bluuebunny/crossref_metadata_2025 huggingface dataset:
Old World sources of the first New World human inhabitants: A comparative craniofacial view
Authors: Brace C. Loring , Nelson A. Russell , Seguchi Noriko , Oe Hiroaki , Sering Leslie , Qifeng Pan , Yongyi Li , Tumen Dashtseveg
Published: July 2002

Human craniofacial data were used to assess the similarities and differences between recent and prehistoric Old World samples, and between these samples and a similar representation of samples from the New World. The data were analyzed by the neighbor-joining clustering procedure, assisted by bootstrapping and by canonical discriminant analysis score plots. The first entrants to the Western Hemisphere of maybe 15,000 years ago gave rise to the continuing native inhabitants south of the U.S.–Canadian border. These show no close association with any known mainland Asian population. Instead they show ties to the Ainu of Hokkaido and their Jomon predecessors in prehistoric Japan and to the Polynesians of remote Oceania. All of these also have ties to the Pleistocene and recent inhabitants of Europe and may represent an extension from a Late Pleistocene continuum of people across the northern fringe of the Old World. With roots in both the northwest and the northeast, these people can be described as Eurasian. The route of entry to the New World was at the northwestern edge. In contrast, the Inuit (Eskimo), the Aleut, and the Na-Dene speakers who had penetrated as far as the American Southwest within the last 1,000 years show more similarities to the mainland populations of East Asia. Although both the earlier and later arrivals in the New World show a mixture of traits characteristic of the northern edge of Old World occupation and the Chinese core of mainland Asia, the proportion of the latter is greater for the more recent entrants.
Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers
Authors: Rekasi Zoltan , Czompoly Tamas , Schally Andrew V. , Halmos Gabor
Published: July 2002

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH)
in vitro
and
in vivo
, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV
1
and SV
2
are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV
1
is identical to that of human pituitary GHRH-R, whereas in SV
2
exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV
1
is identical to that of pituitary GHRH-R. SV
2
may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.
Modulation of HIV-like particle assemblyin vitroby inositol phosphates
Authors: Campbell Stephen , Fisher Robert J. , Towler Eric M. , Fox Stephen , Issaq Haleem J. , Wolfe Tracy , Phillips Lawrence R. , Rein Alan
Published: July 2002

HIV-1 Gag protein assembles into 100- to 120-nm diameter particles in mammalian cells. Recombinant HIV-1 Gag protein assembles in a fully defined systemin vitrointo particles that are only 25–30 nm in diameter and that differ significantly in other respects from authentic particles. However, particles with the size and other properties of authentic virions were obtainedin vitroby addition of inositol phosphates or phosphatidylinsitol phosphates to the assembly system. Thus, the interactions between HIV-1 Gag protein molecules are altered by binding of inositol derivatives; this binding is apparently essential for normal HIV-1 particle assembly. This requirement is not seen in a deleted Gag protein lacking residues 16–99 within the matrix domain.
Incomplete taxon sampling is not a problem for phylogenetic inference
Authors: Rosenberg Michael S. , Kumar Sudhir
Published: July 2002

A major issue in all data collection for molecular phylogenetics is taxon sampling, which refers to the use of data from only a small representative set of species for inferring higher-level evolutionary history. Insufficient taxon sampling is often cited as a significant source of error in phylogenetic studies, and consequently, acquisition of large data sets is advocated. To test this assertion, we have conducted computer simulation studies by using natural collections of evolutionary parameters—rates of evolution, species sampling, and gene lengths—determined from data available in genomic databases. A comparison of the true tree with trees constructed by using taxa subsamples and trees constructed by using all taxa shows that the amount of phylogenetic error per internal branch is similar; a result that holds true for the neighbor-joining, minimum evolution, maximum parsimony, and maximum likelihood methods. Furthermore, our results show that even though trees inferred by using progressively larger taxa subsamples of a real data set become increasingly similar to trees inferred by using the full sample, all inferred trees are equidistant from the true tree in terms of phylogenetic error per internal branch. Our results suggest that longer sequences, rather than extensive sampling, will better improve the accuracy of phylogenetic inference.
Solvent interactions determine carbohydrate conformation
Authors: Kirschner Karl N. , Woods Robert J.
Published: July 2002

The relationship between the three-dimensional structures of oligosaccharides and polysaccharides and their biological properties has been the focus of many recent studies. The overall conformation of an oligosaccharide depends primarily on the orientation of the torsion angles (φ, ψ, and ω) between glycosyl residues. Numerous experimental studies have shown that in glucopyranosides the ω-torsion angle (O
6
-C
6
-C
5
-O
5
) displays a preference for
gauche
orientations, in disagreement with predictions based on gas-phase quantum mechanics calculations. In contrast, the ω-angle in galactopyranosides displays a high proportion of the anti-orientation. For oligosaccharides containing glycosidic linkages at the 6-position (1→6 linked), variations in rotamer population have a direct effect on the oligosaccharides' structure and function, and yet the physical origin of these conformational preferences remains unclear. Although it is generally recognized that the
gauche
effect in carbohydrates is a solvent-dependent phenomenon, the mechanism through which solvent induces the
gauche
preference is not understood. In the present work, quantum mechanics and solvated molecular dynamics calculations were performed on two representative carbohydrates, methyl α-
d
-glucopyranoside and methyl α-
d
-galactopyranoside. We show that correct reproduction of the experimental rotamer distributions about the ω-angles is obtained only after explicit water is included in the molecular dynamics simulations. The primary role of the water appears to be to disrupt the hydrogen bonding within the carbohydrate, thereby allowing the rotamer populations to be determined by internal electronic and steric repulsions between the oxygen atoms. The results reported here provide a quantitative explanation of the conformational behavior of (1→6)-linked carbohydrates.
Mechanisms of pain
Authors: Stucky Cheryl L. , Gold Michael S. , Zhang Xu
Published: July 2002

Persistent or chronic pain is the primary reason people seek medical care, yet current therapies are either inadequate for certain types of pain or cause intolerable side effects. Recently, pain neurobiologists have identified a number of cellular and molecular processes that lead to the initiation and maintenance of pain. Understanding these underlying mechanisms has given significant promise for the development of more effective, more specific pain therapies in the near future.
The classical progesterone receptor mediates
Xenopus
oocyte maturation through a nongenomic mechanism
Authors: Bayaa Mustafa , Booth Ronald A. , Sheng Yinglun , Liu X. Johné
Published: July 2002

Xenopus
laevis
oocytes are
physiologically arrested at G
2
of meiosis I. Resumption of
meiosis, or oocyte maturation, is triggered by progesterone.
Progesterone-induced
Xenopus
oocyte maturation is
mediated via an extranuclear receptor and is independent of gene
transcription. The identity of this extranuclear oocyte progesterone
receptor (PR), however, has remained a longstanding problem. We have
isolated the amphibian homologue of human PR from a
Xenopus
oocyte cDNA library. The cloned
Xenopus
progesterone receptor (xPR) functioned in heterologous cells as a
progesterone-regulated transcription activator. However,
endogenous xPR was excluded from the oocyte nucleus and
instead appeared to be a cytosolic protein not associated with any
membrane structures. Injection of xPR mRNA into
Xenopus
oocytes accelerated the progesterone-induced oocyte maturation and
reduced the required concentrations of progesterone. In enucleated
oocytes, xPR accelerated the progesterone-induced mitogen-activated
protein kinase activation. These data suggest that xPR is the long
sought after
Xenopus
oocyte receptor responsible
for progesterone-induced oocyte maturation.
Synthesis and characterization of selenotrisulfide-derivatives of lipoic acid and lipoamide
Authors: Self William T. , Tsai Lin , Stadtman Thressa C.
Published: July 2002

Thiol-containing compounds, such as glutathione and cysteine,
react with selenite under specific conditions to form
selenotrisulfides. Previous studies have focused on isolation and
characterization of intermolecular selenotrisulfides. This study
describes the preparation and characterization of intramolecular
selenotrisulfide derivatives of lipoic acid and lipoamide. These
derivatives, after separation from other reaction products by
reverse-phase HPLC, exhibit an absorbance maximum at 288 nm with an
extinction coefficient of 1,500
M
−1
⋅cm
−1
. The selenotrisulfide
derivative of lipoic acid was significantly stable at or below pH 8.0
in contrast to several other previously studied selenotrisulfides. Mass
spectral analysis of the lipoic acid and lipoamide derivatives
confirmed both the expected molecular weights and also the presence of
a single atom of selenium as revealed by its isotopic distribution. The
selenotrisulfide derivative of lipoic acid was found to serve as an
effective substrate for recombinant human thioredoxin reductase as well
as native rat thioredoxin reductase in the presence of NADPH. Likewise,
the lipoamide derivative was efficiently reduced by NADH-dependent
bovine lipoamide dehydrogenase. The significant
in vitro
stability of these intramolecular selenotrisulfide derivatives of
lipoic acid can serve as an important asset in the study of such
selenium adducts as model selenium donor compounds for selenophosphate
biosynthesis and as rate enhancement effectors in various redox
reactions.
Chromosomal inversions and the reproductive isolation of species
Authors: Noor Mohamed A. F. , Grams Katherine L. , Bertucci Lisa A. , Reiland Jane
Published: July 2002

Recent genetic studies have suggested that many genes contribute to differences between closely related species that prevent gene exchange, particularly hybrid male sterility and female species preferences. We have examined the genetic basis of hybrid sterility and female species preferences in
Drosophila pseudoobscura
and
Drosophila persimilis
, two occasionally hybridizing North American species. Contrary to findings in other species groups, very few regions of the genome were associated with these characters, and these regions are associated also with fixed arrangement differences (inversions) between these species. From our results, we propose a preliminary genic model whereby inversions may contribute to the speciation process, thereby explaining the abundance of arrangement differences between closely related species that co-occur geographically. We suggest that inversions create linkage groups that cause sterility to persist between hybridizing taxa. The maintenance of this sterility allows the species to persist in the face of gene flow longer than without such inversions, and natural selection will have a greater opportunity to decrease the frequency of interspecies matings.
Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate
Blepharisma
japonicum
Authors: Sugiura Mayumi , Harumoto Terue
Published: July 2002

Conjugation in
Blepharisma japonicum
is induced by
interaction between complementary mating-types I and II, which excrete
blepharmone (gamone 1) and blepharismone (gamone 2), respectively.
Gamone 1 transforms type II cells such that they can unite, and gamone
2 similarly transforms type I cells. Moreover, each gamone promotes the
production of the other gamone. Gamone 2 has been identified as
calcium-3-(2′-formylamino-5′-hydroxy-benzoyl) lactate and has been
synthesized chemically. Gamone 1 was isolated and characterized as a
glycoprotein of 20–30 kDa containing 175 amino acids and 6 sugars.
However, the amino acid sequence and arrangement of sugars in this
gamone are still unknown. To determine partial amino acid sequences of
gamone 1, we established a method of isolation based on the finding
that this glycoprotein can be concentrated by a Con A affinity column.
Gamone 1 is extremely unstable and loses its biological activity once
adsorbed to any of the columns that we tested. By using a Con A
affinity column and native PAGE, we detected a 30-kDa protein
corresponding to gamone 1 activity and determined the partial amino
acid sequences of the four peptides. To isolate gamone 1 cDNA, we
isolated mRNA from mating-type I cells stimulated by synthetic gamone 2
and then performed rapid amplification of cDNA ends procedures by using
gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence
contains an ORF of 305 amino acids and codes a possibly novel protein.
We also estimated the arrangement of sugars by comparing the affinity
to various lectin columns.
Gene characterized for membrane desaturase that produces (
E
)-11 isomers of mono- and diunsaturated fatty acids
Authors: Liu Weitian , Jiao Hongmei , Murray Nancy C. , O'Connor Marion , Roelofs Wendell L.
Published: July 2002

Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth,
Epiphyas postvittana
, that makes
E
11 isomers of monounsaturated (E11-16 and E11-14) fatty acids and a diunsaturated (E9,E11-14) fatty acid. In the pheromone gland, the monoene precursor is made available by β oxidation of E11-16 acid with a subsequent two-carbon loss to E9-14 acid. A functional assay using a baculovirus expression system required addition of myristic acid and E9-14 acid precursors to demonstrate the unusual regiospecificity and stereospecificity of this desaturase. The amino acid sequence of this desaturase has ≈61% identity to that of
Z
11-desaturases from two other insect species, and only ≈48% identity to the metabolic
Z
9-desaturases in those species. A pheromone-gland
Z
9-desaturase gene also was found with the light brown apple moth that differed in its deduced amino acid sequence (66% identity) with the metabolic
Z
9-desaturase from fat body in this species.
Kinetics of duplex formation for individual DNA strands within a single protein nanopore
Authors: Howorka Stefan , Movileanu Liviu , Braha Orit , Bayley Hagan
Published: July 2002

A single oligonucleotide was covalently attached to a genetically engineered subunit of the heptameric protein pore, α-hemolysin, to allow DNA duplex formation inside the pore lumen. Single-channel current recording was used to study the properties of the modified pore. On addition of an oligonucleotide 8 bases in length and with a sequence complementary to the tethered DNA strand, current blockades with durations of hundreds of milliseconds occurred, representing hybridization events of individual oligonucleotides to the tethered DNA strand. Kinetic constants for DNA duplex formation at the single molecule level were derived and found to be consistent with established literature values for macroscopic duplex formation. The resultant equilibrium constant for duplex formation in the nanopore was found to be close to the experimentally derived constant for duplex formation in solution. A good agreement between the equilibrium constants for duplex formation in the nanopore and in solution was also found for two other oligonucleotide pairs. In addition, the nanopore recordings revealed details of the kinetics difficult to obtain by conventional methods, like surface plasmon resonance, which measure ensemble properties. By investigating the temperature dependence of DNA duplex formation at the single molecule level, the standard enthalpy and entropy of the interaction could be obtained.
Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding
Authors: Nollau Peter , Mayer Bruce J.
Published: July 2002

Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosine-phosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.
TheSUR2gene ofArabidopsis thalianaencodes the cytochrome P450 CYP83B1, a modulator of auxin homeostasis
Authors: Barlier Isabelle , Kowalczyk Mariusz , Marchant Alan , Ljung Karin , Bhalerao Rishikesh , Bennett Malcolm , Sandberg Goeran , Bellini Catherine
Published: July 2002

Genetic screens have been performed to identify mutants with altered auxin homeostasis inArabidopsis. A tagged allele of the auxin-overproducing mutantsur2was identified within a transposon mutagenized population. TheSUR2gene was cloned and shown to encode the CYP83B1 protein, which belongs to the large family of the P450-dependent monooxygenases.SUR2expression is up-regulated insur1mutants and induced by exogenous auxin in the wild type. Analysis of indole-3-acetic acid (IAA) synthesis and metabolism insur2plants indicates that the mutation causes a conditional increase in the pool size of IAA through up-regulation of IAA synthesis.
Delayed-onset ataxia in mice lacking α-tocopherol transfer protein: Model for neuronal degeneration caused by chronic oxidative stress
Authors: Yokota Takanori , Igarashi Keiji , Uchihara Toshiki , Jishage Kou-ichi , Tomita Hiroshi , Inaba Akira , Li Yi , Arita Makoto , Suzuki Hiroshi , Mizusawa Hidehiro , Arai Hiroyuki
Published: July 2002

α-Tocopherol transfer protein (α-TTP) maintains
the concentration of serum α-tocopherol (vitamin E), one of the most
potent fat-soluble antioxidants, by facilitating α-tocopherol export
from the liver. Mutations of the α-TTP gene are linked to
ataxia with isolated vitamin E deficiency (AVED). We produced a model
mouse of AVED by deleting the α-TTP gene, which showed ataxia and
retinal degeneration after 1 year of age. Because the brain α-TTP
functions in maintaining α-tocopherol levels in the brain,
α-tocopherol was completely depleted in the
α-TTP
−/−
mouse brain, and the neurological phenotype
of α-TTP
−/−
mice is much more severe than that of
wild-type mice when maintained on an α-tocopherol-deficient diet.
Lipid peroxidation in α-TTP
−/−
mice brains showed a
significant increase, especially in degenerating neurons.
α-Tocopherol supplementation suppressed lipid peroxidation and almost
completely prevented the development of neurological symptoms. This
therapy almost completely corrects the abnormalities in a mouse model
of human neurodegenerative disease. Moreover,
α-TTP
−/−
mice may prove to be excellent animal models
of delayed onset, slowly progressive neuronal degeneration caused by
chronic oxidative stress.
Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.
Authors: Clinton G M , Rougeot C , Derancourt J , Roger P , Defrenne A , Godyna S , Argraves W S , Rochefort H
Published: July 2002

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.
DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.
Authors: Roskey M T , Juhasz P , Smirnov I P , Takach E J , Martin S A , Haff L A
Published: July 2002

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.
Activating transcription from single stranded DNA.
Authors: Tomonaga T , Levens D
Published: July 2002

Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.
Inhibition of sustained gamma oscillations (35-80 Hz) by fast transient responses in cat visual cortex.
Authors: Kruse W , Eckhorn R
Published: July 2002

Interactions between stimulus-induced oscillations (35-80 Hz) and stimulus-locked nonoscillatory responses were investigated in the visual cortex areas 17 and 18 of anaesthetized cats. A single square-wave luminance grating was used as a visual stimulus during simultaneous recordings from up to seven electrodes. The stimulus movement consisted of a superposition of a smooth movement with a sequence of dynamically changing accelerations. Responses of local groups of neurons at each electrode were studied on the basis of multiple unit activity and local slow field potentials (13-120 Hz). Oscillatory and stimulus-locked components were extracted from multiple unit activity and local slow field potentials and quantified by a combination of temporal and spectral correlation methods. We found fast stimulus-locked components primarily evoked by sudden stimulus accelerations, whereas oscillatory components (35-80 Hz) were induced during slow smooth movements. Oscillations were gradually reduced in amplitude and finally fully suppressed with increasing amplitudes of fast stimulus-locked components. It is argued that suppression of oscillations is necessary to prevent confusion during sequential processing of stationary and fast changing retinal images.
Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.
Authors: Tsuzuki T , Fujii Y , Sakumi K , Tominaga Y , Nakao K , Sekiguchi M , Matsushiro A , Yoshimura Y , MoritaT
Published: July 2002

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.
The structure of bovine F1-ATPase complexed with the antibiotic inhibitor aurovertin B.
Authors: van Raaij M J , Abrahams J P , Leslie A G , Walker J E
Published: July 2002

In the structure of bovine mitochondrial F1-ATPase that was previously determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. Adenylyl-imidodiphosphate is bound to one beta-subunit (betaTP), ADP is bound to the second (betaDP), and no nucleotide is bound to the third (betaE). Here we show that the uncompetitive inhibitor aurovertin B binds to bovine F1 at two equivalent sites in betaTP and betaE, in a cleft between the nucleotide binding and C-terminal domains. In betaDP, the aurovertin B pocket is incomplete and is inaccessible to the inhibitor. The aurovertin B bound to betaTP interacts with alpha-Glu399 in the adjacent alphaTP subunit, whereas the aurovertin B bound to betaE is too distant from alphaE to make an equivalent interaction. Both sites encompass betaArg-412, which was shown by mutational studies to be involved in binding aurovertin. Except for minor changes around the aurovertin pockets, the structure of bovine F1-ATPase is the same as determined previously. Aurovertin B appears to act by preventing closure of the catalytic interfaces, which is essential for a catalytic mechanism involving cyclic interconversion of catalytic sites.
Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids.
Authors: Do T Q , Schultz J R , Clarke C F
Published: July 2002

Coenzyme Q (ubiquinone or Q) plays a well known electron transport function in the respiratory chain, and recent evidence suggests that the reduced form of ubiquinone (QH2) may play a second role as a potent lipid-soluble antioxidant. To probe the function of QH2 as an antioxidant in vivo, we have made use of a Q-deficient strain of Saccharomyces cerevisiae harboring a deletion in the COQ3 gene [Clarke, C. F., Williams, W. & Teruya, J. H. (1991) J. Biol. Chem. 266, 16636-16644]. Q-deficient yeast and the wild-type parental strain were subjected to treatment with polyunsaturated fatty acids, which are prone to autoxidation and breakdown into toxic products. In this study we find that Q-deficient yeast are hypersensitive to the autoxidation products of linolenic acid and other polyunsaturated fatty acids. In contrast, the monounsaturated oleic acid, which is resistant to autoxidative breakdown, has no effect. The hypersensitivity of the coq3delta strains can be prevented by the presence of the COQ3 gene on a single copy plasmid, indicating that the sensitive phenotype results solely from the inability to produce Q. As a result of polyunsaturated fatty acid treatment, there is a marked elevation of lipid hydroperoxides in the coq3 mutant as compared with either wild-type or respiratory-deficient control strains. The hypersensitivity of the Q-deficient mutant can be rescued by the addition of butylated hydroxytoluene, alpha-tocopherol, or trolox, an aqueous soluble vitamin E analog. The results indicate that autoxidation products of polyunsaturated fatty acids mediate the cell killing and that QH2 plays an important role in vivo in protecting eukaryotic cells from these products.
Defective forebrain development in mice lacking gp330/megalin.
Authors: Willnow T E , Hilpert J , Armstrong S A , Rohlmann A , Hammer R E , Burns D K , Herz J
Published: July 2002

gp330/megalin, a member of the low density lipoprotein (LDL) receptor gene family, is expressed on the apical surfaces of epithelial tissues, including the neuroepithelium, where it mediates the endocytic uptake of diverse macromolecules, such as cholesterol-carrying lipoproteins, proteases, and antiproteinases. Megalin knockout mice manifest abnormalities in epithelial tissues including lung and kidney that normally express the protein and they die perinatally from respiratory insufficiency. In brain, impaired proliferation of neuroepithelium produces a holoprosencephalic syndrome, characterized by lack of olfactory bulbs, forebrain fusion, and a common ventricular system. Similar syndromes in humans and animals are caused by insufficient supply of cholesterol during development. Because megalin can bind lipoproteins, we propose that the receptor is part of the maternal-fetal lipoprotein transport system and mediates the endocytic uptake of essential nutrients in the postgastrulation stage.
A common major histocompatibility complex class II allele HLA-DQB1* 0301 is present in clinical variants of pemphigoid.
Authors: Delgado J C , Turbay D , Yunis E J , Yunis J J , Morton E D , Bhol K , Norman R , Alper C A , Good R A , Ahmed R
Published: July 2002

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease seen primarily in elderly persons. It is characterized clinically by the development of tense bullae and by the presence of an antibasement membrane antibody. In BP, the antigens involved in the autoimmunity are epidermal basement membrane peptides BPAg1 and BPAg2. We have compared high resolution typing of major histocompatibility complex class II loci (HLA-DRB1, DQB1) in 21 patients with BP, 17 with ocular cicatricial pemphigoid (OCP), and 22 with oral pemphigoid (OP) to a panel of 218 haplotypes of normal individuals. We found that the three diseases (BP, OCP, and OP) have significant association with DQB1*0301 (P = 0.005, P < 0.0001, and P = 0.001, respectively). The frequencies of alleles DQB1*0302, 0303, and 06, which share a specific amino acid sequence from position 71 to 77 (Thr-Arg-Ala-Glu-Leu-Val-Thr) were also increased (P = 0.01). We suggest that an identical major histocompatibility complex class II allele (DQB1*0301) is a common marker for enhanced susceptibility and that the same amino acid residues in positions 71-77 (DQB1*0301, -0302, -0305, -0602, -0603 alleles) are found in patients with BP, OCP and OP. Our findings propose that the autoimmune response in the three different clinical variants of pemphigoid, involves the recognition by T cells of a class II region of DQB1, bound to a peptide from the basement membrane of conjunctiva, oral mucosa, and skin.
An information theory model of hydrophobic interactions.
Authors: Hummer G , Garde S , García A E , Pohorille A , Pratt L R
Published: July 2002

A molecular model of poorly understood hydrophobic effects is heuristically developed using the methods of information theory. Because primitive hydrophobic effects can be tied to the probability of observing a molecular-sized cavity in the solvent, the probability distribution of the number of solvent centers in a cavity volume is modeled on the basis of the two moments available from the density and radial distribution of oxygen atoms in liquid water. The modeled distribution then yields the probability that no solvent centers are found in the cavity volume. This model is shown to account quantitatively for the central hydrophobic phenomena of cavity formation and association of inert gas solutes. The connection of information theory to statistical thermodynamics provides a basis for clarification of hydrophobic effects. The simplicity and flexibility of the approach suggest that it should permit applications to conformational equilibria of nonpolar solutes and hydrophobic residues in biopolymers.
Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.
Authors: Carter R E , Feldman A R , Coyle J T
Published: July 2002

This report demonstrates that the investigational prostatic carcinoma marker known as the prostate-specific membrane antigen (PSM) possesses hydrolytic activity with the substrate and pharmacologic properties of the N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase that has been characterized in the mammalian nervous system on the basis of its catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) to yield glutamate and N-acetylaspartate and that has been hypothesized to influence glutamatergic signaling processes. The immunoscreening of a rat brain cDNA expression library with anti-NAALADase antisera identified a 1428-base partial cDNA that shares 86% sequence identity with 1428 bases of the human PSM cDNA [Israeli, R. S., Powell, C. T., Fair, W. R. & Heston, W.D.W. (1993) Cancer Res. 53, 227-230]. A cDNA containing the entire PSM open reading frame was subsequently isolated by reverse transcription-PCR from the PSM-positive prostate carcinoma cell line LNCaP. Transient transfection of this cDNA into two NAALADase-negative cell lines conferred NAAG-hydrolyzing activity that was inhibited by the NAALADase inhibitors quisqualic acid and beta-NAAG. Thus we demonstrate a PSM-encoded function and identify a NAALADase-encoding cDNA. Northern analyses identify at least six transcripts that are variably expressed in NAALADase-positive but not in NAALADase-negative rat tissues and human cell lines; therefore, PSM and/or related molecular species appear to account for NAAG hydrolysis in the nervous system. These results also raise questions about the role of PSM in both normal and pathologic prostate epithelial-cell function.
Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.
Authors: Savage C , Das P , Finelli A L , Townsend S R , Sun C Y , Baird S E , Padgett R W
Published: July 2002

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.
DNA strand annealing is promoted by the yeast Rad52 protein.
Authors: Mortensen U H , Bendixen C , Sunjevaric I , Rothstein R
Published: July 2002

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.
Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.
Authors: Bennett R J , West S C
Published: July 2002

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.
A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing
Authors: Baeyens Katrien J. , De Bondt Hendrik L. , Pardi Arthur , Holbrook Stephen R.
Published: July 2002

The crystal structure of the RNA dodecamer 5′-GGCC(GAAA)GGCC-3′ has
been determined from x-ray diffraction data to 2.3-Å resolution. In
the crystal, these oligomers form double helices around twofold
symmetry axes. Four consecutive non-Watson–Crick base pairs make up an
internal loop in the middle of the duplex, including sheared G·A
pairs and novel asymmetric A·A pairs. This internal loop sequence
produces a significant curvature and narrowing of the double helix. The
helix is curved by 34° from end to end and the diameter is narrowed
by 24% in the internal loop. A Mn
2+
ion is bound directly
to the N7 of the first guanine in the Watson–Crick region following
the internal loop and the phosphate of the preceding residue. This
Mn
2+
location corresponds to a metal binding site observed
in the hammerhead catalytic RNA.
Direct physical interaction between DnaG primase and DnaB helicase of
Escherichia coli
is necessary for optimal synthesis of primer RNA
Authors: Lu Ya-Bin , Ratnakar Pillarisetty V. A. L. , Mohanty Bidyut K. , Bastia Deepak
Published: July 2002

The primase DnaG of
Escherichia coli
requires the
participation of the replicative helicase DnaB for optimal synthesis of
primer RNA for lagging strand replication. However, previous studies
had not determined whether the activation of the primase or its loading
on the template was accomplished by a helicase-mediated structural
alteration of the single-stranded DNA or by a direct physical
interaction between the DnaB and the DnaG proteins. In this paper we
present evidence supporting direct interaction between the two
proteins. We have mapped the surfaces of interaction on both DnaG and
DnaB and show further that mutations that reduce the physical
interaction also cause a significant reduction in primer synthesis.
Thus, the physical interaction reported here appears to be
physiologically significant.
A critical arginine residue mediates cooperativity in the contact interface between transcription factors NFAT and AP-1
Authors: Peterson Blake R. , Sun Li Jing , Verdine Gregory L.
Published: July 2002

The heterologous transcription factors NFAT and AP-1 coordinately regulate cytokine gene expression through cooperative binding to precisely juxtaposed DNA recognition elements. The molecular origins of cooperativity in the binding of NFAT and AP-1 to DNA are poorly understood. Herein we have used yeast one-hybrid screening and alanine-scanning mutagenesis to identify residues in AP-1 that affect cooperative interactions with NFAT on DNA. Mutation of a single conserved Arg residue to Ala in the cJun spacer region (R285A) led to a virtually complete abolition of cooperative interactions with NFAT. The DNA-binding activity of AP-1 alone was unaffected by the cJun R285A mutation, thus indicating that this residue influences cooperative binding only. Ala-scanning mutations elsewhere in AP-1, including the cFos subunit, revealed no other strongly interacting single positions. We thus conclude that NFAT contacts AP-1 in the spacer region of the cJun subunit, making an especially important contact to R285, and that these interactions drive formation of the cooperative NFAT/AP-1/DNA complex. These results provide a general strategy for selectively ablating cooperativity between transcription factors without affecting their ability to act alone and yield insights into the structural basis for coordinate regulation of gene expression.
Tumor progression to the metastatic state involves structural modifications in DNA markedly different from those associated with primary tumor formation
Authors: Malins Donald C. , Polissar Nayak L. , Gunselman Sandra J.
Published: July 2002

Wavenumber–absorbance relationships of infrared spectra of DNA analyzed by principal components analysis may be expressed as points in space. Each point represents a highly discriminating measure of DNA structure. Structural modifications of DNA, such as those induced by free radicals, alter vibrational and rotational motion and consequently change the spatial location of the points. Using this technology to analyze breast tumor DNA, we revealed a 94° difference in direction between the progression of normal DNA → primary tumor DNA and the progression of primary tumor DNA → metastatic tumor DNA (P< 0.001). This sharp directional change was accompanied by a substantial increase in the structural diversity of the metastatic tumor DNA (P= 0.003), which, on the basis of the volume of the core cluster of points, could comprise as many as 11 × 109different phenotypes. This suggests that the heterogeneity and varied physiological properties known to characterize malignant tumor cell populations may at least partially arise from these diverse phenotypes. The evidence suggests that the progression to the metastatic state involves structural modifications in DNA that are markedly different from the modifications associated with the formation of the primary tumor. Overall, the findings of this and earlier studies imply that the observed DNA alterations are a pivotal factor in the etiology of breast cancer and a formidable barrier to overcome in intervention to control the disease. In terms of cancer etiology and prediction, the technology described has potentially wide application to studies in which the structural status of DNA is an important consideration.
Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA
Authors: Cheung Vivian G. , Nelson Stanley F.
Published: July 2002

Genetic analysis of limiting quantities of genomic DNA play an
important role in DNA forensics, paleoarcheology, genetic disease
diagnosis, genetic linkage analysis, and genetic diversity studies. We
have tested the ability of degenerate oligonucleotide primed polymerase
chain reaction (DOP-PCR) to amplify picogram quantities of human
genomic DNA for the purpose of increasing the amount of template for
genotyping with microsatellite repeat markers. DNA was uniformly
amplified at a large number of typable loci throughout the human genome
with starting template DNAs from as little as 15 pg to as much as 400
ng. A much greater-fold enrichment was seen for the smaller genomic
DOP-PCRs. All markers tested were amplified from starting genomic DNAs
in the range of 0.6–40 ng with amplifications of 200- to 600-fold. The
DOP-PCR-amplified genomic DNA was an excellent and reliable template
for genotyping with microsatellites, which give distinct bands with no
increase in stutter artifact on di-, tri-, and tetranucleotide repeats.
There appears to be equal amplification of genomic DNA from 55 of 55
tested discrete microsatellites implying near complete coverage of the
human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold
whole genome amplification of human genomic DNA for genotypic analysis.