bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal variation of energy reserves in mayfly nymphs (Hexagenia spp.) from Lake St Clair and western Lake Erie
Authors: Cavaletto J.F. , Nalepa T.F. , Fanslow D.L. , Schloesser D.W.
Published: September 2003

1. We analysed changes in energy reserves (lipid and glycogen) and length–weight relationships of burrowing mayflies (Hexagenia spp.) in 1997–99 to compare an established population in Lake St Clair with a recovering population in western Lake Erie of the Laurentian Great Lakes. In addition, we measured changes in water temperature and potential food in both water columns and sediments.

2. Although overall mean values of lipid and glycogen levels of Hexagenia nymphs from Lake St Clair and western Lake Erie were not significantly different, there were differences in seasonal patterns between the two lakes. In Lake St Clair, levels were highest in early spring, declined throughout the year, and reached their lowest levels in fall during all 3 years of study. In contrast, levels in western Lake Erie were lower in spring, increased to a maximum in summer, then declined in fall. Seasonal patterns in length–weight relationships were similar to those for lipid and glycogen.

3. Total lipid as a percentage of dry weight did not increase with developmental stage of nymphs until just prior to metamorphosis and emergence from water. However, the major reserve lipid, triacylglycerols, increased systematically with development stage. In the final stage of development, triacylglycerols declined, probably as a result of energy consumption and its conversion to other biochemical components for metamorphosis and reproduction.

4. Indicators of potential food (algal fluorescence in the water column and chlorophyll a and chlorophyll a/phaeophytin ratio in sediments) suggest that Hexagenia in Lake St Clair have a food source that is benthic based, especially in early spring, whereas in western Lake Erie nymphs have a food source that is water column based and settles to the lake bottom during late spring and summer.
Measuring and controlling data quality in biological assemblage surveys with special reference to stream benthic macroinvertebrates
Authors: Cao Yong , Hawkins Charles P. , Vinson Mark R.
Published: September 2003

1. Biological assemblage surveys primarily aim to characterise species composition and relative abundance at one or more spatial or temporal scales. Data interpretation and conclusions are dependent on how well samples characterise the assemblage of interest.

2. Conventional measures of data quality, e.g. standard deviations or coefficients of variation, were designed for single variable estimation, and they are either insufficient or invalid for assessing the quality of data describing entire assemblages. Similarity indices take species composition and relative abundance into account and may be used to effectively measure and control the quality of data used to characterise assemblage structure.

3. The average Jaccard coefficient (JC) calculated across multiple pairs of replicate samples, i.e. autosimilarity JC (AJC), is conceptually and numerically related to the average coefficient of variation in the densities of all species recorded, a measure of sampling precision, and to the proportion of total species richness sampled, a measure of sampling accuracy.

4. We explored how AJC can be used to assess the effect of different potential sources of error on the quality of assemblage survey data, including the sampling effort used both within regions and at individual sites, the individuals collecting samples, sub‐sampling procedures, and consistency of taxon identification.

5. We found that the autosimilarity‐based approach overcomes most weaknesses associated with conventional measures of data quality and can be used to effectively measure and control the quality of assemblage survey data.
Habitat change in braided flood plains (Tagliamento, NE‐Italy)
Authors: Van Der Nat Dimitry , Tockner Klement , Edwards Peter J. , Ward J.V. , Gurnell Angela M.
Published: September 2003

1. Relative changes and age distribution of habitats were investigated in the active channel of a bar‐braided and an island‐braided reach of the Tagliamento River (NE‐Italy). Between September 1999 and January 2002, six habitat types were delineated with a differential Global Positioning System on five dates following floods of different magnitude. Overlay maps were employed to calculate age and relative change of habitats. We established exponential decay rates (k‐values) for islands and major aquatic habitats.

2. Relative changes of all aquatic habitats combined were up to 82% between survey dates in the bar‐braided flood plain, with a cumulative rate of 85% over the 2.5‐year period. Relative habitat changes in the island‐braided flood plain were lower with a cumulative change of almost 60% during the study period. In the bar‐braided flood plain significant exponential decay relationships were established for channels, alluvial channels, backwaters, and ponds.

3. Half‐lives were particularly short for backwaters and ponds. In the island‐braided reach, significant relationships existed for channels and alluvial channels. The half‐lives of channels and alluvial channels increased with the presence of vegetated islands. Relative habitat composition within the active corridor remained almost constant, supporting the applicability of the shifting mosaic steady state model to braided floodplain ecosystems.

4. Our results indicate that under natural conditions aquatic floodplain habitats can be highly dynamic over short time‐scales. Even small water level fluctuations (‘flow pulses’) can lead to major habitat changes with important consequences for the fauna and flora.
Variation in the temperature preference and growth rate of individual fish reconciles differences between two growth models
Authors: Elliott J.M. , Hurley M.A.
Published: September 2003

1. A growth model, originally developed for brown trout (Salmo trutta), has now been fitted to data for Atlantic salmon (S. salar) and stone‐loach (Barbatula barbatula) from English populations, and Arctic charr (Salvelinus alpinus) from Sweden. The model relates growth rate to temperature for a fish of standard size and the functional relationship has a triangular shape with a sharp peak at the optimal temperature for growth and zero growth at the base of the triangle. It was unsuitable for growth data for Norwegian salmon, and a curvilinear Ratkowsky model provided a better fit, though the experimental protocol was different in the Norwegian and English experiments.

2. The Norwegian salmon were kept in groups in each tank, had to compete for food, and had to be divided into slow, moderate and fast growers before the Ratkowsky model could be fitted. Each English salmon was kept in its own tank and fed individually. For replicate experiments, fish of similar size were selected. Variation among fish kept under similar conditions was therefore small, and the triangular model was essentially for individual fish, not groups of fish.

3. The present simulation study tests the hypothesis that individual differences in the growth response could account for the curvilinear growth‐temperature relationship for the Norwegian salmon. The triangular model was used to generate the growth response to temperature for a group of salmon, each fish having a slightly different temperature preference and growth rate. The result was a curvilinear response, well approximated by the Ratkowsky model (adjusted R2 = 0.96). When the variability in individual temperature preference was increased, the Ratkowsky model was an even better fit (adjusted R2 = 0.98). Therefore, the apparent discrepancy between the two models was reconciled by allowing for individual differences in temperature preference and growth rate within groups of fish.
Are biological classifications of headwater streams concordant across multiple taxonomic groups?
Authors: Paavola Riku , Muotka Timo , Virtanen Risto , Heino Jani , Kreivi Petri
Published: September 2003

1. Studies assessing human impacts on freshwater ecosystems are typically based on a single taxonomic group, often macroinvertebrates or fish. Unfortunately, the degree to which such macroinvertebrate or fish‐based surveys can be generalised across other taxonomic groups remains largely unknown. A prerequisite for useful generalisations is that different taxonomic groups exhibit concordant patterns of community structure across sites.

2. We examined the concordance among fish, benthic macroinvertebrates and bryophytes in 32 streams in a boreal catchment in Finland. Our goal was to test how consistently different taxonomic groups classify stream sites; for example, can site groupings based on macroinvertebrates be used as a surrogate for bryophyte or fish assemblage classification?

3. Our results show that community classifications in headwater streams are not concordant across taxonomic groups, at least not at the within‐river system scale. The lack of concordance reflected the fact that all three groups responded to different environmental factors. Macroinvertebrate community structure was mainly correlated with stream size and pH, whereas bryophytes were related to water colour, nutrient content and in‐stream habitat variability. Fish community structure was best described by stream depth, substrate size and water oxygen concentration.

4. Our results suggest that great care should be taken when typologies based on benthic macroinvertebrates, or any other taxonomic group, are extrapolated to other groups in creating typologies of lotic environments.
Photoheterotrophy and light‐dependent uptake of organic and organic nitrogenous compounds by Planktothrix rubescens under low irradiance
Authors: Zotina Tatiana , Köster Oliver , Jüttner Friedrich
Published: September 2003

1. Planktothrix rubescens is the dominant photoautotrophic organism in Lake Zürich, a prealpine, deep, mesotrophic freshwater lake with an oxic hypolimnion. Over long periods of the year, P. rubescens accumulates at the metalimnion and growth occurs in situ at irradiance near the photosynthesis compensation point. Experiments were conducted to evaluate the contribution of photoheterotrophy, heterotrophy and light‐dependent uptake of nitrogenous organic compounds to the carbon and nitrogen budget of this cyanobacterium under conditions of restricted availability of light quanta.

2. We used both purified natural populations of P. rubescens from the depth of 9 m and an axenic culture grown under low irradiance at 11 μmol m−2 s−1 on a light : dark cycle (10 : 14 h) to determine the uptake rates of various amino acids, urea, glucose, fructose, acetate and inorganic carbon. The components were added to artificial lake water in low amounts that simulated the naturally occurring potential concentrations.

3. The uptake rates of acetate and amino acids (glycine, serine, glutamate and aspartate) were strongly enhanced at low irradiance as compared with the dark. However, no difference was observed in the uptake of arginine, which was taken up at high rates under both treatments. The uptake rates of glucose, fructose and urea were very low under all conditions. Similar results were obtained for both axenic P. rubescens and for purified natural populations of P. rubescens that were separated from bacterioplankton and other phytoplankton.

4. Metalimnetic P. rubescens that was stratified at low irradiance for weeks exhibited much higher uptake rates than filaments that were entrained in the deepening surface mixed layer and experienced higher irradiance. The added organic compounds contributed up to 62% to the total carbon uptake of metalimnetic P. rubescens. On the basis of a molar C : N ratio of 4.9, the nitrogen uptake as organic compounds satisfied up to 84% of the nitrogen demand.

5. The experiments indicate that photoheterotrophy and light‐dependent uptake of nitrogenous organic compounds may contribute significantly to the carbon and nitrogen budget of filaments at low irradiance typical for growth of P. rubescens in the metalimnion and at the bottom of the surface mixed layer.
Systematic analysis of the TGF‐β/Smad signalling pathway in the rhabdomyosarcoma cell line RD
Authors: Wang Hua , Yang Guang‐Hua , Bu Hong , Zhou Qiao , Guo Li‐Xin , Wang Shou‐Li , Ye Lv
Published: September 2003

Summary.  Transforming growth factor‐β (TGF‐β) is a multifunctional regulator of cell growth and differentiation, whose actions are highly cell type specific. To study the role of the TGF‐β1 autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD, an attempt was made to establish a framework for the expression of several components of TGF‐β1/Smad signalling pathway at the mRNA and protein levels by reverse transcription‐polymerase chain reaction (RT‐PCR) and Western blot analysis in RD cells compared with the normal myoblasts. Higher exogenous concentration of TGF‐β1 was necessary to reach a growth‐inhibition effect, whereas TGF‐β1 downregulated the expression of myosin heavy‐chain mRNA at lower concentrations than that was required for growth inhibition. Treatment with TGF‐β1 significantly decreased the number of sarcomeric actin and myosin‐expressing cells. In this study, we have shown that RD cells displayed higher expression of TβRI, TβRII, Smad2 and Smad4 at both the mRNA and protein levels than myoblasts. Smad3 and Smad7 mRNA were expressed at higher level in RD cells than in myoblasts. The staining patterns of TβR and Smads suggest that they may transduce different TGF‐β1 signalling in RD cells than in myoblasts. TGF‐β1 signalling induced a rapid relocation of Smad2 to the nucleus; in contrast, Smad4 remained localized to the cytoplasm unless it was coexpressed with Smad2. These studies suggest that signalling from the cell surface to the nucleus through Smad proteins is a required component of TGF‐β1‐induced cell response in RD cells. The RD cell line is a suitable model to study the TGF‐β autocrine loop involved in growth and differentiation of RMS.
Investigating intellectual disability: A genetic perspective
Authors: Poplawski NK
Published: September 2003

Abstract:  Intellectual disability (ID) is a common paediatric problem. Investigation focused on determining the aetiology of ID is warranted because a specific diagnosis may assist in prognostication, recurrence risk counselling, and identification of therapeutic and educational interventions. Three groups of screening investigations can be justified on the basis of expert opinion, common sense and the small body of published evidence‐based medicine. First, investigations where current evidence justifies routine use in the investigation of ID; second, investigations which should be considered in all children with ID, although there is insufficient current evidence to support routine use; and finally, investigations which currently have an unclear role in children with ID and should be restricted to specific clinical situations unless further evidence suggesting otherwise emerges. There is a great need for systematic evaluation of the diagnostic yield of investigation templates based on this proposed stratification of investigations.
Hospitalization of childhood rotavirus infection from Kuala Lumpur, Malaysia
Authors: Lee WS , Veerasingam PD , Goh AYT , Chua KB
Published: September 2003

Aim:  To determine the epidemiology of rotavirus gastroenteritis in children admitted to an urban hospital in a developing country from South‐East Asia.

Methods:  Retrospective review of cases of acute gastroenteritis admitted to the children's ward of the University of Malaya Medical Centre, Kuala Lumpur, Malaysia, between 1996 and 1999.

Results:  During the study period, 333 cases (24%) of 1362 stool samples, obtained from children admitted with acute diarrhoea, were positive for rotavirus. Acute gastroenteritis constituted 8.2%, and rotavirus infection 1.6% of all the paediatric admissions each year. Of the 271 cases analysed, 72% of the affected population were less than 2 years of age. Peak incidence of admissions was between January to March, and September to October. Dehydration was common (92%) but electrolyte disturbances, lactose intolerance (5.2%), prolonged diarrhoea (2.6%) and cow's milk protein intolerance was uncommon. No deaths were recorded.

Conclusions:  Rotavirus infection was a common cause of childhood diarrhoea that required hospital admission in an urban setting in Malaysia.
Long and short‐term effect of prednisolone in hospitalized infants with acute bronchiolitis
Authors: Zhang L , Ferruzzi E , Bonfanti T , Auler MI , D’avila NE , Faria CS , Costa MM
Published: September 2003

Objective:  To assess long and short‐term effect of prednisolone in hospitalized infants with bronchiolitis

Methodology:  A randomized and controlled trial was carried out at the Federal University of Rio Grande, Rio Grande‐RS, Brazil. Twenty‐eight patients were randomly allocated prednisolone (1 mg/kg/day for 5 days) plus standard care, and 24 patients allocated standard care alone. The primary endpoint was the prevalence of post‐bronchiolitis wheezing at 1, 3, 6 and 12 months after hospital discharge. The secondary endpoints were: length of hospital stay, duration of oxygen therapy and time to clinical improvement during the hospitalization.

Results:  There were no significant differences between the prednisolone and control group in the prevalence of post‐bronchioltis wheezing at 1 month (73.1 vs 83.3%, P = 0.5), 3 months (73.1 vs 79.2%, P = 0.7), 6 months (65.4 vs 66.7%, P = 0.9) and 12 months (50.0 vs 58.3%, P = 0.5) after hospital discharge. No reduction was observed in the prednisolone group, compared with the control group, in terms of length of hospital stay (6.0 vs 5.0 days, P = 0.7), duration of oxygen therapy (24.0 vs 24.0 h, P = 0.4) and time to clinical resolution (4.0 vs 4.0 days, P = 0.8).

Conclusions:  Prednisolone has no significant effect on reducing the prevalence of post‐bronchiolitis wheezing and on improving the acute course of illness in hospitalized infants with bronchiolitis.
Fatal late onset necrotising enterocolitis in a term infant: Atypical Kawasaki disease or polyarteritis nodosa of infancy?
Authors: Munro AR , Beasley SW , Pattemore PK , Fraser R
Published: September 2003

Abstract:  A 3‐month‐old infant died following a 3‐week illness that commenced with diarrhoea and vomiting, and produced progressive infarction of his small bowel. Post‐mortem identified multiorgan arteritis of uncertain aetiology, giving rise to coronary artery occlusion and myocardial infarction as well as necrosis of the entire small bowel and other organs. Kawasaki disease and polyarteritis nodosa of infancy are proposed as possible aetiologies in this case.
Temperature-dependence of carbon acquisition and demand in relation to shoot and fruit growth of fruiting kiwifruit (Actinidia deliciosa) vines grown in controlled environments
Authors: Greer Dennis H. , Cirillo Chiara , Norling Cara L.
Published: September 2003

Fruiting kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson] vines were grown in two controlled temperatures of 28 / 22 and 17 / 12°C (day / night) for 160 and 215 d, to measure shoot and fruit growth and carbon demand, and to examine competition between fruit and the shoot. Leaf area, internode lengths, fruit diameters, photosynthesis and respiration were measured at regular intervals. The net daily carbon balance per shoot was determined from the net carbon acquisition of shoots, and carbon sequestration as shoot biomass. Vines grown at high temperature had 200% more leaf area, similar stem lengths and 100% more biomass than vines grown at low temperature. Leaf area expansion and stem extension were transiently reduced when fruit growth was maximal. Photosynthetic and respiration rates were affected by temperature, leading to net carbon acquisition of 450 g shoot–1 for 28 / 22°C-grown vines and 253 g shoot–1 for 17 / 12°C-grown vines, 54% being used for leaf, stem and fruit growth. Reallocation of carbon occurred from leaves to fruit, and the consequent reduction in leaf area strongly reduced the overall carbon balance compared with vegetative vines at similar temperatures. The data support the conclusion that at low temperatures especially, there is insufficient carbon to meet the full demands of both fruit and shoot growth.
Dissociable neural mechanisms underlying response-based and familiarity-based conflict in working memory
Authors: Nelson James K. , Reuter-Lorenz Patricia A. , Sylvester Ching-Yune C. , Jonides John , Smith Edward E.
Published: September 2003

Cognitive control requires the resolution of interference among competing and potentially conflicting representations. Such conflict can emerge at different points between stimulus input and response generation, with the net effect being that of compromising performance. The goal of this article was to dissociate the neural mechanisms underlying different sources of conflict to elucidate the architecture of the neural systems that implement cognitive control. By using functional magnetic resonance imaging and a verbal working memory task (item recognition), we examined brain activity related to two kinds of conflict with comparable behavioral consequences. In a trial of our item-recognition task, participants saw four letters, followed by a retention interval, and a probe letter that did or did not match one of the letters held in working memory (positive probe and negative probe, respectively). On some trials, conflict arose solely because of the current negative probe having a high familiarity, due to its membership in the immediately preceding trial's target set. On other trials, additional conflict arose because of the current negative probe having also been a positive probe on the immediately preceding trial, producing response-level conflict. Consistent with previous work, conflict due to high familiarity was associated with left prefrontal activation, but not with anterior cingulate activation. The response-conflict condition, when compared with high-familiarity conflict trials, was associated with anterior cingulate cortex activation, but with no additional left prefrontal activation. This double dissociation points to differing contributions of specific cortical areas to cognitive control, which are based on the source of conflict.
Sporormiellaand the late Holocene extinctions in Madagascar
Authors: Burney David A. , Robinson Guy S. , Burney Lida Pigott
Published: September 2003

Fossil spores of the dung fungusSporormiellaspp. in sediment cores from throughout Madagascar provide new information concerning megafaunal extinction and the introduction of livestock.Sporormiellapercentages are very high in prehuman southwest Madagascar, but at the site with best stratigraphic resolution the spore declines sharply by ≈1,720 yr B.P. (radiocarbon years ago). Within a few centuries there is a concomitant rise in microscopic charcoal that probably represents human transformation of the local environment. Reduced megaherbivore biomass in wooded savannas may have resulted in increased plant biomass and more severe fires. Some now-extinct taxa persisted locally for a millennium or more after the inferred megafaunal decline. Sites in closed humid forests of northwest Madagascar and a montane ericoid formation of the central highlands show only low to moderateSporormiellapercentages before humans. A subsequent rise in spore concentrations, thought to be evidence for livestock proliferation, occurs earliest at Amparihibe in the northwest at ≈1,130 yr B.P.
Cholesterol-induced conformational change in SCAP enhanced by Insig proteins and mimicked by cationic amphiphiles
Authors: Adams Christopher M. , Goldstein Joseph L. , Brown Michael S.
Published: September 2003

Sterols mediate feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway by preventing movement of the SREBP cleavage-activating protein (SCAP)/SREBP complex from endoplasmic reticulum (ER) to Golgi, where proteolytic cleavage of SREBPs releases the transcription factor domain that activates genes for lipid biosynthesis. Our laboratory previously used a trypsin cleavage assay to show that the conformation of SCAP is altered
in vitro
by addition of cholesterol to ER membranes. More recently, Insig-1 and Insig-2 were identified as ER resident proteins that bind the SCAP/SREBP complex and promote its ER retention when cells are treated with sterols. Here, we use the trypsin assay to show that Insig proteins reduce the concentration of cholesterol needed
in vitro
to produce the conformational change in SCAP. Insig-1 and Insig-2 also enhance the conformational change in SCAP that occurs upon addition of certain cationic amphiphiles, such as chlorpromazine, trifluoperazine, and imipramine, which mimic the effect of cholesterol. The effects of cationic amphiphiles raise the possibility that SCAP may monitor specifically the composition of the cytoplasmic leaflet of the ER membrane.
Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase
Authors: Mahajan Kiran N. , Mitchell Beverly S.
Published: September 2003

Terminal deoxynucleotidyl transferase (TdT; EC 2.7.7.31) adds nucleotides to DNA ends generated during V(D)J recombination that are subsequently processed by proteins involved in general double-strand break (DSB) repair pathways. We report an association between TdT and a 55-kDa protein in lymphoid cells. This protein, identified as hPso4, is a homolog of the protein encoded by the
PS04
/
PRP19
gene in
Saccharomyces cerevisiae
that has pleiotropic functions in DNA recombination and error-prone repair. Purified hPso4 binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA. hPso4 protein is induced 15- to 30-fold in cells by γ radiation and chemical mutagens but not by UV treatment. Loss of hPso4 expression induced by siRNA results in accumulation of DSBs, apoptosis, and decreased cell survival after DNA damage. We conclude that hPso4 plays a major and previously undefined role in mammalian DNA DSB repair.
Ionizing radiation delivered by specific antibody is therapeutic against a fungal infection
Authors: Dadachova Ekaterina , Nakouzi Antonio , Bryan Ruth A. , Casadevall Arturo
Published: September 2003

There is an urgent need for new antimicrobial therapies to combat drug resistance, new pathogens, and the relative inefficacy of current therapy in compromised hosts. Ionizing radiation can kill microorganisms quickly and efficiently, but this modality has not been exploited as a therapeutic antimicrobial strategy. We have developed methods to target ionizing radiation to a fungal cell by labeling a specific mAb with the therapeutic radioisotopes Rhenium-188 and Bismuth-213. Radiolabeled antibody killed cells of human pathogenic fungusCryptococcus neoformans in vitro, thus converting an antibody with no inherent antifungal activity into a microbicidal molecule. Administration of radiolabeled antibody to mice withC. neoformansinfection delivered213Bi and188Re to the sites of infection, reduced their organ fungal burden, and significantly prolonged their survival without apparent toxicity. This study establishes the principle that targeted radiation can be used for the therapy of an infectious disease, and suggests that it may have wide applicability as an antimicrobial strategy.
Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa
Authors: Jaiswal Bijay S. , Conti Marco
Published: September 2003

In mammals, Ca2+and\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}ions play a critical role in the regulation of sperm function, most likely by regulation of cAMP levels. Mammalian germ cells contain a soluble adenylyl cyclase (sAC) with properties distinct from the well characterized membrane-bound enzymes Here we investigated whether the cyclase expressed in mature spermatozoa has the properties of sAC and whether it is regulated by Ca2+. In addition to an\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}-dependent activation, the cyclase endogenous to human spermatozoa is stimulated 2- to 3-fold by Ca2+in a concentration-dependent manner (EC50≈ 400 nM). In a similar fashion, Ca2+activates the recombinant rat and human full-length sAC with similar EC50values. The Ca2+stimulation was also observed when sAC was activated with\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}, was independent of calmodulin, and was associated with an increase inVmaxwithout changes inKmfor ATP-Mg2+. An increase in intracellular Ca2+by ionophore or by a muscarinic cholinergic receptor agonist increases cAMP in cells transfected with FL-hsAC, but not in mock-transfected cells. Similarly, both Ca2+and\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}stimulate cAMP accumulation in human spermatozoa. These findings provide evidence that human spermatozoa express a cyclase with the properties of sAC and that Ca2+can substitute for\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}in the stimulation of this enzyme, underscoring an important role for sAC in the control of sperm functions.
Strand-biased DNA methylation associated with centromeric regions in
Arabidopsis
Authors: Luo Song , Preuss Daphne
Published: September 2003

The
Arabidopsis
genome project assembled 15 megabases of heterochromatic sequence, facilitating investigations of heterochromatin assembly, maintenance, and structure. In many species, large quantities of methylcytosine decorate heterochromatin; these modifications are typically maintained by methyltransferases that recognize newly replicated hemimethylated DNA. We assessed the extent and patterns of
Arabidopsis
heterochromatin methylation by amplifying and sequencing genomic DNA treated with bisulfite, which converts cytosine, but not methylcytosine, to uracil. This survey revealed unexpected asymmetries in methylation patterns, with one helix strand often exhibiting higher levels of methylation. We confirmed these observations both by immunoprecipitating methylated DNA strands and by restriction enzyme digestion of amplified, bisulfite-treated DNA. We also developed a primer-extension assay that can monitor the methylation status of an entire chromosome, demonstrating that strand-specific methylation occurs predominantly in the centromeric regions. Conventional models for methylation maintenance do not explain these unusual patterns; instead, new models that allow for strand specificity are required. The abundance of
Arabidopsis
strand-specific modifications points to their importance, perhaps as epigenetic signals that mark the heterochromatic regions that confer centromere activity.
The Trojan exosome hypothesis
Authors: Gould Stephen J. , Booth Amy M. , Hildreth James E. K.
Published: September 2003

We propose that retroviruses exploit a cell-encoded pathway of intercellular vesicle traffic, exosome exchange, for both the biogenesis of retroviral particles and a low-efficiency but mechanistically important mode of infection. This Trojan exosome hypothesis reconciles current paradigms of retrovirus-directed transmission with the unique lipid composition of retroviral particles, the host cell proteins present in retroviral particles, the complex cell biology of retroviral release, and the ability of retroviruses to infect cells independently of Envelope protein–receptor interactions. An exosomal origin also predicts that retroviruses pose an unsolvable paradox for adaptive immune responses, that retroviral antigen vaccines are unlikely to provide prophylactic protection, and that alloimmunity is a central component of antiretroviral immunity. Finally, the Trojan exosome hypothesis has important implications for the fight against HIV and AIDS, including how to develop new antiretroviral therapies, assess the risk of retroviral infection, and generate effective antiretroviral vaccines.
Rapid induction of medullary thymocyte phenotypic maturation and egress inhibition by nanomolar sphingosine 1-phosphate receptor agonist
Authors: Rosen Hugh , Alfonso Christopher , Surh Charles D. , McHeyzer-Williams Michael G.
Published: September 2003

Only a small number of T cells generated in the thymus each day are selected to replenish the peripheral T cell pool. Much is known about thymic selection; however, little is known of the mechanisms regulating medullary maturation and the release of mature T cells into the blood. Here we demonstrate a rapid acceleration of medullary thymocyte phenotypic maturation through loss of CD69 induced by sphingosine 1-phosphate (S1P) receptor agonist. Low nanomolar agonist concentrations selectively induce changes in CD69intCD62Lhighsingle positive T cells, resulting in down-modulation of CD69 within 2 h. While CD69 loss is accelerated, egress of mature T cells into blood is inhibited >95% within 2 h. Both processes exhibit parallel sensitivities and dose–responses. Together, these data reveal a potent means for rapidly regulating thymic export where S1P receptor agonism alters both phenotypic maturation and egress of thymocytes into blood during late thymic maturation. The S1P system is now shown to acutely regulate both thymic and lymph node egress. Inhibition of lymphocyte egress from thymus and lymph node can contribute synergistically to clinically useful immunosupression by disrupting recirculation of peripheral T cells.
Mapping
Drosophila
mutations with molecularly defined
P
element insertions
Authors: Zhai R. Grace , Hiesinger P. Robin , Koh Tong-Wey , Verstreken Patrik , Schulze Karen L. , Cao Yu , Jafar-Nejad Hamed , Norga Koenraad K. , Pan Hongling , Bayat Vafa , Greenbaum Michael P. , Bellen Hugo J.
Published: September 2003

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of
Drosophila
genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped
P
element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals <500 kb. We document the extent of distortions between the recombination and molecular maps and describe the required steps to map with an accuracy of <50 kb. Finally, we describe a recently developed method to determine molecular lesions in 50-kb intervals by using a heteroduplex DNA mutation detection system. Our data show that this mapping approach is inexpensive, efficient, and precise, and that it significantly broadens the application of
P
elements in
Drosophila.
Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187
Authors: Classen Scott , Olland Stephane , Berger James M.
Published: September 2003

Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA. To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5′-[β,γ-imino]-triphosphate-bound yeast topo II ATPase region (
Sc
T2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187. The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences. The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers. Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity.
The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion
Authors: Wilson Michael T. , Johansson Cecilia , Olivares-Villagómez Danyvid , Singh Avneesh K. , Stanic Aleksandar K. , Wang Chyung-Ru , Joyce Sebastian , Wick Mary Jo , Van Kaer Luc
Published: September 2003

CD1d-restricted natural killer T (NKT) cells are a subset of regulatory T cells that react with glycolipid antigens. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the earlyin vivoresponse of these cells to antigenic stimulation. We have analyzed the early response of NKT cells to glycolipid antigens and bacterial infection by using specific reagents for tracking these cells. Our results demonstrate dramaticin vivoexpansion and surface phenotype alterations after NKT cell activation with α-galactosylceramide. In addition, we show significant NK1.1 down-modulation on NKT cells in the setting of oralSalmonellainfection. Our results indicate thatin vivoactivation of NKT cells leads to a dynamic response characterized by surface receptor down-modulation and expansion. These findings alter current understanding of NKT cell biology and should aid in the rational design of NKT cell-based immunotherapies.
GPI transamidase of
Trypanosoma brucei
has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits
Authors: Nagamune Kisaburo , Ohishi Kazuhito , Ashida Hisashi , Hong Yeonchul , Hino Jun , Kangawa Kenji , Inoue Norimitsu , Maeda Yusuke , Kinoshita Taroh
Published: September 2003

Glycosylphosphatidylinositol (GPI) anchor is a membrane attachment mechanism for cell surface proteins widely used in eukaryotes. GPIs are added to proteins posttranslationally by a complex enzyme, GPI transamidase. Previous studies have shown that human and
Saccharomyces cerevisiae
GPI transamidases are similar and consist of five homologous components: GAA1, GPI8, PIG-S, PIG-T, and PIG-U in humans and Gaa1p, Gpi8p, Gpi17p, Gpi16p, and Cdc91p in
S. cerevisiae
. We report that GPI transamidase of
Trypanosoma brucei
(
Tb
), a causative agent of African sleeping sickness, shares only three components (
Tb
GAA1,
Tb
GPI8, and
Tb
GPI16) with humans and
S. cerevisiae
but has two other specific components, trypanosomatid transamidase 1 (TTA1) and TTA2. GPI transamidases of both bloodstream form (growing in mammalian blood) and procyclic form (growing in tsetse fly vector) of the parasite have the same five components. Homologues of TTA1 and TTA2 are present in
Leishmania
and
Trypanosoma cruzi
but not in mammals, yeasts, flies, nematodes, plants, or malaria parasites, suggesting that these components may play unique roles in attachment of GPI anchors in trypanosomatid parasites and provide good targets for antitrypanosome drugs.
Horizontal gene transfer from flowering plants to Gnetum
Authors: Won Hyosig , Renner Susanne S.
Published: September 2003

Although horizontal gene transfer is well documented in microbial genomes, no case has been reported in higher plants. We discovered horizontal transfer of the mitochondrial
nad1
intron 2 and adjacent exons b and c from an asterid to
Gnetum
(Gnetales, gymnosperms).
Gnetum
has two copies of intron 2, a group II intron, that differ in their exons, nucleotide composition, domain lengths, and structural characteristics. One of the copies, limited to an Asian clade of
Gnetum
, is almost identical to the homologous locus in angiosperms, and partial sequences of its exons b and c show characteristic substitutions unique to angiosperms. Analyses of 70 seed plant
nad1
exons b and c and intron 2 sequences, including representatives of all angiosperm clades, support that this copy originated from a euasterid and was horizontally transferred to
Gnetum
. Molecular clock dating, using calibrations provided by gnetalean macrofossils, suggests an age of 5 to 2 million years for the Asian clade that received the horizontal transfer.
A quantitative characterization of the yeast heterotrimeric G protein cycle
Authors: Yi Tau-Mu , Kitano Hiroaki , Simon Melvin I.
Published: September 2003

The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct
in vivo
measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the
in vivo
rates of heterotrimeric G protein activation and deactivation in yeast.
CD4+CD25+T regulatory cells control anti-islet CD8+T cells through TGF-β–TGF-β receptor interactions in type 1 diabetes
Authors: Green E. Allison , Gorelik Leonid , McGregor Catrin M. , Tran Elise H. , Flavell Richard A.
Published: September 2003

Pancreatic lymph node-derived CD4+CD25+T regulatory (Treg) cells inhibitin situdifferentiation of islet-reactive CD8+T cells into cytotoxic T lymphocytes, thereby preventing diabetes progression. The mechanism by which these Treg cells suppress anti-islet CD8+T cells is unknown. Here, we show by using a CD8+T cell-mediated model of type 1 diabetes that transforming growth factor (TGF)-β–TGF-β receptor signals are critical for CD4+CD25+Treg cell regulation of autoreactive islet-specific cytotoxic T lymphocytes. Transgenic expression of tumor necrosis factor α from birth to 25 days of age in the islets of B6 mice that constitutively express CD80 on their β cells results in accumulation of CD4+CD25+TGF-β+cells exclusively in the islets and pancreatic lymph nodes, which delays diabetes progression. In contrast, expression of tumor necrosis factor α until 28 days of age prevents islet accumulation of CD4+CD25+TGF-β+Treg cells, resulting in acceleration to diabetes. Furthermore, adoptive transfer experiments demonstrated that CD4+CD25+Treg cells could not control naïve or activated isletreactive CD8+T cells bearing a dominant negative TGF-β receptor type II. Our data demonstrate that,in vivo, TGF-β signaling in CD8+T cells is critical for CD4+CD25+Treg cell suppression of isletreactive CD8+T cells in type 1 diabetes.
Rho-dependent formation of epithelial “leader” cells during wound healing
Authors: Omelchenko T. , Vasiliev J. M. , Gelfand I. M. , Feder H. H. , Bonder E. M.
Published: September 2003

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special “leader” cells at the tops of outgrowths and “follower” cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell–cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively, transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.
Evidence for an enantioselective pumiliotoxin 7-hydroxylase in dendrobatid poison frogs of the genus
Dendrobates
Authors: Daly John W. , Garraffo H. Martin , Spande Thomas F. , Clark Valerie C. , Ma Jingyuan , Ziffer Herman , Cover John F.
Published: September 2003

Dendrobatid poison frogs readily accumulate alkaloids from diet into skin, where such compounds serve as a chemical defense against predators. Arthropods seem to be the source of decahydroquinolines (DHQs), several izidines, coccinellines, spiropyrrolizidines, pumiliotoxins (PTXs), and allopumiliotoxins (aPTXs). A DHQ iso-223F, and PTX (+)-251D were fed to poison frogs of the dendrobatid genera
Dendrobates, Epipedobates
, and
Phyllobates
. The two alkaloids were accumulated in skin unchanged except for the three species of
Dendrobates
, where ≈80% of accumulated PTX (+)-251D was stereoselectively hydroxylated to aPTX (+)-267A. The unnatural enantiomer PTX (-)-251D was accumulated efficiently when fed to
Dendrobates auratus
, but was not hydroxylated. The enantiomers of PTX 251D and their desmethyl analogs were synthesized from
N
-Boc-protected (-)- and (+)-proline methyl esters. Both PTX (+)-251D and aPTX (+)-267A proved to be potent convulsants in mice, with (+)-267A being ≈5-fold more toxic than (+)-251D. Both alkaloids were hyperalgesic at the site of injection. The unnatural PTX (-)-251D caused no overt effect in mice. Thus, the evolutionary development of a pumiliotoxin 7-hydroxylase would have provided frogs of the genus
Dendrobates
with a means of enhancing the antipredator potency of ingested PTXs.