bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural analysis of glucoamylase encoded by the STA1 gene of Saccharomyces cerevisiae (var. diastaticus)
Authors: Adam Ana Cristina , Latorre‐García Lorena , Polaina Julio
Published: March 2004

The sequence of the STA1‐encoded glucoamylase of amylolytic Saccharomyces cerevisiae (var. diastaticus) strains shows two well‐defined regions: an amino‐terminal part rich in serine and threonine residues and a carboxy‐terminal part very similar to the catalytic domain of other fungal glucoamylases. A version of the enzyme in which most of the amino‐terminal region was deleted still has glucoamylase activity, indicating that the remaining carboxy‐terminal part forms a functional catalytic domain. Homology‐based models of the two parts of the protein have been obtained. As expected, the shortened form of the enzyme is very similar to the catalytic domain of related glucoamylases of known structure. However, the amino‐terminal part yielded a structure revealing an unexpected similarity to bacterial invasins, suggesting functional connections between several yeast proteins homologous to STA1‐encoded glucoamylase and invasins. A characteristic of Saccharomyces glucoamylase in its native form is its extreme degree of glycosylation. Despite its high molecular mass (about 300 kDa), and in contrast with what occurs with other extracellular glycoproteins produced by yeast, the enzyme does not remain attached to the cell wall, being fully and efficiently secreted into the medium, even when it is produced in large amounts by overexpression of its gene. Copyright © 2004 John Wiley & Sons, Ltd.
Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae
Authors: Boorsma André , Nobel Hans de , Riet Bas ter , Bargmann Bastiaan , Brul Stanley , Hellingwerf Klaas J. , Klis Frans M.
Published: March 2004

The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1–Rho1‐controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall‐related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes. Transcript profiles were analysed using two recently developed algorithms, viz. REDUCE, which correlates upstream regulatory motifs with expression, and Quontology, which compares expression of genes from functional groups with overall gene expression. Both methods indicated upregulation of Rlm1p‐controlled cell wall genes and STRE‐controlled genes, and downregulation of ribosomal genes and rRNA genes. Comparison of these expression profiles with the published profiles of two constitutively active upstream activators of the Slt2p–MAP kinase pathway, viz. Pkc1‐R398A and Rho1‐Q68A, revealed significant similarity. In addition, a new putative regulatory motif, CCC(N)10GGC, was found. In Zymolyase ‐treated cells a regulatory site was identified, ATGACGT, which resembles the AFT/CRE binding site. Interestingly, Sko1p, a downstream regulator of the high osmolarity pathway is known to bind to the AFT/CRE binding site, suggesting a possible role for the Hog1 pathway in the response to cell wall stress. Finally, using REDUCE, an improved version of the Rlm1 binding motif, viz. TA(W)4TAGM, was discovered. We propose that this version can be used in combination with REDUCE as a sensitive indicator of cell wall stress. Taken together, our data indicate that cell wall stress results in activation of various signalling pathways including the cell wall integrity pathway. Copyright © 2004 John Wiley & Sons, Ltd.
Molecular characterization of the glutathione‐dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica
Authors: Nakagawa Tomoyuki , Ito Takashi , Fujimura Shuki , Chikui Mika , Mizumura Tasuku , Miyaji Tatsuro , Yurimoto Hiroya , Kato Nobuo , Sakai Yasuyoshi , Tomizuka Noboru
Published: March 2004

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione‐dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica. The P. methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80–88%). In P. methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose. Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium. These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol‐grown cells according to the methanol concentration on growth. The sequence has been submitted to the DDBJ/EMBL/GenBank databases under Accession No. AB112935. Copyright © 2004 John Wiley & Sons, Ltd.
Animal Ototoxicity of Topical Antibiotics and the Relevance to Clinical Treatment of Human Subjects
Authors: Roland Peter S. , Rybak Leonard , Hannley Maureen , Matz Greg , Stewart Michael G. , Manolidis Spiros , Friedman Rick , Weber Peter , Owens Fred
Published: March 2004

OBJECTIVE

The objective of this study was to systematically review the literature on animal ototoxicity from ototopical medications. A secondary objective was to assess the relevance of animal data to the use of ototopical drops in clinical situations involving humans.

STUDY DESIGN

We performed a MEDLINE search of the published literature using appropriate search terms to identify pertinent articles, which were reviewed, summarized, and tabulated.

RESULTS

One hundred seventy‐three articles were reviewed; 61 articles were appropriate to the study question and were further analyzed.

CONCLUSIONS

Virtually all studies demonstrate that aminoglycoside antibiotics, when applied topically into the middle ear space, are ototoxic in experimental animals.
The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells
Authors: Holappa Katja , Muñoz Maria Teresa , Egea Gustavo , Kellokumpu Sakari
Published: March 2004

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin‐based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi‐associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin‐based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS‐7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin195, but were accompanied by the disappearance of the well‐focused microtubule‐organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP‐AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.
Disruption of type 5 adenylyl cyclase negates the developmental increase in Gαolf expression in the striatum
Authors: Iwamoto Tamio , Iwatsubo Kousaku , Okumura Satoshi , Hashimoto Yoko , Tsunematsu Takashi , Toya Yoshiyuki , Hervé Denis , Umemura Satoshi , Ishikawa Yoshihiro
Published: March 2004

The two stimulatory G protein α subunits, Gαs and Gαolf, activate adenylyl cyclase in a similar way. We examined whether type 5 adenylyl cyclase knockout, the major striatal isoform, can differentially and/or developmentally change the expression of these G proteins in the striatum. Gαs and Gαolf expressions at birth were unaffected in knockouts, which, however, demonstrated a blunted developmental increase in Gαolf, but not Gαs. Adenylyl cyclase activity was unaffected at birth, but subsequently became lower in knockouts. These findings suggest that type 5 adenylyl cyclase does not contribute to striatal cAMP signaling at birth. However, it may play an important role in developmental changes in the expression of Gαolf, but not Gαs.
Complicated Grief in Survivors of Suicide
Authors: Mitchell Ann M. , Kim Yookyung , Prigerson Holly G. , Mortimer-Stephens MaryKay
Published: March 2004

Summary: Complicated grief is a newly defined and distinctive psychiatric disorder that occurs in response to a significant loss through death. New findings suggest that survivors who were close to the deceased are at heightened risk for complicated grief. Little is known about whether close kinship (spouses, parents, children, siblings, vs. in-laws, aunts/uncles, nieces/nephews, friends, or coworkers) to a suicide victim also represents a heightened risk for complicated grief. Assessing for complicated grief is important, especially with survivors of suicide, because of the potential for associated health risks. This report contains preliminary data from an exploratory, descriptive pilot study examining complicated grief in adult survivors of suicide. Sixty bereaved subjects, within one month after the suicide of a family member or significant other, were assessed for complicated grief symptoms. Statistically significant differences, as measured with the Inventory of Complicated Grief, were noted between closely related and distantly related survivors of the suicide victim. These preliminary results indicate that health care professional's assessments and interventions for complicated grief should take into consideration the bereaved's familial and/or social relationship to the deceased. The closely related survivors of suicide had higher levels of complicated grief and could be at risk of developing physical and/or mental health problems, including suicidal ideation, in the future.
Graft‐versus‐Host‐Disease: Ein interdisziplinäres Problem aus der Sicht des Dermatologen
Authors: Rupec Rudolf A. , Plewig Gerd
Published: March 2004

Die Graft‐versus‐Host‐Disease (GVHD) der Haut ist eine häufige Komplikation im Anschluß an eine Transplantation hämatopoetischer Stammzellen. In Abhängigkeit vom Zeitpunkt ihres Auftretens wird zwischen der akuten und chronischen GVHD unterschieden. Stellt sich die akute GVHD relativ einheitlich, wenn auch sehr unterschiedlich in ihrer Ausprägung dar, so zeichnet sich die chronische GVHD durch ein breites Spektrum unterschiedlicher Manifestationen an der Haut aus. Diese Übersichtsarbeit soll neben den pathophysiologischen Vorgängen, die der GVHD zugrunde liegen, ihre unterschiedlichen Erscheinungsformen an der Haut, sowie die derzeit zur Verfügung stehenden Therapiemöglichkeiten wiedergeben.
Either/or → and
Authors: Knight Terry W
Published: March 2004

The writings of Wassily Kandinsky and Paul Klee, Bauhaus theorists and pioneers of nonrepresentational art, are the framework for a discussion of contemporary issues in computational design. Kandinsky and Klee wrote of the many misguided dualisms in the art theory and pedagogy of their time. They sought to reconcile the seemingly mutually exclusive issues associated with abstraction in art. Some of these dualisms, and new ones, arise today in discussions of computational design. How and whether these dualisms can be reconciled in computational design are considered here.
Evaluating Design Alternatives Using Conjoint Experiments in Virtual Reality
Authors: Dijkstra Jan , van Leeuwen Jos , Timmermans Harry
Published: March 2004

In this paper the authors describe the design of an experiment based on conjoint measurement that explores the possibility of using the Internet to evaluate design alternatives. These design alternatives are presented as panoramic views, and preferences are measured by asking subjects which alternative they prefer from a choice set of design alternatives. The approach is illustrated by using the design of office spaces as an example.
Analysing the Visual Dynamics of Spatial Morphology
Authors: Turner Alasdair
Published: March 2004

Recently there has been a revival of interest in visibility analysis of architectural configurations. The new analyses rely heavily on computing power and statistical analysis, two factors which, according to the postpositivist school of geography, should immediately cause us to be wary. The danger, they would suggest, is in the application of a reductionist formal mathematical description in order to ‘explain’ multilayered sociospatial phenomena. The author presents an attempt to rationalise how we can use visibility analysis to explore architecture in this multilayered context by considering the dynamics that lead to the visual experience. In particular, it is recommended that we assess the visual process of inhabitation, rather than assess the visibility in vacuo. In order to investigate the possibilities and limitations of the methodology, an urban environment is analysed by means of an agent-based model of visual actors within the configuration. The results obtained from the model are compared with actual pedestrian movement and other analytic measurements of the area: the agents correlate well both with human movement patterns and with configurational relationship as analysed by space-syntax methods. The application of both methods in combination improves on the correlation with observed movement of either, which in turn implies that an understanding of both the process of inhabitation and the principles of configuration may play a crucial role in determining the social usage of space.
Effects of Planning on the Garbage-Can Decision Processes: A Reformulation and Extension
Authors: Lai Shih-Kung
Published: March 2004

A computer simulation, based on the garbage-can decisionmaking process, is presented in order to investigate effects of making plans and of two other organizational characteristics—namely, decision cost and problem disutility—on the behavior of complex systems. In this simulation a three-factor factorial design was implemented where decision cost, problem disutility, and planning investment were considered in terms of combinations of six levels of these factors. The results suggest that all three factors have statistically significant effects on the behavior of the complex system. In particular, decision cost and problem disutility tended to have effects counter to each other, whereas planning resulted in more efficient decisionmaking, but at the cost of resolving fewer problems.
Changes in Dutch Land Policy: From Monopoly towards Competition in the Building Market
Authors: Priemus Hugo , Louw Erik
Published: March 2004

Dutch local authorities used to acquire land long before urban development of the land took place. Recently, private development companies have become active in the Dutch land market. The public monopoly of the land market has been transformed into private monopolies of the building market. One option to introduce more competition into the building market is to separate land-ownership from development-and-construction rights. A radical amendment of the expropriation legislation seems to be necessary to promote the transparent and effective implementation of spatial policies. Property rights could be limited to promote competition in the building market, and to safeguard the public dimension of urban development.
Advances in Visual Diversity and Entropy
Authors: Stamps Arthur E
Published: March 2004

Visual diversity is an important component of contemporary environmental design. Current issues on visual diversity include (a) how to measure it?, (b) what is the function between visual diversity and pleasure?, and (c) is the function between visual diversity and pleasure the same for different kinds of stimuli? Data are reported from twenty experiments covering 1126 stimuli and 1027 participants. The concept of statistical entropy is proposed as a measure of visual diversity. The correlation of ratings of visual diversity and entropy for environmental scenes was r = 0.87. Tenable relationships between pleasure and entropy for visual stimuli include linear and asymptotic but not the inverted U function. The type of stimulus strongly mediates the relationship between pleasure and entropy. Results from simple laboratory stimuli do not generalize to environmental scenes and, even within environmental scenes, the relationship between pleasure and entropy is positive for some types of scenes but negative for other types of scenes.
Measures of Spatial Accessibility to Health Care in a GIS Environment: Synthesis and a Case Study in the Chicago Region
Authors: Luo Wei , Wang Fahui
Published: March 2004

This article synthesizes two GIS-based accessibility measures into one framework, and applies the methods to examining spatial accessibility to primary health care in the Chicago ten-county region. The floating catchment area (FCA) method defines the service area of physicians by a threshold travel time while accounting for the availability of physicians by their surrounded demands. The gravity-based method considers a nearby physician more accessible than a remote one and discounts a physician's availability by a gravity-based potential. The former is a special case of the latter. Based on the 2000 Census and primary care physician data, this research assesses the variation of spatial accessibility to primary care in the Chicago region, and analyzes the sensitivity of results by experimenting with ranges of threshold travel times in the FCA method and travel friction coefficients in the gravity model. The methods may be used to help the US Department of Health and Human Services and state health departments improve designation of Health Professional Shortage Areas.
Visibility Studies in Archaeology: A Review and Case Study
Authors: Lake Mark W , Woodman Patricia E
Published: March 2004

This paper describes the history and current state of archaeological visibility studies. The first part is a survey of both GIS (geographic information systems) and non-GIS studies of visibility by archaeologists, which demonstrates how advances in GIS visibility studies have tended to recapitulate, albeit over a compressed timescale, theoretically driven developments in non-GIS studies. The second part presents an example of the kind of methodological development required for the use of GIS to contribute to the agenda set by certain strands of a more humanistic archaeology. An algorithm developed to retrieve various summaries of the inclination at which points on the horizon are visible from a specified viewpoint was applied to nineteen recumbent stone circles in the Grampian region of Scotland. The results suggest that these summaries provide a useful tool for ‘unpacking’ what archaeologists mean when they claim that the topographic setting of certain stone circles creates an ‘impression of circularity’.
The Use of Scenarios in Land-Use Planning
Authors: Xiang Wei-Ning , Clarke Keith C
Published: March 2004

Land-development scenarios as a means of representing the future have been in the planner's toolkit for several decades. In this paper we provide a systematic view of four basic issues that concern scenarists and scenario users—the concepts, functions, credentials, and efficacy of land-development scenarios. Drawing upon the wealthy and expanding pool of knowledge and experience as reported in the literature, we put forward the notion that a land-development scenario set is both a bridge that connects the process of modeling with that of planning and a cognitive apparatus that stretches people's thinking and broadens their views in planning. The dual function entitles a scenario set to be a favored member of a family of innate instruments that humans operate in making decisions. Under this overarching framework, we propose three credentials that are by no means exhaustive yet are claimed to be essential for a scenario set to perform best the dual function. These are plausible unexpectedness, informational vividness, and cognitively ergonomic design. After exploring the efficacy issue of a scenario set with respect to its impacts on communities at large, we suggest that basic research efforts be underway that aim at the development of unified theories of land-development scenarios, or even scenarios in general, under a possible name of scenariology—the study of scenarios.
Visibility in Computer Graphics
Authors: Bittner Jiří , Wonka Peter
Published: March 2004

Visibility computation was crucial for computer graphics from its very beginning. The first visibility algorithms in computer graphics aimed to determine visible surfaces in a synthesized image of a three-dimensional scene. Nowadays there are many different visibility algorithms for various visibility problems. We propose a new taxonomy of visibility problems that is based on a classification according to the problem domain. We provide a broad overview of visibility problems and algorithms in computer graphics grouped by the proposed taxonomy. We survey visible surface algorithms, visibility culling algorithms, visibility algorithms for shadow computation, global illumination, point-based and image-based rendering, and global visibility computations. Finally, we discuss common concepts of visibility algorithm design and several criteria for the classification of visibility algorithms.
Radiowave Propagation, Building Databases, and GIS: Anything in Common? A Radio Engineer's Viewpoint
Authors: Wagen Jean-Frédéric , Rizk Karim
Published: March 2004

Mobile cellular communication has already entered the mass market, and mobile Internet services will soon become a reality. The frequent use of mobile radio technologies wherever people are has a direct impact on the deployment of base stations or radio access points, including antennas. Put simply, to serve an increasing number of users requires an increasing number of base stations. Thus, operators must carefully plan the deployment and configurations of radio base stations in order to support voice and data traffic at a level of quality expected by customers. Planning tools are used to help radio engineers in their difficult tasks of balancing requirements or radio coverage and quality with economic and other practical aspects. These planning tools make extensive use of terrain databases and of visualisation tools that lead some tool vendors to offer functionalities very similar to a geographical information system (GIS) or even to base their product on a GIS. Futhermore, because radio communication between base stations and users is crucial, all computations in a planning tool are based on the use of radio-propagation predictions. Until recently, empirical propagation prediction seemed sufficient. However, more efficient planning and the planning of nonvoice services or of a mixture of voice and nonvoice services require more accurate propagation-prediction models. These propagation models are usually based on the computation of the physical interaction of ratio waves and the environment. Thus more detailed databases are required, especially in urban environments where most users are located. Although the authors are not GIS specialists, they aim to expose some of the relationship between radio-propagation models used for mobile radio network planning and databases for terrain and buildings. For example, an accuracy of about 1 m on the coordinates of building corners is found to be acceptable in terms of accuracy in radio coverage. As another example, simulation results show that the use of conventional propagation models and rough geographical databases for the planning of future cellular systems like UMTS might cause serious difficulties.
Hepatocyte growth factor/c-metsignaling pathway is required for efficient liver regeneration and repair
Authors: Huh Chang-Goo , Factor Valentina M. , Sánchez Aránzazu , Uchida Koichi , Conner Elizabeth A. , Thorgeirsson Snorri S.
Published: March 2004

Hepatocyte growth factor/scatter factorc-metsignaling pathway is of central importance during development as well as in tumorigenesis. Because homozygous null mice for eitherhgf/sforc-metdiein utero, we used Cre/loxP-mediated gene targeting to investigate the function ofc-metspecifically in the adult liver. Loss ofc-metappeared not to be detrimental to hepatocyte function under physiological conditions. Nonetheless, the adaptive responses of the liver to injury were dramatically affected. Mice lackingc-metgene in hepatocytes were hypersensitive to Fas-induced apoptosis. When injected with a low dose of anti-Fas antibody, the majority of these mice died from massive apoptosis and hemorrhagic necrosis, whereas all wild-type mice survived with signs of minor injury. After a challenge with a single necrogenic dose of CCl4,c-metconditional knockout mice exhibited impaired recovery from centrolobular lesions rather than a deficit in hepatocyte proliferation. The delayed healing was associated with a persistent inflammatory reaction, over-production of osteopontin, early and prominent dystrophic calcification, and impaired hepatocyte scattering/migration into diseased areas. These studies provide direct genetic evidence in support of the critical role ofc-metin efficient liver regeneration and suggest that disruption ofc-metaffects primarily hepatocyte survival and tissue remodeling.
Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones
Authors: Dvorak Melita M. , Siddiqua Ashia , Ward Donald T. , Carter D. Howard , Dallas Sarah L. , Nemeth Edward F. , Riccardi Daniela
Published: March 2004

We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca2+]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca2+]oand treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3β, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor α1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [Ca2+]o, as was mineralized nodule formation. Alkaline phosphatase activity was maximal for [Ca2+]obetween 1.2 and 1.8 mM. Inhibition of CaR by NPS 89636 blocked responses to the CaR agonists. In conclusion, we show that small deviations of [Ca2+]ofrom physiological values have a profound impact on bone cell fate, by means of the CaR and independently of systemic calciotropic peptides.
13-
cis
-retinoic acid suppresses hippocampal cell division and hippocampal-dependent learning in mice
Authors: Crandall James , Sakai Yasuo , Zhang Jinghua , Koul Omanand , Mineur Yann , Crusio Wim E. , McCaffery Peter
Published: March 2004

The active component of the acne drug Accutane is 13-
cis
-retinoic acid (RA), and it is highly teratogenic for the developing central nervous system. Very little is known, however, regarding the effect of this drug on the adult brain. Regions of the brain that may be susceptible to RA are those that continue to generate new neurons. In the adult mouse, neurogenesis is maintained in the hippocampus and subventricular zone. This report demonstrates that a clinical dose (1 mg/kg/day) of 13-
cis
-RA in mice significantly reduces cell proliferation in the hippocampus and the subventricular zone, suppresses hippocampal neurogenesis, and severely disrupts capacity to learn a spatial radial maze task. The results demonstrate that the regions of the adult brain where cell proliferation is ongoing are highly sensitive to disruption by a clinical dose of 13-
cis
-RA.
Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells
Authors: Chou Wen-Chien , Jie Chunfa , Kenedy Andrew A. , Jones Richard J. , Trush Michael A. , Dang Chi V.
Published: March 2004

Arsenic has played a key medicinal role against a variety of ailments for several millennia, but during the past century its prominence has been displaced by modern therapeutics. Recently, attention has been drawn to arsenic by its dramatic clinical efficacy against acute promyelocytic leukemia. Although toxic reactive oxygen species (ROS) induced in cancer cells exposed to arsenic could mediate cancer cell death, how arsenic induces ROS remains undefined. Through the use of gene expression profiling, interference RNA, and genetically engineered cells, we report here that NADPH oxidase, an enzyme complex required for the normal antibacterial function of white blood cells, is the main target of arsenic-induced ROS production. Because NADPH oxidase enzyme activity can also be stimulated by phorbol myristate acetate, a synergism between arsenic and the clinically used phorbol myristate acetate analog, bryostatin 1, through enhanced ROS production can be expected. We show that this synergism exists, and that the use of very low doses of both arsenic and bryostatin 1 can effectively kill leukemic cells. Our findings pinpoint the arsenic target of ROS production and provide a conceptual basis for an anticancer regimen.
Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by
Plasmodium falciparum
Authors: Sijwali Puran S. , Rosenthal Philip J.
Published: March 2004

Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite food vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining food vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2′). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites.
Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins
Authors: Si Zhihai , Madani Navid , Cox Jason M. , Chruma Jason J. , Klein Jeffrey C. , Schön Arne , Phan Ngoc , Wang Liping , Biorn Alyssa C. , Cocklin Simon , Chaiken Irwin , Freire Ernesto , Smith Amos B. , Sodroski Joseph G.
Published: March 2004

When interacting with the CD4 receptor, the HIV gp120 envelope glycoprotein undergoes conformational changes that allow binding to the chemokine receptor. Receptor binding is proposed to lead to conformational changes in the gp41 transmembrane envelope glycoprotein involving the creation and/or exposure of a coiled coil consisting of three heptad repeat (HR) sequences. The subsequent interaction of the HR2 region of gp41 with this coiled coil results in the assembly of a six-helix bundle that promotes the fusion of the viral and target cell membranes. Here we show that CD4 binding to gp120 induces the formation and/or exposure of the gp41 HR1 coiled coil in a process that does not involve gp120 shedding and that depends on the proteolytic maturation of the gp160 envelope glycoprotein precursor. Importantly, BMS-806 and related HIV-1 entry inhibitors bind gp120 and block the CD4 induction of HR1 exposure without significantly affecting CD4 binding. Moreover, these compounds do not disrupt gp120-chemokine receptor binding or the HR1-HR2 interaction within gp41. These studies thus define a receptor-induced conformational rearrangement of gp120-gp41 that is important for both CD4-dependent and CD4-independent HIV-1 entry and is susceptible to inhibition by low-molecular-weight compounds.
A transgenic mouse model of metastatic carcinoma involving transdifferentiation of a gastric epithelial lineage progenitor to a neuroendocrine phenotype
Authors: Syder Andrew J. , Karam Sherif M. , Mills Jason C. , Ippolito Joseph E. , Ansari Habib R. , Farook Vidya , Gordon Jeffrey I.
Published: March 2004

Human neuroendocrine cancers (NECs) arise in various endoderm-derived epithelia, have diverse morphologic features, exhibit a wide range of growth phenotypes, and generally have obscure cellular origins and ill-defined molecular mediators of initiation and progression. We describe a transgenic mouse model of metastatic gastric cancer initiated by expressing simian virus 40 large tumor antigen (SV40 TAg), under control of regulatory elements from the mouse
Atp4b
gene, in the progenitors of acid-producing parietal cells. Parietal cells normally do not express endocrine or neural features, and
Atp4b
-Cre bitransgenic mice with a Cre reporter confirmed that the
Atp4b
regulatory elements are not active in gastric enteroendocrine cells. GeneChip analyses were performed on laser capture microdissected SV40 TAg-expressing cells in preinvasive foci and invasive tumors. Genes that distinguish invasive from preinvasive cells were then hierarchically clustered with DNA microarray datasets obtained from human lung and gastric cancers. The results, combined with immunohistochemical and electron microscopy studies of
Apt4b-
SV40 TAg stomachs, revealed that progression to invasion was associated with transdifferentiation of parietal cell progenitors to a neuroendocrine phenotype, and that invasive cells shared molecular features with NECs arising in the human pulmonary epithelium, including transcription factors that normally regulate differentiation of various endocrine lineages and maintain neural progenitors in an undifferentiated state. The 399 mouse genes identified as regulated during acquisition of an invasive phenotype and concomitant neuroendocrine transdifferentiation, plus their human orthologs associated with lung NECs, provide a foundation for molecular classification of NECs arising in other tissues and for genetic tests of the molecular mechanisms underlying NEC pathogenesis.
Peroxisome proliferator-activated receptor γ is required in mature white and brown adipocytes for their survival in the mouse
Authors: Imai Takeshi , Takakuwa Reiko , Marchand Sandra , Dentz Emilie , Bornert Jean-Marc , Messaddeq Nadia , Wendling Olivia , Mark Manuel , Desvergne Béatrice , Wahli Walter , Chambon Pierre , Metzger Daniel
Published: March 2004

The peroxisome proliferator-activated receptor γ (PPARγ) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARγ functions in mature adipocytes is precluded by lethality of PPARγ
–/–
fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARγ in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER
T2
recombination system. We show that mature PPARγ-null white and brown adipocytes die within a few days and are replaced by newly formed PPARγ-positive adipocytes, demonstrating that PPARγ is essential for the
in vivo
survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARγ antagonists could be used to acutely reduce obesity.
Quantitative analysis of the formation and diffusion of A
1
-adenosine receptor-antagonist complexes in single living cells
Authors: Briddon S. J. , Middleton R. J. , Cordeaux Y. , Flavin F. M. , Weinstein J. A. , George M. W. , Kellam B. , Hill S. J.
Published: March 2004

The A
1
-adenosine receptor (A
1
-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A
1
-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A
1
-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A
1
-AR-mediated inhibition of cAMP accumulation [log
10
of the affinity constant (pK
b
) = 6.7)] and stimulation of inositol phosphate accumulation (pK
b
= 6.5). Specific binding of XAC-BY630 to cell surface A
1
-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (τ

D

) of 62 μs (τ

D
1

, free ligand), 17 ms (τ

D
2

, A
1
-AR-ligand), and 320 ms (τ

D
3

). Confirmation that τ

D
2

resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A
1
-AR-Topaz fusion protein (15 ms). Quantification of τ

D
2

showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A
1
-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A
1
-AR interactions in single living cells in health and disease.
A Ca
2+
/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning
Authors: Mitra Raka M. , Gleason Cynthia A. , Edwards Anne , Hadfield James , Downie J. Allan , Oldroyd Giles E. D. , Long Sharon R.
Published: March 2004

In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule. Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth. A number of plant mutants with defects in the Nod factor signaling pathway have been identified. One such
Medicago truncatula
mutant,
dmi3
, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation. On the basis of these data, it is thought that the
dmi3
mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking. Additionally, the
dmi3
mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses. We report the identification of the
DMI3
gene, using a gene cloning method based on transcript abundance. We show that transcript-based cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants.
DMI3
encodes a calcium/calmodulin-dependent protein kinase. Mutants in pea
sym9
have phenotypes similar to
dmi3
and have alterations in this gene. The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in
Arabidopsis thaliana
, which does not participate in the mycorrhizal symbiosis.
Caenorhabditis elegans
functional orthologue of human protein h-mucolipin-1 is required for lysosome biogenesis
Authors: Treusch Sebastian , Knuth Sarah , Slaugenhaupt Susan A. , Goldin Ehud , Grant Barth D. , Fares Hanna
Published: March 2004

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disease characterized by severe psychomotor retardation, achlorhydria, and ophthalmological abnormalities. Cells from several tissues in MLIV patients accumulate large vacuoles that are presumed to be lysosomes, but whose exact nature remains to be determined. Other defects include the deterioration of neuronal integrity in the retina and the cerebellum.
MCOLN1
, the gene mutated in MLIV patients, encodes a protein called h-mucolipin-1 that has six predicted transmembrane domains and functions as a Ca
2+
-permeable channel that is modulated by changes in Ca
2+
concentration. CUP-5 is the
Caenorhabditis elegans
functional orthologue of h-mucolipin-1. Mutations in
cup-5
result in the accumulation of large vacuoles in several cells, in increased cell death, and in embryonic lethality. We demonstrate here that CUP-5 functions in the biogenesis of lysosomes originating from hybrid organelles. We also show that at least two h-mucolipin family members rescue
cup-5
mutant endocytic defects, indicating that there may be functional redundancy among the human proteins. Finally, we propose a model that relates the lysosome biogenesis defect in the absence of CUP-5/h-mucolipin-1 to cellular phenotypes in worms and in humans.
Mammalian GLD-2 homologs are poly(A) polymerases
Authors: Kwak Jae Eun , Wang Liaoteng , Ballantyne Scott , Kimble Judith , Wickens Marvin
Published: March 2004

GLD-2 is a cytoplasmic poly(A) polymerase present in the
Caenorhabditis elegans
germ line and embryo. It is a divergent member of the DNA polymerase β nucleotidyl transferase superfamily, which includes CCA-adding enzymes, DNA polymerases and eukaryotic nuclear poly(A) polymerases. The polyadenylation activity of GLD-2 is stimulated by physical interaction with an RNA binding protein, GLD-3. To test whether GLD-3 might stimulate GLD-2 by recruiting it to RNA, we tethered
C. elegans
GLD-2 to mRNAs in
Xenopus
oocytes by using MS2 coat protein. Tethered GLD-2 adds poly(A) and stimulates translation of the mRNA, demonstrating that recruitment is sufficient to stimulate polyadenylation activity. We use the same tethered assay to identify human and mouse poly(A) polymerases related to GLD-2. This may provide entrees to previously uncharacterized modes of polyadenylation in mammalian cells.
Circle-to-circle amplification for precise and sensitive DNA analysis
Authors: Dahl Fredrik , Banér Johan , Gullberg Mats , Mendel-Hartvig Maritha , Landegren Ulf , Nilsson Mats
Published: March 2004

We present a tightly controlled process for strand-specific amplification of circularized DNA molecules. Tandem repeated complements of DNA circles are generated by rolling-circle replication, and converted to monomer circles of opposite polarity to that of the starting material. These circles are then subjected to one more round of rolling-circle replication and circularization, and the process can be further repeated. The method can be directed to produce single-stranded circular or linear monomers, or linear concatemers of the desired polarity. The reaction is not product inhibited, and can yield ≈100-fold higher concentrations of monomer products than PCR. Each generation of the amplification process proceeds in a linear fashion, ensuring precise quantification. The procedure is suitable for parallel amplification of large numbers of DNA circles, because the few cycles and the robust reaction mechanism preserves the proportion of amplified molecules. We demonstrate the utility of the method for multiplexed genotyping of polymorphic loci and for quantitative DNA analysis.
mAbs toBacillus anthraciscapsular antigen for immunoprotection in anthrax and detection of antigenemia
Authors: Kozel Thomas R. , Murphy William J. , Brandt Suzanne , Blazar Bruce R. , Lovchik Julie A. , Thorkildson Peter , Percival Ann , Lyons C. Rick
Published: March 2004

Bacillus anthracisis surrounded by an antiphagocytic polypeptide capsule composed of poly γ-d-glutamic acid (γDPGA). γDPGA has been identified recently as a potential target for vaccine development. Studies of the role of γDPGA in disease have been hampered by the poor Ab response to this antigen and the lack of immunochemical reagents. As a consequence, neither the extent of γDPGA production during anthrax nor the protective activity of γDPGA Abs in inhalation anthrax are known. Here we report production of IgG Abs to γDPGA in mice following an immunization regimen using γDPGA in combination with agonist mAbs to CD40. mAbs were produced that are specific for γDPGA. Passive immunization with γDPGA mAbs protected >90% of mice in a pulmonary model of anthrax that was lethal in control mice (P< 0.0001). Use of γDPGA mAb in an antigen detection immunoassay found that the appearance of γDPGA in serum coincided with the emergence of bacteremia. These studies identify CD40 stimulation as a means for production of Ab and generation of mAbs against a weakly immunogenic antigen and demonstrate that the capsule is an effective target for immunoprotection and for antigen detection in the diagnosis of anthrax.
Embryonic Stem Cells As an Alternate Marrow Donor Source
Authors: Burt Richard K. , Verda Larissa , Kim Duck-An , Oyama Yu , Luo Kehuan , Link Charles
Published: March 2004

A single embryonic stem cell (ESC) line can be repetitively cryopreserved, thawed, expanded, and differentiated into various cellular components serving as a potentially renewable and well-characterized stem cell source. Therefore, we determined whether ESCs could be used to reconstitute marrow and blood in major histocompatibility complex (MHC)-mismatched mice. To induce differentiation toward hematopoietic stem cells (HSCs) in vitro, ESCs were cultured in methylcellulose with stem cell factor, interleukin (IL)-3, and IL-6. ESC-derived, cytokine-induced HSCs (c-kit+/CD45+) were isolated by flow cytometry and injected either intra bone marrow or intravenously into lethally irradiated MHC-mismatched recipient mice. From 2 wk to 6 mo after injection, the peripheral blood demonstrated increasing ESC-derived mononuclear cells that included donor-derived T and B lymphocytes, monocytes, and granulocytes without clinical or histologic evidence of graft-versus-host disease (GVHD). Mixed lymphocyte culture assays demonstrated T cell tolerance to both recipient and donor but intact third party proliferative responses and interferon γ production. ESCs might be used as a renewable alternate marrow donor source that reconstitutes hematopoiesis with intact immune responsiveness without GVHD despite crossing MHC barriers.