bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and Antimicrobial Resistance of Salmonella spp. in Pet Mammals, Reptiles, Fish Aquarium Water, and Birds in Trinidad
Authors: Seepersadsingh N. , Adesiyun A. A.
Published: December 2003

The prevalence of Salmonella spp. was determined in 970 animals comprising 423 pet birds, 485 fish aquaria water and 62 other pets (40 pet mammals, 14 reptiles, eight others – crustaceans, snail, stingray) from both pet shops and households throughout Trinidad. The serotypes of Salmonella spp. isolated were identified and the resistance to various antimicrobial agents was determined. Overall nine (0.9%) of 970 pet animals were positive for Salmonella spp. Six isolates of Salmonella spp. were recovered from all pet birds with two isolates of serotype Aberdeen and one isolate each of Thompson, Rubislaw, Panama and Newport. The prevalence of Salmonella spp. in birds was 0.9%. Four isolates of Salmonella spp. were recovered from fish aquaria water, serotypes included Panama (two isolates), Newport (one isolate) and Virchow (one isolate). Prevalence of Salmonella spp. from fish aquaria was 0.4%. No isolate of Salmonella spp. was detected in pet mammals sampled while two isolates were recovered from reptiles, S. Enteritidis and S. Montevideo. One isolate of Salmonella spp. was recovered from a stingray, serotype unknown. Antimicrobial resistance was present is all animal types. The highest prevalence of resistance was to streptomycin among isolates from birds (83.3%) and other pets (100.0%) while isolates from fish aquarium water exhibited comparatively high resistance to cephalothin (50.0%). It was concluded that the isolation of Salmonella spp. from apparently healthy birds, fish aquarium water and other pet animals may pose a health risk to their owners and contacts as all serotypes are known to be potentially pathogenic depending on the oral dosage of the organism and the immune status of those in contact. The high prevalence of resistance to antimicrobial agents among Salmonella isolates across pet species may pose chemotherapeutic consequences to their owners and contacts.
Microgeographic population structure of brook charr: a comparison of microsatellite and mark‐recapture data
Authors: Adams B. K. , Hutchings J. A.
Published: December 2003

Polymorphism at five microsatellite genetic markers (genotyped n = 496) and mark‐recapture tagging data (tagged n = 9813) were used to define the population structure of brook charr, Salvelinus fontinalis from the Indian Bay watershed, Newfoundland, Canada. Despite the absence of physical barriers to migration among lakes, both genetic and tagging data suggest that brook charr in each lake represent reproductively isolated populations. Exact tests comparing allele frequencies, θ (global value = 0·063), Rst (global value = 0·052), individual assignment tests, and Nei's genetic distance provided congruent estimates of population subdivision in agreement with the tagging data (only 2·2% of recaptures were lake‐to‐lake). The genetic structure of the brook charr populations corresponded with the geographic structure of the drainage basin on a qualitative level, although linear distance over water was not significantly correlated with the tagging data or the genetic distance measures. The agreement between the tagging and the genetic data suggest that microsatellite markers can be useful tools for defining real biological units. The results also suggest that brook charr exhibit microgeographic population structure at the watershed scale, and that this is the scale at which conservation and management of this salmonid might best be implemented.
The importance of measuring biotic and abiotic factors in the lower egg pocket to predict coho salmon egg survival
Authors: Meyer C. B.
Published: December 2003

Based on results from simulated redds of coho salmon Oncorhynchus kisutch, the amount of fine sediment <0·5 mm in the lower half of the egg pocket, rather than the entire egg pocket of the redd, was a strong predictor of egg survival to hatching ( r2 = 0·62). The relationship was much stronger than observed in other studies, which typically ignore egg pocket structure. Abundance of a fish egg‐eating worm, Haplotaxis ichthyophagous, an oligochaete that may have been attracted to fine sediment and dead eggs in the egg pocket, was also associated with a decrease in egg survival. The worm, however, accounted for little of the variance in survival compared to fine sediment. Only 10% fine sediment (<0·5 mm) in the lower pocket was required to decrease survival from 100 to 5%. Other abiotic factors had weaker (gravel permeability) or non‐existent (dissolved oxygen) correlations with survival.
Learning from unsuccessful radio tagging of common carp in a Namibian reservoir
Authors: Økland F. , Hay C. J. , Næsje T. F. , Nickandor N. , Thorstad E. B.
Published: December 2003

Radio tagged common carp Cyprinus carpio in a Namibian reservoir, where this species has been introduced, experienced 100% mortality or tag loss ( n = 13) with surgically implanted transmitters and 100% survival ( n = 5) with externally attached transmitters at water temperatures of 24–25° C. It seems that the negative effects of the tagging were greater for surgical implantation than for external attachment. The body shape of common carp make them well suited for external tagging, and this tagging method is recommended for common carp studies at high water temperatures or under other stressful conditions.
Neonatal and juvenile sandbar sharks in the northern Adriatic Sea
Authors: Costantini M. , Affronte M.
Published: December 2003

The occurrence of neonatal sandbar sharks Carcharhinus plumbeus in the Adriatic Sea is reported; however, nursery areas still need to be identified.
Mitochondrial DNA sequence divergence between two cryptic species of Lethenteron, with reference to an improved identification technique
Authors: Yamazaki Y. , Goto A. , Nishida M.
Published: December 2003

Genetic divergences and population structures were examined in the cryptic Lethenteron sp. N and sp. S, based on mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (CO I) region sequences. An improved method of discrimination between L. sp. N and sp. S was found using PCR, with diagnostic primers for each species‐specific sequence in the mtDNA CO I region. Identification of 50 individuals of each species by this analysis was consistent with that by allozyme analysis of nuclear DNA. L. sp. N and sp. S, identified on the basis of diagnostic alleles at five allozymic loci, were independently grouped in a neighbour‐joining (NJ) tree, with a large sequence difference (mean ± s.d. = 9·10 ± 0·36%) between them. Within each species, the values of sequence divergence among localities were significantly higher in L. sp. S (1·61 ± 0·44%) than in L. sp. N (1·10 ± 0·48%). On the tree and nested clade analyses, several phylogenetic groups comprising geographically close localities were detected in the former, although scarcely detected in the latter, probably resulting from dispersal pattern differences between them.
Topics
Authors: Taylor Gordon
Published: December 2003

‘The strata passed through on the whole line were Shale, Clay, Washing and Boulders, Red Sandstone, Sand and Mar’. Wildman's Almanac (Settle) 1873.
Primary nurses’ performance: role of supportive management
Authors: Drach‐Zahavy Anat
Published: December 2003

Background.  Most studies examining primary nursing focus on outcome variables such as enhanced patient and staff satisfaction with care, perceived autonomy and quality of care, whereas only limited research has examined processes of implementing and maintaining primary nursing. In addition, the few studies that have explored process variables discuss only direct relationships between the design of the primary nursing care‐delivery system, process and nurse outcomes, and disregard how such variables interact.

Aim.  This study sought to address previous inconsistent findings about the impact of primary nursing care‐delivery models on the performance of nurses by incorporating the moderating role of supportive management practices.

Method.  Three hundred and sixty‐eight nurses from a variety of wards completed questionnaires that assessed the degree of primary nursing on their unit, the support practices of their direct supervisor and their perceptions of the cost of seeking support from their supervisor. In addition, the direct supervisor evaluated the nurse's performance in the unit.

Results.  Primary nursing did not exert a direct impact on nurses’ performance. Rather, the interaction of primary nursing with supervisor support was more predictive of performance: if supervisor support was high, performance was substantially higher than if supervisor support was low. In addition, nurses’ perceptions of the costs of seeking support had a detrimental impact on their performance.

Conclusions and limitations.  The study was cross‐sectional and relied on self‐report data only. Nevertheless, the findings show that supportive management practices are crucial to the success of implementing primary nursing.
Health visiting: the public health role
Authors: Smith Martin A.
Published: December 2003

Background.

United Kingdom (UK) government policy advocates the development of a family‐centred public health role for health visitors. Studies have suggested that barriers and constraints exist to hinder the development of a public health role. However, few of these have considered the issues from a practitioner perspective.

Aim.  The aim of the study was to examine the constraints on developing a family‐centred public health role in a health visiting service.

Methods.  An exploratory study was carried out using focus groups with 27 health visitors in one primary health care trust in North West England. Data were transcribed and analysed to identify themes.

Findings.  Elements of practice that contributed to public health were identified, along with constraints on the development of a family‐centred public health role. Three themes were generated from the health visitor focus group discussions as a framework for addressing the constraints: connecting to public health, clarifying the role and changing practice.

Conclusions.  The study sets out the challenge faced in developing the public health roles of practitioners. Leadership is essential for clarifying the public health role, developing networks between public health and practitioners, and supporting change in practice. Despite the limitations of the study, its findings, together with evidence from previous literature, suggest that there are common issues that face health visiting services in the UK as they seek to develop the public health role.
Coping strategies of people with kidney transplants
Authors: Lindqvist Ragny , Carlsson Marianne , Sjödén Per‐Olow
Published: December 2003

Background.  There has been little research into coping process following renal transplantation, but it has been shown that health‐related stress is not eliminated and that fear of rejection and economic factors are the most stressful issues. Coping strategies used have included prayer, looking at problems objectively, and trying to main control over the situation.

Aim.  The study was concerned with kidney transplant patients and their efforts to cope with problems posed by the transplantation and treatment. The aim was to describe and compare the use and perceived effectiveness of different coping strategies in people after kidney transplant with an age, gender and civil status‐matched sample of the general Swedish population.

Methods.  Coping was assessed by the Jalowiec Coping Scale and self‐rated efficiency with visual analogue scales. From a total of 73 post‐transplant patients, a consecutive series of 30 was compared with an age, gender and civil status‐individually matched sample of the general Swedish population (n = 30). Data were collected from 1994 to 1997.

Results.  The patient sample used significantly more optimistic, self‐reliant, supportive and emotive coping than the general population sample. Evasive, emotive, and fatalistic coping were associated with low perceived efficiency in handling various aspects of the condition.

Study limitations.  The study design does not permit causal conclusions and the patient sample was relatively small and non‐randomly chosen, which could affect its external validity.

Conclusions.  The results suggest that assessment of coping strategies should be explored in clinical nursing practice so that nurses can encourage the use of those likely to be more useful, and discourage those with possible negative effects.
Randomized controlled trials of socially complex nursing interventions: creating bias and unreliability?
Authors: Lindsay Bruce
Published: December 2003

Background.  The randomized controlled trial is viewed by many researchers as the ‘gold standard’ research design. It is used increasingly to evaluate the effectiveness of socially complex activities such as nursing interventions. This use is seen by many commentators as problematic, while others are concerned about the quality of many published trial reports. One area of concern is that of intervention bias: the impact that a sentient intervention, such as a nursing one, may have consciously or unconsciously on study outcomes. This paper reports on an analysis of intervention definitions and possible intervention bias in 47 reports of randomized controlled trials of nursing interventions published in 2000 or 2001.

Aims.  This study evaluates four characteristics of the included reports: intervention sample size, intervention definition, involvement of intervention nurses in other aspects of the trial, and the claimed generalizability of results.

Methods.  Reports of randomized controlled trials published in 2000 or 2001 were identified. Full‐text versions of 47 papers were obtained and information about the four characteristics was extracted and analysed.

Results.  Problems relating to possible intervention bias were identified in each of the papers. Inadequate intervention definition was the commonest problem, leading to difficulties in calculating the ‘intervention dose’ and in replicating or generalizing from the studies.

Discussion.  None of the included studies met the requirements of the Consolidated Standards of Reporting Trials. Four types of intervention bias were identified, and their possible implications for the reporting of trials of nursing interventions are discussed. This was a small‐scale study, limited by time and resources. Its results are suggestive of a major problem of intervention bias but larger‐scale investigations are necessary to quantify its extent.

Conclusions.  Intervention bias is potentially a problem in randomized controlled trials. Lack of detail about interventions in published papers could be corrected by stricter adherence to guidelines such as the Consolidated Standards of Reporting Trials, but this will not correct the underlying problem of inadequate study design that appears to be widespread in randomized controlled trials of nursing interventions.
A hyperparasitic microsporidian infecting the salmon louse, Lepeophtheirus salmonis: an rDNA‐based molecular phylogenetic study
Authors: Freeman M A , Bell A S , Sommerville C
Published: December 2003

The sea louse, Lepeophtheirus salmonis, is an obligate ectoparasitic copepod that lives on the external surface of salmonid fish. It is the most common ectoparasite of marine cage‐reared salmonids, causing major economic loss to the aquaculture industry. During a sea louse monitoring programme, samples of L. salmonis were found to harbour an unreported microsporidian parasite. The microsporidian was observed in pre‐adult and adult stages of both male and female copepods, with a prevalence of up to 5%. Unfixed spores were slightly pyriform in shape measuring 2.34 μm by 1.83 μm (±0.01 μm) and were not observed to be enclosed by a sporophorous vesicle. The microsporidian infection was observed in all areas of the copepods’ body, xenoma‐like cysts forming directly under the cuticle in the epidermal tissue layer. In the present study, rDNA (530f–580r) sequence data gathered from the unidentified microsporidian parasite isolated from infected sea lice were compared with equivalents available in the databases in an attempt to identify its systematic position. The microsporidian was found to group within the phylogenetic clade containing the family Enterocytozoonidae, being most similar to members of the intranuclear genus Nucleospora. This is the first report of a hyperparasitic microsporidian infecting a caligid copepod.
The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia
Authors: Aust Sylvia , Thalhammer Theresia , Humpeler Susanne , Jäger Walter , Klimpfinger Martin , Tucek Gerhard , Obrist Peter , Marktl Wolfgang , Penner Edward , Ekmekcioglu Cem
Published: December 2003

Abstract: Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT‐PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine‐N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT‐PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin‐embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5‐hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1‐mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.
Does water status of Eucalyptus largiflorens (Myrtaceae) affect infection by the mistletoe Amyema miquelii (Loranthaceae)?
Authors: Miller Anthony C. , Watling Jennifer R. , Overton Ian C. , Sinclair Russell
Published: December 2003

The impact of soil salinity and host water status on the host / parasite association between Eucalyptus largiflorens (F.Muell.) and Amyema miquelii (Lehm. ex Miq.) Tiegh. was investigated in a semi-arid floodplain environment in southern Australia. Water status of potential hosts (i.e. uninfected E. largiflorens) was assessed at a range of sites with different soil salinities and then compared with mistletoe infection at the same sites. Pre-dawn leaf water potentials (Ψ) of uninfected E. largiflorens declined with increasing salinity, while leaf δ13C values increased. The proportion of infected E. largiflorens at each site decreased significantly with increasing soil salinity. A significant relationship was found between the proportion of infected trees at each site and leaf δ13C values, but not pre-dawn Ψ, of potential hosts. The impact of mistletoes on water status of infected trees was also investigated. Among infected trees, we found no significant relationship between mistletoe volume and either pre-dawn Ψ or leaf δ13C values of hosts. However, there was a significant relationship between host midday leaf Ψ and mistletoe volume, with hosts exhibiting increased stress as mistletoe volume increased. The data suggest that increasing water and / or salinity stress make E. largiflorens a less suitable host for A. miquelii.
Buoyancy-driven flow in a peat moss layer as a mechanism for solute transport
Authors: Rappoldt Cornelis , Pieters Gert-Jan J. M. , Adema Erwin B. , Baaijens Gerrit J. , Grootjans Ab P. , van Duijn Cornelis J.
Published: December 2003

Transport of nutrients, CO
2
, methane, and oxygen plays an important ecological role at the surface of wetland ecosystems. A possibly important transport mechanism in a water-saturated peat moss layer (usually
Sphagnum cuspidatum
) is nocturnal buoyancy flow, the downward flow of relatively cold surface water, and the upward flow of warm water induced by nocturnal cooling. Mathematical stability analysis showed that buoyancy flow occurs in a cooling porous layer if the system's Rayleigh number (
Ra
) exceeds 25. For a temperature difference of 10 K between day and night, a typical
Ra
value for a peat moss layer is 80, which leads to quickly developing buoyancy cells. Numerical simulation demonstrated that fluid flow leads to a considerable mixing of water. Temperature measurements in a cylindrical peat sample of 50-cm height and 35-cm diameter were in agreement with the theoretical results. The nocturnal flow and the associated mixing of the water represent a mechanism for solute transport in water-saturated parts of peat land and in other types of terrestrializing vegetation. This mechanism may be particularly important in continental wetlands, where
Ra
values in summer are often much larger than the threshold for fluid flow.
Turning immunological memory into amnesia by depletion of dividing T cells
Authors: Bellier Bertrand , Thomas-Vaslin Véronique , Saron Marie-Françoise , Klatzmann David
Published: December 2003

Immunological memory, defined as more efficient immune responses on antigen reexposure, can last for decades. The current paradigm is that memory is maintained by antigen-experienced “memory T cells” that can be long-lived quiescent or dividing. The contribution of T cell division to memory maintenance is poorly known and has important clinical implications. In this study, we directly addressed the role of dividing T cells in immunological memory maintenance by evaluating the consequences of their elimination. The specific ablation of dividing T cells was obtained by administration of ganciclovir to immune mice expressing the herpes simplex type 1 thymidine kinase suicide gene in T cells. We show that depletion of dividing T cells for 5 or 2 weeks suffices to abolishin vitroandin vivomemory responses against the male H-Y transplantation alloantigen or against lymphocytic choriomeningitis virus antigens, respectively. Similar results were obtained after the nonspecific elimination of all dividing cells by using hydroxyurea, a cytostatic toxic agent commonly used for cancer chemotherapy. This immune amnesia occurred in otherwise immunocompetent mice and despite the persistence of functional quiescent T cells displaying a “memory” phenotype. Thus, division of antigen-experienced T cells is an absolute requirement for immunological memory maintenance and the current concept of memory T cells is challenged.
Recursions for statistical multiple alignment
Authors: Hein Jotun , Jensen Jens Ledet , Pedersen Christian N. S.
Published: December 2003

Algorithms are presented that allow the calculation of the probability of a set of sequences related by a binary tree that have evolved according to the Thorne–Kishino–Felsenstein model for a fixed set of parameters. The algorithms are based on a Markov chain generating sequences and their alignment at nodes in a tree. Depending on whether the complete realization of this Markov chain is decomposed into the first transition and the rest of the realization or the last transition and the first part of the realization, two kinds of recursions are obtained that are computationally similar but probabilistically different. The running time of the algorithms is

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\begin{equation*}O({\Pi}_{i}^{d}=1~L_{i})\end{equation*}\end{document}

, where

L
i

is the length of the
i
th observed sequences and
d
is the number of sequences. An alternative recursion is also formulated that uses only a Markov chain involving the inner nodes of a tree.
A detailed predictive model of the mammalian circadian clock
Authors: Forger Daniel B. , Peskin Charles S.
Published: December 2003

Experimental data on the circadian (≈24-h) clock in mammalian cells are vast, diverse, and detailed. Mathematical models are therefore needed to piece these data together and to study overall clock behavior. Previous models have focused on
Neurospora
or
Drosophila
or can be converted to a
Drosophila
model simply by renaming variables. Those models used Hill-type terms for transcription regulation and Michaelis–Menten type or delay terms for posttranslation regulation. Recent mammalian experimental data call into question some of the assumptions in these approaches. Moreover, gene duplication has led to more proteins in the mammalian system than in lower organisms. Here we develop a detailed distinctly mammalian model by using mass action kinetics. Parameters for our model are found from experimental data by using a coordinate search method. The model accurately predicts the phase of entrainment, amplitude of oscillation, and shape of time profiles of clock mRNAs and proteins and is also robust to parameter changes and mutations.
Evolution of genomic diversity and sex at extreme environments: Fungal life under hypersaline Dead Sea stress
Authors: Kis-Papo Tamar , Kirzhner Valery , Wasser Solomon P. , Nevo Eviatar
Published: December 2003

We have found that genomic diversity is generally positively correlated with abiotic and biotic stress levels (
1
–
3
). However, beyond a high-threshold level of stress, the diversity declines to a few adapted genotypes. The Dead Sea is the harshest planetary hypersaline environment (340 g·liter
–1
total dissolved salts, ≈10 times sea water). Hence, the Dead Sea is an excellent natural laboratory for testing the “rise and fall” pattern of genetic diversity with stress proposed in this article. Here, we examined genomic diversity of the ascomycete fungus
Aspergillus versicolor
from saline, nonsaline, and hypersaline Dead Sea environments. We screened the coding and noncoding genomes of
A. versicolor
isolates by using >600 AFLP (amplified fragment length polymorphism) markers (equal to loci). Genomic diversity was positively correlated with stress, culminating in the Dead Sea surface but dropped drastically in 50- to 280-m-deep seawater. The genomic diversity pattern paralleled the pattern of sexual reproduction of fungal species across the same southward gradient of increasing stress in Israel. This parallel may suggest that diversity and sex are intertwined intimately according to the rise and fall pattern and adaptively selected by natural selection in fungal genome evolution. Future large-scale verification in micromycetes will define further the trajectories of diversity and sex in the rise and fall pattern.
TRF3, a TATA-box-binding protein-related factor, is vertebrate-specific and widely expressed
Authors: Persengiev Stephan P. , Zhu Xiaochun , Dixit Bharat L. , Maston Glenn A. , Kittler Ellen L. W. , Green Michael R.
Published: December 2003

TATA-box-binding protein (TBP) is a highly conserved RNA polymerase II general transcription factor that binds to the core promoter and initiates assembly of the preinitiation complex. Two proteins with high homology to TBP have been found: TBP-related factor 1 (TRF1), described only in
Drosophila melanogaster
, and TRF2, which is broadly distributed in metazoans. Here, we report the identification and characterization of an additional TBP-related factor, TRF3. TRF3 is virtually identical to TBP in the C-terminal core domain, including all residues involved in DNA binding and interaction with other general transcription factors. Like other TBP family members, the N-terminal region of TRF3 is divergent. The
TRF3
gene is present and expressed in vertebrates, from fish through humans, but absent from the genomes of the urochordate
Ciona intestinalis
and the lower eukaryotes
D. melanogaster
and
Caenorhabditis elegans
. TRF3 is a nuclear protein that is present in all human and mouse tissues and cell lines examined. Despite the highly homologous TBP-like C-terminal core domain, gel filtration analysis indicates that the native molecular weight of TRF3 is substantially less than that of TFIID. Interestingly, after mitosis, reimport of TRF3 into the nucleus occurs subsequent to TBP and other basal transcription factors. In summary, TRF3 is a highly conserved vertebrate-specific TRF whose phylogenetic conservation, expression pattern, and other properties are distinct from those of TBP and all other TRFs.
Cancerous stem cells can arise from pediatric brain tumors
Authors: Hemmati Houman D. , Nakano Ichiro , Lazareff Jorge A. , Masterman-Smith Michael , Geschwind Daniel H. , Bronner-Fraser Marianne , Kornblum Harley I.
Published: December 2003

Pediatric brain tumors are significant causes of morbidity and mortality. It has been hypothesized that they derive from self-renewing multipotent neural stem cells. Here, we tested whether different pediatric brain tumors, including medulloblastomas and gliomas, contain cells with properties similar to neural stem cells. We find that tumor-derived progenitors form neurospheres that can be passaged at clonal density and are able to self-renew. Under conditions promoting differentiation, individual cells are multipotent, giving rise to both neurons and glia, in proportions that reflect the tumor of origin. Unlike normal neural stem cells, however, tumor-derived progenitors have an unusual capacity to proliferate and sometimes differentiate into abnormal cells with multiple differentiation markers. Gene expression analysis reveals that both whole tumors and tumor-derived neurospheres express many genes characteristic of neural and other stem cells, including CD133,
Sox2
, musashi-1,
bmi-1
, maternal embryonic leucine zipper kinase, and phosphoserine phosphatase, with variation from tumor to tumor. After grafting to neonatal rat brains, tumor-derived neurosphere cells migrate, produce neurons and glia, and continue to proliferate for more than 4 weeks. The results show that pediatric brain tumors contain neural stem-like cells with altered characteristics that may contribute to tumorigenesis. This finding may have important implications for treatment by means of specific targeting of stem-like cells within brain tumors.
Imaging
Tetrahymena
ribozyme splicing activity in single live mammalian cells
Authors: Hasegawa Sumitaka , Jackson W. Coyt , Tsien Roger Y. , Rao Jianghong
Published: December 2003

Tetrahymena
ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high
in vivo
activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, β-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing
Tetrahymena thermophila
group I intron ribozyme that is inserted into the ORF of the mRNA of β-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of
Tetrahymena
ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final
in vivo
activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile
in vivo
selection of desired ribozyme variants.
Chlorophyll breakdown: Pheophorbide
a
oxygenase is a Rieske-type iron–sulfur protein, encoded by the
accelerated cell death 1
gene
Authors: Pružinská Adriana , Tanner Gaby , Anders Iwona , Roca Maria , Hörtensteiner Stefan
Published: December 2003

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide)
a
followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide
a
oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of
Arabidopsis thaliana
(
AtPaO
). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to
Arabidopsis
accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide
a
and molecular oxygen. By using a maize
lls1
mutant, the
in vivo
function of PaO, i.e., degradation of pheide
a
during senescence, could be confirmed. Thus,
lls1
leaves stayed green during dark incubation and accumulated pheide
a
that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and
lls1
, a chl-binding protein was selectively retained in the mutant.
PaO
expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.
Coding potential of laboratory and clinical strains of human cytomegalovirus
Authors: Murphy Eain , Yu Dong , Grimwood Jane , Schmutz Jeremy , Dickson Mark , Jarvis Michael A. , Hahn Gabriele , Nelson Jay A. , Myers Richard M. , Shenk Thomas E.
Published: December 2003

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs.
Hyperactivated sperm motility driven by CatSper2 is required for fertilization
Authors: Quill Timothy A. , Sugden Sarah A. , Rossi Kristen L. , Doolittle Lynda K. , Hammer Robert E. , Garbers David L.
Published: December 2003

Elevations of sperm Ca
2+
seem to be responsible for an asymmetric form of motility called hyperactivation, which is first seen near the time of fertilization. The mechanism by which intracellular Ca
2+
concentrations increase remains unknown despite considerable investigation. Although several prototypical voltage-gated calcium channels are present in spermatozoa, they are not essential for motility. Furthermore, the forward velocity and percentage of motility of spermatozoa are associated with infertility, but their importance relative to hyperactivation also remains unknown. We show here that disruption of the gene for a recently described sperm-specific voltage-gated cation channel, CatSper2, fails to significantly alter sperm production, protein tyrosine phosphorylation that is associated with capacitation, induction of the acrosome reaction, forward velocity, or percentage of motility, yet CatSper2
–/–
males are completely infertile. The defect that we identify in the null sperm cells is a failure to acquire hyperactivated motility, which seems to render spermatozoa incapable of generating the “power” needed for penetration of the extracellular matrix of the egg. A loss of power is suggested also by experiments in which the viscosity of the medium was increased after incubation of spermatozoa in normal capacitating conditions. In high-viscosity medium, CatSper2-null spermatozoa lost the ability to swim forward, whereas wild-type cells continued to move forward. Thus, CatSper2 is responsible for driving hyperactivated motility, and, even with typical sperm forward velocities, fertilization is not possible in the absence of this highly active form of motility.
Crystal structure of human α-tocopherol transfer protein bound to its ligand: Implications for ataxia with vitamin E deficiency
Authors: Min K. Christopher , Kovall Rhett A. , Hendrickson Wayne A.
Published: December 2003

Human α-tocopherol (α-T) transfer protein (ATTP) plays a central role in vitamin E homeostasis, preventing degradation of α-T by routing this lipophilic molecule for secretion by hepatocytes. Mutations in the gene encoding ATTP have been shown to cause a severe deficiency in α-T, which results in a progressive neurode-generative spinocerebellar ataxia, known as ataxia with vitamin E deficiency (AVED). We have determined the high-resolution crystal structure of human ATTP with (2
R
,4′
R
,8′
R
)-α-T in the binding pocket. Surprisingly, the ligand is sequestered deep in the hydrophobic core of the protein, implicating a large structural rearrangement for the entry and release of α-T. A comparison to the structure of a related protein, Sec14p, crystallized without a bona fide ligand, shows a possibly relevant open conformation for this family of proteins. Furthermore, of the known mutations that cause AVED, one mutation, L183P, is located directly in the binding pocket. Finally, three mutations associated with AVED involve arginine residues that are grouped together on the surface of ATTP. We propose that this positively charged surface may serve to orient an interacting protein, which might function to regulate the release of α-T through an induced change in conformation of ATTP.
Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion
Authors: Takeda Makoto , Leser George P. , Russell Charles J. , Lamb Robert A.
Published: December 2003

Lipid raft microdomains are enriched in sphingomyelin and cholesterol and function as platforms for signal transduction and as the site of budding of several enveloped viruses, including influenza virus. The influenza virus hemagglutinin (HA) glycoprotein, which mediates both viral-cell attachment and membrane fusion, associates intrinsically with lipid rafts. Residues in the HA transmembrane (TM) domain are important for raft association as sequence substitutions in the HA TM domain ablate HA association with rafts (nonraft HA). Cells expressing either WT or nonraft HA cause complete fusion (lipid mixing and content mixing) over widely varying HA expression levels. However, the number of fusion events measured for nonraft HA mutant protein at all HA surface densities was reduced to ≈55% of the events for WT HA protein. Mutant influenza viruses were generated that contain the nonraft HA TM domain alterations. Electron microscopy experiments showed that WT HA was distributed at the cell surface in clusters of 200–280 nm in diameter, whereas nonraft HA was distributed mostly randomly at the plasma membrane. Nonraft HA virus showed reduced budding, contained reduced amounts of HA protein, was greatly reduced in infectivity, and exhibited decreased virus–membrane fusion activity. Cholesterol depletion of virus did not affect the ability of virions to cause either virus–cell lipid mixing or virus-mediated hemolysis, a surrogate for content mixing. Taken together, the data suggest that HA clusters in rafts to provide a sufficient concentration of HA in budding virus to mediate efficient virus–cell fusion.
TRPM5 is a transient Ca
2+
-activated cation channel responding to rapid changes in [Ca
2+
]
i
Authors: Prawitt Dirk , Monteilh-Zoller Mahealani K. , Brixel Lili , Spangenberg Christian , Zabel Bernhard , Fleig Andrea , Penner Reinhold
Published: December 2003

Transient receptor potential (TRP) proteins are a diverse family of proteins with structural features typical of ion channels. TRPM5, a member of the TRPM subfamily, plays an important role in taste receptors, although its activation mechanism remains controversial and its function in signal transduction is unknown. Here we characterize the functional properties of heterologously expressed human TRPM5 in HEK-293 cells. TRPM5 displays characteristics of a calcium-activated, nonselective cation channel with a unitary conductance of 25 pS. TRPM5 is a monovalent-specific, nonselective cation channel that carries Na
+
, K
+
, and Cs
+
ions equally well, but not Ca
2+
ions. It is directly activated by [Ca
2+
]
i
at concentrations of 0.3–1 μM, whereas higher concentrations are inhibitory, resulting in a bell-shaped dose–response curve. It activates and deactivates rapidly even during sustained elevations in [Ca
2+
]
i
, thereby inducing a transient membrane depolarization. TRPM5 does not simply mirror levels of [Ca
2+
]
i
, but instead responds to the rate of change in [Ca
2+
]
i
in that it requires rapid changes in [Ca
2+
]
i
to generate significant whole-cell currents, whereas slow elevations in [Ca
2+
]
i
to equivalent levels are ineffective. Moreover, we demonstrate that TRPM5 is not limited to taste signal transduction, because we detect the presence of TRPM5 in a variety of tissues and we identify endogenous TRPM5-like currents in a pancreatic beta cell line. TRPM5 can be activated physiologically by inositol 1,4,5-trisphosphate-producing receptor agonists, and it may therefore couple intracellular Ca
2+
release to electrical activity and subsequent cellular responses.
Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells
Authors: Tone Masahide , Tone Yukiko , Adams Elizabeth , Yates Stephen F. , Frewin Mark R. , Cobbold Stephen P. , Waldmann Herman
Published: December 2003

Recently, agonist antibodies to glucocorticoid-induced tumor necrosis factor receptor (GITR) (tumor necrosis factor receptor superfamily 18) have been shown to neutralize the suppressive activity of CD4+CD25+regulatory T cells. It was anticipated that this would be the role of the physiological ligand. We have identified and expressed the gene for mouse GITR ligand and have confirmed that its interaction with GITR reverses suppression by CD4+CD25+T cells. It also, however, provides a costimulatory signal for the antigen-driven proliferation of naïve T cells and polarized T helper 1 and T helper 2 clones. RT-PCR and mAb staining revealed mouse GITR ligand expression in dendritic cells, macrophages, and B cells. Expression was controlled by the transcription factor NF-1 and potentially by alternative splicing of mRNA destabilization sequences.
A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation
Authors: Hang Howard C. , Yu Chong , Kato Darryl L. , Bertozzi Carolyn R.
Published: December 2003

Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide
N
-acetyl-α-galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (
N
-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.
Natural killer cells in HIV-1 infection: Dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates
Authors: Mavilio Domenico , Benjamin Janet , Daucher Marybeth , Lombardo Gabriella , Kottilil Shyam , Planta Marie A. , Marcenaro Emanuela , Bottino Cristina , Moretta Lorenzo , Moretta Alessandro , Fauci Anthony S.
Published: December 2003

Natural killer (NK) cells play a central role in host defense against various pathogens. Functional defects of NK cells in HIV-1 infection as a direct effect of abnormal expression or function of inhibitory NK receptors (iNKRs), activating natural cytotoxicity receptors (NCRs), and NKG2D have not yet been described. This study demonstrates an expansion of the functionally defective CD56-/CD16+population of NK cells in viremic versus aviremic patients. We also demonstrate that in HIV-infected viremic patients, expression of iNKRs was well conserved and that in most cases, there was a trend toward increased expression on NK cells as compared with healthy donors. It was also demonstrated that the major activating NK receptors, with the exception of NKG2D, were significantly down-regulated. In contrast, the expression of iNKRs and activating receptors in HIV-infected individuals whose viremia was suppressed to below detectable levels by highly active antiretroviral therapy for 2 years or longer was comparable to that of healthy donors. Functional tests confirmed that the abnormal expression of the activating receptors and of iNKRs was associated with a markedly impaired NK cytolytic function. This phenomenon is not attributed to a direct HIV-1 infection of NK cells; thus, this study may provide insight into the mechanisms of impaired host defenses in HIV-1 viremic patients.
The thermodynamics of template-directed DNA synthesis: Base insertion and extension enthalpies
Authors: Minetti Conceição A. S. A. , Remeta David P. , Miller Holly , Gelfand Craig A. , Plum G. Eric , Grollman Arthur P. , Breslauer Kenneth J.
Published: December 2003

We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension. Specifically, we used families of hairpin self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues. Our results reveal exothermic heats between –9.8 and –16.0 kcal/bp for template-directed enzymatic polymerization. These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base. Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies. We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes. It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template–primer level. Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.