bluuebunny/crossref_metadata_2025 huggingface dataset:
Richness of semantic encoding in a hypermedia-assisted instructional environment for ESP:
effects on incidental vocabulary retention among learners with low ability in the target language
Authors: COLL JUAN FRANCISCO
Published: December 2002

The objective of this study is to investigate how low-proficiency English for Specific
Purposes (ESP) students benefit from a hypermedia-enhanced learning environment, specifically in terms of
incidental acquisition of lexical items in the target language. Additionally, this study aims at finding
out how frequently the comprehension tools available from within the application were used by the learners.
The hypermedia-assisted learning environment used in this study provides a rich environment where learners
gain exposure to foreign language texts by listening and reading in the target language. Learners are invited
to explore Chemistry-related video segments through listening comprehension questions in combination with
access to various comprehension tools, for example, L1 translations of questions and answers, L2 video
manuscript, translation of manuscript sentences, specific video segments containing answers to questions
and video control tools. A total of 40 subjects with a low-intermediate proficiency level were exposed to
the treatment. An achievement test was administered before the treatment as a pre-test, and two weeks following
the completion of the treatment as a post-test, to evaluate retention. The courseware was set up by the
researcher to record every user action and compile these data into user logs of behaviour data which were
subsequently submitted to analysis. The findings of this experimental study are conclusive in suggesting
that hypermedia-based instruction can provide an effective learning environment to build vocabulary among
ESP students. It can be claimed that the access to a variety of comprehension tools results in a deep
processing of the lexical items and can therefore contribute to favour retention.
Habitat mapping for bird conservation in North America
Authors: Taulman James F. , Smith Kimberly G.
Published: December 2002

In view of the continuing appropriation and conversion of natural land areas in North America for human uses, there is growing concern about the impacts of changing land use on terrestrial bird species. In order to promote conservation of critical remaining habitats for birds, Partners in Flight (PIF) initiated a project in 1997 in which bird conservation plans were prepared by members in each of 60 ecologically defined physiographical areas throughout the United States. Accurate, nationwide information on the location and extent of vegetative cover types, as well as lands under state and federal management, are critically important elements in the creation of effective bird conservation plans. The National Fish and Wildlife Foundation (NFWF) awarded a challenge grant to The Nature Conservancy's (TNC) Wings of the Americas Program to assist Partners in Flight in acquiring land cover data to serve as the foundation of the planning effort. Canon U.S.A., Inc. and the American Bird Conservancy also contributed support toward this goal. The Center for Advanced Spatial Technology at the University of Arkansas was contracted to produce the needed land cover maps and associated tabular products. Digital land cover databases created by the U.S. Forest Service, the U.S. Geological Survey, the Canada Centre for Remote Sensing, the University of California-Santa Barbara Department of Geography, and the U.S. Department of Transportation, Bureau of Transportation Statistics were used in this project. The final spatial products were produced during 1998–1999 and are described in this paper. This effort represents the first nationwide habitat mapping project in the United States aimed at supporting and enhancing conservation of terrestrial bird species.
Breeding and mortality of Oriental White-backed Vulture Gyps bengalensis in Punjab Province, Pakistan
Authors: Gilbert Martin , Virani Munir Z. , Watson Richard T. , Oaks J. Lindsay , Benson Patrick C. , Khan Aleem A. , Ahmed Shakeel , Chaudhry Jamshed , Arshad Muhammad , Mahmood Shahid , Ali Shah Qaswar
Published: December 2002

Populations of Oriental White-backed Vulture Gyps bengalensis and Long-billed Vulture G. indicus declined in India between the mid 1980s and late 1990s. Regional reports from India described declines of 95–100% across a wide area. This study was conducted to investigate the breeding success and pattern of mortality in two vulture colonies (Dholewala and Changa Manga) within Punjab Province, Pakistan between December 2000 and June 2001. Breeding success was found to be 62% in Dholewala and 59% in Changa Manga. A total of 668 sick and dead vultures were collected of which 591 were less than one month post mortem. No significant variation was found in the weekly mortality rate of adult and subadult vultures during the study period spanning winter through summer. A peak in mortality rate was observed during late April and early May that corresponded to mortality of newly fledged juveniles. Minimum annual mortality rate in the adult breeding population was calculated to be 11.4% and 18.6% in Dholewala and Changa Manga respectively. In a subsample of dead vultures (n = 185) visceral gout was found in 80% of adults, 63% of subadults, 19% of juveniles and 13% of nestlings. These mortality rates were consistent with a rapid population decline. Results imply that the mortality factor responsible for the decline in Gyps vultures described in India is also present in Pakistan and will potentially lead to a population decline of a comparable magnitude.
Status and habitat preference of White-bellied Shortwing Brachypteryx major in the Western Ghats (Kerala and Tamilnadu), India
Authors: Robin V. V. , Sukumar R.
Published: December 2002

White-bellied Shortwing is a globally threatened species found in the Western Ghats of India. There have been few records of this species over the past 119 years and its population is thought to have fallen due to habitat loss and fragmentation. The present study was conducted to assess the habitat preferences and distribution of this species in the Western Ghats of Kerala and Tamilnadu states as a prelude to a long-term study on the biology and causes of decline of this species. Several areas across varying vegetation type and altitude were surveyed after an intensive reconnaissance. There were 214 sightings from evergreen and shola forests during the study. They were found to prefer shola forests to other vegetation types. The species was also found in pine, wattle and tea plantations near shola forests. Although it was found to prefer an altitude above 1,500 m, it was also found in shola forests between 1,000 and 1,500 m elevations. White-bellied Shortwing was found to occupy an elevation range from 1,000 m to 2,200 m. Encounter rates in shola forests fragmented by human interference were four times higher than those in natural shola forests. Most encounters of shortwings were near streams that were wet (running or damp). Emphasis is laid on the need for long-term studies to unravel the basic biology of the species in order to aid active conservation efforts.
A Paradigm Shift in Classical Archaeology?
Authors: Snodgrass Anthony
Published: December 2002

This article, a revised version of the 13th McDonald Lecture given on 21 November 2001, sets the recent and partial transformation in the content and practice of Classical archaeological against the background of Kuhn's well-known work, first published in 1962, on paradigm and revolution in the scientific disciplines. Perhaps the most important question in this context — how would we know when a change in paradigm had taken place? — is harder to answer for a humanities discipline than for a science. But the attempt is made, first to set out a traditional paradigm for the subject; then to give examples of new approaches which seem to satisfy many of Kuhn's criteria for the introduction of a new paradigm; and, more briefly, to show that other approaches, innovatory though they may be, by their nature cannot bring about such a change. Whether a true paradigm shift has been set in motion, the future alone will show.
Evolution of the Physiological and Neurological Capacities for Music
Authors: Morley Iain
Published: December 2002

Neuropsychological and developmental studies suggest human musical ability has a deep evolutionary history; but we do not find evidence of the manufacture and use of instruments, with which musical behaviours have often been assumed to be equated, until 70,000 years after the advent of Homo sapiens. This anomaly is addressed by examining the evidence from the fossil record for the evolution of the physiology and neurology required for musical behaviours, with the aim of identifying the development of the physiological and neurological capacity to produce and process melody and/or rhythm. Aural and vocal sophistication appear to have developed in tandem, beginning with full bipedalism around 1.75 million years ago, until a vocal apparatus similar to the modern was present in Homo heidelbergensis 400,000–300,000 years ago. Prosodic and structural aspects of both speech and music production and processing are lateralized in the brain in similar ways suggesting evolutionarily-shared foundations for these mechanisms.
Objects with Attitude: Biographical Facts and Fallacies in the Study of Late Bronze Age and Early Iron Age Warrior Graves
Authors: Whitley James
Published: December 2002

Aegean prehistory still has to deal with the legacy of ‘Homeric archaeology’. One of these legacies is the ‘warrior grave’, or practice of burying individuals (men?) with weapons which we find both in the Late Bronze Age and the Early Iron Age in the Aegean. This article suggests that the differences between the ‘weapon burial rituals’ in these two periods can tell us much about the kind of social and cultural changes that took place across the Bronze Age/Iron Age ‘divide’ of c. 1100 BC. In neither period, however, can items deposited in ‘warrior graves’ be seen as straightforward biographical facts that tell us what the individual did and suffered in life. Rather, the pattern of grave goods should be seen as a metaphor for a particular kind of identity and ideal. It is only in the Early Iron Age that this identity begins to correspond to the concept of the ‘hero’ as described in the Iliad. One means towards our better understanding of this new identity is to follow up work in anthropology on the biography of objects. It is argued that the ‘life cycle’ of ‘entangled objects’, a cycle which ends in deposition in a grave, provides us with indispensable clues about the nature of new social identities in Early Iron Age Greece.
Ritual and Ideology in the Pre-Pottery Neolithic B of the Levant and Southeast Anatolia
Authors: Verhoeven Marc
Published: December 2002

The study of ritual is an important and indeed essential part of prehistoric archaeology, and is reported at several early Neolithic sites in the Levant and Anatolia. In this study, the evidence for ritual at five Pre-Pottery Neolithic B sites (‘Ain Ghazal, Kfar HaHoresh, Nevali Çori, Çayönü and Göbekli Tepe) is compared. This comparison serves as the basis for an interpretation which focuses on the ideology of PPNB ritual rather than on its social function. It is argued that there are four basic structuring principles in PPNB ritual and ideology: communality, dominant symbolism, vitality (including the notions of domestication, fecundity and life-force), and human–animal linkage. These concepts are related in an attempt to delineate the PPNB ritual system and the place of these specific sites therein.
Modelling a coastal ridging event over south‐eastern Australia
Authors: Reason C. J. C. , Jackson P. L.
Published: December 2002

Coastal ridging is an important component of the weather of south‐eastern Australia during the summer. The mesoscale aspects of such an event are often not well forecast, and this can have serious implications for aviation, shipping, air quality and bushfire control. A previous study by Speer & Leslie (1997) identified three types of coastal ridging according to the associated synoptic conditions. In this study, a coastal ridging event, which appears to have aspects of all three types, is examined with the aid of mesoscale manual analyses, surface and radiosonde data and simulations with a mesoscale numerical model (Colorado State University Regional Atmospheric Modeling System).

It is found that the model is able to capture the salient features of the event but, on the east coast, tends to produce a weaker event than that observed. The model ridging also tends to lag the observed feature on the east coast by a few hours. It is suggested that these model deficiencies may relate to deficiencies in the lower atmosphere air mass characteristics, which, in turn, may relate to the surface parameterisations in the model. Copyright © 2002 Royal Meteorological Society.
Areal PMP distribution of one‐day to three‐day duration over India
Authors: Clark Rakhecha C P R
Published: December 2002

There is a need to assess the areal depth of the probable maximum precipitation (PMP) over specified catchment areas for the safe construction of dam spillways. The large number of dams in India, coupled with their risk of failure, makes this need imperative both for the maximum use of water resources and for public safety. On the basis of rainfall data for the heaviest storms that occurred in different parts of India during the period 1880–1983, improved estimates of one‐, two‐, and three‐day point PMP for India have been made. In this paper the distribution of areal PMP over specified catchment sizes is provided for the first time. The areal reduction factors (ARF) were based on envelope curves of major storms to give the ARF for areas of 10–20,000 km2 . These factors were found to vary from 1.0 to 0.41, though there was no real difference between durations of rainfall. These values of ARF were then multiplied by values of one‐ to three‐day PMP. The resulting maps allow a broad description of the spatial distribution of areal PMP and also provide a rapid and consistent estimate of the probable maximum flood (PMF) from the PMP.

For 500 km2 the areal PMP varies from 40 to 120 cm for one‐day duration; from 70 to 200 cm for twoday duration; and from 75 to 270 cm for three‐day duration. The pattern of PMP is consistent with the geography and available moisture. Copyright © 2002 Royal Meteorological Society.
Two simple wind speed models for practical application under stable conditions
Authors: Cvitan Lidija , S̆inik Nadez̆da , Klaić Zvjezdana Bencetić
Published: December 2002

This paper deals with the utility of Monin‐Obukhov similarity theory for wind speed simulation in stable conditions over flat terrain in northern Croatia. Wind speed profiles observed through a 150‐m deep layer are compared with those obtained from two simple wind speed models. For this purpose, the similarity functions for two stable classes based on Monin‐Obukhov theory are locally determined. They are tested in wind speed simulations using a model based on the Monin‐Obukhov theory (MO) and a model which combines the surface‐layer model of Monin‐Obukhov theory and the Ekman‐Taylor type model (MOE). Under less stable conditions the combined model (MOE) produces better skill, whereas the model based on the Monin‐Obukhov theory (MO) performed better under more stable conditions. Copyright © 2002 Royal Meteorological Society.
A study of the theory and operation of a resonance fluorescence water vapour sensor for upper tropospheric humidity measurements
Authors: Keramitsoglou I , Harries J E , Colling D J , Barker R A , Foot J S
Published: December 2002

In‐situ methods for measuring upper tropospheric humidity are important for two reasons: (i) they can be used as accurate spot measurements with which to calibrate more extensive data from satellites and other sensors; (ii) they can provide high accuracy measurements from aircraft or balloon with which individual processes of transport, phase change or chemistry can be studied.

In either case the accuracy of the in‐situ measurement is of paramount importance. This study compares the performance and accuracy of a resonance fluorescence type of sensor (the Fluorescence Water Vapour Sensor) with a standard frost‐point hygrometer.

An intercomparison of these two hygrometers has confirmed a long‐standing difference between these two types of sensors (the FWVS overestimates the water vapour volume mixing ratio by 10–20%, depending on pressure). Testing the FWVS experimentally in the laboratory, along with a modelling study of the sensor has revealed that a significant source of error is due to contamination of the FWVS source emission, and the subsequent underestimation of the oxygen absorption cross‐section. Copyright © 2002 Royal Meteorological Society.
On the relationship between Clayton's skill score and expected value for forecasts of binary events
Authors: Wandishin Matthew S. , Brooks Harold E.
Published: December 2002

Following upon previous efforts to associate standard measures of forecast skill to relative economic value, the connection is established between the Clayton skill score and the range of users who realise positive value from the forecasts. It is also shown that, whereas the maximum relative value that can be obtained from a set of forecasts is based on the probabilities conditioned on the observations, the range of users for whom the forecasts provide positive relative value is based on the probabilities conditioned on the forecasts. Copyright © 2002 Royal Meteorological Society.
Chaperoning brain degeneration
Authors: Bonini Nancy M.
Published: December 2002

Drosophila
has emerged as a première model system for the study of human neurodegenerative disease. Genes associated with neurodegeneration can be expressed in flies, causing phenotypes remarkably similar to those of the counterpart human diseases. Because human neurodegenerative diseases, including Huntington's and Parkinson's diseases, are disorders for which few cures or treatments are available,
Drosophila
brings to bear powerful genetics to the problem of these diseases. The molecular chaperones were the first modifiers defined that interfere in the progression of such disease phenotypes in
Drosophila
. Hsp70 is a potent suppressor of both polyglutamine disease and Parkinson's disease in
Drosophila
. These studies provide the promise of treatments for human neurodegeneration through the up-regulation of stress and chaperone pathways.
Conservation of a portion of the
S. cerevisiae
Ure2p prion domain that interacts with the full-length protein
Authors: Edskes Herman K. , Wickner Reed B.
Published: December 2002

The [URE3] prion of
Saccharomyces cerevisiae
is a self-propagating inactive amyloid form of the Ure2 protein. Ure2p residues 1–65 constitute the prion domain, and the remaining C-terminal portion regulates nitrogen catabolism. We have examined the
URE2
genes of wild-type isolates of
S. cerevisiae
and those of several pathogenic yeasts and a filamentous fungus. We find that the normal function of the
S. cerevisiae
Ure2p in nitrogen regulation is fully complemented by the Ure2p of
Candida albicans
,
Candida glabrata
,
Candida kefyr
,
Candida maltosa
,
Saccharomyces bayanus
, and
Saccharomyces paradoxus
, all of which have high homology in the C-terminal nitrogen regulation domain. However, there is considerable divergence of their N-terminal domains from that of Ure2p of
S. cerevisiae
. [URE3
Sc
] showed efficient transmission into
S. cerevisiae
ure2Δ cells if expressing a Ure2p of species within
Saccharomyces
. However, [URE3
Sc
] did not seed self-propagating inactivation of the Ure2p's from the other yeasts. When overexpressed as a fusion with green fluorescent protein, residues 5–47 of the
S. cerevisiae
prion domain are necessary for curing the [URE3] prion. Residues 11–39 are necessary for an inactivating interaction with the full-length Ure2p. A nearly identical region is highly conserved among many of the yeasts examined in this study, despite the wide divergence of sequences found in other parts of the N-terminal domains.
RNA-directed DNA methylation inArabidopsis
Authors: Aufsatz Werner , Mette M. Florian , van der Winden Johannes , Matzke Antonius J. M. , Matzke Marjori
Published: December 2002

In plants, double-stranded RNA that is processed to short RNAs ≈21–24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNA-directed DNA methylation involvesde novomethylation of almost all cytosine residues within a region of RNA–DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing andde novomethylation of homologous promoters intranscan occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established inArabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure.
Locus-specific control of asymmetric and CpNpG methylation by the
DRM
and
CMT3
methyltransferase genes
Authors: Cao Xiaofeng , Jacobsen Steven E.
Published: December 2002

Many plant, animal, and fungal genomes contain cytosine DNA methylation in asymmetric sequence contexts (CpHpH, H = A, T, C). Although the enzymes responsible for this methylation are unknown, it has been assumed that asymmetric methylation is maintained by the persistent activity of
de novo
methyltransferases (enzymes capable of methylating previously unmodified DNA). We recently reported that the
DOMAINS REARRANGED METHYLASE
(
DRM
) genes are required for
de novo
DNA methylation in
Arabidopsis thaliana
because
drm1 drm2
double mutants lack the
de novo
methylation normally associated with transgene silencing. In this study, we have used bisulfite sequencing and Southern blot analysis to examine the role of the
DRM
loci in the maintenance of asymmetric methylation. At some loci,
drm1 drm2
double mutants eliminated all asymmetric methylation. However, at the
SUPERMAN
locus, asymmetric methylation was only completely abolished in
drm1 drm2
chromomethylase
3
(
cmt3
) triple mutant plants.
drm1 drm2
double mutants also showed a strong reduction of CpNpG (
n
= A, T, C, or G) methylation at some loci, but not at others. The
drm1 drm2 cmt3
triple mutant plants did not affect CpG methylation at any locus tested, suggesting that the primary CpG methylases are encoded by the
MET1
class of genes. Although neither the
drm1 drm2
double mutants nor the
cmt3
single mutants show morphological defects,
drm1 drm2 cmt3
triple mutant plants show pleiotropic effects on plant development. Our results suggest that the
DRM
and
CMT3
genes act in a partially redundant and locus-specific manner to control asymmetric and CpNpG methylation.
Transmission of prions
Authors: Weissmann C. , Enari M. , Klöhn P.-C. , Rossi D. , Flechsig E.
Published: December 2002

The “protein only” hypothesis states that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain, and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. A striking feature of prions is their extraordinary resistance to conventional sterilization procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity. This property, first observed in a clinical setting, is now being investigated in experimental settings, both in animals and in cell culture.
Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington's disease by using an automated filter retardation assay
Authors: Heiser Volker , Engemann Sabine , Bröcker Wolfgang , Dunkel Ilona , Boeddrich Annett , Waelter Stephanie , Nordhoff Eddi , Lurz Rudi , Schugardt Nancy , Rautenberg Susanne , Herhaus Christian , Barnickel Gerhard , Böttcher Henning , Lehrach Hans , Wanker Erich E.
Published: December 2002

Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation
in vitro
. Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of ≈184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethyl-benzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation
in vivo
. These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders.
Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity
Authors: Sakahira Hideki , Breuer Peter , Hayer-Hartl Manajit K. , Hartl F. Ulrich
Published: December 2002

The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by proteins with expanded polyglutamine (polyQ) repeats. However, the mechanistic relationship between polyQ aggregation and its toxic effects on neurons remains unclear. Two main hypotheses have been put forward for how polyQ expansions may cause cellular dysfunction. In one model neurotoxicity results from the ability of polyQ-expanded proteins to recruit other important cellular proteins with polyQ stretches into the aggregates. In the other model, aggregating polyQ proteins partially inhibit the ubiquitin–proteasome system for protein degradation. These two mechanisms are not exclusive but may act in combination. In general, protein misfolding and aggregation are prevented by the machinery of molecular chaperones. Some chaperones such as the members of the Hsp70 family also modulate polyQ aggregation and suppress its toxicity. These recent findings suggest that an imbalance between the neuronal chaperone capacity and the production of potentially dangerous polyQ proteins may trigger the onset of polyQ disease.
Histone H3 lysine 4 methylation is mediated by Set1 and promotes maintenance of active chromatin states in fission yeast
Authors: Noma Ken-ichi , Grewal Shiv I. S.
Published: December 2002

Methylation of histone H3 at lysine 4 (H3 Lys-4) or lysine 9 (H3 Lys-9) is known to define active and silent chromosomal domains respectively from fission yeast to humans. However, in budding yeast, H3 Lys-4 methylation is also necessary for silent chromatin assembly at telomeres and ribosomal DNA. Here we demonstrate that deletion of
set1,
which encodes a protein containing an RNA recognition motif at its amino terminus and a SET domain at the carboxy terminus, abolishes H3 Lys-4 methylation in fission yeast. Unlike in budding yeast, Set1-mediated H3 Lys-4 methylation is not required for heterochromatin assembly at the silent mating-type region and centromeres in fission yeast. Our analysis suggests that H3 Lys-4 methylation is a stable histone modification present throughout the cell cycle, including mitosis. The loss of H3 Lys-4 methylation in
set1
Δ cells is correlated with a decrease in histone H3 acetylation levels, suggesting a mechanistic link between H3 Lys-4 methylation and acetylation of the H3 tail. We suggest that methylation of H3 Lys-4 primarily acts in the maintenance of transcriptionally poised euchromatic domains, and that this modification is dispensable for heterochromatin formation in fission yeast, which instead utilizes H3 Lys-9 methylation.
Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA)
Authors: Lanz Rainer B. , Razani Bahram , Goldberg Aaron D. , O'Malley Bert W.
Published: December 2002

Steroid receptor RNA activator (SRA) is an RNA transcript that functions as a eukaryotic transcriptional coactivator for steroid hormone receptors. We report here the isolation and functional characterization of distinct RNA substructures within the SRA molecule that constitute its coactivation function. We used comparative sequence analysis and free energy calculations to systematically study SRA RNA subdomains for identification of structured regions and base pairings, and we used site-directed mutagenesis to assess their functional consequences. Together with genetic deletion analysis, this approach identified six RNA motifs in SRA important for coactivation. Because all nucleotide changes in the mutants that disrupted SRA function were silent mutations presumed not to alter deduced encoded amino acid sequence, our analysis provides strong evidence that SRA-mediated coactivation is executed by distinct RNA motifs and not by an encoded protein.
Immunization and immunotherapy for cancers involving infection by a human papillomavirus in a mouse model
Authors: Li Jin , Sun Ying , Garen Alan
Published: December 2002

We describe a protocol for generating a potent cellular immune response against viral-infected cells, and demonstrate its efficacy and safety in a mouse model of human cancer associated with infection by a human papillomavirus (HPV). In the mouse model, the mouse tumor TC-1, which expresses the E7 oncoprotein from HPV-16, is used as a surrogate for human tumors infected with HPV-16. The antigen for the protocol is composed of the E7 oncoprotein conjugated to the Fc region of a mouse IgG1 Ig (E7-mFc). The mFc domain should bind to Fc receptors on dendritic cells, enhancing the processing and presentation of E7 peptides by dendritic cells to T cells, which mediate a cellular immune attack against tumors expressing E7. The E7-mFc antigen was encoded in a replication-incompetent adenoviral vector, called Ad(E7-mFc), for infection of the human kidney cell line 293. The infected 293 cells synthesize the E7-mFc antigen and also infectious Ad(E7-mFc) vector particles for ≈2 days, until the cells lyse and the vector particles are released. To test the protocol for immunization against formation of a TC-1 tumor, the mice first were injected s.c. with 293 cells infected with Ad(E7-mFc), followed by two challenges with TC-1 cells. The immunized mice remained healthy and tumor-free for the 6-month duration of the experiment, and the autopsies showed no toxicity. In the control mice immunized with 293 cells infected with an adenoviral vector that does not encode the E7-mFc antigen, the TC-1 tumor grew continuously and the mice had to be killed within 1 month. To test the protocol for immunotherapy, the mice first were injected with TC-1 cells, followed by s.c. injections of 293 cells infected with Ad(E7-mFc). Tumor growth was prevented or strongly retarded in these mice, in contrast to the continuous tumor growth in the controls. These results suggest that the protocol could be adapted for immunization against human cancers associated with an HPV infection, notably cervical cancer, and for immunotherapy to prevent recurrence of a tumor after surgery.
Homology modeling of the cation binding sites of Na
+
K
+
-ATPase
Authors: Ogawa Haruo , Toyoshima Chikashi
Published: December 2002

Homology modeling of the α-subunit of Na
+
K
+
-ATPase, a representative member of P-type ion transporting ATPases, was carried out to identify the cation (three Na
+
and two K
+
) binding sites in the transmembrane region, based on the two atomic models of Ca
2+
-ATPase (Ca
2+
-bound form for Na
+
, unbound form for K
+
). A search for potential cation binding sites throughout the atomic models involved calculation of the valence expected from the disposition of oxygen atoms in the model, including water molecules. This search identified three positions for Na
+
and two for K
+
at which high affinity for the respective cation is expected. In the models presented, Na
+
- and K
+
-binding sites are formed at different levels with respect to the membrane, by rearrangements of the transmembrane helices. These rearrangements ensure that release of one type of cation coordinates with the binding of the other. Cations of different radii are accommodated by the use of amino acid residues located on different faces of the helices. Our models readily explain many mutational and biochemical results, including different binding stoichiometry and affinities for Na
+
and K
+
.
Studies of the aggregation of mutant proteins
in vitro
provide insights into the genetics of amyloid diseases
Authors: Chiti Fabrizio , Calamai Martino , Taddei Niccolò , Stefani Massimo , Ramponi Giampietro , Dobson Christopher M.
Published: December 2002

Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt–Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.
AGAMOUS-LIKE 24, a dosage-dependent mediator of the flowering signals
Authors: Yu Hao , Xu Yifeng , Tan Ee Ling , Kumar Prakash P.
Published: December 2002

The most dramatic phase change in plants is the transition from vegetative to reproductive growth. This flowering process is regulated by several interacting pathways that monitor both the developmental state of the plants and environmental cues such as light and temperature. The flowering-time genes
FLOWERING LOCUS T
(
FT
) and
SUPPRESSOR OF OVEREXPRESSION OF CO1
(
SOC1
), together with the floral meristem identity gene
LEAFY
(
LFY
), are three essential regulators integrating floral signals from multiple pathways in
Arabidopsis thaliana
. Part of the crosstalk among these genes is mediated by a putative transcription factor,
AGAMOUS-LIKE 24
(
AGL24
). This gene is gradually activated in shoot apical meristems during the floral transition and later located in the whole zone of both inflorescence and floral meristems. Loss and reduction of
AGL24
activity by double-stranded RNA-mediated interference result in late flowering, whereas constitutive overexpression of
AGL24
causes precocious flowering. The correlation between the level of
AGL24
accumulation and the alteration of flowering time suggests that
AGL24
is a dosage-dependent flowering promoter. Analysis of
AGL24
expression in various flowering-time mutants shows that it is regulated in several floral inductive pathways. Further genetic analyses of epistasis indicate that
AGL24
may act downstream of
SOC1
and upstream of
LFY
.
Combining computational and experimental screening for rapid optimization of protein properties
Authors: Hayes Robert J. , Bentzien Jörg , Ary Marie L. , Hwang Marian Y. , Jacinto Jonathan M. , Vielmetter Jost , Kundu Anirban , Dahiyat Bassil I.
Published: December 2002

We present a combined computational and experimental method for the rapid optimization of proteins. Using β-lactamase as a test case, we redesigned the active site region using our Protein Design Automation technology as a computational screen to search the entire sequence space. By eliminating sequences incompatible with the protein fold, Protein Design Automation rapidly reduced the number of sequences to a size amenable to experimental screening, resulting in a library of ≈200,000 mutants. These were then constructed and experimentally screened to select for variants with improved resistance to the antibiotic cefotaxime. In a single round, we obtained variants exhibiting a 1,280-fold increase in resistance. To our knowledge, all of the mutations were novel, i.e., they have not been identified as beneficial by random mutagenesis or DNA shuffling or seen in any of the naturally occurring TEM β-lactamases, the most prevalent type of Gram-negative β-lactamases. This combined approach allows for the rapid improvement of any property that can be screened experimentally and provides a powerful broadly applicable tool for protein engineering.
Disulfide bond-mediated dimerization of HLA-G on the cell surface
Authors: Boyson Jonathan E. , Erskine Robert , Whitman Mary C. , Chiu Michael , Lau Julie M. , Koopman Louise A. , Valter Markus M. , Angelisova Pavla , Horejsí Václav , Strominger Jack L.
Published: December 2002

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-β2-microglobulin mAb BBM.1 revealed the presence of an ≈78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-G/receptor interactions and for the search for specific receptors that bind HLA-G.
Phospholipase A2 group IIA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis
Authors: Leung Suet Y. , Chen Xin , Chu Kent M. , Yuen Siu T. , Mathy Jonathan , Ji Jiafu , Chan Annie S. Y. , Li Rui , Law Simon , Troyanskaya Olga G. , Tu I-Ping , Wong John , So Samuel , Botstein David , Brown Patrick O.
Published: December 2002

We analyzed gene expression patterns in human gastric cancers by using cDNA microarrays representing ≈30,300 genes. Expression of PLA2G2A, a gene previously implicated as a modifier of theApcMin/+(multiple intestinal neoplasia 1) mutant phenotype in the mouse, was significantly correlated with patient survival. We confirmed this observation in an independent set of patient samples by using quantitative RT-PCR. Beyond its potential diagnostic and prognostic significance, this result suggests the intriguing possibility that the activity of PLA2G2A may suppress progression or metastasis of human gastric cancer.
Sets meeting isometric copies of the lattice
Z
2
in exactly one point
Authors: Jackson Steve , Mauldin R. Daniel
Published: December 2002

The construction of a subset
S
of ℝ
2
such that each isometric copy of ℤ
2
(the lattice points in the plane) meets
S
in exactly one point is indicated. This provides a positive answer to a problem of H. Steinhaus [Sierpiński, W. (1958)
Fund. Math.
46, 191–194].
The membrane protein FeoB contains an intramolecular G protein essential for Fe(II) uptake in bacteria
Authors: Marlovits Thomas C. , Haase Winfried , Herrmann Christian , Aller Stephen G. , Unger Vinzenz M.
Published: December 2002

G proteins are critical for the regulation of membrane protein function and signal transduction. Nevertheless, coupling between G proteins and membrane proteins with multiple membrane-spanning domains has so far been observed only in higher organisms. Here we show that the polytopic membrane protein FeoB, which is essential for Fe(II) uptake in bacteria, contains a guanine-nucleotide-specific nucleotide binding site. We identify the G4-motif, NXXD, responsible for guanine nucleotide specificity, and show that GTP hydrolysis occurs very slowly. In contrast to typical G proteins, the association and dissociation of GDP were found to be faster than for GTP, suggesting that in the absence of additional factors, FeoB's G protein domain may exist mostly in the GTP-bound form. Furthermore, the binding of GTP is required for efficient Fe(II) uptake through the FeoB-dependent system. Notably, even in bacteria, this covalent linkage between a G protein and a polytopic membrane protein appears, to our knowledge, to be unique. These findings raise the intriguing question whether FeoB represents a primordial archetype of G protein-regulated membrane proteins.
Molecular characterization of lymphatic endothelial cells
Authors: Podgrabinska Simona , Braun Pascal , Velasco Paula , Kloos Bryan , Pepper Michael S. , Jackson David G. , Skobe Mihaela
Published: December 2002

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.
Crystal structure of a DNA decamer containing a
cis-syn
thymine dimer
Authors: Park HaJeung , Zhang Kaijiang , Ren Yingjie , Nadji Sourena , Sinha Nanda , Taylor John-Stephen , Kang ChulHee
Published: December 2002

It is well known that exposure to UV induces DNA damage, which is the first step in mutagenesis and a major cause of skin cancer. Among a variety of photoproducts, cyclobutane-type pyrimidine photodimers (CPD) are the most abundant primary lesion. Despite its biological importance, the precise relationship between the structure and properties of DNA containing CPD has remained to be elucidated. Here, we report the free (unbound) crystal structure of duplex DNA containing a CPD lesion at a resolution of 2.0 Å. Our crystal structure shows that the overall helical axis bends ≈30° toward the major groove and unwinds ≈9°, in remarkable agreement with some previous theoretical and experimental studies. There are also significant differences in local structure compared with standard B-DNA, including pinching of the minor groove at the 3′ side of the CPD lesion, a severe change of the base pair parameter in the 5′ side, and serious widening of both minor and major groves both 3′ and 5′ of the CPD. Overall, the structure of the damaged DNA differs from undamaged DNA to an extent that DNA repair proteins may recognize this conformation, and the various components of the replicational and transcriptional machinery may be interfered with due to the perturbed local and global structure.
A self-organizing principle for learning nonlinear manifolds
Authors: Agrafiotis Dimitris K. , Xu Huafeng
Published: December 2002

Modern science confronts us with massive amounts of data: expression profiles of thousands of human genes, multimedia documents, subjective judgments on consumer products or political candidates, trade indices, global climate patterns, etc. These data are often highly structured, but that structure is hidden in a complex set of relationships or high-dimensional abstractions. Here we present a self-organizing algorithm for embedding a set of related observations into a low-dimensional space that preserves the intrinsic dimensionality and metric structure of the data. The embedding is carried out by using an iterative pairwise refinement strategy that attempts to preserve local geometry while maintaining a minimum separation between distant objects. In effect, the method views the proximities between remote objects as lower bounds of their true geodesic distances and uses them as a means to impose global structure. Unlike previous approaches, our method can reveal the underlying geometry of the manifold without intensive nearest-neighbor or shortest-path computations and can reproduce the true geodesic distances of the data points in the low-dimensional embedding without requiring that these distances be estimated from the data sample. More importantly, the method is found to scale linearly with the number of points and can be applied to very large data sets that are intractable by conventional embedding procedures.