bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein
Authors: Folli Claudia , Calderone Vito , Ottonello Simone , Bolchi Angelo , Zanotti Giuseppe , Stoppini Monica , Berni Rodolfo
Published: July 2002

Two cellular retinol-binding proteins (CRBP I and II) with distinct
tissue distributions and retinoid-binding properties have been
recognized thus far in mammals. Here, we report the identification of a
human retinol-binding protein resembling type I (55.6% identity) and
type II (49.6% identity) CRBPs, but with a unique H residue in the
retinoid-binding site and a distinctively different tissue
distribution. Additionally, this binding protein (CRBP III) exhibits a
remarkable sequence identity (62.2%) with the recently identified
ι-crystallin/CRBP of the diurnal gecko
Lygodactylus
picturatus
[Werten, P. J. L., Röll, B., van
Alten, D. M. F. & de Jong, W. W. (2000)
Proc.
Natl. Acad. Sci. USA
97, 3282–3287 (First Published March 21,
2000; 10.1073/pnas.050500597)]. CRBP III and
all-trans
-retinol form a complex
(
K
d
≈ 60 nM), the absorption spectrum
of which is characterized by the peculiar fine structure typical of the
spectra of holo-CRBP I and II. As revealed by a 2.3-Å x-ray molecular
model of apo-CRBP III, the amino acid residues that line the
retinol-binding site in CRBP I and II are positioned nearly identically
in the structure of CRBP III. At variance with the human CRBP I and II
mRNAs, which are most abundant in ovary and intestine, respectively,
the CRBP III mRNA is expressed at the highest levels in kidney and
liver thus suggesting a prominent role for human CRBP III as an
intracellular mediator of retinol metabolism in these tissues.
RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human
Authors: Moorefield Beth , Greene Elizabeth A. , Reeder Ronald H.
Published: July 2002

We have cloned a human cDNA that is related to the RNA polymerase I transcription factor Rrn3 of
Saccharomyces cerevisiae
. The recombinant human protein displays both sequence similarity and immunological crossreactivity to yeast Rrn3 and is capable of rescuing a yeast strain carrying a disruption of the RRN3 gene
in vivo
. Point mutation of an amino acid that is conserved between the yeast and human proteins compromises the function of each factor, confirming that the observed sequence similarity is functionally significant. Rrn3 is the first RNA polymerase I-specific transcription factor shown to be functionally conserved between yeast and mammals, suggesting that at least one mechanism that regulates ribosomal RNA synthesis is conserved among eukaryotes.
IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression
Authors: Spanbroek Rainer , Hildner Markus , Köhler André , Müller Angelika , Zintl Felix , Kühn Hartmut , Rådmark Olof , Samuelsson Bengt , Habenicht Andreas J. R.
Published: July 2002

Dendritic cell (DC) differentiation from human CD34
+
hematopoietic progenitor cells (HPCs) can be triggered
in
vitro
by a combination of cytokines consisting of stem cell
factor, granulocyte-macrophage colony-stimulating factor, and tumor
necrosis factor α. The immune response regulatory cytokines, IL-4 and
IL-13, promote DC maturation from HPCs, induce monocyte-DC
transdifferentiation, and selectively up-regulate 15-lipoxygenase 1
(15-LO-1) in blood monocytes. To gain more insight into
cytokine-regulated eicosanoid production in DCs we studied the effects
of IL-4/IL-13 on LO expression during DC differentiation. In the
absence of IL-4, DCs that had been generated from CD34
+
HPCs in response to stem cell factor/granulocyte-macrophage
colonystimulating factor/tumor necrosis factor α expressed
high levels of 5-LO and 5-LO activating protein. However, a small
subpopulation of eosinophil peroxidase
+
(EOS-PX) cells
significantly expressed 15-LO-1. Addition of IL-4 to differentiating
DCs led to a marked and selective down-regulation of 5-LO but not of
5-LO activating protein in DCs and in EOS-PX
+
cells and,
when added at the onset of DC differentiation, also prevented 5-LO
up-regulation. Similar effects were observed during IL-4- or
IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of
5-LO was accompanied by up-regulation of 15-LO-1, yielding
15-LO-1
+
5-LO-deficient DCs. However, transforming growth
factor β1 counteracted the IL-4-dependent inhibition of 5-LO but only
minimally affected 15-LO-1 up-regulation. Thus, transforming growth
factor β1 plus IL-4 yielded large mature DCs that coexpress both LOs.
Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was
maintained at all cytokine combinations in all DC phenotypes and in
EOS-PX
+
cells. In the absence of IL-4, major eicosanoids of
CD34
+
-derived DCs were 5S-hydroxyeicosatetraenoic acid
(5S-HETE) and leukotriene B
4
, whereas the major eicosanoids
of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of
IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of
the inhibition of one and the stimulation of another LO in a single
leukocyte lineage.
Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: Sensitivity of human thymocytes
Authors: Simon Anna Katharina , Williams Owen , Mongkolsapaya Juthathip , Jin Boquan , Xu Xiao Ning , Walczak Henning , Screaton Gavin R.
Published: July 2002

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.
Developmental expression of functional cyclooxygenases in zebrafish
Authors: Grosser Tilo , Yusuff Shamila , Cheskis Ellina , Pack Michael A. , FitzGerald Garret A.
Published: July 2002

Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E
2
is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation
ex vivo
and hemostasis
in vivo
are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development.
Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase
Authors: Hall Diane A. , Vander Kooi Craig W. , Stasik Chad N. , Stevens Shawn Y. , Zuiderweg Erik R. P. , Matthews Rowena G.
Published: July 2002

Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. InEscherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.
What visual perception tells us about mind and brain
Authors: Shimojo Shinsuke , Paradiso Michael , Fujita Ichiro
Published: July 2002

Recent studies of visual perception have begun to reveal the connection between neuronal activity in the brain and conscious visual experience. Transcranial magnetic stimulation of the human occipital lobe disrupts the normal perception of objects in ways suggesting that important aspects of visual perception are based on activity in early visual cortical areas. Recordings made with microelectrodes in animals suggest that the perception of the lightness and depth of visual surfaces develops through computations performed across multiple brain areas. Activity in earlier areas is more tightly correlated with the physical properties of objects whereas neurons in later areas respond in a manner more similar to visual perception.
A method for fine mapping quantitative trait loci in outbred animal stocks
Authors: Mott Richard , Talbot Christopher J. , Turri Maria G. , Collins Allan C. , Flint Jonathan
Published: July 2002

High-resolution mapping of quantitative trait loci (QTL) in
animals has proved to be difficult because the large effect sizes
detected in crosses between inbred strains are often caused by numerous
linked QTLs, each of small effect. In a study of fearfulness in mice,
we have shown it is possible to fine map small-effect QTLs in a
genetically heterogeneous stock (HS). This strategy is a powerful
general method of fine mapping QTLs, provided QTLs detected in crosses
between inbred strains that formed the HS can be reliably detected in
the HS. We show here that single-marker association analysis identifies
only two of five QTLs expected to be segregating in the HS and
apparently limits the strategy's usefulness for fine mapping. We solve
this problem with a multipoint analysis that assigns the probability
that an allele descends from each progenitor in the HS. The analysis
does not use pedigrees but instead requires information about the HS
founder haplotypes. With this method we mapped all three previously
undetected loci [chromosome (Chr.) 1 logP 4.9, Chr. 10 logP 6.0, Chr.
15 logP 4.0]. We show that the reason for the failure of single-marker
association to detect QTLs is its inability to distinguish opposing
phenotypic effects when they occur on the same marker allele. We have
developed a robust method of fine mapping QTLs in genetically
heterogeneous animals and suggest it is now cost effective to undertake
genomewide high-resolution analysis of complex traits in parallel on
the same set of mice.
Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors.
Authors: Uren A G , Pakusch M , Hawkins C J , Puls K L , Vaux D L
Published: July 2002

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.
BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.
Authors: Chang C C , Ye B H , Chaganti R S , Dalla-Favera R
Published: July 2002

Approximately 40% of diffuse large cell lymphoma are associated with chromosomal translocations that deregulate the expression of the BCL6 gene by juxtaposing heterologous promoters to the BCL-6 coding domain. The BCL6 gene encodes a 95-kDa protein containing six C-terminal zinc-finger motifs and an N-terminal POZ domain, suggesting that it may function as a transcription factor. By using a DNA sequence selected for its ability to bind recombinant BCL-6 in vitro, we show here that BCL-6 is present in DNA-binding complexes in nuclear extracts from various B-cell lines. In transient transfectin experiments, BCL6 can repress transcription from promoters linked to its DNA target sequence and this activity is dependent upon specific DNA-binding and the presence of an intact N-terminal half of the protein. We demonstrate that this part of the BCL6 molecule contains an autonomous transrepressor domain and that two noncontiguous regions, including the POZ motif, mediate maximum transrepressive activity. These results indicate that the BCL-6 protein can function as a sequence-specific transcriptional repressor and have implications for the role of BCL6 in normal lymphoid development and lymphomagenesis.
Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.
Authors: Kelly J J , Baird E E , Dervan P B
Published: July 2002

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.
Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.
Authors: Finnegan E J , Peacock W J , Dennis E S
Published: July 2002

Arabidopsis plants transformed with an antisense construct of an Arabidopsis methyltransferase cDNA (METI) have reduced cytosine methylation in CG dinucleotides. Methylation levels in progeny of five independent transformants ranged from 10% to 100% of the wild type. Removal of the antisense construct by segregation in sexual crosses did not fully restore methylation patterns in the progeny, indicating that methylation patterns are subject to meiotic inheritance in Arabidopsis. Plants with decreased methylation displayed a number of phenotypic and developmental abnormalities, including reduced apical dominance, smaller plant size, altered leaf size and shape, decreased fertility, and altered flowering time. Floral organs showed homeotic transformations that were associated with ectopic expression of the floral homeotic genes AGAMOUS and APETALA3 in leaf tissue. These observations suggest that DNA methylation plays an important role in regulating many developmental pathways in plants and that the developmental abnormalities seen in the methyltransferase antisense plants may be due to dysregulation of gene expression.
Fibroblast growth factor 2 can replace ectodermal signaling for feather development.
Authors: Song H , Wang Y , Goetinck P F
Published: July 2002

The initiation and morphogenesis of cutaneous appendages depend on a series of reciprocal signaling events between the epithelium and mesenchyme of the embryonic skin. In the development of feather germs, early dermal signals induce the formation of epidermal placodes that in turn signal the mesoderm to form dermal condensations immediately beneath them. We find a spatially and temporally restricted pattern of transcription for the genes that encode fibroblast growth factor (FGF) 2 and FGF receptor (FGFR) 1 in developing feather germs of the chicken embryo. FGF-2 expression is restricted to the epidermal placodes, whereas FGFR-1 expression is limited to the dermal condensations. Transcription of these genes could not be detected in skins of scaleless (sc/sc) embryos that fail to develop feathers as a result of an ectodermal defect. Treatment of sc/sc skins with FGF-2 results in the formation of feathers at the site of application of the growth factor and the induced feathers express FGFR-1 in their dermal condensations. Thus, we have established FGF-2 as an epidermal signal in early feather germ formation. The observation that FGF-2 can rescue the mutant phenotype of sc/sc embryos suggests that FGF-2 either is, or is downstream from, the signal that the sc/sc mutant ectoderm fails to generate.
How flowering plants discriminate between self and non-self pollen to prevent inbreeding.
Authors: Kao T H , McCubbin A G
Published: July 2002

Flowering plants have evolved various genetic mechanisms to circumvent the tendency for self-fertilization created by the close proximity of male and female reproductive organs in a bisexual flower. One such mechanism is gametophytic self-incompatibility, which allows the female reproductive organ, the pistil, to distinguish between self pollen and non-self pollen; self pollen is rejected, whereas non-self pollen is accepted for fertilization. The Solanaceae family has been used as a model to study the molecular and biochemical basis of self/non-self-recognition and self-rejection. Discrimination of self and non-self pollen by the pistil is controlled by a single polymorphic locus, the S locus. The protein products of S alleles in the pistil, S proteins, were initially identified based on their cosegregation with S alleles. S proteins have recently been shown to indeed control the ability of the pistil to recognize and reject self pollen. S proteins are also RNases, and the RNase activity has been shown to be essential for rejection of self pollen, suggesting that the biochemical mechanism of self-rejection involves the cytotoxic action of the RNase activity. S proteins contain various numbers of N-linked glycans, but the carbohydrate moiety has been shown not to be required for the function of S proteins, suggesting that the S allele specificity determinant of S proteins lies in the amino acid sequence. The male component in self-incompatibility interactions, the pollen S gene, has not yet been identified. The possible nature of the pollen S gene product and the possible mechanism by which allele-specific rejection of pollen is accomplished are discussed.
Evidence for a recommended dietary allowance for vitamin C from pharmacokinetics: A comment and analysis
Authors: Young Vernon R.
Published: July 2002

The current recommended dietary allowance (RDA) for vitamin C, as
proposed by the Food and Nutrition Board/National Research Council in
1980 and reconfirmed in 1989, is 60 mg daily for nonsmoking adult
males. Levine
et al.
[Levine, M., Conry-Cantilena, C.,
Wang, Y., Welch, R. W., Washko, P. W.,
et al.
(1996)
Proc. Natl. Acad. Sci. USA
93, 3704–3709], based on a
study of vitamin C pharmacokinetics in seven healthy men, have now
proposed that the RDA should be increased to 200 mg daily. I have
examined, in brief, the experimental and conceptual bases for this new
recommendation and its implications for public health and nutrition
policy and programs. Using, for illustrative purposes only, data
extracted from each of two recent dietary surveys of
noninstitutionalized adult males living in households in the
Netherlands and the United States, it is predicted that the prevalence
of intakes inadequate to meet the individual’s own requirement would
be about 96% or 84%, respectively, if the criteria of adequacy used
for derivation of the 200 mg RDA are accepted. Depending upon the
particular average requirement value for ascorbic acid that might be
derived from their data, the proposal by Levine
et al.
would mean a desirable increase in mean intakes in these two
populations by as much about 2- to 3-fold. Hence, before an action of
this kind is to be recommended, an answer must be sought to the
question whether current experimental data including the criteria
selected (saturation kinetics) are adequate to establish a new set of
requirements for vitamin C, which then carry such profound policy
implications. This will require critical assessment of all of the
available evidence emerging from laboratory, clinical, and
epidemiological studies to determine whether it provides a sufficient
rationale for accepting criteria of vitamin C adequacy such as those
proposed by Levine
et al.
and the requirement estimates
so derived.
Length suppression in histone messenger RNA 3′-end maturation: Processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA
Authors: Scharl Elizabeth C. , Steitz Joan A.
Published: July 2002

Efficient 3′-end processing of cell cycle-regulated mammalian
histone premessenger RNAs (pre-mRNAs) requires an upstream stem–loop
and a histone downstream element (HDE) that base pairs with the U7
small ribonuclearprotein. Insertions between these elements have two
effects: the site of cleavage moves in concert with the HDE and
processing efficiency declines. We used
Xenopus
oocytes
to ask whether compensatory length insertions in the human U7 RNA could
restore the fidelity and efficiency of processing of mouse histone
insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its
complementary to the HDE compensated for both defects in processing of
a 5-nt insertion substrate; a noncomplementary insertion into U7 did
not. Yet, the noncomplementary insertion mutant U7 was shown to be
active on insertion substrates further mutated to allow base pairing.
Our results suggest that the histone pre-mRNA becomes rigidified
upstream of its HDE, allowing the bound U7 small ribonucleoprotein to
measure from the HDE to the cleavage site. Such a mechanism may be
common to other RNA measuring systems. To our knowledge, this is the
first demonstration of length suppression in an RNA processing system.
A transgenic mouse model with an inducible skin blistering disease phenotype
Authors: Takahashi Kenzo , Coulombe Pierre A.
Published: July 2002

One of the current limitations of gene transfer protocols involving
mammalian genomes is the lack of spatial and temporal control over the
desired gene manipulation. Starting from a human keratin gene showing a
complex regulation as a template, we identified regulatory sequences
that confer inducible gene expression in a subpopulation of
keratinocytes in stratified epithelia of adult transgenic mice. We used
this cassette to produce transgenic mice with an inducible skin
blistering phenotype mimicking a form of epidermolytic hyperkeratosis,
a keratin gene disorder. Upon induction by topical application of a
phorbol ester, the mutant keratin transgene product accumulates in the
differentiating layers of epidermis, leading to keratinocyte lysis
after application of mechanical trauma. This mouse model will allow for
a better understanding of the complex relationship between keratin
mutation, keratinocyte cytoarchitecture, and hypersensitivity to
trauma. The development of an inducible expression vector showing an
exquisite cellular specificity has important implications for
manipulating genes in a spatially and temporally controlled fashion in
transgenic mice, and for the design of gene therapy strategies using
skin as a tissue source for the controlled delivery of foreign
substances.
The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins.
Authors: Wang X , Zafian P , Choudhary M , Lawton M
Published: July 2002

We have isolated an Arabidopsis thaliana gene that codes for a receptor related to antifungal pathogenesis-related (PR) proteins. The PR5K gene codes for a predicted 665-amino acid polypeptide that comprises an extracellular domain related to the PR5 proteins, a central transmembrane-spanning domain, and an intracellular protein-serine/threonine kinase. The extracellular domain of PR5K (PR5-like receptor kinase) is most highly related to acidic PR5 proteins that accumulate in the extracellular spaces of plants challenged with pathogenic microorganisms. The kinase domain of PR5K is related to a family of protein-serine/threonine kinases that are involved in the expression of self-incompatibility and disease resistance. PR5K transcripts accumulate at low levels in all tissues examined, although particularly high levels are present in roots and inflorescence stems. Treatments that induce authentic PR5 proteins had no effect on the level of PR5K transcripts, suggesting that the receptor forms part of a preexisting surveillance system. When the kinase domain of PR5K was expressed in Escherichia coli, the resulting polypeptide underwent autophosphorylation, consistent with its predicted enzyme activity. These results are consistent with PR5K encoding a functional receptor kinase. Moreover, the structural similarity between the extracellular domain of PR5K and the antimicrobial PR5- proteins suggests a possible interaction with common or related microbial targets.
Cells that register logical relationships among proteins
Authors: Xu C. Wilson , Mendelsohn Andrew R. , Brent Roger
Published: July 2002

Two-hybrid methods have augmented the classical genetic techniques biologists use to assign function to genes. Here, we describe construction of a two-bait interaction trap that uses yeast cells to register more complex protein relationships than those detected in existing two-hybrid systems. We show that such cells can identify bridge or connecting proteins and peptide aptamers that discriminate between closely related allelic variants. The protein relationships detected by these cells are analogous to classical genetic relationships, but lend themselves to systematic application to the products of entire genomes and combinatorial libraries. We show that, by performing logical operations on the phenotypic outputs of these complex cells and existing two-hybrid cells, we can make inferences about the topology and order of protein interactions. Finally, we show that cells that register such relationships can perform logical operations on protein inputs. Thus these cells will be useful for analysis of gene and allele function, and may also define a path for construction of biological computational devices.
Preliminary description of the cranium of
Proteopithecus sylviae
, an Egyptian late Eocene anthropoidean primate
Authors: Simons Elwyn L.
Published: July 2002

Recent discovery of crania, dentitions, and postcrania of a primitive anthropoidean primate,
Proteopithecus sylviae
, at the late Eocene L-4l quarry in the Fayum, Egypt, provides evidence of a new taxonomic family of early African higher primates, the Proteopithecidae. This family could be part of the basal radiation that produced the New World platyrrhine primates, or it could be unrelated to any subsequent lineages. Although no larger than a small callitrichid or a dwarf lemur, this tiny primate already possessed many of the derived features of later anthropoids and was a diurnal and probably dimorphic species. In dental formula and other dental proportions, as well as in known postcranial features,
Proteopithecus
more nearly resembles platyrrhines than does any other Old World higher primate. The small size of the
Proteopithecus
cranium demonstrates that the defining cranial characteristics of Anthropoidea did not arise as a consequence of an increase in size during derivation from earlier prosimians.
Microtubule-mediated transport of organelles and localization of β-catenin to the future dorsal side of
Xenopus
eggs
Authors: Rowning Brian A. , Wells Jonathan , Wu Mike , Gerhart John C. , Moon Randall T. , Larabell Carolyn A.
Published: July 2002

The dorsal–ventral axis in frog embryos is specified during the first cell cycle, when the cortex rotates relative to the cytoplasmic core along parallel microtubules associated with the core. Cytoplasmic transfer experiments suggest that dorsal determinants are transported 90° from the vegetal pole to the dorsal equator, even though the cortex rotates only 30°. Here we show that, during rotation, small endogenous organelles are rapidly propelled along the subcortical microtubules toward the future dorsal side and that fluorescent carboxylated beads injected into the vegetal pole are transported at least 60° toward the equator. We also show that deuterium oxide, which broadens the zone of dorsalization even though it reduces the extent of rotation and is known to randomize the microtubules, also randomizes the direction of organelle transport. Moreover, β-catenin, a component of the Wnt signaling pathway that possesses dorsalizing activity in
Xenopus
, colocalizes with subcortical microtubules at the dorsal side of the egg at the end of rotation. We propose that cortical rotation functions to align subcortical microtubules, which then mediate the transport of dorsal determinants toward their plus ends on one side of the egg.
Identification of a novel negative retinoic acid responsive element in the promoter of the human matrix Gla protein gene
Authors: Kirfel Jutta , Kelter Manuela , Cancela Leonor M. , Price Paul A. , Schüle Roland
Published: July 2002

The vitamin K-dependent matrix Gla protein (MGP) is synthesized in a wide variety of tissues such as lung, heart, kidney, cartilage, and bone. Expression of the MGP gene is regulated by various growth factors, steroid hormones, and the vitamin A metabolite retinoic acid (RA). In this report, we present evidence that RA down-regulates MGP gene expression in different rat and human cell lines via endogenous retinoid receptors [RA receptor (RAR) and retinoid X receptor (RXR)]. Repression of the human MGP (hMGP) gene is specifically mediated by ligand-activated RAR and RXR. Deletion analysis led to the identification of a novel negative response element (NRE) within the hMGP promoter. DNA binding studies performed with bacterially expressed RAR/RXR reveal the formation of a specific heterodimer/NRE complex. Furthermore, electrophoretic mobility-shift assays performed with proteins from RA-treated cells show that endogenous RAR/RXR binds to the NRE. We demonstrate that the NRE contains a CCAAT box and that both RAR/RXR and CCAAT-binding proteins such as c/EBPβ recognize this common regulatory sequence in the hMGP promoter. Our results indicate that RA-mediated repression of the hMGP gene is due to binding of liganded RAR/RXR to a novel negative RA response element.
The La antigen binds 5′ noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation
Authors: Ali Naushad , Siddiqui Aleem
Published: July 2002

Translation initiation of the hepatitis C virus (HCV) RNA genome occurs through an internal ribosome entry site in a cap-independent manner. Here, we have examined the interaction between La antigen and the HCV 5′ noncoding region (5′NCR). In this analysis, competitor RNAs derived from HCV 5′NCR carrying deletions and a point mutation were used to identify the site(s) of La antigen binding during UV cross-linking assay. These studies suggest that La antigen recognizes the intact HCV 5′NCR structure. Further, these interactions occurred in the context of the initiator AUG. The latter view is supported by an analysis in which mutants of the HCV 5′NCR RNA with deletion or substitution in the initiator AUG codon failed to compete for La antigen binding to the wild-type 5′NCR. The evidence for the interaction between liver cell-derived La antigen and the HCV 5′NCR is provided by immunoprecipitation of a UV cross-linked species from the S100 fraction of Huh7 cell lysates. The functional relevance of this interaction was demonstrated by the stimulation of the HCV internal ribosome entry site-mediated translation in the presence of La protein. These results suggest an important functional role of La protein in the regulation of internal initiation of translation of the HCV RNA genome.
RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins
Authors: Chandler Sharon D. , Mayeda Akila , Yeakley Joanne M. , Krainer Adrian R. , Fu Xiang-Dong
Published: July 2002

Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.
Astrocyte apoptosis induced by HIV-1 transactivation of the c
-kit
protooncogene
Authors: He Jianglin , deCastro Carlos M. , Vandenbark George R. , Busciglio Jorge , Gabuzda Dana
Published: July 2002

HIV-1 infection of the central nervous system (CNS) frequently causes dementia and other neurological disorders. The mechanisms of CNS injury in HIV-1 infection are poorly understood. Apoptosis of neurons and astrocytes is induced by HIV-1 infection
in vitro
and in brain tissue from AIDS patients, but the apoptotic stimuli have not been identified. We report herein that HIV-1 infection of primary brain cultures induces the receptor tyrosine kinase protooncogene c-
kit
and that high levels of c-Kit expression are associated with astrocyte apoptosis. Overexpression of c-Kit in an astrocyte-derived cell line in the absence of HIV-1 induces rapid apoptotic death. The apoptotic mechanism requires the c-Kit tyrosine kinase domain. The mechanism of c-
kit
induction by HIV-1 involves transactivation of the c-
kit
promoter by the HIV-1 Nef protein. These studies demonstrate that c-Kit can induce astrocyte apoptosis and suggest that this mechanism may play a role in CNS injury caused by HIV-1 infection. We propose that c-Kit can serve dual functions as a growth factor receptor or apoptosis inducer.
Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells
Authors: Siegel D. S. , Zhang X. , Feinman R. , Teitz T. , Zelenetz A. , Richon V. M. , Rifkind R. A. , Marks P. A. , Michaeli J.
Published: July 2002

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12–48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.
MMS2
, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway
Authors: Broomfield Stacey , Chow Barbara L. , Xiao Wei
Published: July 2002

Among the three
Saccharomyces cerevisiae
DNA repair epistasis groups, the
RAD6
group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The
rad6
and
rad18
mutants are defective in both pathways, and the
rev3
mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the
MMS2
gene from a yeast genomic library by functional complementation of the
mms2-1
mutant [Prakash, L. & Prakash, S. (1977)
Genetics
86, 33–55].
MMS2
encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the
mms2
mutation is hypostatic to
rad6
and
rad18
but is synergistic with the
rev3
mutation, and the
mms2
mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a
pol30-46
mutant known to be impaired in postreplication repair. The
mms2
mutant also displayed a
REV3
-dependent mutator phenotype, strongly suggesting that the
MMS2
gene functions in the error-free postreplication repair pathway, parallel to the
REV3
mutagenesis pathway. Furthermore, with respect to UV sensitivity,
mms2
was found to be hypostatic to the
rad6
Δ1–9
mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the
mms2
null mutation and two other allele-specific mutations,
rad6
Δ1–9
and
pol30-46,
define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.
Tissue factor gene expression in the adipose tissues of obese mice
Authors: Samad Fahumiya , Pandey Manjula , Loskutoff David J.
Published: July 2002

Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. We previously reported that plasminogen activator inhibitor 1 (PAI-1) is dramatically up-regulated in the plasma and adipose tissues of genetically obese mice. This change may disturb normal hemostatic balance and create a severe hypofibrinolytic state. Here we show that tissue factor (TF) gene expression also is significantly elevated in the epididymal and subcutaneous fat pads from
ob/ob
mice compared with their lean counterparts, and that its level of expression in obese mice increases with age and the degree of obesity. Cell fractionation and
in situ
hybridization analysis of adipose tissues indicate that TF mRNA is increased in adipocytes and in unidentified stromal vascular cells. Transforming growth factor β (TGF-β) is known to be elevated in the adipose tissue of obese mice, and administration of TGF-β increased TF mRNA expression in adipocytes
in vivo
and
in vitro
. These observations raise the possibility that TF and TGF-β may contribute to the increased cardiovascular disease that accompanies obesity and related non-insulin-dependent diabetes mellitus, and that the adipocyte plays a key role in this process. The recent demonstration that TF also influences angiogenesis, cell adhesion, and signaling suggests that its exact role in adipose tissue physiology/pathology, may be complex.
Identification of genes expressed in human CD34+hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning
Authors: Mao Mao , Fu Gang , Wu Ji-Sheng , Zhang Qing-Hua , Zhou Jun , Kan Li-Xin , Huang Qiu-Hua , He Kai-Li , Gu Bai-Wei , Han Ze-Guang , Shen Yu , Gu Jian , Yu Ya-Ping , Xu Shu-Hua , Wang Ya-Xin , Chen Sai-Juan , Chen Zhu
Published: July 2002

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34+HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34+cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.
The
Optx
2
homeobox gene is expressed in early precursors of the eye and activates retina-specific genes
Authors: Toy Jeffrey , Yang Jun-Ming , Leppert Gregory S. , Sundin Olof H.
Published: July 2002

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens.
Optx2
, a homeobox gene of the
sine oculis-Six
family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium.
Optx2
expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an
Optx2
-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that
Optx2
functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated
optix
, a
Drosophila
gene that is the closest insect homologue of
Optx2
and
Six3. Optix
is expressed during early development of the fly head and eye primordia.
Identification of 5′-deoxyribose phosphate lyase activity in human DNA polymerase γ and its role in mitochondrial base excision repair
in vitro
Authors: Longley Matthew J. , Prasad Rajendra , Srivastava Deepak K. , Wilson Samuel H. , Copeland William C.
Published: July 2002

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol γ is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol γ to participate in uracil-provoked base excision repair reconstituted
in vitro
with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol γ was able to fill a single nucleotide gap in the presence of a 5′ terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol γ catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH
4
strongly implicates a Schiff base intermediate in a β-elimination reaction mechanism.
Knock-out mutants from anEn-1mutagenizedArabidopsis thalianapopulation generate phenylpropanoid biosynthesis phenotypes
Authors: Wisman Ellen , Hartmann Ulrike , Sagasser Martin , Baumann Elvira , Palme Klaus , Hahlbrock Klaus , Saedler Heinz , Weisshaar Bernd
Published: July 2002

A collection of 8,000Arabidopsis thalianaplants carrying 48,000 insertions of the maize transposable elementEn-1has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes.En-1showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases,En-1was inserted within a few kilobases upstream or downstream of the gene.En-1was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained anEn-1insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containingEn-1insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated thatTRANSPARENT TESTA 6(TT6) encodes flavanone 3-hydroxylase. Thef3handflsnull mutants complete the set ofA. thalianalines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.