bluuebunny/crossref_metadata_2025 huggingface dataset:
Neighboring-Nucleotide Effects on Single Nucleotide Polymorphisms: A Study of 2.6 Million Polymorphisms Across the Human Genome
Authors: Zhao Zhongming , Boerwinkle Eric
Published: November 2002

We investigated substitution patterns and neighboring-nucleotide effects for 2,576,903 single nucleotide polymorphisms (SNPs) publicly available through the National Center for Biotechnology Information (NCBI). The proportions of substitutions were A/G, 32.77%; C/T, 32.81%; A/C, 8.98%; G/T, 9.06%; A/T, 7.46%; and C/G, 8.92%. The two nucleotides immediately neighboring the variable site showed major deviation from genome-wide and chromosome-specific expectations, although lesser biases extended as far as 200 bp. On the 5′ side, the biases for A, C, G, and T were 1.43%, 4.91%, −1.70%, and −4.62%, respectively. These biases were −4.44%, −1.59%, 5.05%, and 0.99%, respectively, on the 3′ side. The neighboring-nucleotide patterns for transitions were dominated by the hypermutability effects of CpG dinucleotides. Transitions were more common than transversions, and the probability of a transversion increased with increasing A + T content at the two adjacent sites. Neighboring-nucleotide biases were not consistent among chromosomes, with Chromosomes 19 and 22 standing out as different from the others. These data provide genome-wide information about the effects of neighboring nucleotides on mutational and evolutionary processes giving rise to contemporary patterns of nucleotide occurrence surrounding SNPs.
Genome-wide Co-occurrence of Promoter Elements Reveals a cis-Regulatory Cassette of rRNA Transcription Motifs in Saccharomyces cerevisiae
Authors: Sudarsanam Priya , Pilpel Yitzhak , Church George M.
Published: November 2002

Combinatorial regulation is an important feature of eukaryotic transcription. However, only a limited number of studies have characterized this aspect on a whole-genome level. We have conducted a genome-wide computational survey to identify cis-regulatory motif pairs that co-occur in a significantly high number of promoters in theS. cerevisiae genome. A pair of novel motifs, mRRPE and PAC, co-occur most highly in the genome, primarily in the promoters of genes involved in rRNA transcription and processing. The two motifs show significant positional and orientational bias with mRRPE being closer to the ATG than PAC in most promoters. Two additional rRNA-related motifs, mRRSE3 and mRRSE10, also co-occur with mRRPE and PAC. mRRPE and PAC are the primary determinants of expression profiles while mRRSE3 and mRRSE10 modulate these patterns. We describe a new computational approach for studying the functional significance of the physical locations of promoter elements that combine analyses of genome sequence and microarray data. Applying this methodology to the regulatory cassette containing the four rRNA motifs demonstrates that the relative promoter locations of these elements have a profound effect on the expression patterns of the downstream genes. These findings provide a function for these novel motifs and insight into the mechanism by which they regulate gene expression. The methodology introduced here should prove particularly useful for analyzing transcriptional regulation in more complex genomes.
Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA
Authors: Dunn John J. , McCorkle Sean R. , Praissman Laura A. , Hind Geoffrey , van der Lelie Daniel , Bahou Wadie F. , Gnatenko Dmitri V. , Krause Maureen K.
Published: November 2002

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site forMmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.

[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: W. Crockett, K. Pellechi, J. Paparelli, J. Romeo, and K. Thompson.]
Segmental Polymorphisms in the Proterminal Regions of a Subset of Human Chromosomes
Authors: Der-Sarkissian Hera , Vergnaud Gilles , Borde Yves-Marie , Thomas Gilles , Londoño-Vallejo José-Arturo
Published: November 2002

The subtelomeric domains of chromosomes are probably the most rapidly evolving structures of the human genome. The highly variable distribution of large duplicated subtelomeric segments has indicated that frequent exchanges between nonhomologous chromosomes may have been taking place during recent genome evolution. We have studied the extent and variability of such duplications using in situ hybridization techniques and a set of well-defined subtelomeric cosmid probes that identify discrete regions within the subtelomeric domain. In addition to reciprocal translocation and illegitimate recombination events that could explain the observed mosaic pattern of subtelomeric regions, it is likely that homology-based recombination mechanisms have also contributed to the spread of distal subtelomeric sequences among particular groups of nonhomologous chromosome arms. The frequency and distribution of large-scale subtelomeric polymorphisms may have direct implications for the design of chromosome-specific probes that are aimed at the identification of cryptic subtelomeric deletions. Furthermore, our results indicate that the relevance of some of the telomere closures proposed within the present Human Genome Sequence draft are restricted to specific allelic variants of unknown frequencies.

[The sequence of cosmid ICRF10 (carrying DNF92) was deposited in GenBank under accession no. Y13543.]
In Silico Model-Driven Assessment of the Effects of Single Nucleotide Polymorphisms (SNPs) on Human Red Blood Cell Metabolism
Authors: Jamshidi Neema , Wiback Sharon J. , Palsson Bernhard Ø.
Published: November 2002

The completion of the human genome project and the construction of single nucleotide polymorphism (SNP) maps have lead to significant efforts to find SNPs that can be linked to pathophysiology. In silico models of complete biochemical reaction networks relate a cell's individual reactions to the function of the entire network. Sequence variations can in turn be related to kinetic properties of individual enzymes, thus allowing an in silico model-driven assessment of the effects of defined SNPs on overall cellular functions. This process is applied to defined SNPs in two key enzymes of human red blood cell metabolism: glucose-6-phosphate dehydrogenase and pyruvate kinase. The results demonstrate the utility of in silico models in providing insight into differences between red cell function in patients with chronic and nonchronic anemia. In silico models of complex cellular processes are thus likely to aid in defining and understanding key SNPs in human pathophysiology.
Genomic Structure and Evolution of the Ancestral Chromosome Fusion Site in 2q13–2q14.1 and Paralogous Regions on Other Human Chromosomes
Authors: Fan Yuxin , Linardopoulou Elena , Friedman Cynthia , Williams Eleanor , Trask Barbara J.
Published: November 2002

Human chromosome 2 was formed by the head-to-head fusion of two ancestral chromosomes that remained separate in other primates. Sequences that once resided near the ends of the ancestral chromosomes are now interstitially located in 2q13–2q14.1. Portions of these sequences had duplicated to other locations prior to the fusion. Here we present analyses of the genomic structure and evolutionary history of >600 kb surrounding the fusion site and closely related sequences on other human chromosomes. Sequence blocks that closely flank the inverted arrays of degenerate telomere repeats marking the fusion site are duplicated at many, primarily subtelomeric, locations. In addition, large portions of a 168-kb centromere-proximal block are duplicated at 9pter, 9p11.2, and 9q13, with 98%–99% average sequence identity. A 67-kb block on the distal side of the fusion site is highly homologous to sequences at 22qter. A third ∼100-kb segment is 96% identical to a region in 2q11.2. By integrating data on the extent and similarity of these paralogous blocks, including the presence of phylogenetically informative repetitive elements, with observations of their chromosomal distribution in nonhuman primates, we infer the order of the duplications that led to their current arrangement. Several of these duplicated blocks may be associated with breakpoints of inversions that occurred during primate evolution and of recurrent chromosome rearrangements in humans.

[Supplemental material is available online at http://www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: T. Newman, C. Harris, and J. Young.]
Gene Content and Function of the Ancestral Chromosome Fusion Site in Human Chromosome 2q13–2q14.1 and Paralogous Regions
Authors: Fan Yuxin , Newman Tera , Linardopoulou Elena , Trask Barbara J.
Published: November 2002

Various portions of the region surrounding the site where two ancestral chromosomes fused to form human chromosome 2 are duplicated elsewhere in the human genome, primarily in subtelomeric and pericentromeric locations. At least 24 potentially functional genes and 16 pseudogenes reside in the 614-kb of sequence surrounding the fusion site and paralogous segments on other chromosomes. By comparing the sequences of genomic copies and transcripts, we show that at least 18 of the genes in these paralogous regions are transcriptionally active. Among these genes are new members of the cobalamin synthetase W domain (CBWD) and forkhead domain FOXD4 gene families. Copies of RPL23A and SNRPA1 on chromosome 2 are retrotransposed-processed pseudogenes that were included in segmental duplications; we find 53 RPL23A pseudogenes in the human genome and map the functional copy of SNRPA1 to 15qter. The draft sequence of the human genome also provides new information on the location and intron–exon structure of functional copies of other 2q-fusion genes (PGM5, retina-specific F379, helicaseCHLR1, and acrosin). This study illustrates that the duplication and rearrangement of subtelomeric and pericentromeric regions have functional relevance to human biology; these processes can change gene dosage and/or generate genes with new functions.

[Supplemental material is available online athttp://www.genome.org. Sequence data reported in this paper have been deposited in GenBank and assigned the following accession nos.:AF452722, AF452723, and AF452724.]
Gene Expression Analysis Using Oligonucleotide Arrays Produced by Maskless Photolithography
Authors: Nuwaysir Emile F. , Huang Wei , Albert Thomas J. , Singh Jaz , Nuwaysir Kate , Pitas Alan , Richmond Todd , Gorski Tom , Berg James P. , Ballin Jeff , McCormick Mark , Norton Jason , Pollock Tim , Sumwalt Terry , Butcher Lawrence , Porter DeAnn , Molla Michael , Hall Christine , Blattner Fred , Sussman Michael R. , Wallace Rodney L. , Cerrina Franco , Green Roland D.
Published: November 2002

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized toDrosophila melanogasterand mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.

[The sequence data from this study have been submitted to GEO under accession nos. GPL208, GSM2409, GSM2410, GSM2411, GSM2412, GSM2413, GSM2414, GSE81, GSE82.]
The Automatic Detection of Homologous Regions (ADHoRe) and Its Application to Microcolinearity Between Arabidopsis and Rice
Authors: Vandepoele Klaas , Saeys Yvan , Simillion Cedric , Raes Jeroen , Van de Peer Yves
Published: November 2002

It is expected that one of the merits of comparative genomics lies in the transfer of structural and functional information from one genome to another. This is based on the observation that, although the number of chromosomal rearrangements that occur in genomes is extensive, different species still exhibit a certain degree of conservation regarding gene content and gene order. It is in this respect that we have developed a new software tool for the Automatic Detection of Homologous Regions (ADHoRe). ADHoRe was primarily developed to find large regions of microcolinearity, taking into account different types of microrearrangements such as tandem duplications, gene loss and translocations, and inversions. Such rearrangements often complicate the detection of colinearity, in particular when comparing more anciently diverged species. Application of ADHoRe to the complete genome of Arabidopsis and a large collection of concatenated rice BACs yields more than 20 regions showing statistically significant microcolinearity between both plant species. These regions comprise from 4 up to 11 conserved homologous gene pairs. We predict the number of homologous regions and the extent of microcolinearity to increase significantly once better annotations of the rice genome become available.
Signatures of Domain Shuffling in the Human Genome
Authors: Kaessmann Henrik , Zöllner Sebastian , Nekrutenko Anton , Li Wen-Hsiung
Published: November 2002

To elucidate the role of exon shuffling in shaping the complexity of the human genome/proteome, we have systematically analyzed intron phase distributions in the coding sequence of human protein domains. We found that introns at the boundaries of domains show high excess of symmetrical phase combinations (i.e., 0–0, 1–1, and 2–2), whereas nonboundary introns show no excess symmetry. This suggests that exon shuffling has primarily involved rearrangement of structural and functional domains as a whole. Furthermore, we found that domains flanked by phase 1 introns have dramatically expanded in the human genome due to domain shuffling and that 1–1 symmetrical domains and domain families are nonrandomly distributed with respect to their age. The predominance and extracellular location of 1–1 symmetrical domains among domains specific to metazoans suggests that they are associated with the rise of multicellularity. On the other hand, 0–0 symmetrical domains tend to be over-represented among ancient protein domains that are shared between the eukaryotic and prokaryotic kingdoms, which is compatible with the suggestion of primordial domain shuffling in the progenote. To see whether the human data reflect general genomic patterns of metazoans, similar analyses were done for the nematodeCaenorhabditis elegans. Although the C. elegans data generally concur with the human patterns, we identified fewer intron-bounded domains in this organism, consistent with the lower complexity of C. elegans genes.

[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: Z. Gu and R. Stevens.]
Early Gene Expression Associated with the Commitment and Differentiation of a Plant Tracheary Element Is Revealed by cDNA–Amplified Fragment Length Polymorphism Analysis[W]
Authors: Milioni Dimitra , Sado Pierre-Etienne , Stacey Nicola J. , Roberts Keith , McCann Maureen C.
Published: November 2002

Isolated mesophyll cells from Zinnia elegans are induced by auxin and cytokinin to form tracheary elements (TEs) in vitro with high synchrony. To reveal the changing patterns of gene expression during the 48 h of transdifferentiation from meso-phyll to TE cell fate, we used a cDNA–amplified fragment length polymorphism approach to generate expression profiles of >30,000 cDNA fragments. Transcriptional changes of 652 cDNA fragments were observed, of which 304 have no previously described function or sequence identity. Sixty-eight genes were upregulated within 30 min of induction and represent key candidates for the processes that underlie the early stages of commitment and differentiation to a TE cell fate.
TheChlamydomonas reinhardtiiPlastid Chromosome
Authors: Maul Jude E. , Lilly Jason W. , Cui Liying , dePamphilis Claude W. , Miller Webb , Harris Elizabeth H. , Stern David B.
Published: November 2002

Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of ∼80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.
Do Cardiac Neurons Play a Role in the Intrinsic Control of Heart Rate in the Rat?
Authors: Wilson Suzanne J. , Bolter Chris P.
Published: November 2002

Three experiments were performed to see whether cardiac neurons contribute to the intrinsic control of heart rate in right atria of adult rats. The intrinsic heart rate response (IRR) was examined by raising right atrial pressure from 2 to 8 mmHg for 3 min. In isolated preparations of the right atrium, the IRR was not significantly altered by the addition of either 1 μM atropine (n = 6; control +19 ± 3 min−1; atropine +18 ± 3 min−1 (mean ± S.E.M.)) or 1 μM propranolol (n = 5; control +22 ± 4 min−1; propranolol +21 ± 3 min−1). Tetrodotoxin (0.5 μM) had no effect on the IRR (n = 6; control +37 ± 5 min−1; tetrodotoxin 38 ± 5 min−1). In another experiment, 2‐day‐old rat pups were injected with capsaicin (50 mg kg−1; treated) or with vehicle (control). There was no difference in the IRR of right atrial preparations taken from control and treated animals after they reached adulthood (control (n = 7) and treated (n = 8): +30 ± 4 and +32 ± 4 min−1). The influence of right atrial pressure on the efficacy of vagal stimulation was examined. The rate response to vagal stimulation was reduced similarly in control and treated preparations when pressure was elevated from 2 to 4 mmHg (control and treated: ‐34 ± 5% and ‐33 ± 3%). The effectiveness of the capsaicin treatment was confirmed by the depletion of substance P‐immunoreactive nerve fibres in cardiac tissues. Together, these results strongly suggest that cardiac neurons are not involved in intrinsic heart rate control.
Neuropeptides, Amines and Amino Acids as Mediators of the Sympathetic Effects of Paraventricular Nucleus Activation in the Rat
Authors: Yang Zhuo , Wheatley Mark , Coote John H.
Published: November 2002

The aim of the present study was to determine the influence on renal sympathetic nerve activity of the different chemically coded neuronal phenotypes that project from the paraventricular nucleus (PVN) to the spinal cord. Experiments were carried out on male Wistar rats anaesthetised with chloralose and urethane. Changes in renal sympathetic nerve activity were measured following activation of neurones in the PVN with D,L‐homocysteic acid (100 nl, 200 mM), before and following intrathecal application of glutamate, vasopressin, oxytocin, dopamine and their receptor antagonists. Excitatory and inhibitory effects on renal sympathetic nerve activity were elicited by PVN stimulation. PVN excitatory effects were mimicked by intrathecal administration of glutamate and vasopressin and selectively antagonised by intrathecal administration of kynurenic acid and a V1a receptor antagonist, respectively. A low dose of dopamine increased renal sympathetic activity and this was selectively antagonised by haloperidol; however, the latter was without effect on PVN excitatory responses. A high dose of dopamine decreased renal sympathetic nerve activity and this was selectively blocked by a D1 dopamine receptor antagonist (SCH 23390), which also antagonised a minority of inhibitory responses obtained from the caudal extension of the PVN. Oxytocin also had two actions: in 5 rats it inhibited and in 10 rats it increased renal sympathetic nerve activity, both actions being blocked selectively by oxytocin receptor antagonists. Neither of the PVN effects on renal sympathetic nerve activity appeared to be dependent on oxytocin pathways. Tests with intrathecal administration of bicuculline showed that PVN inhibition of renal sympathetic nerve activity was not dependent on spinal GABAA receptor activation. The results show that PVN‐induced excitation of sympathetic activity to the kidney is mainly mediated by glutamate or vasopressin neurones whereas dopamine via D1 receptors may mediate some of the PVN inhibitory effects.
Multiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp‐Stimulated Rat Brown Adipocytes
Authors: Omatsu‐Kanbe Mariko , Isono Takahiro , Matsuura Hiroshi
Published: November 2002

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca2+ concentration ([Ca2+]i), which arises primarily from a release of Ca2+ from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca2+]i elevation during exposure to ATP by using fura‐2 fluorescence measurements together with the P2 receptor antagonists pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) and suramin. Extracellular ATP (10 μM) almost completely depressed the thapsigargin (100 nM)‐evoked [Ca2+]i elevation mediated through store‐operated Ca2+ entry. The inhibitory effect of ATP was antagonized by PPADS with IC50 of 0.7 μM. In the presence of PPADS at concentrations of more than 5 μM, the ATP‐induced [Ca2+]i elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca2+]i elevation was reduced in a concentration‐dependent manner by PPADS with an IC50 of 200 μM. In contrast, the ATP‐induced [Ca2+]i elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store‐operated pathway. These results suggest that the [Ca2+]i responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS‐resistant P2 receptor to mobilize intracellular Ca2+ stores, which is probably followed by the activation of store‐operated Ca2+ entry. Extracellular ATP, however, would inhibit this Ca2+ entry process through the stimulation of the PPADS‐sensitive P2‐receptor, which may underlie the transient nature of [Ca2+]i elevation in response to extracellular ATP.
Calcium Dynamics in Spines: Link to Synaptic Plasticity
Authors: Holthoff Knut , Tsay David
Published: November 2002

Dendritic spines are morphologically and functionally heterogeneous. Here, we use two‐photon imaging of layer V pyramidal neurons in slices from mouse visual cortex to characterize differences in spine calcium dynamics between individual spines. By measuring action potential‐evoked [Ca2+]i transients in spines, we find different calcium dynamics in spines from proximal apical and distal apical dendrites. Using a mathematical multi‐compartmental model, we demonstrate that these differences are even more pronounced in the absence of exogenous calcium buffers. We also find that these different calcium dynamics cause different susceptibility to synaptic depression in proximal and distal apical synapses, and that modifying calcium decay kinetics in spines changes the expression of long‐term depression. We conclude that the location of the spine determines its time window of calcium compartmentalization and degree of calcium‐dependent synaptic plasticity. Our results highlight the precision of the design of neocortical neurons.
Advantages and Risks of Multiphoton Microscopy in Physiology
Authors: Tauer Ulrike
Published: November 2002

Multiphoton microscopy is based on the simultaneous absorption of two photons emitted by a pulsed infrared laser source. In this technique, the excitation is restricted to a very small focus and thus results in optical sectioning a priori without the need of a confocal aperture. Multiphoton microscopy was introduced in live cell imaging as an alternative to confocal microscopy due to its superior qualities, such as the deep penetration depth, the reduced photodamage and the lack of out‐of‐focus bleaching. However, during the past years, examinations revealed severe limitations to the initial expectations. In the focal plane, photodamage and photobleaching can be worse than in single photon microscopy. However, studies showed that with low excitation intensity and by special technical adaptations photodamage could be avoided successfully. For functional biological imaging, multiphoton excitation provides an excellent tool such as the release of caged compounds in a diffraction‐limited volume combined with multiphoton or confocal imaging.
Protein Kinase C Regulation of Rat Jejunal Transport Systems: Mechanisms Involved in Lactate Movement
Authors: Tosco Marisa , Orsenigo Maria Novella , Gastaldi Giulia , Faelli Alide
Published: November 2002

We examined whether protein kinase C (PKC) modulates the transport systems involved in lactate movements across the plasma membranes of rat jejunum. In vitro phosphorylated membrane vesicles were used to perform uptake studies, the results of which suggested that PKC activation exerts an inhibitory effect on basolateral H+‐lactate symport, as well as on apical Na+‐glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4‐α‐PMA. Experiments performed using the whole tissue incubated in vitro confirmed the reduction of lactate transport elicited by PKC and gave evidence for an associated inhibition of fluid transport. Na+,K+‐ATPase activity seems to be unaffected by the kinase and inhibited by Ca2+. Taken together, our results suggest that the overall action of PKC results from the simultananeous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis.
Combined Confocal Microscopy and Stereology: a Highly Efficient and Unbiased Approach to Quantitative Structural Measurement in Tissues
Authors: Howell Katherine , Hopkins Natalie , McLoughlin Paul
Published: November 2002

Understanding the relationship of the structure of organs to their function is a key component of integrative physiological research. The structure of the organs of the body is not constant but changes, both during growth and development and under conditions of sustained stress (e.g. high altitude exposure and disease). Recently, powerful new techniques have become available in molecular biology, which promise to provide novel insights into the mechanisms and consequences of these altered structure‐function relationships. Conventionally structure‐function relationships are studied by microscopic examination of tissue sections. However, drawing conclusions about the three‐dimensional structure of an organ based on this two‐dimensional information frequently leads to serious errors. The techniques of stereology allow precise and accurate quantification of structural features within three‐dimensional organs that relate in a meaningful way to integrated function. For example, knowledge of changes in the total surface area of the capillary endothelium in an organ can be related directly to changes in fluid filtration and permeability, or knowledge of total vessel length and mean radius allows deductions about vascular resistance. Confocal microscopy adds enormously to the power of stereological approaches. It reduces the difficulties and labour involved in obtaining suitable images. Moreover, when used in conjunction with new analytical software, it allows convenient application of stereology to small samples and those in which it is essential to maintain a specific orientation for interpretation. The information obtained will allow us to examine in a quantitative manner the altered structure‐function relationships produced by manipulation of single genes and regulatory pathways in whole organisms.
Miniaturization of Fluorescence Microscopes Using Fibre Optics
Authors: Helmchen Fritjof
Published: November 2002

In both medical research and diagnostics characterization of biological tissue on the cellular level relies on high‐resolution optical microscopy. In most cases, however, tissue is excised for microscopic investigation, in part because conventional microscopes are bulky instruments. Imaging of cells in the intact living organism has been difficult. Over the last decade several groups have developed miniature confocal microscopes that use fibre optics to deliver light to the specimen and to measure either reflected or excited fluorescence light. In addition, two‐photon excitation recently has been employed in a small portable ‘fibrescope’. A potential clinical application of these microscope probes is their endoscopic use for optical biopsy of inner organs or guidance of conventional biopsy. As a mobile research tool they may permit imaging of neuronal activity in the brain of awake, behaving animals. Here, we review technological approaches to build miniaturized fluorescence microscopes and discuss their potential applications.
Dynamic Confocal Imaging in Acute Brain Slices and Organotypic Slice Cultures Using a Spectral Confocal Microscope with Single Photon Excitation
Authors: Kasparov S. , Teschemacher A. G. , Paton J. F. R.
Published: November 2002

Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca2+ concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca2+, cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane‐permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (∼≤50 μm), while multiphoton excitation has a much greater depth of penetration. However, the overall optical resolution achievable in single photon mode is at least as good as when using multiphoton excitation.
Functional Imaging of Single Synapses in Brain Slices
Authors: Oertner Thomas G.
Published: November 2002

The strength of synaptic connections in the brain is not fixed, but can be modulated by numerous mechanisms. Traditionally, electrophysiology has been used to characterize connections between neurons. Electrophysiology typically reports the activity of populations of synapses, while most mechanisms of plasticity are thought to operate at the level of single synapses. Recently, two‐photon laser scanning microscopy has enabled us to perform optical quantal analysis of individual synapses in intact brain tissue. Here we introduce the basic principle of the two‐photon microscope and discuss its main differences compared to the confocal microscope. Using calcium imaging in dendritic spines as an example, we explain the advantages of simultaneous dual‐dye imaging for quantitative calcium measurements and address two common problems, dye saturation and background fluorescence subtraction.
Dynamic Confocal Imaging of Living Brain
Authors: Paton Julian F. R. , Kasparov Sergey
Published: November 2002

Introduction

This special issue contains an exciting series of papers that are based on presentations made at a workshop entitled ‘Dynamic Confocal Imaging of Living Brain’ which was held on the 30th June 2001 at the University of Bristol. The paper by Howell et al. was not presented at this symposium but is included to further portray the versatility of confocal imaging.

As the saying goes: ‘Seeing is believing’ and in this day and age imaging of the brain has become an important modern research tool. With the recent technological advances in single and multi‐photon microscopy, structures beneath the surface of the brain can be imaged with sub‐micrometre resolution to further enhance our understanding of inter‐ and intra‐cellular signalling. With the development and employment of calcium indicators and naturally occurring fluorescent proteins the field has advanced dramatically into imaging of living processes. The following papers encapsulate the types of research that can now be performed with confocal microscopes for imaging dynamic events in living brain both in vitro and in vivo.

We are grateful to the Physiological Society, Leica Microsystems and Molecular Probes for their financial support.
Impact of differential energy allocation in Atlantic salmon (Salmo salar) precocious males on otolithsomatic size proportionality: a longitudinal approach
Authors: Aubin-Horth Nadia , Dodson Julian J
Published: November 2002

We studied juvenile Atlantic salmon (Salmo salar) males that become precociously mature or not at age-1+ to test the hypothesis that differential energy allocation affects the relationship between otolith size and fish size and to validate the use of a back-calculation method to estimate size over 30 weeks. We used a longitudinal approach by repeatedly measuring marked fish and obtaining corresponding otolith radius measurements. Differential energy allocation of mature males did not affect the proportionality ratio between otolith and somatic size. Short-term otolith growth varied with short-term somatic growth, but only weakly with temperature. Some correlation coefficients of the covariation of otolith growth estimated over a longer time interval with somatic growth were significantly greater than the short-term estimate. For mature and immature males, back-calculated lengths accurately estimated the observed individual length on practically all occasions. These results indicate that back-calculation can be used to estimate size for Atlantic salmon with different energy allocation patterns. Variable strength of coupling of otolith and somatic growth depending on time interval suggests that these processes are completed on different time scales.
Methyl mercury in zooplanktonthe role of size, habitat, and food quality
Authors: Kainz Martin , Lucotte Marc , Parrish Christopher C
Published: November 2002

Pathways of methyl mercury (MeHg) accumulation in zooplankton include ingestion of organic matter (OM). We analyzed fatty acid (FA) biomarkers in zooplankton to (i) investigate the effect of allochthonous and autochthonous OM ingestion on MeHg concentrations ([MeHg]) in zooplankton and (ii) examine how algal and bacterial food sources affect MeHg bioaccumulation. We partitioned bulk zooplankton samples (i.e., >500, 202, 100, and 53 μm) from Lake Lusignan (Québec) and measured [MeHg] and [FA] in each fraction. [MeHg] increased with increasing body size and was significantly higher in pelagic than in littoral macrozooplankton (>500 μm). The amount of the ingested terrestrial FA biomarker 24:0 indicated that less than 1% of the total FA in zooplankton was derived from allochthonous sources. More than 60% of the ingested FA originated from algal biomarkers and <10% from bacterial biomarkers. Relative amounts of algal-derived essential FA and bacterial FA were not associated with [MeHg] in any size fraction. In pelagic zones, the amount of MeHg in zooplankton related positively to the number of large organisms such as Calanoid copepods and Daphnia. We propose that the accumulation of MeHg in lacustrine zooplankton depends on the zooplankton habitat rather than on the quality of ingested food.
Pacific salmon (Oncorhynchus spp.) early marine feeding patterns based on 15N/14N and 13C/12C in Prince William Sound, Alaska
Authors: Kline, Jr. Thomas C , Willette T Mark
Published: November 2002

Nitrogen and carbon mass and stable isotope composition among cohorts of Pacific salmon (Oncorhynchus spp.) released from Prince William Sound, Alaska, hatcheries in 1994 varied widely, suggesting a range in early marine feeding patterns. Analyses consisted of whole-body stable carbon and nitrogen mass and stable isotope composition of selected release-date cohorts that had been identified by implanted coded wire tags (CWT). Nitrogen isotopic and mass shifts suggested that the initial protein pool within individual fish was replaced at different rates among cohorts. There was a notable difference in carbon source dependency among hatcheries. Salmon from the hatchery closest to the Gulf of Alaska had a 13C-depleted carbon signature consistent with Gulf carbon, whereas salmon from the other hatcheries had Sound signatures. Differences in early marine feeding histories among 1994 hatchery-release-date cohorts reconstructed from the stable isotope composition of fry bore no relationship to marine survival pattern. Varied survival rates of 1994 Prince William Sound hatchery salmon were more likely related to the fry size at time of release, the observed differences in growth rate among release cohorts, and predation refuge effects of pen-rearing.
Molecular pedigree analysis in natural populations of fishes: approaches, applications, and practical considerations
Authors: Wilson A J , Ferguson M M
Published: November 2002

Molecular markers can provide information on the family structure of natural fish populations through molecular pedigree analysis. This information, which is otherwise difficult to obtain, can give important insights into the expression and evolution of phenotypic traits. We review the literature to provide examples of how molecular pedigree analysis has been used extensively to examine patterns of distribution, dispersal, and social behaviour in fishes and how it provides a tool for the estimation of quantitative genetic parameters. Although multiple methodologies can be used to examine family structure, the efficacy of any molecular pedigree analysis is generally dependent on prior consideration of interrelated statistical and biological factors. Statistical issues stem from the choice of molecular marker type and marker set used, in addition to sampling strategy. We discuss these considerations and additionally emphasize the utility of supplemental nongenetic data for increasing the efficacy of pedigree analysis. We advocate that, where possible, a priori knowledge of the study system's biology should be used to inform study design and further highlight the need for additional empirical testing of methodologies.
Influence of food web structure on the growth and bioenergetics of lake trout (Salvelinus namaycush)
Authors: Pazzia Ivano , Trudel Marc , Ridgway Mark , Rasmussen Joseph B
Published: November 2002

In this study, we compared energy budgets of lake trout (Salvelinus namaycush) populations in contrasting food webs. Nonpiscivorous lake trout (NPLT) reached a much smaller size and grew at a much slower rate than piscivorous lake trout (PLT) populations. Food consumption rates were, on average, 23 times higher in NPLT when they were expressed on a wet weight basis. However, only a slight (less than 10%) difference in their energy intake was detected once consumption rates were corrected for differences in prey caloric content. Growth efficiency was approximately two times lower in NPLT compared with PLT, while their metabolic costs were higher and their assimilation efficiency was lower. It is most likely that the increased metabolic costs were associated with higher foraging costs, since more feeding attempts must be made to acquire a given quantity of food when fish are feeding on smaller prey. Furthermore, the portion of indigestible matter is likely to be higher in the diet of NPLT than in PLT (i.e., chitin vs. bone). These results are consistent with theoretical models of fish growth that show that lake trout must have access to larger prey, even if they are rare, to reach larger body sizes.
Ice storm impacts on woody debris and debris dam formation in northeastern U.S. streams
Authors: Kraft Clifford E , Schneider Rebecca L , Warren Dana R
Published: November 2002

In January 1998, an ice storm damaged forests in northeastern United States and eastern Canada, causing coarse woody debris (CWD) deposition in riparian areas and associated streams. During 1999 and 2000, tree canopy damage, stream physical habitat, and wood deposition were evaluated within 51 first-, second-, and third-order streams located within five eastern Adirondack Mountain watersheds (New York, U.S.A.). In first- through third-order streams, the number and volume of stream debris dams increased in response to streamside trees with canopy damage. Tree canopy damage was not a significant predictor for individual pieces of stream CWD but was correlated with CWD >10 cm in diameter in third-order, but not first-order, streams. At debris dam locations, bankfull width was greater and stream substrates consisted of increased fines. Woody debris resulting from the 1998 ice storm was not associated with increased pool formation; instead, boulders and rocky substrate were the dominant pool-forming elements. CWD length in first-order streams generally exceeded bankfull width, but in third-order streams, CWD length was shorter than bankfull width and therefore was subject to greater transport and accumulation into debris dams. Our results indicate that ice storm disturbances can increase wood inputs to first- through third-order forested stream ecosystems.
Distinguishing between contaminant and reef effects on meiofauna near offshore hydrocarbon platforms in the Gulf of Mexico
Authors: Montagna Paul A , Jarvis Stephen C , Kennicutt, II Mahlon C
Published: November 2002

Offshore hydrocarbon production effects are localized in the Gulf of Mexico extending only 200 m from platforms. Effects are caused by the contaminant gradient or the fouling community on platform legs. To distinguish between contaminant and reef effects, meiofauna were sampled at production platforms (reef and contaminant effects), artificial reefs (reef, but no contaminant effects), platform removal sites (contaminant, but no reef effects), and controls (no contaminants or reef effects), and replicated in three blocks. The removal sites had higher concentrations of many contaminants than reef or control sites, but lower concentrations than platform sites. Reduced meiobenthic abundances and altered Harpacticoida community structure were primarily a function of reef effects, not contaminant effects. The habitat influence is likely a result of complex ecological interactions near platforms. The reef effect appears to be important in controlling meiofauna near platforms where contaminants are low because of drilling and production techniques used in the Gulf of Mexico. The finding that contaminants alone do not explain faunal patterns around offshore platforms indicates that future studies should include appropriate artificial reef control sites.
Evidence of recovery from phosphorus enrichment in Lake Michigan
Authors: Barbiero Richard P , Tuchman Marc L , Warren Glenn J , Rockwell David C
Published: November 2002

During the last century, diatom production responses to increased phosphorus loading in Lake Michigan resulted in increased silica sedimentation losses and a consequent decline in the silica content of the lake. In light of recent reductions in phosphorus loading to the lake, we examined long-term monitoring data to determine if this trend might be reversing. Spring total phosphorus concentrations, although highly variable, declined approximately 1 μg·L1 throughout the lake between 1983 and 2000. Spring soluble silica concentrations, an indicator of total in-lake reserves, increased from 1.1 to 1.6 mg·L1 during this period. Summer epilimnetic silica concentrations in the southern basin were at apparently limiting levels of approximately 0.15 mg·L1 until 1991 and then increased sharply. A similar trend was seen in the northern basin. Summer diatom populations increased in both basins after 1991, and ordination analyses suggest that the species composition of both spring and summer communities has responded to these silica increases. These results document for the first time a reversal of the silica depletion sequence and provide the most compelling evidence to date that phosphorus load reductions are having an impact on the Lake Michigan ecosystem.