bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypothalamic integration of body fluid regulation.
Authors: Denton D A , McKinley M J , Weisinger R S
Published: July 2002

The progression of animal life from the paleozoic ocean to rivers and diverse econiches on the planet's surface, as well as the subsequent reinvasion of the ocean, involved many different stresses on ionic pattern, osmotic pressure, and volume of the extracellular fluid bathing body cells. The relatively constant ionic pattern of vertebrates reflects a genetic "set" of many regulatory mechanisms--particularly renal regulation. Renal regulation of ionic pattern when loss of fluid from the body is disproportionate relative to the extracellular fluid composition (e.g., gastric juice with vomiting and pancreatic secretion with diarrhea) makes manifest that a mechanism to produce a biologically relatively inactive extracellular anion HCO3- exists, whereas no comparable mechanism to produce a biologically inactive cation has evolved. Life in the ocean, which has three times the sodium concentration of extracellular fluid, involves quite different osmoregulatory stress to that in freshwater. Terrestrial life involves risk of desiccation and, in large areas of the planet, salt deficiency. Mechanisms integrated in the hypothalamus (the evolutionary ancient midbrain) control water retention and facilitate excretion of sodium, and also control the secretion of renin by the kidney. Over and above the multifactorial processes of excretion, hypothalamic sensors reacting to sodium concentration, as well as circumventricular organs sensors reacting to osmotic pressure and angiotensin II, subserve genesis of sodium hunger and thirst. These behaviors spectacularly augment the adaptive capacities of animals. Instinct (genotypic memory) and learning (phenotypic memory) are melded to give specific behavior apt to the metabolic status of the animal. The sensations, compelling emotions, and intentions generated by these vegetative systems focus the issue of the phylogenetic emergence of consciousness and whether primal awareness initially came from the interoreceptors and vegetative systems rather than the distance receptors.
Self-recognition and abstraction abilities in the common chimpanzee studied with distorting mirrors.
Authors: Kitchen A , Denton D , Brent L
Published: July 2002

The reactions of chimpanzees to regular mirrors and the results of the standard Gallup mark test have been well documented. In addition to using the mark test to demonstrate self-recognition in a regular mirror, we exposed six female chimpanzees to mirrors that produced distorted or multiplied self-images. Their reactions to their self-images, in terms of mirror-guided self-referenced behaviors, indicated that correct assessment of the source of the mirror image was made by each subject in each of the mirrors. Recognition of a distorted self-image implies an ability for abstraction in the subjects in that the distortion must be rationalized before self-recognition occurs. The implications of these results in terms of illuminating the relative importance of feature and contingency of movement cues to self-recognition are discussed.
Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.
Authors: Cassetti M A , Moss B
Published: July 2002

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.
Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population.
Authors: FitzGerald M G , Harkin D P , Silva-Arrieta S , MacDonald D J , Lucchina L C , Unsal H , O'Neill E , Koh J , Finkelstein D M , Isselbacher K J , Sober A J , Haber D A
Published: July 2002

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.
Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction.
Authors: Gautvik K M , de Lecea L , Gautvik V T , Danielson P E , Tranque P , Dopazo A , Bloom F E , Sutcliffe J G
Published: July 2002

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.
Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.
Authors: Ishitani R , Chuang D M
Published: July 2002

Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells.
Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.
Authors: Yao R , Schneider E , Ryan T J , Galivan J
Published: July 2002

A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence. The cDNA encodes a 318-amino acid protein of Mr 35,960. The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase. In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence. There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme. Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates. With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product. The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate. Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity. Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate. These properties are consistent with the enzymes derived from human and rat sources.
Molecular biology of retinal ganglion cells.
Authors: Xiang M , Zhou H , Nathans J
Published: July 2002

Retinal ganglion cells are the output neurons that encode and transmit information from the eye to the brain. Their diverse physiologic and anatomic properties have been intensively studied and appear to account well for a number of psychophysical phenomena such as lateral inhibition and chromatic opponency. In this paper, we summarize our current view of retinal ganglion cell properties and pose a number of questions regarding underlying molecular mechanisms. As an example of one approach to understanding molecular mechanisms, we describe recent work on several POU domain transcription factors that are expressed in subsets of retinal ganglion cells and that appear to be involved in ganglion cell development.
Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.
Authors: Venkataraman G , Sasisekharan V , Herr A B , Ornitz D M , Waksman G , Cooney C L , Langer R , Sasisekharan R
Published: July 2002

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.
An immunoglobulin mutator that targets G.C base pairs.
Authors: Bachl J , Wabl M
Published: July 2002

Hypermutation can be defined as an enhancement of the spontaneous mutation rate which the organism uses in certain types of differentiated cells where a high mutation rate is advantageous. At the immunoglobulin loci this process increases the mutation rate > 10(5)-fold over the normal, spontaneous rate. Its proximate cause is called the immunoglobulin mutator system. The most important function of this system is to improve antibody affinity in an ongoing response; it is turned on and off during the differentiation of B lymphocytes. We have established an in vitro system to study hypermutation by transfecting a rearranged mu gene into a cell line in which an immunoglobulin mutator has been demonstrated. A construct containing the mu gene and the 3' kappa enhancer has all the cis-acting elements necessary for hypermutation of the endogenous gene segments encoding the variable region. The activity of the mutator does not seem to depend strongly on the position of the transfected gene in the genome. The mutator is not active in transformed cells of a later differentiation stage. It is also not active on a transfected lacZ gene. These results are consistent with the specificity of the mutator system being maintained and make it possible to delineate cis and trans mutator elements in vitro. Surprisingly, the mutator preferentially targets G-C base pairs. Two hypotheses are discussed: (i) the immunoglobulin mutator system in mammals consists of several mutators, of which the mutator described here is only one; or (ii) the primary specificity of the system is biased toward mutation of G-C base pairs, but this specificity is obscured by antigenic selection.
Acyl tunichlorins: a new class of nickel chlorins isolated from the Caribbean tunicate Trididemnum solidum.
Authors: Sings H L , Bible K C , Rinehart K L
Published: July 2002

A new class of nickel-containing chlorins (acyl tunichlorins) has been isolated from the Caribbean tunicate Trididemnum solidum. The structures of 28 of these nickel (II) hydroporphyrins were elucidated using mass spectrometry, one- and two-dimensional NMR spectroscopy, and chemical degradation/derivatization. Unique structural features of these compounds include the diversity of aliphatic side chains, which are derived from C14:0 to C22:6 fatty acids, and their location at an unprecedented position at C-2a on the hydroporphyrin nucleus. No chlorins with ester-linked acyl side chains at C-2a have been reported previously. Although the exact biological role that these compounds play in T. solidum remains unknown, acyl tunichlorins represent the only nickel-containing chlorins to be isolated from a living system and are the C-2a acyl derivatives of tunichlorin, a nickel chlorin reported by this laboratory in 1988.
Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.
Authors: Tzavara E T , Pouille Y , Defer N , Hanoune J
Published: July 2002

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.
Profluorescent protease substrates: intramolecular dimers described by the exciton model.
Authors: Packard B Z , Toptygin D D , Komoriya A , Brand L
Published: July 2002

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.
The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage.
Authors: Frise E , Knoblich J A , Younger-Shepherd S , Jan L Y , Jan Y N
Published: July 2002

Specification of unequal daughter cell fates in the Drosophila external sense organ lineage requires asymmetric localization of the intrinsic determinant Numb as well as cell-cell interactions mediated by the Delta ligand and Notch receptor. Previous genetic studies indicated that numb acts upstream of Notch, and biochemical studies revealed that Numb can bind Notch. For a functional assay of the action of Numb on Notch signaling, we expressed these proteins in cultured Drosophila cells and used nuclear translocation of Suppressor of Hairless [Su(H)] as a reporter for Notch activity. We found that Numb interfered with the ability of Notch to cause nuclear translocation of Su(H); both the C-terminal half of the phosphotyrosine binding domain and the C terminus of Numb are required to inhibit Notch. Overexpression of Numb during wing development, which is sensitive to Notch dosage, revealed that Numb is also able to inhibit the Notch receptor in vivo. In the external sense organ lineage, the phosphotyrosine binding domain of Numb was found to be essential for the function but not for asymmetric localization of Numb. Our results suggest that Numb determines daughter cell fates in the external sense organ lineage by inhibiting Notch signaling.
The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.
Authors: Xu J , Nawaz Z , Tsai S Y , Tsai M J , O'Malley B W
Published: July 2002

Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.
Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.
Authors: Curtis S W , Washburn T , Sewall C , DiAugustine R , Lindzey J , Couse J F , Korach K S
Published: July 2002

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.
Toward a molecular definition of long-term memory storage
Authors: Bailey Craig H. , Bartsch Dusan , Kandel Eric R.
Published: July 2002

The storage of long-term memory is associated with a cellular
program of gene expression, altered protein synthesis, and the growth
of new synaptic connections. Recent studies of a variety of memory
processes, ranging in complexity from those produced by simple forms of
implicit learning in invertebrates to those produced by more complex
forms of explicit learning in mammals, suggest that part of the
molecular switch required for consolidation of long-term memory is the
activation of a cAMP-inducible cascade of genes and the recruitment of
cAMP response element binding protein-related transcription factors.
This conservation of steps in the mechanisms for learning-related
synaptic plasticity suggests the possibility of a molecular biology of
cognition.
BAX enhances paclitaxel-induced apoptosis through a p53-independent pathway
Authors: Strobel Thomas , Swanson Linda , Korsmeyer Stanley , Cannistra Stephen A.
Published: July 2002

To investigate the role of BAX in chemotherapy-induced
apoptosis, we transfected the SW626 human ovarian cancer cell
line, which lacks functional p53, with a cDNA encoding for murine BAX.
Immunoblotting revealed that BAX transfectants expressed a mean of
10-fold increased levels of BAX compared with neo-transfected control
clones, with similar levels of BCL-2 and BCL-x
L
. The
cytotoxicity of paclitaxel, vincristine, and doxorubicin was
significantly enhanced in BAX transfectants compared with control
clones, whereas the cytotoxicity profile of carboplatin, etoposide, and
hydroxyurea was unchanged. Increased paclitaxel-induced cytotoxicity of
BAX clones was associated with enhanced apoptosis, as assessed
by morphologic and flow cytometric criteria. These data suggest that
sufficient levels of BAX may bypass the need for upstream molecules
such as p53 in the process of chemotherapy-induced apoptosis.
Determinant for β-subunit regulation in high-conductance voltage-activated and Ca
2+
-sensitive K
+
channels: An additional transmembrane region at the N terminus
Authors: Wallner Martin , Meera Pratap , Toro Ligia
Published: July 2002

The pore-forming α subunit of large conductance voltage- and
Ca
2+
-sensitive K (MaxiK) channels is regulated by a β
subunit that has two membrane-spanning regions separated by an
extracellular loop. To investigate the structural determinants in the
pore-forming α subunit necessary for β-subunit modulation, we made
chimeric constructs between a human MaxiK channel and the
Drosophila
homologue, which we show is insensitive to
β-subunit modulation, and analyzed the topology of the α subunit. A
comparison of multiple sequence alignments with hydrophobicity plots
revealed that MaxiK channel α subunits have a unique hydrophobic
segment (S0) at the N terminus. This segment is in addition to the six
putative transmembrane segments (S1–S6) usually found in
voltage-dependent ion channels. The transmembrane nature of this unique
S0 region was demonstrated by
in vitro
translation
experiments. Moreover, normal functional expression of signal sequence
fusions and
in vitro
N-linked glycosylation experiments
indicate that S0 leads to an exoplasmic N terminus. Therefore, we
propose a new model where MaxiK channels have a seventh transmembrane
segment at the N terminus (S0). Chimeric exchange of 41 N-terminal
amino acids, including S0, from the human MaxiK channel to the
Drosophila
homologue transfers β-subunit regulation to
the otherwise unresponsive
Drosophila
channel. Both the
unique S0 region and the exoplasmic N terminus are necessary for this
gain of function.
Heteromeric connexons in lens gap junction channels.
Authors: Jiang J X , Goodenough D A
Published: July 2002

Gap junction channels are formed by paired oligomeric membrane hemichannels called connexons, which are composed of proteins of the connexin family. Experiments with transfected cell lines and paired Xenopus oocytes have demonstrated that heterotypic intercellular channels which are formed by two connexons, each composed of a different connexin, can selectively occur. Studies by Stauffer [Stauffer, K. A. (1995) J. Biol. Chem. 270, 6768-6772] have shown that recombinant Cx26 and Cx32 coinfected into insect cells may form heteromeric connexons. By solubilizing and subfractionating individual connexons from ovine lenses, we show by immunoprecipitation that connexons can contain two different connexins forming heteromeric assemblies in vivo.
Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.
Authors: Clark G J , Drugan J K , Terrell R S , Bradham C , Der C J , Bell R M , Campbell S
Published: July 2002

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.
Structure of a gene encoding a cytosolic acetyl-CoA carboxylase of hexaploid wheat.
Authors: Podkowinski J , Sroga G E , Haselkorn R , Gornicki P
Published: July 2002

An entire gene encoding wheat (var. Hard Red Winter Tam 107) acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] has been cloned and sequenced. Comparison of the 12-kb genomic sequence with the 7.4-kb cDNA sequence reported previously revealed 29 introns. Within the coding region, the exon sequence is 98% identical to the known wheat cDNA sequence. A second ACCase gene was identified by sequencing fragments of genomic clones that include the first two exons and the first intron. Additional transcripts were detected by 5' and 3' RACE analysis (rapid amplification of cDNA ends). One set of transcripts had a 5' end sequence identical to the cDNA found previously and another set was identical to the gene reported here. The 3' RACE clones fall into four distinguishable sequence sets, bringing the number of ACCase sequences to six. None of these cDNA or genomic clones encodes a chloroplast targeting signal. Identification of six different sequences suggests that either the cytosolic ACCase genes are duplicated in the three chromosome sets in hexaploid wheat or that each of the six alleles of the cytosolic ACCase gene has a readily distinguishable DNA sequence.
Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.
Authors: Lee J , Gray A , Yuan J , Luoh S M , Avraham H , Wood W I
Published: July 2002

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.
Spectroscopic recognition of guanine dimeric hairpin quadruplexes by a carbocyanine dye.
Authors: Chen Q , Kuntz I D , Shafer R H
Published: July 2002

Isolated guanine quadruplex structures have been described at high resolution both in solution and in the solid state. The existence of this unusual DNA structure in vivo and its biological significance remain to be determined. We describe the binding of 3,3'-diethyloxadicarbocyanine to dimeric hairpin guanine quadruplexes. This interaction results in a set of unique spectrophotometric signatures, none of which arises from binding to single strands or Watson-Crick duplexes. These unique signatures include a new absorbance peak (lambda max = 534 nm), an induced circular dichroism (lambda = 534-626 nm), a quenching of the dye fluorescence upon excitation with visible light, and strong energy transfer from DNA. This last effect provides the basis for detecting hairpin quadruplex structures in the presence of excess amounts of nonquadruplex DNA structures, such as single strands and Watson-Crick duplexes. The mechanism of quadruplex recognition by this dye is discussed, along with the possibility of using this dye as a probe for hairpin quadruplex structures in vitro and in vivo.
Thermal unfolding of human high-density apolipoprotein A-1: implications for a lipid-free molten globular state.
Authors: Gursky O , Atkinson D
Published: July 2002

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximately 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.
Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase.
Authors: Finley R L , Thomas B J , Zipursky S L , Brent R
Published: July 2002

During Drosophila development, nuclear and cell divisions are coordinated in response to developmental signals. In yeast and mammalian cells, signals that control cell division regulate the activity of cyclin-dependent kinases (Cdks) through proteins such as cyclins that interact with the Cdks. Here we describe two Drosophila cyclins identified from a set of Cdk-interacting proteins. One, cyclin J, is of a distinctive sequence type; its exclusive maternal expression pattern suggests that it may regulate oogenesis or the early nuclear divisions of embryogenesis. The other belongs to the D class of cyclins, previously identified in mammalian cells. We show that Drosophila cyclin D is expressed in early embryos and in imaginal disc cells in a pattern that anticipates cell divisions. Expression in the developing eye disc at the anterior edge of the morphogenetic furrow suggests that cyclin D acts early, prior to cyclin E, in inducing G1-arrested cells to enter S phase. Our results also suggest that, although cyclin D may be necessary, its expression alone is not sufficient to initiate the events leading to S phase.
High-resolution physical mapping by combined Alu-hybridization/PCR screening: construction of a yeast artificial chromosome map covering 31 centimorgans in 3p21-p14.
Authors: Aburatani H , Stanton V P , Housman D E
Published: July 2002

We describe an integrated approach to large-scale physical mapping using an Alu-PCR hybridization screening strategy in conjunction with direct PCR-based screening to construct a continuous yeast artificial chromosome map covering >20 mb in human chromosome 3, bands p14-p21, composed of 205 loci, connected by 480 yeast artificial chromosomes, with average interlocus distance of approximately equal to 100 kb. We observe an inverse distribution of Alu-PCR and (CA)n markers. These results suggest that the two screening methods may be complementary and demonstrate the utility of Alu-PCR hybridization screening in the closure of high-resolution human physical maps.
Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone
Authors: Rose Ken A. , Stapleton Genevieve , Dott Karin , Kieny Marie Paule , Best Ruth , Schwarz Margrit , Russell David W. , Björkhem Ingemar , Seckl Jonathan , Lathe Richard
Published: July 2002

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (
K
m
, 13.6 μM) and pregnenolone (
K
m
, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-
3
H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-
3
H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.
Prion-inducing domain 2–114 of yeast Sup35 protein transforms
in vitro
into amyloid-like filaments
Authors: King Chih-Yen , Tittmann Peter , Gross Heinz , Gebert Roland , Aebi Markus , Wüthrich Kurt
Published: July 2002

The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in
Saccharomyces cerevisiae
, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2–114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments
in vitro
. The filaments show a β-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element
in vivo
.
Dwarfism and age-associated spinal degeneration of heterozygote
cmd
mice defective in aggrecan
Authors: Watanabe Hideto , Nakata Ken , Kimata Koji , Nakanishi Isao , Yamada Yoshihiko
Published: July 2002

Mouse cartilage matrix deficiency (
cmd
) is an autosomal recessive disorder caused by a genetic defect of aggrecan, a large chondroitin sulfate proteoglycan in cartilage. The homozygotes (−/−) are characterized by cleft palate and short limbs, tail, and snout. They die just after birth because of respiratory failure, and the heterozygotes (+/−) appear normal at birth. Here we report that the heterozygotes show dwarfism and develop spinal misalignment with age. Within 19 months of age, they exhibit spastic gait caused by misalignment of the cervical spine and die because of starvation. Histological examination revealed a high incidence of herniation and degeneration of vertebral discs. Electron microscopy showed a degeneration of disc chondrocytes in the heterozygotes. These findings may facilitate the identification of mutations in humans predisposed to spinal degeneration.
Membrane-associated molecules regulate the formation of layer-specific cortical circuits
Authors: Castellani Valerie , Bolz Jürgen
Published: July 2002

The columnar organization of the mammalian neocortex is based on radially oriented axon collaterals which precisely link cells from distinct cortical layers. During development, these interlaminar connections are specific from their initial outgrowth: collaterals form only in the target layers and there are no transient axonal collaterals in the nontarget layers. To examine whether positional cues within individual cortical layers regulate the laminar specificity of collateral formation, explants of cells destined for different cortical layers were cultured on membranes prepared from target and nontarget layers. Axonal growth and branching were examined on homogeneous membrane substrates and on alternating stripes of membranes from different layers. Results show that axons branch preferentially on membrane substrates from those layers that they would target
in vivo
. In addition, when cortical axons were given a choice to grow on membranes from either their target or their nontarget layer, they exhibited a clear preference for the target layers. This indicates that membrane-associated cues confined to individual layers regulate the formation of collaterals of cortical axons and restrict their growth to their target layers. Heat inactivation of membranes from target layers resulted in reduced axonal branching. The same manipulation of membranes from nontarget layers increased axonal branching for one population of cortical neurons. Taken together, these results suggest that membrane-associated molecules confined to individual layers induce and prevent the formation of axon collaterals in distinct populations of cortical neurons. Thus, the expression of layer-specific cues provides important constraints for the remodeling of local circuits during cortical development.
A 10-amino acid sequence of fibroblast growth factor 2 is sufficient for its mitogenic activity on neural progenitor cells
Authors: Ray Jasodhara , Baird Andrew , Gage Fred H.
Published: July 2002

During development of the central nervous system, neurons and glia are generated from immature neural progenitor cells (NPCs). Basic fibroblast growth factor (FGF-2) is a mitogen for these cells both
in vitro
and
in vivo
. However, it is not known whether other members of the FGF family have similar mitogenic effects on NPCs. We have found that FGF-4, in addition to FGF-2, is a mitogen for NPCs isolated from fetal and adult central nervous systems. Other family members have no proliferative effects on these cells. FGFs transduce signals to the cytoplasm through a family of transmembrane tyrosine kinase receptors (FGFR-1–4) or their isoforms. The high-affinity receptor binding sites are found in two regions of the FGF-2 molecule. We have examined the involvement of these sites in mitogenic signaling. Synthetic peptides corresponding to sequences in FGF-2 receptor binding sites were examined in [
3
H]thymidine incorporation assays for their agonist or antagonist activity. A 10-aa sequence present in the first receptor binding domain has been found to act as an antagonist, blocking the mitogenic effects of FGF-2. Chemical crosslinking studies using
125
I-labeled FGF-2 showed specific reduction in binding of radiolabeled FGF-2 to its receptors present on the membranes of NPCs. The identification of this sequence will assist in the study of pathways involved in signal transduction for mitogenesis in these cells and elucidate the role of FGF-2 and FGF-4 during normal development and in the pathogenesis of disease.
Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action
Authors: Eldar-Finkelman Hagit , Krebs Edwin G.
Published: July 2002

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity
in vitro
. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.