bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex
Authors: Suzuki Masatoshi , Wright Lynda S. , Marwah Padma , Lardy Henry A. , Svendsen Clive N.
Published: March 2004

Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans. However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system. Furthermore, the mechanism of its action is unknown. Here, we show that DHEA significantly increased the growth rates of human neural stem cells derived from the fetal cortex and grown with both epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). However, it had no effect on cultures grown in either factor alone, suggesting a specific action on the EGF/LIF-responsive cell. Precursors of DHEA such as pregnenolone or six of its major metabolites, had no significant effect on proliferation rates. DHEA did not alter the small number (<3%) of newly formed neuroblasts or the large number (>95%) of nestin-positive precursors. However, the number of glial fibrillary acidic protein-positive cells, its mRNA, and protein were significantly increased by DHEA. We found both
N
-methyl-
d
-aspartate and sigma 1 antagonists, but not GABA antagonists, could completely eliminate the effects of DHEA on stem cell proliferation. Finally we asked whether the EGF/LIF/DHEA-responsive stem cells had an increased potential for neurogenesis and found a 29% increase in neuronal production when compared to cultures grown in EGF/LIF alone. Together these data suggest that DHEA is involved in the maintenance and division of human neural stem cells. Given the wide availability of this neurosteroid, this finding has important implications for future use.
Whole-genome annotation by using evidence integration in functional-linkage networks
Authors: Karaoz Ulas , Murali T. M. , Letovsky Stan , Zheng Yu , Ding Chunming , Cantor Charles R. , Kasif Simon
Published: March 2004

The advent of high-throughput biology has catalyzed a remarkable improvement in our ability to identify new genes. A large fraction of newly discovered genes have an unknown functional role, particularly when they are specific to a particular lineage or organism. These genes, currently labeled “hypothetical,” might support important biological cell functions and could potentially serve as targets for medical, diagnostic, or pharmacogenomic studies. An important challenge to the scientific community is to associate these newly predicted genes with a biological function that can be validated by experimental screens. In the absence of sequence or structural homology to known genes, we must rely on advanced biotechnological methods, such as DNA chips and protein–protein interaction screens as well as computational techniques to assign putative functions to these genes. In this article, we propose an effective methodology for combining biological evidence obtained in several high-throughput experimental screens and integrating this evidence in a way that provides consistent functional assignments to hypothetical genes. We use the visualization method of propagation diagrams to illustrate the flow of functional evidence that supports the functional assignments produced by the algorithm. Our results contain a number of predictions and furnish strong evidence that integration of functional information is indeed a promising direction for improving the accuracy and robustness of functional genomics.
Cardiac hypertrophy and sudden death in mice with a genetically clamped renin transgene
Authors: Caron Kathleen M. I. , James Leighton R. , Kim Hyung-Suk , Knowles Josh , Uhlir Rick , Mao Lan , Hagaman John R. , Cascio Wayne , Rockman Howard , Smithies Oliver
Published: March 2004

Several mouse models have already proved valuable for investigating hypertrophic responses to cardiac stress. Here, we characterize one caused by a well defined single copy transgene, RenTgMK, that genetically clamps plasma renin and thence angiotensin II at high levels. All of the transgenic males develop concentric cardiac hypertrophy with fibrosis but without dilatation. Over half die suddenly aged 6-8 months. Telemetry showed disturbances in diurnal rhythms a few days before death and, later, electrocardiographic disturbances comparable to those in humans with congestive heart failure. Expression of seven hypertrophy-related genes in this and two categorically different models (lack of atrial natriuretic peptide receptor A; overexpression of calsequestrin) were compared. Statistical analyses show that ventricular expressions of the genes coding for atrial natriuretic peptide, β myosin heavy chain, medium chain acyl-CoA dehydrogenase, and adrenomedullin correlate equally well with the degree of hypertrophy, although their ranges of expression are, respectively, 50-, 30-, 10-, and 3-fold.
Competition for self-peptide-MHC complexes and cytokines between naïve and memory CD8
+
T cells expressing the same or different T cell receptors
Authors: Ge Qing , Bai Ailin , Jones Brendan , Eisen Herman N. , Chen Jianzhu
Published: March 2004

To study competition between naïve and memory T cells, we examined proliferation of adoptively transferred naïve CD8
+
T cells in lymphopenic recipients or recipients containing a clonal population of CD8
+
T cells. We find a hierarchy in the extent of T cell proliferation that appears to correlate with the strength of T cell receptor (TCR)-self-peptide-MHC (pepMHC) interactions. CD8
+
T cells also proliferate in recipients containing a full complement of CD8
+
cells with a different TCR if the transferred T cells experience stronger TCR-self-pepMHC interactions than the resident T cells. Furthermore, CD8
+
T cells proliferate in recipients that contain memory CD8
+
cells with a different TCR, but in this case the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, CD8
+
T cells do not proliferate significantly in recipients harboring naïve or memory CD8
+
cells that bear the same TCR as the transferred cells. These results suggest that, among naïve T cells and between naïve and memory T cells, CD8
+
cells having the same TCR compete for both self-pepMHC and cytokines, whereas TCR-different CD8
+
cells compete for cytokines. These competitive relationships probably help maintain the size and TCR diversity of naïve and memory T cell populations required for optimal immune responses.
Intersubunit signal transmission in integrins by a receptor-like interaction with a pull spring
Authors: Yang Wei , Shimaoka Motomu , Salas Azucena , Takagi Junichi , Springer Timothy A.
Published: March 2004

The function of some multidomain proteins is regulated by interdomain communication. We use second-site suppressor cysteine mutations to test a hypothesis on how the inserted (I)-like domain in the integrin β-subunit regulates ligand binding by the neighboring I domain in the integrin α-subunit [Huth, J. R., Olejniczak, E. T., Mendoza, R., Liang, H., Harris, E. A.,et al. (2000)Proc. Natl. Acad. Sci. USA97, 5231–5236; and Alonso, J. L., Essafi, M., Xiong, J. P., Stehle, T. & Arnaout, M. A. (2002)Curr. Biol.12, R340–R342]. The hypothesis is that an interaction between the β I-like metal ion-dependent adhesion site (MIDAS) and an intrinsic ligand in the linker following the α I domain, Glu-310, exerts a pull that activates the α I domain. Individual mutation of αLlinker residue Glu-310 or β2MIDAS residues Ala-210 or Tyr-115 to cysteine abolishes I domain activation, whereas the double mutation of αL-E310C with either β2-A210C or β2-Y115C forms a disulfide bond that constitutively activates ligand binding. The disulfide-bonded mutant is resistant to small molecule antagonists that bind to the β I-like domain near its interface with the α I domain and inhibit communication between these domains but remains susceptible to small molecule antagonists that bind underneath the I domain α7-helix and certain allosteric antagonistic antibodies. Thus, the α7-helix and its linker are better modeled as a pull spring than a bell rope. The results suggest that αLresidue Glu-310, which is universally conserved in all I domain-containing integrins, functions as an intrinsic ligand for the β I-like domain, and that when integrins are activated, the β I-like MIDAS binds to Glu-310, pulls the spring, and thereby activates the α I domain.
Recycling of a regulatory protein by degradation of the RNA to which it binds
Authors: Deikus Gintaras , Babitzke Paul , Bechhofer David H.
Published: March 2004

When
Bacillus subtilis
is grown in the presence of excess tryptophan, transcription of the
trp
operon is regulated by binding of tryptophan-activated TRAP to
trp
leader RNA, which promotes transcription termination in the
trp
leader region. Transcriptome analysis of a
B. subtilis
strain lacking polynucleotide phosphorylase (PNPase; a 3′-to-5′ exoribonuclease) revealed a striking overexpression of
trp
operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of
trp
leader RNA in the PNPase
-
strain showed accumulation of a stable, TRAP-protected fragment of
trp
leader RNA. Loss of
trp
operon transcriptional regulation in the PNPase
-
strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the
B. subtilis trp
operon, specific ribonuclease degradation of RNA in an RNA–protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.
Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of β-catenin
Authors: Cong Feng , Varmus Harold
Published: March 2004

Wnt signaling regulates many aspects of development by increasing the signaling activity of β-catenin. Axin is a negative regulator of the Wnt signaling pathway, and it is responsible for the formation of the β-catenin degradation complex. Genetic studies with
Drosophila
suggest that Axin promotes cytoplasmic localization of β-catenin independent of Axin's known role of enhancing degradation of β-catenin. Here, we show that Axin is a nuclear-cytoplasmic shuttling protein. Nuclear export of Axin depends on the chromosome maintenance region 1 nuclear receptor; treatment with the chromosome maintenance region 1 inhibitor leptomycin B induces nuclear accumulation of ectopically expressed or endogenous Axin. Functional nuclear localization and nuclear export signals have been mapped within Axin. Significantly, overexpression of an Axin fragment shifts coexpressed stabilized β-catenin to the cytoplasm, and this effect requires shuttling of Axin between the cytoplasm and the nucleus. Our results suggest that Axin functions as a molecular chaperone for β-catenin and that nuclear-cytoplasmic shuttling of Axin regulates the nuclear-cytoplasmic distribution of β-catenin.
Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors
Authors: Padilla Philip Ian , Pacheco-Rodriguez Gustavo , Moss Joel , Vaughan Martha
Published: March 2004

Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an ≈200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions.
Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system
Authors: Fernández-Solari Javier , Scorticati Camila , Mohn Claudia , De Laurentiis Andrea , Billi Silvia , Franchi Ana , McCann Samuel M. , Rettori Valeria
Published: March 2004

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the
N
-methyl-
d
-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: β-endorphin and γ-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10
-9
M) on
N
-methyl-
d
-aspartic acid-induced LHRH release, but naltrexone, a μ-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on β-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not β-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10
-1
M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.
Integrating the genotype and phenotype in hominid paleontology
Authors: Hlusko Leslea J.
Published: March 2004

Competing interpretations of human origins and evolution have recently proliferated despite the accelerated pace of fossil discovery. These controversies parallel those involving other vertebrate families and result from the difficulty of studying evolution among closely related species. Recent advances in developmental and quantitative genetics show that some conventions routinely used by hominid and other mammalian paleontologists are unwarranted. These same advances provide ways to integrate knowledge of the genotype into the study of the phenotype. The result is an approach that promises to yield a fuller understanding of evolution below the family level.
Expression of an active tobacco mitogen-activated protein kinase kinase kinase enhances freezing tolerance in transgenic maize
Authors: Shou Huixia , Bordallo Patricia , Fan Jian-Bing , Yeakley Joanne M. , Bibikova Marina , Sheen Jen , Wang Kan
Published: March 2004

Cold acclimation is the major process that prepares plants for freezing tolerance. In addition to extensive transcription regulation by cold-inducible master transcription factors, oxidative stress signaling has been postulated to play a role in freezing tolerance. Activation of oxidative signaling through the expression of an active mitogen-activated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing temperature bypassing cold acclimation. Because involvement of the mitogen-activated protein kinase cascade in oxidative stress signaling is evolutionarily conserved in eukaryotes from yeast to mammals, we tested the effect of expressing a heterologous tobacco mitogen-activated protein kinase kinase kinase (
Nicotiana
PK1), which can mimic H
2
O
2
signaling, in a major cereal crop. We demonstrate that low-level but constitutive expression of the
Nicotiana
PK1 gene enhances freezing tolerance in transgenic maize plants that are normally frost sensitive. Our results suggest that a new molecular approach can be designed to genetically enhance freezing tolerance in important crops.
The vortex patch problem for the surface quasi-geostrophic equation
Authors: Rodrigo José Luis
Published: March 2004

In this article, the analog of the Euler vortex patch problem for the surface quasi-geostrophic equation is considered.
Transition state stabilization by general acid catalysis, water expulsion, and enzyme reorganization in
Medicago savita
chalcone isomerase
Authors: Hur Sun , Newby Zachary E. R. , Bruice Thomas C.
Published: March 2004

In aqueous solution,
Medicago savita
chalcone isomerase (CHI) enhances the reaction rate for the unimolecular rearrangement of chalcone (CHN) into flavanone by seven orders of magnitude. Conformations of CHN and their relative free energies in water and CHI were investigated by the thermodynamic perturbation method. In water, CHN adopts two conformations (I and II) with conformation I being higher in energy than conformation II by 3 kcal/mol. Only I can give rise to a near attack conformer (NAC) where the nucleophile O2′ and the electrophile C9 are placed in proximity. In CHI, I binds less tightly than II by ≈2 kcal/mol, resulting in the free energy for NAC formation being ≈2 kcal/mol higher in the enzyme than in water. This unfavorable feature in the ground state of the CHI reaction requires the predominant catalytic advantage to be taken in the step of NAC → transition state (TS). From the molecular dynamics simulations of apo-CHI, CHI complexed with CHN (CHI·CHN) and CHI·TS, we found: (
i
) Lys-97-general-acid catalysis of the O2′
-
nucleophilic addition; (
ii
) expulsion of three water molecules in the process of TS formation; (
iii
) release of enzyme structural distortion on TS formation. In the conclusion, CHI's remarkable efficiency of stabilizing the TS and its relatively poor ability in organizing the ground state is compared with chorismate mutase whose catalytic prowess, when compared with water, originates predominantly from the enhanced NAC population at the active site.
Genetic evidence for the bidirectional modulation of synaptic plasticity in the prefrontal cortex by D1 receptors
Authors: Huang Yan-You , Simpson Eleanor , Kellendonk Christoph , Kandel Eric R.
Published: March 2004

To address the role of D1 receptors in the medial prefrontal cortex, we combined pharmacological and genetic manipulations to examine long-term synaptic potentiation (LTP)/long-term synaptic depression (LTD) in brain slices of rats and mice. We found that the D1 antagonist SCH23390 selectively blocked the maintenance but not the induction of LTP in the prefrontal cortex. Conversely, activation of D1 receptors facilitated the maintenance of LTP, and this effect is impaired in heterozygous D1 receptor knockout mice. Low-frequency stimulation induced a transient depression in the medial prefrontal cortex. This depression could be transformed into LTD by coapplication of dopamine. Coapplication of dopamine, however, shows no facilitating effect on LTD in heterozygous D1 receptor knockout mice. These results provide pharmacological and genetic evidence for a role of D1 receptors in the bidirectional modulation of synaptic plasticity in the medial prefrontal cortex. The absence of this modulation in heterozygous knockout mice shows that a dysregulation of dopamine receptor expression levels can have dramatic effects on synaptic plasticity in the prefrontal cortex.
The nonconserved wrapping of conserved protein folds reveals a trend toward increasing connectivity in proteomic networks
Authors: Fernández Ariel , Scott Ridgway , Berry R. Stephen
Published: March 2004

Although protein folding domains are generally conserved for function across distant homologous sequences, one crucial structural feature is not conserved: the wrapping of backbone hydrogen bonds, that is, the extent to which they are intramolecularly desolvated and thereby protected from water attack. Extensive data on protein complex interfaces led us to postulate that insufficiently wrapped backbone hydrogen bonds in monomeric domains must be adhesive, and therefore determinants of interactivity, a result that has been experimentally confirmed. Here, we show that the wrapping of certain conserved folds becomes progressively poorer as species diverge in some lineages. This trend is thus concurrent with a progressive enhancement of the interactivity of individual domains sharing the conserved fold. Such increase in interactivity is predicted to impose an “evolutionary brake” on the overall speed of sequence divergence. This phenomenon follows when more and more residues become engaged in protein associations and thus become functionally indispensable. For complete proteomes for which statistically significant structural data are available, scale-free network statistics based solely on the distribution of folding domains, catalogued by their number of wrapping defects, best describe the proteomic connectivity. Thus, the intermolecular connectivity may be effectively used as a measure of species complexity. Our results might contribute to explaining how interactome complexity may be achieved without a dramatic increase in genome size.
Global analysis of nutrient control of gene expression inSaccharomyces cerevisiaeduring growth and starvation
Authors: Wu Jian , Zhang Nianshu , Hayes Andrew , Panoutsopoulou Kalliope , Oliver Stephen G.
Published: March 2004

Global gene expression in yeast was examined in five different nutrient-limited steady states and in their corresponding starvation-induced stationary phases. The use of chemostats, with their ability to generate defined and reproducible physiological conditions, permitted the exclusion of the confounding variables that frequently complicate transcriptome analyses. This approach allowed us to dissect out effects on gene expression that are specific to particular physiological states. Thus, we discovered that a large number of ORFs involved in protein synthesis were activated under ammonium limitation, whereas the expression of ORFs concerned with energy and metabolism was enhanced by carbon limitation. Elevated transcription of genes in high-affinity glucose uptake, the trichloroacetic acid cycle, and oxidative phosphorylation were observed in glucose-limiting, but not glucose-abundant, conditions. In contrast, genes involved in gluconeogenesis and, interestingly, genes subject to nitrogen catabolite repression increased their transcription when ethanol was the carbon source, even though ammonium was in excess. This result suggests that up-regulation of genes sensitive to nitrogen catabolite repression may contribute anapleurotic intermediates in ethanol-grown cells. The different starvation conditions produced two general types of transcription profiles, with carbon-starved cells transcribing far fewer genes than cells starved for any of the other macronutrients. Nonetheless, each starvation condition induced its own peculiar set of genes, and only 17 genes were induced >5-fold by all five starvations. In all cases, analysis of the upstream sequences of clusters of coregulated genes identified motifs that may be recognized by transcription factors specific for controlling gene expression in each of the physiological conditions examined.
Pathogenicity and immunogenicity of influenza viruses with genes from the 1918 pandemic virus
Authors: Tumpey Terrence M. , García-Sastre Adolfo , Taubenberger Jeffery K. , Palese Peter , Swayne David E. , Basler Christopher F.
Published: March 2004

The 1918 influenza A H1N1 virus caused the worst pandemic of influenza ever recorded. To better understand the pathogenesis and immunity to the 1918 pandemic virus, we generated recombinant influenza viruses possessing two to five genes of the 1918 influenza virus. Recombinant influenza viruses possessing the hemagglutinin (HA), neuraminidase (NA), matrix (M), nonstructural (NS), and nucleoprotein (NP) genes or any recombinant virus possessing both the HA and NA genes of the 1918 influenza virus were highly lethal for mice. Antigenic analysis by hemagglutination inhibition (HI) tests with ferret and chicken H1N1 antisera demonstrated that the 1918 recombinant viruses antigenically most resembled A/Swine/Iowa/30 (Sw/Iowa/30) virus but differed from H1N1 viruses isolated since 1930. HI and virus neutralizing (VN) antibodies to 1918 recombinant and Sw/Iowa/30 viruses in human sera were present among individuals born before or shortly after the 1918 pandemic. Mice that received an intramuscular immunization of the homologous or Sw/Iowa/30-inactivated vaccine developed HI and VN antibodies to the 1918 recombinant virus and were completely protected against lethal challenge. Mice that received A/PR/8/34, A/Texas/36/91, or A/New Caledonia/20/99 H1N1 vaccines displayed partial protection from lethal challenge. In contrast, control-vaccinated mice were not protected against lethal challenge and displayed high virus titers in respiratory tissues. Partial vaccine protection mediated by baculovirus-expressed recombinant HA vaccines suggest common cross-reactive epitopes on the H1 HA. These data suggest a strategy of vaccination that would be effective against a reemergent 1918 or 1918-like virus.
Surface hydrocarbons of queen eggs regulate worker reproduction in a social insect
Authors: Endler Annett , Liebig Jürgen , Schmitt Thomas , Parker Jane E. , Jones Graeme R. , Schreier Peter , Hölldobler Bert
Published: March 2004

A hitherto largely unresolved problem in behavioral biology is how workers are prevented from reproducing in large insect societies with high relatedness. Signals of the queen are assumed to inform the nestmates about her presence in the colony, which leads to indirect fitness benefits for workers. In the ant
Camponotus floridanus
, we found such a signal located on queen-laid eggs. In groups of workers that were regularly provided with queen-laid eggs, larvae, and cocoons, with larvae and cocoons alone, or with no brood, only in the groups with queen-laid eggs did workers not lay eggs. Thus, the eggs seem to inform the nestmates about the queen's presence, which induces workers to refrain from reproducing. The signal on queen-laid eggs is presumably the same that enables workers to distinguish between queen- and worker-laid eggs. Despite their viability, the latter are destroyed by workers when given a choice between both types. Queen- and worker-laid eggs differ in their surface hydrocarbons in a way similar to the way fertile queens differ from workers in the composition of their cuticular hydrocarbons. When we transferred hydrocarbons from the queen cuticle to worker-laid eggs, the destruction of those eggs was significantly mitigated. We conclude that queen-derived hydrocarbon labels inform workers about the presence of a fertile queen and thereby regulate worker reproduction.
Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120
Authors: Huang Chih-chin , Venturi Miro , Majeed Shahzad , Moore Michael J. , Phogat Sanjay , Zhang Mei-Yun , Dimitrov Dimiter S. , Hendrickson Wayne A. , Robinson James , Sodroski Joseph , Wyatt Richard , Choe Hyeryun , Farzan Michael , Kwong Peter D.
Published: March 2004

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective VH-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.
Differential utilization of upstream AUGs in the β-secretase mRNA suggests that a shunting mechanism regulates translation
Authors: Rogers George W. , Edelman Gerald M. , Mauro Vincent P.
Published: March 2004

β-Secretase [also known as the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)] is an enzyme involved in the production of Aβ-amyloid plaques in the brains of patients with Alzheimer's disease. The enhanced production of this enzyme occurs without corresponding changes in BACE1 mRNA levels. The complex 5′ leader of BACE1 mRNA contains three upstream ORFs (uORFs) preceding the BACE1 initiation codon. In this study, we investigated how this 5′ leader affects translation efficiency as a first step in understanding the enhanced production of the enzyme in the disease. Using reporter constructs in transfected mammalian cell lines and cell-free lysates, we showed that the translation mediated by the BACE1 5′ leader is cap-dependent and inhibited by cis-acting segments contained within the 5′ leader. Disruption of the uORFs had no effect on translation in B104 cells, which was surprising because the first two AUGs reside in contexts able to function as initiation codons. Possible mechanisms to explain how ribosomes bypass the uORFs, including reinitiation, leaky scanning, and internal initiation of translation were found to be inconsistent with the data. The data are most consistent with a model in which ribosomes shunt uORF-containing segments of the 5′ leader as the ribosomes move from the 5′ end of the mRNA to the initiation codon. In PC12 cells, however, the second uORF appears to be translated. We hypothesize that the translation efficiency of the BACE1 initiation codon may be increased in patients with Alzheimer's disease by molecular mechanisms that enhance shunting or increase the relative accessibility the BACE1 initiation codon.
Stereochemistry in self-assembled encapsulation complexes: Constellational isomerism
Authors: Yamanaka Masamichi , Shivanyuk Alexander , Rebek Julius
Published: March 2004

A previously uncharacterized form of stereochemistry, constellational isomerism, is described. The isomerism arises from different arrangements of small-molecule guests in the space of a self-assembled, cylindrical host. The cylindrical host detains three molecules each of CHCl
3
, 1,2-dichloroethane, or isopropyl chloride. The exchange of guests in and out of the host is slow on the NMR time scale. The dimensions of the capsular host and the sizes of the guests hinder the mobility of molecules inside, and separate NMR signals are seen for guests at the ends of the capsule and those near its center. When two different guests are encapsulated, the spectra show up to four additional species: two sets of constellational isomers. In every pairwise combination of the three guests, all isomers could be identified. The equilibrium distributions of isomers depended on the concentrations of the guests in the bulk solution. The relative stability of the constellational isomers was a function of the polarity of the guest molecule and its ability to interact with the components of the capsule. The different arrangements represent information, and some possibilities for their use in data storage are proposed.
Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes
Authors: Paz Arnon , Mester David , Baca Ivan , Nevo Eviatar , Korol Abraham
Published: March 2004

The mechanism of an organism's adaptation to high temperatures has been investigated intensively in recent years. It was suggested that the macromolecules of thermophilic microorganisms (especially proteins) have structural features that enhance their thermostability. We compared mRNA sequences of 72 fully sequenced prokaryotic proteomes (14 thermophilic and 58 mesophilic species). Although the differences between the percentage of adenine plus guanine content of whole mRNAs of different prokaryotic species are much lower than those of guanine plus cytosine content, the thermophile purine-pyrimidine (R/Y) ratio within their mRNAs is significantly higher than that of the mesophiles. The first and third codon positions of both thermophiles and mesophiles are purine-biased, with the bias more pronounced by the thermophiles. Thermophile mRNAs that display the highest R/Y ratio (1.43–1.69) are those of the ribosomal proteins, histone-like proteins, DNA-dependent RNA polymerase subunits, and heat-shock proteins. Within mesophilic prokaryotes and five eukaryotic species, the R/Y ratio of the mRNAs of heat-shock proteins is higher than their average over coding part of the genome. Polypurine tracts (R)n(withn≥ 5) are much more abundant within the thermophile mRNAs compared with mesophiles. Between two sequential pure-purinic codons of thermophile mRNAs, there is a rather strong tendency for the occurrence of adenine but not guanine tracts. The data suggest that mixed adenine·guanine and polyadenine tracts in mRNAs increase the thermostability beyond the contribution of amino acids encoded by purine tracts, which highlights the importance of ecological stress in the evolution of genome architecture.
Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: A major site of neurogenesis
Authors: Haubensak Wulf , Attardo Alessio , Denk Winfried , Huttner Wieland B.
Published: March 2004

Neurons of the mammalian CNS are thought to originate from progenitors dividing at the apical surface of the neuroepithelium. Here we use mouse embryos expressing GFP from the
Tis21
locus, a gene expressed throughout the neural tube in most, if not all, neuron-generating progenitors, to specifically reveal the cell divisions that produce CNS neurons. In addition to the apical, asymmetric divisions of neuroepithelial (NE) cells that generate another NE cell and a neuron, we find, from the onset of neurogenesis, a second population of progenitors that divide in the basal region of the neuroepithelium and generate two neurons. Basal progenitors are most frequent in the telencephalon, where they outnumber the apically dividing neuron-generating NE cells. Our observations reconcile previous data on the origin and lineage of CNS neurons and show that basal, rather than apical, progenitors are the major source of the neurons of the mammalian neocortex.
Ghrelin cells replace insulin-producing β cells in two mouse models of pancreas development
Authors: Prado Catherine L. , Pugh-Bernard Aimee E. , Elghazi Lynda , Sosa-Pineda Beatriz , Sussel Lori
Published: March 2004

The pancreatic islet is necessary for maintaining glucose homeostasis. Within the pancreatic islet, the homeodomain protein Nkx2.2 is essential for the differentiation of all insulin-producing β cells and a subset of glucagon-producing α cells (
1
). Mice lacking
Nkx2.2
have relatively normal sized islets, but a large number of cells within the mutant islet fail to produce any of the four major islet hormones. In this study we demonstrate that
Nkx2.2
mutant endocrine cells have been replaced by cells that produce ghrelin, an appetite-promoting peptide predominantly found in the stomach. Intriguingly, normal mouse pancreas also contains a small population of ghrelin-producing cells, defining a new islet “ε” cell population. The expansion of ghrelin-producing cells at the expense of β cells may be a general phenomenon, because we demonstrate that
Pax4
mutant mice display a similar phenotype. We propose that insulin and ghrelin cells share a common progenitor and that Nkx2.2 and Pax4 are required to specify or maintain differentiation of the β cell fate. This finding also suggests that there is a genetic component underlying the balance between insulin and ghrelin in regulating glucose metabolism.
Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene
Authors: Liu Zhipei , Hergenhahn Manfred , Schmeiser Heinz H. , Wogan Gerald N. , Hong Amanda , Hollstein Monica
Published: March 2004

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m2) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4-9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single- and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C →T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A → T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.
Trends between gene content and genome size in prokaryotic species with larger genomes
Authors: Konstantinidis Konstantinos T. , Tiedje James M.
Published: March 2004

Although the evolution process and ecological benefits of symbiotic species with small genomes are well understood, these issues remain poorly elucidated for free-living species with large genomes. We have compared 115 completed prokaryotic genomes by using the Clusters of Orthologous Groups database to determine whether there are changes with genome size in the proportion of the genome attributable to particular cellular processes, because this may reflect both cellular and ecological strategies associated with genome expansion. We found that large genomes are disproportionately enriched in regulation and secondary metabolism genes and depleted in protein translation, DNA replication, cell division, and nucleotide metabolism genes compared to medium- and small-sized genomes. Furthermore, large genomes do not accumulate noncoding DNA or hypothetical ORFs, because the portion of the genome devoted to these functions remained constant with genome size. Traits other than genome size or strain-specific processes are reflected by the dispersion around the mean for cell functions that showed no correlation with genome size. For example, Archaea had significantly more genes in energy production, coenzyme metabolism, and the poorly characterized category, and fewer in cell membrane biogenesis and carbohydrate metabolism than Bacteria. The trends we noted with genome size by using Clusters of Orthologous Groups were confirmed by our independent analysis with The Institute for Genomic Research's Comprehensive Microbial Resource and Kyoto Encyclopedia of Genes and Genomes' Orthology annotation databases. These trends suggest that larger genome-sized species may dominate in environments where resources are scarce but diverse and where there is little penalty for slow growth, such as soil.
Early protection against pathogenic virus infection at a mucosal challenge site after vaccination with attenuated simian immunodeficiency virus
Authors: Tenner-Racz Klara , Hennig Christiane Stahl , Überla Klaus , Stoiber Heribert , Ignatius Ralf , Heeney Jonathan , Steinman Ralph M. , Racz Paul
Published: March 2004

Atraumatic application of attenuated SIVmac239Δ
nef
vaccine to the tonsils of rhesus macaques provided protection against challenge 26 weeks later with infectious SIVmac251 applied through this route. Early events at the mucosal portal of entry of challenge virus were followed. Wild-type virus was detected in nonvaccinated controls by day 4, and then simian immunodeficiency virus (SIV) replicated vigorously at days 7 and 14. In contrast, a challenge of 10 of 10 vaccinees with SIV did not significantly raise RNA levels in the plasma or increase infected cells in lymphoid tissues, as assessed by single-cell labeling for viral RNA and
nef
protein. Vaccine virus was found in the tonsils of all vaccinees, but challenge virus was only detected at this portal of entry in 4 of 10 monkeys. In the tonsil, the challenge virus did not induce an expansion of perforin
+
killer cells. However, there was a significant increase in γδ T cells and mature dendritic cells relative to unvaccinated controls. Therefore, during tonsillar SIVΔ
nef
vaccination, infection is blocked early at the entry portal, which we propose is due in part to innate functions of γδ T and dendritic cells.
Structural and functional divergence of insect CYP6B proteins: From specialist to generalist cytochrome P450
Authors: Li Xianchun , Baudry Jerome , Berenbaum May R. , Schuler Mary A.
Published: March 2004

How polyphagous herbivores cope with the diversity and unpredictability of plant defenses remains largely unknown at both the genetic and molecular levels. To examine whether generalist counterdefense enzymes are structurally more flexible and functionally more diverse, two counterdefensive allelochemical-metabolizing cytochrome P450 proteins, CYP6B1 from the specialist
Papilio polyxenes
, feeding on furanocoumarin-containing plants, and CYP6B8 from the generalist
Helicoverpa zea
, feeding on hundreds of host plant species, are compared structurally and functionally. Molecular modeling indicates that CYP6B8 has more flexible overall folding, a more elastic catalytic pocket, and one more substrate access channel than CYP6B1. Baculovirus-mediated expression of the CYP6B8 and CYP6B1 proteins demonstrates that CYP6B8 metabolizes six biosynthetically diverse plant allelochemicals (xanthotoxin, quercetin, flavone, chlorogenic acid, indole-3-carbinol, and rutin) and three insecticides (diazinon, cypermethrin, and aldrin), whereas CYP6B1 metabolizes only two allelochemicals (xanthotoxin and flavone) and one insecticide (diazinon) of those tested. These results indicate that generalist counterdefense proteins are capable of accepting a more structurally diverse array of compounds compared with specialist counterdefense proteins.
A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts
Authors: Hansson Anna , Hance Nicole , Dufour Eric , Rantanen Anja , Hultenby Kjell , Clayton David A. , Wibom Rolf , Larsson Nils-Göran
Published: March 2004

We performed global gene expression analyses in mouse hearts with progressive respiratory chain deficiency and found a metabolic switch at an early disease stage. The tissue-specific mitochondrial transcription factor A (
Tfam
) knockout mice of this study displayed a progressive heart phenotype with depletion of mtDNA and an accompanying severe decline of respiratory chain enzyme activities along with a decreased mitochondrial ATP production rate. These characteristics were observed after 2 weeks of age and became gradually more severe until the terminal stage occurred at 10-12 weeks of age. Global gene expression analyses with microarrays showed that a metabolic switch occurred early in the progression of cardiac mitochondrial dysfunction. A large number of genes encoding critical enzymes in fatty acid oxidation showed decreased expression whereas several genes encoding glycolytic enzymes showed increased expression. These alterations are consistent with activation of a fetal gene expression program, a well-documented phenomenon in cardiac disease. An increase in mitochondrial mass was not observed until the disease had reached an advanced stage. In contrast to what we have earlier observed in respiratory chain-deficient skeletal muscle, the increased mitochondrial biogenesis in respiratory chain-deficient heart muscle did not increase the overall mitochondrial ATP production rate. The observed switch in metabolism is unlikely to benefit energy homeostasis in the respiratory chain-deficient hearts and therefore likely aggravates the disease. It can thus be concluded that at least some of the secondary gene expression alterations in mitochondrial cardiomyopathy do not compensate but rather directly contribute to heart failure progression.
Sister grouping of chimpanzees and humans as revealed by genome-wide phylogenetic analysis of brain gene expression profiles
Authors: Uddin Monica , Wildman Derek E. , Liu Guozhen , Xu Wenbo , Johnson Robert M. , Hof Patrick R. , Kapatos Gregory , Grossman Lawrence I. , Goodman Morris
Published: March 2004

Gene expression profiles from the anterior cingulate cortex (ACC) of human, chimpanzee, gorilla, and macaque samples provide clues about genetic regulatory changes in human and other catarrhine primate brains. The ACC, a cerebral neocortical region, has human-specific histological features. Physiologically, an individual's ACC displays increased activity during that individual's performance of cognitive tasks. Of ≈45,000 probe sets on microarray chips representing transcripts of all or most human genes, ≈16,000 were commonly detected in human ACC samples and comparable numbers, 14,000–15,000, in gorilla and chimpanzee ACC samples. Phylogenetic results obtained from gene expression profiles contradict the traditional expectation that the non-human African apes (i.e., chimpanzee and gorilla) should be more like each other than either should be like humans. Instead, the chimpanzee ACC profiles are more like the human than like the gorilla; these profiles demonstrate that chimpanzees are the sister group of humans. Moreover, for those unambiguous expression changes mapping to important biological processes and molecular functions that statistically are significantly represented in the data, the chimpanzee clade shows at least as much apparent regulatory evolution as does the human clade. Among important changes in the ancestry of both humans and chimpanzees, but to a greater extent in humans, are the up-regulated expression profiles of aerobic energy metabolism genes and neuronal function-related genes, suggesting that increased neuronal activity required increased supplies of energy.