B1 transmembrane complex strongly suggests the direct interaction of actin and a cell surface component. The high‐speed supernates contain soluble actin. By gel filtration or rate‐zonal sucrose density gradient centrifugation about 30% of the microvillar actin is found as small oligomers and about 10% as G‐actin in this extraction buffer. We suggest that the actin‐containing transmembrane complexes may serve as membrane‐association sites for oligomeric actin segments and microfilaments and as initiation sites for actin polymerization.
Calcium regulation of the actin‐mediated cytoskeletal transformation of sea urchin coelomocytes
Authors: Henson J. H. , Schatten G.
Published: April 2005

Coelomocytes from several echinoderm species undergo an actin‐mediated cytoskeletal transformation once subjected to hypotonic shock. In this study, coelomocytes from the sea urchins Lytechinus variegatus and Arbacia punctulata were induced to “transform” by treatment with > 5 μM of the calcium ionophore A23187 in the presence of external Ca++. The dependence of ionophore transformation on external Ca++ and the lack of chlorotetracycline staining indicates that these cells rely on external Ca++ sources. NBD‐phallacidin (7‐Nitrobenz‐2‐oxa‐1,3‐diazole‐phallacidin) staining of lysolecithin permeabilized cells and wholemount transmission electron microscopy (TEM) show that similar reorganizations of the actin cytoskeleton take place during hypotonic shock and ionophore transformation, although actin filament bundling is less apparent in A23187‐treated cells. As has been shown with hypotonic shock transformation, the ionophore elicited shape change is inhibited by anticalmodulin drugs. Greater than 10 μM concentrations of W 13 inhibit filopod formation, while this drug's less active structural analogue, W 12, exhibits no effects. W 13 also appears to disrupt actin filament‐membrane associations in the cells. Fluorescent localization of calmodulin using a photooxidized derivative of trifluoperazine indicates a general cytoplasmic distribution with some concentration in filopod core bundles. Coelomocyte transformation may be an example of a cellular shape change regulated by Ca++ through the action of calmodulin modulation of actin‐membrane interactions.
Effect of cytochalasin D on smooth muscle contraction
Authors: Adler Kenneth B. , Krill Judith , Alberghini Tod V. , Evans John N.
Published: April 2005

Cylindrical segments of extraparenchymal pulmonary artery (essentially a preparation of smooth muscle with regard to contractile capability) were isolated from adult male rats. They were mounted in an isometric muscle bath in physiological salt solution (PSS) in an environment of 95% O2/ CO2. After allowing 1 h for equilibration, the maximum force generated by the tissue in response to a depolarizing solution was determined. After relaxation, vessels were incubated for 1 h in one of several concentrations of cytochalasin D (CD) (0.01, 0.05, 0.5, 1, 10 μg/ml) and the response to stimulation retested immediately after returning to PSS, and then at 30 minute intervals up to 2 h.

CD inhibited the ability of vascular smooth muscle to generate force (contract) in a concentration‐dependent manner. The inhibitory effect was reversible within a short period of time. Quantitative electron microscopic examination of these vessels suggested that CD disrupts the integrity of myofilaments, especially at sites of “dense bodies.” Our results indicate that a percentage of actin in smooth muscle cells is not permanently in the filamentous “F” form, but is part of the G:F actin system of the cell, labile to polymerization:depolymerization. The ability of smooth muscle cells to generate force could depend on the proper functioning of the F:G actin “treadmill”.
Intracellular movement of fodrin
Authors: Cheney Richard , Hirokawa Nobutaka , Levine Joel , Willard Mark
Published: April 2005

Fodrin is an actin/calmodulin‐binding protein with similarities to spectrin (erythrocytes) and TW 260/240 (brush border). It is concentrated beneath the plasma membranes of neurons and other cells. We have observed translocations of fodrin in both neurons and lymphocytes. Newly synthesized, radiolabeled fodrin moves down axons at a maximum velocity (about 50 mm/day) that is slower than the most rapidly axonally transported proteins (group I). A portion of fodrin appears to move more slowly at velocities (1–10 mm/day) resembling those of actin and myosin (group IV) and tubulin and neurofilament proteins (group V). In lymphocytes, when certain surface antigens are induced by cross‐linking agents to migrate to one pole of the cell and form a cap, fodrin redistributes beneath the membrane and forms a subcap. The movements of fodrin in lympohocyte capping and in the axonal transport of group IV polypeptides have certain similarities. In both cases, the redistribution of fodrin is accompanied by concomitant redistributions of actin, myosin, and calmodulin, and both processes proceed at similar velocities. We consider the possibilities that these two processes are related, both being driven by a submembrane force‐generating system comprising in part actin, myosin, and fodrin, and that fodrin serves to link various organelles or proteins to this system.
Amino acid alterations in the benA (β‐tubulin) gene of Aspergillus nidulans that confer benomyl resistance
Authors: Jung M. Katherine , Wilder Isaac B. , Oakley Berl R.
Published: April 2005

We report the cloning and sequencing of 18 mutant alleles of the benA, β‐tubulin gene of Aspergillus nidulans that confer resistance to the benzimidazole antifungal, antimicrotubule compounds benomyl, carbendazim, nocodazole, and thia‐bendazole. In 12 cases, amino acid 6 was changed from histidine to tyrosine or leucine. In four cases, amino acid 198 was changed from glutamic acid to aspartic acid, glutamine, or lysine. In two cases, amino acid 200 was altered from phenylalanine to tyrosine. These data, along with previous data indicating that amino acid 165 is involved in the binding of the R2 group of these compounds [Jung and Oakley, 1990: Cell Motil. Cytoskeleton 17:87‐94], suggest that regions of β‐tubulin containing amino acids 6, 165, and 198‐200 interact to form the binding site of benzimidazole antimicrotubule agents. These results also suggest that the presence of phenylalanine at amino acid 200 contributes to the great sensitivity of many fungi to benzimidazole antimicrotubule agents. © 1992 Wiley‐Liss, Inc.
Control of organelle transport in melanophores: Regulation of CA2+ and cAMP levels
Authors: Thaler Catherine D. , Haimo Leah T.
Published: April 2005

Melanophores of the cichlid Tilapia mossambica can be induced to aggregate pigment by addition of epinephrine to the medium, suggesting adrenergic control of this transport. The melanophore response to adrenergic stimulation was examined using agonists and antagonists that are highly specific for each alpha‐adrenoceptor subclass. The signal transduction mechanism of each subclass is unique: stimulation of alpha1 receptors results in a rise in intracellular free Ca2+, while alpha2 stimulation results in decreased cAMP levels [Exton, 1985: Am. J. Physiol. 248:E633–E647 ]. Each alpha1 or alpha2 specific agonist tested showed a dose dependent ability to induce aggregation and each was able to effect complete aggregation of pigment, suggesting that aggregation can be mediated either by elevating Ca2+ or by lowering cAMP. However, in the presence of either an alpha1 or an alpha2 receptor antagonist, none of the agonists were able to induce significant aggregation, suggesting that changes in levels of both messengers are required for pigment aggregation in the melanophores. Moreover, experiments in which intracellular levels of Ca2+ or cAMP were perturbed, using BAPTA and forskolin, respectively, indicated that elevating Ca2+ in the presence of high cAMP is not sufficient to induce aggregation and, conversely, that lowering cAMP levels in the presence of reduced Ca2+ is not sufficient to induce pigment aggregation. These data indicate that the concentrations of both cAMP and Ca2+ are important in regulating pigment aggregation in teleost melanophores, and suggest that maximal aggregation of pigment requires altering the levels of both messengers. © 1992 Wiley‐Liss, Inc.
Three‐dimensional motility cycle in leukocytes
Authors: Murray John , Vawter‐Hugart Holly , Voss Edward , Soll David R.
Published: April 2005

A 3‐dimensional dynamic image analyzing system (3D‐DIAS) has been developed in which a translocating cell is optically sectioned in the z‐axis within a 2 sec period; the perimeter of the cell in each section is digitized into the 3D‐DIAS data file, and the digitized perimeters are wrapped in order to reconstruct the cell image in three dimensions. Using 3D‐DIAS, we have obtained the first dynamic 3‐dimensional description of human polymorphonuclear leukocytes (PMN) translo‐cating on a glass surface. A general behavior cycle has emerged which includes two phases. In the first, an ellipsoidal PMN with significant z‐axis extends anteriorly and descends to the substratum. When the ventral surface of the anterior end contacts the substratum, there is rapid anterior expansion, which correlates with velocity peaks. In the second phase, the elongate PMN stops translocating along the substratum, the anterior end lifts off of the substratum, sometimes to heights greater than the length of the PMN at the substratum, and finally the PMN retracts into an ellipsoidal morphology still capable of random protrusions. During this second phase, which correlates with velocity troughs, turning usually occurs. The degree of turning is restricted by the continuous integrity of the posterior uropod. The period of the behavior cycle varies from roughly 0.5 to 2 min between PMNs, but is relatively constant within each individual PMN. © 1992 Wiley‐Liss, Inc.
Preparation and characterization of calibration beads for sorting cells expressing a β‐lactamase gene reporter
Authors: Cunningham Michael E. , Kapitskaya Marianna , Petrukhin Konstantin , Bednar Bohumil
Published: April 2005

Background

Modern drug discovery has been based on high‐throughput screening using whole‐cell assays. A prominent role has been assigned to the reporter gene technology based on a β‐lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence‐activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the β‐lactamase gene using the CCF2 substrate.

Methods

To model Förster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer.

Results

We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate.

Conclusion

We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence‐activated cell sorting to sort cells with CCF2 reporter substrate and the β‐lactamase reporter gene. © 2005 Wiley‐Liss, Inc.
Aberrant methylation of secreted frizzled‐related protein genes in esophageal adenocarcinoma and Barrett's esophagus
Authors: Zou Hongzhi , Molina Julian R. , Harrington Jonathan J. , Osborn Neal K. , Klatt Kristie K. , Romero Yvonne , Burgart Lawrence J. , Ahlquist David A.
Published: April 2005

Hypermethylation of secreted frizzled‐related proteins (SFRP) genes frequently occurs with several cancers but has not been studied in esophageal adenocarcinoma or its precursor—Barrett's esophagus. To explore the role of SFRP methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated SFRP genes as biomarkers for Barrett's esophagus and cancer, methylation of SFRP genes was determined in esophageal adenocarcinomas, Barrett's esophagus and normal epithelia using methylation‐specific PCR. Protein expression of SFRP genes was then assessed in these tissues by immunohistochemistry. The mRNA expression of SFRP genes was quantified by real‐time reverse‐transcription PCR in esophageal adenocarcinoma cell lines with and without demethylation by 5‐aza‐2′deoxycytidine and inhibition of deacetylation by trichostatin A treatment. Hypermethylation of SFRP1, 2, 4 and 5 was detected in 93%, 83%, 73% and 85% of 40 cancers; 81%, 89%, 78% and 73% of 37 Barrett's epithelia; 25%, 64%, 32% and 21% of 28 adjacent normal epithelia from Barrett's patients; and 10%, 67%, 0% and 13% of 30 normal esophagogastric epithelia from healthy individuals, respectively (p < 0.001 for SFRP1, 4 and 5; p < 0.05 for SFRP2). Protein expression of SFRP1, 2 and 4 was downregulated in 87%, 67% and 90% of cancers, and expression correlated inversely with grade and stage of cancers and with grade of dysplasia. Expression of SFRP2 and SFRP4 proteins was lower in cancers with corresponding gene methylation (p < 0.05). Demethylation treatment effectively re‐expressed SFRP mRNA in cancer cell lines. Thus, hypermethylation of SFRP genes is a common early event in the evolution of esophageal adenocarcinoma, and methylation of SFRP1, 4 and 5 might serve as biomarkers for Barrett's neoplasia. Aberrant promoter methylation appears to functionally silence SFRP gene expression in esophageal adenocarcinoma. © 2005 Wiley‐Liss, Inc.
Functional analysis of p53 gene and the prognostic impact of dominant‐negative p53 mutation in endometrial cancer
Authors: Sakuragi Noriaki , Watari Hidemichi , Ebina Yasuhiko , Yamamoto Ritsu , Steiner Eric , Koelbl Heinz , Yano Masahiro , Tada Mitsuhiro , Moriuchi Tetsuya
Published: April 2005

In addition to the loss of function, mutant p53 can possess a dominant‐negative effect on wild‐type p53 and may also exert gain‐of‐function activity. It is not clear whether the functional status of p53 mutation contributes to differences in outcome in endometrial cancer. We collected a total of 92 RNA samples of high quality from endometrial cancer tissues, and the samples were subjected to yeast functional assay and sequencing for p53 mutations. The detected mutant p53 genes were further investigated for their dominant‐negative activity using a yeast‐based transdominance assay. p53 mutation was found in 24 out of 92 (26.1%) tumors, of which 10 exhibited no dominant‐negative activity (recessive mutation) and 14 showed dominant‐negative activity. Dominant‐negative p53 mutation was related to advanced stages (p = 0.01), nonendometrioid type tumors (p = 0.01) and grade 3 tumors (p = 0.04). The patients with dominant‐negative mutation had significantly shorter survival than patients with no mutation (p < 0.0001) and those with a recessive mutation (p = 0.01) in the p53 gene. No difference in survival was found between the patients with tumors harboring a recessive p53 mutation and those with tumors harboring a wild‐type p53. Multivariate analysis revealed that dominant‐negative p53 mutation (p = 0.019), FIGO stage (p = 0.0037) and histologic subtype (p = 0.014) were independently related to patient survival. Dominant‐negative p53 mutation was the most important prognostic factor for stage III/IV endometrial cancer (p = 0.0023). In conclusion, dominant‐negative p53 mutation is often found in advanced stages and aggressive histologic subtypes of endometrial cancer and it is a strong predictor of survival of patients with advanced endometrial cancer. To elucidate further the role of p53 mutation in endometrial cancer, it is necessary to investigate gain‐of‐function activity involving dominant‐negative p53 mutant proteins. © 2005 Wiley‐Liss, Inc.