bluuebunny/crossref_metadata_2025 huggingface dataset:
Trans
-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: A possible mechanism for lipid peroxidation-induced carcinogenesis
Authors: Feng Zhaohui , Hu Wenwei , Tang Moon-shong
Published: June 2004

Lipid peroxidation (LPO) is a cellular process that commonly takes place under normal physiological conditions. Under excessive oxidative stress, the level of LPO becomes very significant, and a growing body of evidence has shown that excessive LPO may be involved in carcinogenesis.
Trans
-4-hydroxy-2-nonenal (4-HNE) is a major product of LPO, and its level becomes relatively high in cells under oxidative stress. 4-HNE is able to react readily with various cellular components, including DNA and proteins. We previously found that the 4-HNE-DNA adduct is a potent mutagen in human cells and is preferentially formed at codon 249 of the
p53
gene, a mutational hotspot in human cancers. To further understand the role of 4-HNE in carcinogenesis, we addressed the question of whether 4-HNE affects DNA repair in human cells. We found that the repair capacity for benzo[
a
]pyrene diol epoxide and UV light-induced DNA damage was greatly compromised in human cells or human cell extracts treated with 4-HNE, which is mainly through interaction of 4-HNE with cellular repair proteins. We also found that 4-HNE greatly sensitizes cells to benzo[
a
]pyrene diol epoxide- and UV-induced killing. Together these results strongly suggest that this LPO metabolite damages not only DNA but also DNA repair mechanisms in human cells. We propose that these two detrimental effects of LPO may contribute synergistically to human carcinogenesis.
Chemical genetics to identify NFAT inhibitors: Potential of targeting calcium mobilization in immunosuppression
Authors: Venkatesh Natarajan , Feng Yan , DeDecker Brian , Yacono Patrick , Golan David , Mitchison Timothy , McKeon Frank
Published: June 2004

The development of more selective immunosuppressive agents to mitigate transplant rejection and autoimmune diseases requires effective strategies of blocking signaling pathways in T cells. Current immunosuppressive strategies use cyclosporin A (CsA) or FK506 to inhibit calcineurin, which dephosphorylates and promotes the nuclear import of nuclear factor of activated T cells (NFAT) transcription factors. These nuclear NFATs then transactivate cytokine genes that regulate proliferative responses of T cells. Both CsA and FK506 have debilitating side effects, including nephrotoxicity, hypertension, diabetes, and seizures, that argue for the development of alternative or complementary agents. To this end, we developed cell-based assays for monitoring NFAT dynamics in nonlymphoid cells to identify small molecules that inhibit NFAT nuclear import. Interestingly, we found that the majority of these small molecules suppress NFAT signaling by interfering with “capacitative” or “store-operated” calcium mobilization, thus raising the possibility that such mobilization processes are relevant targets in immunosuppression therapy. Further, these small molecules also show dose-dependent suppression of cytokine gene expression in T cells. Significantly, the IC
50
of CsA in primary T cells was reduced by the addition of suboptimal concentrations of these compounds, suggesting the possibility that such small molecules, in combination with CsA, offer safer means of immunosuppression.
Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson–Gilford progeria syndrome
Authors: Goldman Robert D. , Shumaker Dale K. , Erdos Michael R. , Eriksson Maria , Goldman Anne E. , Gordon Leslie B. , Gruenbaum Yosef , Khuon Satya , Mendez Melissa , Varga Renée , Collins Francis S.
Published: June 2004

Hutchinson–Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LAΔ50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LAΔ50. Introduction of LAΔ50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LAΔ50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.
Polarized type 1 cytokine response and cell-mediated immunity determine genetic resistance to mousepox
Authors: Chaudhri Geeta , Panchanathan Vijay , Buller R. Mark L. , van den Eertwegh Alfons J. M. , Claassen Eric , Zhou Jie , de Chazal Rosalind , Laman Jon D. , Karupiah Gunasegaran
Published: June 2004

Ectromelia virus (ECTV), a natural mouse pathogen and an orthopoxvirus, has been used to investigate the correlation between polarized type 1 or type 2 immune responses and resistance to disease in poxvirus infections by using well defined resistant and susceptible mouse strains. Our data show that distinct differences exist in the cytokine profiles expressed in resistant and susceptible mice infected with ECTV. Resistant C57BL/6 mice generate a type 1 cytokine response [IFN-γ, IL-2, and tumor necrosis factor (TNF)], within the first few days of infection, which is associated with strong cytotoxic T lymphocyte response (CTL) and recovery from ECTV infection. Susceptible strains of mice (BALB/c and A/J) on the other hand generate a type 2 cytokine response (IL-4 but little or no IFN-γ and IL-2), which is associated with a weak or an absent CTL response, resulting in uncontrolled virus replication and death. Although deletion of IL-4 function alone did not change the outcome of infection in susceptible mice, the loss of IFN-γ function in resistant mice abrogated natural killer (NK) cell and CTL effector functions resulting in fulminant disease and 100% mortality. Therefore, a clear link exists between the early production of specific type 1 cytokines, in particular, IFN-γ, the nature of the cellular immune response, and disease outcome in this virus model. This finding in the mousepox model raises the possibility that inappropriate cytokine responses may result in increased susceptibility to smallpox in humans.
Interplay between
Drosophila
Bloom's syndrome helicase and Ku autoantigen during nonhomologous end joining repair of
P
element-induced DNA breaks
Authors: Min Bosun , Weinert Brian T. , Rio Donald C.
Published: June 2004

P
transposable elements in
Drosophila
are mobilized via a cut-and-paste mechanism. The broken DNA ends generated during transposition can be repaired via the homology-directed synthesis-dependent strand annealing or by nonhomologous end joining (NHEJ). Genetic studies have demonstrated an interaction between the gene (
mus309
, for mutagen-sensitive) encoding the
Drosophila
Bloom's syndrome helicase homolog (DmBLM) and the
Ku70
gene, which is involved in NHEJ. We have used RNA interference (RNAi) to knock down expression of DmBLM and one or both of the
Drosophila
Ku subunits, DmKu70 or DmKu80. Our results show that upon reduction of DmKu, an increase in small deletions (1–49 bp) and large deletions (≥50 bp) flanking the site of
P
element-induced breaks is observed, and a reduction in large deletions at these sites is found upon reduction of DmBLM. Moreover, double RNAi of DmKu and DmBLM results in an increase in small deletions characteristic of the DmKu RNAi and also partially suppresses the reduction in repair efficiency observed with DmKu RNAi. These results suggest that there are DNA double-strand break recognition and/or processing events involving DmKu and DmBLM that, when eliminated by RNAi, lead to deletions. Finally, these results raise the possibility that, unlike the situation in mammals, where BLM appears to function exclusively in the homologous repair pathway, in
Drosophila
, DmBLM may be directly involved in, or at least influence the double-strand break recognition that leads to the NHEJ repair pathway.
Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding
Authors: Risal Dipesh , Gourinath S. , Himmel Daniel M. , Szent-Györgyi Andrew G. , Cohen Carolyn
Published: June 2004

Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-Å x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm =45° to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called “front door.” In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-Å nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.
Three-dimensional phase-encoded chemical shift MRI in the presence of inhomogeneous fields
Authors: Demas Vasiliki , Sakellariou Dimitris , Meriles Carlos A. , Han Songi , Reimer Jeffrey , Pines Alexander
Published: June 2004

A pulse sequence consisting of an excitation pulse and two adiabatic full-passage pulses with scaled relative peak amplitudes is combined with phase encoding to recover chemical shift information within 3D images in a 1D inhomogeneous static magnetic field with a matched
rf
field gradient. The results are discussed in the context of
ex situ
magnetic resonance and imaging. The future directions of our research in implementing the
ex situ
technique in a real one-sided system are also discussed.
Silencing of a gene adjacent to the breakpoint of a widespread
Drosophila
inversion by a transposon-induced antisense RNA
Authors: Puig Marta , Cáceres Mario , Ruiz Alfredo
Published: June 2004

Adaptive changes in nature occur by a variety of mechanisms, and
Drosophila
chromosomal inversions was one of the first studied examples. However, the precise genetic causes of the adaptive value of inversions remain uncertain. Here we investigate the impact of the widespread inversion
2j
of
Drosophila buzzatii
on the expression of the
CG13617
gene, whose coding region is located only 12 bp away from the inversion proximal breakpoint. This gene is transcribed into a 2.3-kb mRNA present in all
D. buzzatii
developmental stages. More importantly, the expression level of
CG13617
is reduced 5-fold in embryos of lines homozygous for the
2j
inversion compared with lines without the inversion. An antisense RNA that originates in the
Foldback
-like transposon
Kepler
inserted at the breakpoint junction in all of the
2j
lines and that forms duplexes with the
CG13617
mRNA in
2j
embryos is most likely responsible for the near silencing of the gene. Few examples of RNA interference caused by transposable elements (TEs) have been previously described, but this mechanism might be prevalent in many organisms and illustrates the potential of TEs as a major source of genetic variation. In addition, because chromosomal rearrangements are usually induced by TEs, position effects might be more common than previously recognized and contribute significantly to the evolutionary success of inversions.
Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in
Saccharomyces cerevisiae
Authors: Smith Stephanie , Hwang Ji-Young , Banerjee Soma , Majeed Anju , Gupta Amitabha , Myung Kyungjaem
Published: June 2004

Different types of gross chromosomal rearrangements (GCRs), including translocations, interstitial deletions, terminal deletions with
de novo
telomere additions, and chromosome fusions, are observed in many cancers. Multiple pathways, such as S-phase checkpoints, DNA replication, recombination, chromatin remodeling, and telomere maintenance that suppress GCRs have been identified. To experimentally expand our knowledge of other pathway(s) that suppress GCRs, we developed a generally applicable genome-wide screening method. In this screen, we identified 10 genes (
ALO1, CDC50, CSM2, ELG1, ESC1, MMS4, RAD5, RAD18, TSA1
, and
UFO1
) that encode proteins functioning in the suppression of GCRs. Moreover, the breakpoint junctions of GCRs from these GCR mutator mutants were determined with modified breakpoint-mapping methods. We also identified nine genes (
AKR1, BFR1, HTZ1, IES6, NPL6, RPL13B, RPL27A, RPL35A
, and
SHU2
) whose mutations generated growth defects with the
pif1
Δ mutation. In addition, we found that some of these mutations changed the telomere size.
Origins and dispersals of Pacific peoples: Evidence from mtDNA phylogenies of the Pacific rat
Authors: Matisoo-Smith E. , Robins J. H.
Published: June 2004

The human settlement of the Pacific in general, and the origin of the Polynesians in particular, have been topics of debate for over two centuries. Polynesian origins are most immediately traced to people who arrived in the Fiji, Tonga, and Samoa region ≈3,000 B.P. and are clearly associated with the Lapita Cultural Complex. Although this scenario of the immediate origins of the Polynesians is generally accepted, the debate on the ultimate origin of the Polynesians and the Lapita cultural complex continues. Our previous research has shown that analyses of mtDNA variation in the Pacific rat (
Rattus exulans
), often transported as a food item in the colonizing canoes, are valuable for tracing prehistoric human migration within Polynesia. Here we present mtDNA phylogenies based on ≈240 base pairs of the
d
-loop from both archaeological and modern samples collected from Island Southeast Asia and the Pacific. We identify three major haplogroups, two of which occur in the Pacific. Comparing our results with Lapita models of Oceanic settlement, we are able to reject two often cited but simplistic models, finding support instead for multifaceted models incorporating a more complex view of the Lapita intrusion. This study is unique and valuable in that
R. exulans
is the only organism associated with the Lapita dispersal for which there are sufficient ancient and extant populations available for genetic analysis. By tracking population changes through time, we can understand more fully the settlement process and population interactions in both Near and Remote Oceania.
Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21
Authors: Yin Yuxin , Zhu Aiping , Jin Yan J. , Liu Yu-Xin , Zhang Xia , Hopkins Kevin M. , Lieberman Howard B.
Published: June 2004

When human cells incur DNA damage, two fundamental responses can follow, cell cycle arrest or apoptosis. Human RAD9 (hRAD9) and p53 function in both processes, but the mechanistic relationship between their activities is unknown. p53 mediates checkpoint control at G
1
by transcriptional regulation of
p21
. In this report, we show that hRAD9, like p53, can also regulate
p21
at the transcriptional level. We demonstrate that overexpression of
hRAD9
leads to increased
p21
RNA and encoded protein levels. The promoter region of
p21
fused to a luciferase reporter can be transactivated by either hRAD9 or p53, indicating that hRAD9 regulates the
p21
promoter for transcriptional control of expression. Using an electrophoretic mobility-shift assay, we show that hRAD9 specifically binds to a p53-consensus DNA-binding sequence in the
p21
promoter. Microarray screening coupled with Northern analysis reveals that hRAD9 regulates the abundance of other messages in addition to
p21
. Our data reveal a previously undescribed mechanism for regulation of
p21
and demonstrate that hRAD9 can control gene transcription. We suggest that hRAD9 and p53 co-regulate
p21
to direct cell cycle progression by similar molecular mechanisms. Furthermore, hRAD9 might regulate other cellular processes as well by modulating transcription of multiple down-stream target genes.
Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P
Authors: Zhu Jiaming , Trang Phong , Kim Kihoon , Zhou Tianhong , Deng Hongyu , Liu Fenyong
Published: June 2004

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2′-
O
-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of ≈150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.
Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynamin-like GTPase MxB
Authors: King Megan C. , Raposo Graça , Lemmon Mark A.
Published: June 2004

Mx proteins form a subfamily of the dynamin-like GTPases, which have well established roles in cellular trafficking. Some Mx proteins (e.g., human MxA) have antiviral activity and are tightly regulated by type I IFNs. Others (e.g., human MxB) lack antiviral activity and are thought to have normal cellular functions that remain undefined. Consistent with this hypothesis, we report that MxB is expressed without IFN treatment. MxB seems to be exclusively extranuclear and is concentrated at the cytoplasmic face of nuclear pores, suggesting a role in their regulation. We find that expression of dominant negative (GTPase-defective) MxB mutants efficiently blocks nuclear import and causes a delay in G
1
/S cell-cycle progression. MxB depletion using RNA interference (RNAi) leads to a similar cell-cycle defect but does not block nuclear import. MxB therefore seems not to be required for nuclear import
per se
but may instead regulate its efficiency and/or kinetics. These studies indicate an unexpected role for a dynamin-like protein in nucleocytoplasmic trafficking and suggest that a related function might be usurped by its antiviral relatives.
Tumor-specific Ab-mediated targeting of MHC-peptide complexes induces regression of human tumor xenograftsin vivo
Authors: Lev Avital , Noy Roy , Oved Kfir , Novak Hila , Segal Dina , Walden Peter , Zehn Dietmar , Reiter Yoram
Published: June 2004

A cancer immunotherapy strategy is described herein that combines the advantage of the well established tumor targeting capabilities of high-affinity recombinant fragments of Abs with the known efficient, specific, and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC-peptide complexes. Structurally, it consists of a previously uncharacterized class of recombinant chimerical molecules created by the genetic fusion of single-chain (sc) Fv Ab fragments, specific for tumor cell surface antigens, to monomeric scHLA-A2 complexes containing immunodominant tumor- or viral-specific peptides. The fusion protein can induce very efficiently tumor cell lysis, regardless of the expression of self peptide-MHC complexes. Moreover, these molecules exhibited very potent antitumor activityin vivoin nude mice bearing preestablished human tumor xenografts. Thesein vitroandin vivoresults suggest that recombinant scFv-MHC-peptide fusion molecules could represent an approach to immunotherapy, bridging Ab and T lymphocyte attack on cancer cells.
Bers embedding of the Teichmüller space of a once-punctured torus
Authors: Komori Yohei , Sugawa Toshiyuki
Published: June 2004

In this note, we present a method of computing monodromies of projective structures on a once-punctured torus. This leads to an algorithm numerically visualizing the shape of the Bers embedding of a one-dimensional Teichmüller space. As a by-product, the value of the accessory parameter of a four-times punctured sphere will be calculated in a numerical way as well as the generators of a Fuchsian group uniformizing it. Finally, we observe the relation between the Schwarzian differential equation and Heun’s differential equation in this special case.
Gene Silencing of CENP-E by Small Interfering RNA in HeLa Cells Leads to Missegregation of Chromosomes after a Mitotic Delay
Authors: Tanudji Marcel , Shoemaker John , L'Italien Lawrence , Russell Loren , Chin Gregory , Schebye Xiao Min
Published: June 2004

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.
Splicing Factor hSlu7 Contains a Unique Functional Domain Required to Retain the Protein within the Nucleus
Authors: Shomron Noam , Reznik Mika , Ast Gil
Published: June 2004

Precursor-mRNA splicing removes the introns and ligates the exons to form a mature mRNA. This process is carried out in a spliceosomal complex containing >150 proteins and five small nuclear ribonucleoproteins. Splicing protein hSlu7 is required for correct selection of the 3′ splice site. Here, we identify by bioinformatics and mutational analyses three functional domains of the hSlu7 protein that have distinct roles in its subcellular localization: a nuclear localization signal, a zinc-knuckle motif, and a lysine-rich region. The zinc-knuckle motif is embedded within the nuclear localization signal in a unique functional structure that is not required for hSlu7's entrance into the nucleus but rather to maintain hSlu7 inside it, preventing its shuttle back to the cytoplasm via the chromosomal region maintenance 1 pathway. Thus, the zinc-knuckle motif of hSlu7 determines the cellular localization of the protein through a nucleocytoplasmic-sensitive shuttling balance. Altogether, this indicates that zinc-dependent nucleocytoplasmic shuttling might be the possible molecular basis by which hSlu7 protein levels are regulated within the nucleus.
NG2 Proteoglycan Promotes Endothelial Cell Motility and Angiogenesis via Engagement of Galectin-3 and α3β1 Integrin
Authors: Fukushi Jun-ichi , Makagiansar Irwan T. , Stallcup William B.
Published: June 2004

The NG2 proteoglycan is expressed by microvascular pericytes in newly formed blood vessels. We have used in vitro and in vivo models to investigate the role of NG2 in cross-talk between pericytes and endothelial cells (EC). Binding of soluble NG2 to the EC surface induces cell motility and multicellular network formation in vitro and stimulates corneal angiogenesis in vivo. Biochemical data demonstrate the involvement of both galectin-3 and α3β1 integrin in the EC response to NG2 and show that NG2, galectin-3, and α3β1 form a complex on the cell surface. Transmembrane signaling via α3β1 is responsible for EC motility and morphogenesis in this system. Galectin-3–dependent oligomerization may potentiate NG2-mediated activation of α3β1. In conjunction with recent studies demonstrating the early involvement of pericytes in angiogenesis, these data suggest that pericyte-derived NG2 is an important factor in promoting EC migration and morphogenesis during the early stages of neovascularization.
Rab22a Regulates the Recycling of Membrane Proteins Internalized Independently of Clathrin
Authors: Weigert Roberto , Yeung Albert Chi , Li Jean , Donaldson Julie G.
Published: June 2004

Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.
A statistical framework for the design of microarray experiments and effective detection of differential gene expression
Authors: Zhang Shu-Dong , Gant Timothy W.
Published: June 2004

Motivation: Microarray experiments generate a high data volume. However, often due to financial or experimental considerations, e.g. lack of sample, there is little or no replication of the experiments or hybridizations. These factors combined with the intrinsic variability associated with the measurement of gene expression can result in an unsatisfactory detection rate of differential gene expression (DGE). Our motivation was to provide an easy to use measure of the success rate of DGE detection that could find routine use in the design of microarray experiments or in post-experiment assessment.

Results: In this study, we address the problem of both random errors and systematic biases in microarray experimentation. We propose a mathematical model for the measured data in microarray experiments and on the basis of this model present a t-based statistical procedure to determine DGE. We have derived a formula to determine the success rate of DGE detection that takes into account the number of microarrays, the number of genes, the magnitude of DGE, and the variance from biological and technical sources. The formula and look-up tables based on the formula, can be used to assist in the design of microarray experiments. We also propose an ad hoc method for estimating the fraction of non-differentially expressed genes within a set of genes being tested. This will help to increase the power of DGE detection.

Availability: The functions to calculate the success rate of DGE detection have been implemented as a Java application, which is accessible at http://www.le.ac.uk/mrctox/microarray_lab/Microarray_Softwares/Microarray_Softwares.htm

Supplementary information at ftp://alcyone.mrc.le.ac.uk/Pub/twg1/BioInf03-0661suppl.pdf
Reconstruction of gene networks using Bayesian learning and manipulation experiments
Authors: Pournara Iosifina , Wernisch Lorenz
Published: June 2004

Motivation: The analysis of high-throughput experimental data, for example from microarray experiments, is currently seen as a promising way of finding regulatory relationships between genes. Bayesian networks have been suggested for learning gene regulatory networks from observational data. Not all causal relationships can be inferred from correlation data alone. Often several equivalent but different directed graphs explain the data equally well. Intervention experiments where genes are manipulated can help to narrow down the range of possible networks.

Results: We describe an active learning algorithm that suggests an optimized sequence of intervention experiments. Simulation experiments show that our selection scheme is better than an unguided choice of interventions in learning the correct network and compares favorably in running time and results with methods based on value of information calculations.

Availability: Algorithms are available from the authors on request.
Bayesian integrated functional analysis of microarray data
Authors: Bhattacharjee Madhuchhanda , Pritchard Colin C. , Nelson Peter S. , Arjas Elja
Published: June 2004

Motivation: The statistical analysis of microarray data usually proceeds in a sequential manner, with the output of the previous step always serving as the input of the next one. However, the methods currently used in such analyses do not properly account for the fact that the intermediate results may not always be correct, then leading to cumulating error in the inferences drawn based on such steps.

Results: Here we show that, by an application of hierarchical Bayesian methodology, this sequential procedure can be replaced by a single joint analysis, while systematically accounting for the uncertainties in this process. Moreover, we can also integrate relevant functional information available from databases into such an analysis, thereby increasing the reliability of the biological conclusions that are drawn. We illustrate these points by analysing real data and by showing that the genes can be divided into categories of interest, with the defining characteristic depending on the biological question that is considered. We contend that the proposed method has advantages at two levels. First, there are gains in the statistical and biological results from the analysis of this particular dataset. Second, it opens up new possibilities in analysing microarray data in general.

Supplementary information:  http://www.rni.helsinki.fi/~mab/
Java Treeview—extensible visualization of microarray data
Authors: Saldanha Alok J.
Published: June 2004

Summary: Open source software encourages innovation by allowing users to extend the functionality of existing applications. Treeview is a popular application for the visualization of microarray data, but is closed-source and platform-specific, which limits both its current utility and suitability as a platform for further development. Java Treeview is an open-source, cross-platform rewrite that handles very large datasets well, and supports extensions to the file format that allow the results of additional analysis to be visualized and compared. The combination of a general file format and open source makes Java Treeview an attractive choice for solving a class of visualization problems. An applet version is also available that can be used on any website with no special server-side setup.

Availability:  http://jtreeview.sourceforge.net under GPL.
An entity tagger for recognizing acquired genomic variations in cancer literature
Authors: McDonald Ryan T. , Winters R. Scott , Mandel Mark , Jin Yang , White Peter S. , Pereira Fernando
Published: June 2004

Summary: VTag is an application for identifying the type, genomic location and genomic state-change of acquired genomic aberrations described in text. The application uses a machine learning technique called conditional random fields. VTag was tested with 345 training and 200 evaluation documents pertaining to cancer genetics. Our experiments resulted in 0.8541 precision, 0.7870 recall and 0.8192 F-measure on the evaluation set.

Availability: The software is available at http://www.cis.upenn.edu/group/datamining/software_dist/biosfier/.
Protein complex prediction via cost-based clustering
Authors: King A. D. , Pržulj N. , Jurisica I.
Published: June 2004

Motivation: Understanding principles of cellular organization and function can be enhanced if we detect known and predict still undiscovered protein complexes within the cell's protein–protein interaction (PPI) network. Such predictions may be used as an inexpensive tool to direct biological experiments. The increasing amount of available PPI data necessitates an accurate and scalable approach to protein complex identification.

Results: We have developed the Restricted Neighborhood Search Clustering Algorithm (RNSC) to efficiently partition networks into clusters using a cost function. We applied this cost-based clustering algorithm to PPI networks of Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans to identify and predict protein complexes. We have determined functional and graph-theoretic properties of true protein complexes from the MIPS database. Based on these properties, we defined filters to distinguish between identified network clusters and true protein complexes.

Conclusions: Our application of the cost-based clustering algorithm provides an accurate and scalable method of detecting and predicting protein complexes within a PPI network.

Availability: The RNSC algorithm and data processing code are available upon request from the authors.

Supplementary Information: Supplementary data are available at http://www.cs.utoronto.ca/~juris/data/ppi04/
Automatic annotation of organellar genomes with DOGMA
Authors: Wyman Stacia K. , Jansen Robert K. , Boore Jeffrey L.
Published: June 2004

Summary: The Dual Organellar GenoMe Annotator (DOGMA) automates the annotation of organellar (plant chloroplast and animal mitochondrial) genomes. It is a Web-based package that allows the use of BLAST searches against a custom database, and conservation of basepairing in the secondary structure of animal mitochondrial tRNAs to identify and annotate genes. DOGMA provides a graphical user interface for viewing and editing annotations. Annotations are stored on our password-protected server to enable repeated sessions of working on the same genome. Finished annotations can be extracted for direct submission to GenBank.

Availability:  http://phylocluster.biosci.utexas.edu/dogma/

Supplementary information: Detailed documentation and tutorials for annotating both animal mitochondrial and plant chloroplast genomes can be found on the DOGMA home page.
Traditional and Modern Biomedical Prospecting: Part I—the History
Authors: Müller Werner E. G. , Batel Renato , Schröder Heinz C. , Müller Isabel M.
Published: June 2004

Nature, especially the marine environment, provides the most effective drugs used in human therapy. Among the metazoans, the marine sponges (phylum Porifera), which are sessile filter feeders, produce the most potent and highly selective bioactive secondary metabolites. These animals (or their associated symbiotic microorganisms) synthesize secondary metabolites whose activity and selectivity has developed during their long evolutionary history (evochemistry). The exploitation of these resources has become possible due to the progress in molecular and cell biology. BIOTECmarin, the German Center of Excellence follows this rationale. In the past, these animals have been successfully and extensively utilized to isolate bioactive compounds and biomaterials for human benefit. Pharmaceuticals prepared from marine animals, primarily sponges, have been applied since ancient times (Hippocrates, Aristotle and later Plinius). It has been reported that extracts and/or components from sponges can be used for the treatment of specific diseases. For a systematic and applied‐oriented exploitation, the successful development of effective compounds largely depends on quality of the institutional infrastructure of marine stations and more so on the biodiversity. The Center for Marine Research in Rovinj (Croatia) fulfils these prerequisites. Founded in 1891, this institute has to its credit major discoveries related to exploitation of secondary metabolites/biomaterials from sponges for therapeutical application and to obtain biomaterials for general wellbeing.This is the first part of a review focusing on biomedical prospecting. Here, we have mainly described the historic background. The details of techniques, substances, approaches and outlooks will be discussed in the second part.
An Alternative Approach to Atopic Dermatitis: Part I—Case‐Series Presentation
Authors: Kobayashi Hiromi , Takahashi Kuniaki , Mizuno Nobuyuki , Kutsuna Haruo , Ishii Masamitsu
Published: June 2004

Atopic dermatitis (AD) is a complex disease of obscure pathogenesis. A substantial portion of AD patients treated with conventional therapy become intractable after several cycles of recurrence. Over the last 20 years we have developed an alternative approach to treat many of these patients by diet and Kampo herbal medicine. However, as our approach is highly individualized and the Kampo formulae sometimes complicated, it is not easy to provide evidence to establish usefulness of this approach. In this Review, to demonstrate the effectiveness of the method of individualized Kampo therapy, results are presented for a series of patients who had failed with conventional therapy but were treated afterwards in our institution. Based on these data, we contend that there exist a definite subgroup of AD patients in whom conventional therapy fails, but the ‘Diet and Kampo’ approach succeeds, to heal. Therefore, this approach should be considered seriously as a second‐line treatment for AD patients. In the Discussion, we review the evidential status of the current conventional strategies for AD treatment in general, and then specifically discuss the possibility of integrating Kampo regimens into it, taking our case‐series presented here as evidential basis. We emphasize that Kampo therapy for AD is more ‘art’ than technology, for which expertise is an essential pre‐requisite.
Functional Somatic Syndromes: Emerging Biomedical Models and Traditional Chinese Medicine
Authors: Tan Steven , Tillisch Kirsten , Mayer Emeran
Published: June 2004

The so‐called functional somatic syndromes comprise a group of disorders that are primarily symptom‐based, multisystemic in presentation and probably involve alterations in mind‐brain‐body interactions. The emerging neurobiological models of allostasis/allostatic load and of the emotional motor system show striking similarities with concepts used by Traditional Chinese Medicine (TCM) to understand the functional somatic disorders and their underlying pathogenesis. These models incorporate a macroscopic perspective, accounting for the toll of acute and chronic traumas, physical and emotional stressors and the complex interactions between the mind, brain and body. The convergence of these biomedical models with the ancient paradigm of TCM may provide a new insight into scientifically verifiable diagnostic and therapeutic approaches for these common disorders.
Neurobiology of Acupuncture: Toward CAM
Authors: Ma Sheng-Xing
Published: June 2004

It has long been accepted that acupuncture, puncturing and scraping needles at certain points on the body, can have analgesic and anesthetic effects, as well as therapeutic effects in the treatment of various diseases. This therapy, including acupuncture anesthesia, has drawn the attention of many investigators and become a research subject of international interest around the world. Numerous studies have demonstrated that the nervous system, neurotransmitters, endogenous substances and Jingluo (meridians) may respond to needling stimulation and electrical acupuncture. An abundance of information has now accumulated concerning the neurobiological mechanisms of acupuncture, in relation to both neural pathways and neurotransmitters/hormonal factors that mediate autonomic regulation, pain relief and other therapeutics. Early studies demonstrated that the analgesic effects of electroacupuncture (EA) are mediated by opioid peptides in the periaqueductal gray. Recent evidence shows that nitric oxide plays an important role in mediating the cardiovascular responses to EA stimulation through the gracile nucleus‐thalamic pathway. Other substances, including serotonin, catecholamines, inorganic chemicals and amino acids such as glutamate and α‐aminobutyric acid (GABA), are proposed to mediate certain cardiovascular and analgesic effects of acupuncture, but at present their role is poorly understood. The increased interest in acupuncture health care has led to an ever‐growing number of investigators pursuing research in the processes of the sense of needling touch, transduction of needling stimulation signals, stimulation parameters and placebos. In this Review, the evidence and understanding of the neurobiological processes of acupuncture research have been summarized with an emphasis on recent developments of nitric oxide mediating acupuncture signals through the dorsal medulla‐thalamic pathways.
Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics
Authors: Aslanian Aaron , Iaquinta Phillip J. , Verona Raluca , Lees Jacqueline A.
Published: June 2004

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16Ink4a–cycD/cdk4–pRB–E2F and p19Arf–mdm2–p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21Cip1. Thus, E2F3 is a key repressor of the p19Arf–p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21Cip1 in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3-/- cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation.
The loss of circadian PAR bZip transcription factors results in epilepsy
Authors: Gachon Frédéric , Fonjallaz Philippe , Damiola Francesca , Gos Pascal , Kodama Tohru , Zakany Jozsef , Duboule Denis , Petit Brice , Tafti Mehdi , Schibler Ueli
Published: June 2004

DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Here we show that mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions.
Interaction of prolactin, ANPergic, oxytocinergic and adrenal systems in response to extracellular volume expansion in rats
Authors: Durlo F. V. , Castro M. , Elias L. L. K. , Antunes‐Rodrigues J.
Published: June 2004

The present study evaluated the effect of acute extracellular volume expansion (EVE) induced by intravenous injection of isotonic (0.15 m NaCl) or hypertonic saline (0.3 m NaCl) on prolactin, corticosterone, vasopressin, oxytocin and atrial natriuretic peptide (ANP) secretion. Male Wistar rats were treated with bromocriptine, sulpiride or dexamethasone. After isotonic and hypertonic EVE, the control group showed a significant increase in the plasma concentrations of prolactin, corticosterone, ANP and oxytocin. The increase in ANP and oxytocin levels in response to hypertonic EVE was more pronounced than to isotonic EVE. Bromocriptine and sulpiride treatments did not modify corticosterone, ANP and oxytocin responses to either isotonic or hypertonic EVE. The increases in prolactin and oxytocin, but not ANP, were blocked in dexamethasone pretreated rats. In conclusion, isotonic or hypertonic EVE induced an increase in the plasma concentrations of prolactin, corticosterone, ANP and oxytocin. The increases in ANP and oxytocin were independent of plasma concentrations of prolactin. The increases in prolactin and oxytocin were blocked by the inhibition of the hypothalamo–pituitary–adrenal (HPA) axis by dexamethasone. However, dexamethasone did not alter the increase in ANP secretion induced by isotonic or hypertonic EVE. Therefore, prolactin might participate in regulation of the hydroelectrolytic balance in mammals; however, in the present study, there was no evidence for direct interaction with ANPergic and oxytocinergic systems. In addition, the responses of prolactin and oxytocin induced by isotonic or hypertonic EVE are modulated by the HPA axis.