bluuebunny/crossref_metadata_2025 huggingface dataset:
A synthetic all
d
-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion
Authors: Pritsker Moshe , Jones Philip , Blumenthal Robert , Shai Yechiel
Published: July 2002

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997)
J. Biol. Chem.
272, 13496–13505]. Here, we show that a synthetic all
d
-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance–fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides’ chirality is not necessarily required for peptide–peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.
Brain mechanisms of quantity are similar in 5-year-old children and adults
Authors: Temple Elise , Posner Michael I.
Published: July 2002

Both 5-year-old children and adults determine the quantity of a number by the use of a similar parietal lobe mechanism. Event related potentials indicate that input from Arabic digits and from dot patterns reach areas involved in determining quantity about 200 ms after input. However, voluntary key presses indicating the relation of the input to the quantity five take almost three times as long in children. The ability to trace the networks of brain areas involved in the learning of school subjects should aid in the design and testing of educational methods.
Activity of the yeast
MNN1
α-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases
Authors: Wiggins Christine A. R. , Munro Sean
Published: July 2002

A wide diversity of biological molecules are modified by the addition of sugar residues, and a large number of glycosyltransferases have been identified that are responsible for these reactions. Despite catalyzing closely related reactions, many of these transferases show little apparent sequence homology. By comparing two apparently unrelated families of yeast Golgi mannosyltransferases, a short motif containing two aspartate residues was observed that was conserved in both groups of proteins. Mutagenesis of one of the members of these families, the α-1,3-mannosyltransferase Mnn1p, showed that altering either of these aspartates eliminates all enzymatic activity. These changes do not appear to affect the overall folding and assembly of Mnn1p. A similar aspartate-containing sequence was found to be conserved in a diverse range of other glycosyltransferase families, much more frequently than would be expected by chance, suggesting that it is a feature of the catalytic site, or an element of a structural fold, shared by many glycosyltransferases.
Disruption of a PEX1–PEX6 interaction is the most common cause of the neurologic disorders Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease
Authors: Geisbrecht Brian V. , Collins Cynthia S. , Reuber Bernadette E. , Gould Stephen J.
Published: July 2002

Peroxisomal matrix protein import requires the action of two AAA ATPases, PEX1 and PEX6. Mutations in either the
PEX1
or
PEX6
gene are the most common cause of the lethal neurologic disorders Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease and account for disease in 80% of all such patients. We report here that overexpression of
PEX6
can suppress the phenotypes of certain
PEX1
-deficient cells, that overexpression of
PEX1
can suppress the phenotypes of certain
PEX6
-deficient cells, and that these instances of suppression are allele-specific and require partial activity of the mutated gene. In addition to genetic evidence for interaction between
PEX1
and
PEX6
, we find that the PEX1 and PEX6 proteins interact in the yeast two-hybrid assay and physically associate with one another
in vitro
. We previously identified a missense mutation in
PEX1
, G843D, which attenuates
PEX1
function and is the most common cause of these diseases, present in one-third of all such patients. The G843D mutation attenuates the interaction between PEX1 and PEX6 in both the two-hybrid system and
in vitro
and appears to be suppressed by overexpression of
PEX6
. We conclude that PEX1 and PEX6 form a complex of central importance to peroxisome biogenesis and that mutations affecting this complex constitute the most common cause of the Zellweger syndrome spectrum of diseases.
Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas
Authors: Coste Alix de La , Romagnolo Béatrice , Billuart Pierre , Renard Claire-Angélique , Buendia Marie-Annick , Soubrane Olivier , Fabre Monique , Chelly Jamel , Beldjord Cherif , Kahn Axel , Perret Christine
Published: July 2002

Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-β-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. We have examined the status of the β-catenin gene in different transgenic mouse lines of HCC obtained with the oncogenes c-
myc
or H-
ras
. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the β-catenin gene similar to those found in colon cancers and melanomas. These alterations in the β-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of β-catenin and thus to carcinogenesis. We then analyzed human HCCs and found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations led to the accumulation of β-catenin in the nucleus. Thus alterations in the β-catenin gene frequently are selected for during liver tumorigenesis and suggest that disregulation of the Wnt-β-catenin pathway is a major event in the development of HCC in humans and mice.
Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation
Authors: Chan Jonah R. , Phillips Lornie J. , Glaser Michael
Published: July 2002

Dexamethasone and progesterone have been found to accelerate the time of initiation and enhance the rate of myelin synthesis in Schwann cell/neuronal cocultures. The expression of mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3β-hydroxysteroid dehydrogenase (converts pregnenolone to progesterone), and the progesterone receptor were detected and markedly induced during peak myelin formation in the cocultures. The mRNA for the glucocorticoid receptor was detected, but was found to be constituitively expressed. In addition, the specific activity of 3β-hydroxysteroid dehydrogenase was measured and found to increase by 10-fold. The mRNA for cytochrome P450scc and 3β-hydroxysteroid dehydrogenase also were found to be induced during the differentiation of O-2A precursor cells to oligodendrocytes. Fibroblast growth factor and platelet-derived growth factor were found to have proliferative effects on Schwann cells, but they had no effect on the initiation or the rate of myelin formation. These results demonstrate that myelin-forming cells have inducible enzymes responsible for steroid biosynthesis and suggest a critical role for endogenous steroid hormones in signaling the initiation and enhancing the rate of myelin formation.
Structure elucidation and chemical synthesis of stigmolone, a novel type of prokaryotic pheromone
Authors: Hull William E. , Berkessel Albrecht , Plaga Wulf
Published: July 2002

Approximately 2 μmol of a novel prokaryotic pheromone, involved in starvation-induced aggregation and formation of fruiting bodies by the myxobacterium
Stigmatella aurantiaca
, were isolated by a large-scale elution procedure. The pheromone was purified by HPLC, and high-resolution MS, IR,
1
H-NMR, and
13
C-NMR were used to identify the active substance as the hydroxy ketone 2,5,8-trimethyl-8-hydroxy-nonan-4-one, which has been named stigmolone. The analysis was complicated by a solvent-dependent equilibrium between stigmolone and the cyclic enol-ether 3,4-dihydro-2,2,5-trimethyl-6-(2-methylpropyl)-2
H
-pyran formed by intramolecular nucleophilic attack of the 8-OH group at the ketone C4 followed by loss of H
2
O. Both compounds were synthesized chemically, and their structures were confirmed by NMR analysis. Natural and synthetic stigmolone have the same biological activity at
ca.
1 nM concentration.
A family of human receptors structurally related to
Drosophila
Toll
Authors: Rock Fernando L. , Hardiman Gary , Timans Jackie C. , Kastelein Robert A. , Bazan J. Fernando
Published: July 2002

The discovery of sequence homology between the cytoplasmic domains of
Drosophila
Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to
Drosophila
Toll in both intra- and extracellular segments. Five human Toll-like receptors—named TLRs 1–5—are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role—akin to Toll in the dorsoventralization of the
Drosophila
embryo—as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence
in situ
hybridization and sequence-tagged site database analyses, we also show that the cognate
Tlr
genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel β/α fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.
Genome scan of human systemic lupus erythematosus: Evidence for linkage on chromosome 1q in African-American pedigrees
Authors: Moser Kathy L. , Neas Barbara R. , Salmon Jane E. , Yu Hua , Gray-McGuire Courtney , Asundi Neeraj , Bruner Gail R. , Fox Jerome , Kelly Jennifer , Henshall Stephanie , Bacino Debra , Dietz Myron , Hogue Robert , Koelsch Gerald , Nightingale Lydia , Shaver Tim , Abdou Nabih I. , Albert Daniel A. , Carson Craig , Petri Michelle , Treadwell Edward L. , James Judith A. , Harley John B.
Published: July 2002

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies against intracellular antigens including DNA, ribosomal P, Ro (SS-A), La (SS-B), and the spliceosome. Etiology is suspected to involve genetic and environmental factors. Evidence of genetic involvement includes: associations with HLA-DR3, HLA-DR2, FcÎ³ receptors (FcÎ³R) IIA and IIIA, and hereditary complement component deficiencies, as well as familial aggregation, monozygotic twin concordance >20%, Î»
s
> 10, purported linkage at 1q41â42, and inbred mouse strains that consistently develop lupus. We have completed a genome scan in 94 extended multiplex pedigrees by using model-based linkage analysis. Potential [log
10
of the odds for linkage (lod) > 2.0] SLE loci have been identified at chromosomes 1q41, 1q23, and 11q14â23 in African-Americans; 14q11, 4p15, 11q25, 2q32, 19q13, 6q26â27, and 12p12â11 in European-Americans; and 1q23, 13q32, 20q13, and 1q31 in all pedigrees combined. An effect for the FcÎ³RIIA candidate polymorphism) at 1q23 (lod = 3.37 in African-Americans) is syntenic with linkage in a murine model of lupus. Sib-pair and multipoint nonparametric analyses also support linkage (
P
< 0.05) at nine loci detected by using two-point lod score analysis (lod > 2.0). Our results are consistent with the presumed complexity of genetic susceptibility to SLE and illustrate racial origin is likely to influence the specific nature of these genetic effects.
Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation
Authors: Nishizawa Kazuhisa , Freund Christian , Li Jing , Wagner Gerhard , Reinherz Ellis L.
Published: July 2002

An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also found in several yeast andCaenorhabditis elegansproteins of unknown function. In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains regulates protein–protein interactions.
The
Arabidopsis thaliana RPM1
disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response
Authors: Boyes Douglas C. , Nam Jaesung , Dangl Jeffery L.
Published: July 2002

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (
avr
) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (
R
) gene products. Members of the NBS-LRR class of
R
genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from
Arabidopsis thaliana
that confers resistance to
Pseudomonas syringae
expressing either
avrRpm1
or
avrB
. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.
Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies
Authors: O’Donnell Kerry , Kistler H. Corby , Cigelnik Elizabeth , Ploetz Randy C.
Published: July 2002

Panama disease of banana, caused by the fungus
Fusarium oxysporum
f. sp.
cubense
, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has indicated that
F. oxysporum
f. sp.
cubense
consists of several clonal lineages that may be genetically distant. In this study we tested whether lineages of the Panama disease pathogen have a monophyletic origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1α and the mitochondrial small subunit ribosomal RNA genes for
F. oxysporum
strains from banana, pathogenic strains from other hosts and putatively nonpathogenic isolates of
F. oxysporum
. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The tree inferred from the combined dataset resolved five lineages corresponding to “
F. oxysporum
f. sp.
cubense
” with a large dichotomy between two taxa represented by strains most commonly isolated from bananas with Panama disease. The results also demonstrate that the latter two taxa have significantly different chromosome numbers.
F. oxysporum
isolates collected as nonpathogenic or pathogenic to other hosts that have very similar or identical elongation factor 1α and mitochondrial small subunit genotypes as banana pathogens were shown to cause little or no disease on banana. Taken together, these results indicate Panama disease of banana is caused by fungi with independent evolutionary origins.
Engineering an enzyme to resist boiling
Authors: Van den Burg Bertus , Vriend Gert , Veltman Oene R. , Venema Gerard , Eijsink Vincent G. H.
Published: July 2002

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from
Bacillus stearothermophilus
, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100°C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state (“rigidifying” mutations such as Gly → Ala, Ala → Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37°C.
Rabies virus quasispecies: Implications for pathogenesis
Authors: Morimoto Kinjiro , Hooper D. Craig , Carbaugh Heather , Fu Zhen Fang , Koprowski Hilary , Dietzschold Bernhard
Published: July 2002

Passage of the mouse-adapted rabies virus strain CVS-24 (where CVS is challenge virus standard) in BHK cells results in the rapid selection of a dominant variant designated CVS-B2c that differs genotypically and phenotypically from the dominant variant CVS-N2c present in mouse-brain- or neuroblastoma-cell-passaged CVS-24. The glycoprotein of CVS-B2c has 10 amino acid substitutions compared with that of CVS-N2c. Because CVS-B2c can be reproducibly selected in BHK cells, it is likely to be a conserved minor subpopulation of CVS-24. CVS-N2c is more neurotropic
in vitro
and
in vivo
than CVS-B2c, which replicates more readily in nonneuronal cells
in vitro
and
in vivo
. These characteristics appear to be relevant to the pathogenicity of the two variants. CVS-N2c is more pathogenic for adult mice than CVS-B2c. In contrast, CVS-B2c is more pathogenic for neonatal mice. These differences in pathogenicity are reflected in the selection pattern when mixtures of CVS-N2c and CVS-B2c were used to infect neonatal and adult mice. Although CVS-N2c was highly selected in adult mice, no selection for either variant was seen in neonates, suggesting that certain aspects of development, such as maturation of the nervous and immune systems, may contribute to the selection process. We speculate that the existence of different variants within a rabies virus strain may facilitate the virus in overcoming barriers to its spread, both within the host and between species.
Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment
Authors: Hobbs Susan K. , Monsky Wayne L. , Yuan Fan , Roberts W. Gregory , Griffith Linda , Torchilin Vladimir P. , Jain Rakesh K.
Published: July 2002

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (
i
) spatially, by growing tumors in subcutaneous or cranial locations and (
ii
) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (
i
) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (
ii
) delivery may be reduced during tumor regression induced by hormonal ablation, and (
iii
) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.
Sucrose is a signal molecule in assimilate partitioning
Authors: Chiou Tzyy-Jen , Bush Daniel R.
Published: July 2002

The proton–sucrose symporter mediates the key transport step in the resource distribution system that allows many plants to function as multicellular organisms. In the results reported here, we identify sucrose as a signaling molecule in a previously undescribed signal-transduction pathway that regulates the symporter. Sucrose symporter activity declined in plasma membrane vesicles isolated from leaves fed exogenous sucrose via the xylem transpiration stream. Symporter activity dropped to 35–50% of water controls when the leaves were fed 100 mM sucrose and to 20–25% of controls with 250 mM sucrose. In contrast, alanine symporter and glucose transporter activities did not change in response to sucrose treatments. Decreased sucrose symporter activity was detectable after 8 h and reached a maximum by 24 h. Kinetic analysis of transport activity showed a decrease in
V
max
. RNA gel blot analysis revealed a decrease in symporter message levels, suggesting a drop in transcriptional activity or a decrease in mRNA stability. Control experiments showed that these responses were not the result of changing osmotic conditions. Equal molar concentrations of hexoses did not elicit the response, and mannoheptulose, a hexokinase inhibitor, did not block the sucrose effect. These data are consistent with a sucrose-specific response pathway that is not mediated by hexokinase as the sugar sensor. Sucrose-dependent changes in the sucrose symporter were reversible, suggesting this sucrose-sensing pathway can modulate transport activity as a function of changing sucrose concentrations in the leaf. These results demonstrate the existence of a signaling pathway that can control assimilate partitioning at the level of phloem translocation.
The force exerted by a molecular motor
Authors: Fisher Michael E. , Kolomeisky Anatoly B.
Published: July 2002

The stochastic driving force exerted by a single molecular motor (e.g., a kinesin, or myosin) moving on a periodic molecular track (microtubule, actin filament, etc.) is discussed from a general viewpoint open to experimental test. An elementary “barometric” relation for the driving force is introduced that (
i
) applies to a range of kinetic and stochastic models, (
ii
) is consistent with more elaborate expressions entailing explicit representations of externally applied loads, and (
iii
) sufficiently close to thermal equlibrium, satisfies an Einstein-type relation in terms of the velocity and diffusion coefficient of the (load-free) motor. Even in the simplest two-state models, the velocity-vs.-load plots exhibit a variety of contrasting shapes (including nonmonotonic behavior). Previously suggested bounds on the driving force are shown to be inapplicable in general by analyzing discrete jump models with waiting time distributions.
Supraspinal contributions to hyperalgesia
Authors: Urban M. O. , Gebhart G. F.
Published: July 2002

Tissue injury is associated with sensitization of nociceptors and
subsequent changes in the excitability of central (spinal) neurons,
termed central sensitization. Nociceptor sensitization and central
sensitization are considered to underlie, respectively, development of
primary hyperalgesia and secondary hyperalgesia. Because central
sensitization is considered to reflect plasticity at spinal synapses,
the spinal cord has been the principal focus of studies of mechanisms
of hyperalgesia. Not surprisingly, glutamate, acting at a spinal
N
-methyl-
d
-aspartate (NMDA) receptor, has
been implicated in development of secondary hyperalgesia associated
with somatic, neural, and visceral structures. Downstream of NMDA
receptor activation, spinal nitric oxide (NO

⋅

),
protein kinase C, and other mediators have been implicated in
maintaining such hyperalgesia. Accumulating evidence, however, reveals
a significant contribution of supraspinal influences to development and
maintenance of hyperalgesia. Spinal cord transection prevents
development of secondary, but not primary, mechanical and/or thermal
hyperalgesia after topical mustard oil application, carrageenan
inflammation, or nerve-root ligation. Similarly, inactivation of the
rostral ventromedial medulla (RVM) attenuates hyperalgesia and central
sensitization in several models of persistent pain. Inhibition of
medullary NMDA receptors or NO

⋅

generation
attenuates somatic and visceral hyperalgesia. In support, topical
mustard oil application or colonic inflammation increases expression of
NO

⋅

synthase in the RVM. These data suggest a
prominent role for the RVM in mediating the sensitization of spinal
neurons and development of secondary hyperalgesia. Results to date
suggest that peripheral injury and persistent input engage
spinobulbospinal mechanisms that may be the prepotent contributors to
central sensitization and development of secondary hyperalgesia.
Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17
Authors: Varani Luca , Hasegawa Masato , Spillantini Maria Grazia , Smith Michael J. , Murrell Jill R. , Ghetti Bernardino , Klug Aaron , Goedert Michel , Varani Gabriele
Published: July 2002

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splice-donor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10.
Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity
Authors: Storms Robert W. , Trujillo Aliana P. , Springer James B. , Shah Lisa , Colvin O. Michael , Ludeman Susan M. , Smith Clay
Published: July 2002

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity, we developed a fluorescent substrate for ALDH, termed BODIPY aminoacetaldehyde (BAAA), and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSCloALDHbrcells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSCloALDHbrpopulation was depleted of lineage-committed cells, 40–90% pure for CD34+CD38lo/−cells, and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together, these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well.
Enzymatic activation of oleuropein: A protein crosslinker used as a chemical defense in the privet tree
Authors: Konno Kotaro , Hirayama Chikara , Yasui Hiroe , Nakamura Masatoshi
Published: July 2002

Leaves of the privet tree,
Ligustrum obtusifolium
, contain a large amount of oleuropein, a phenolic secoiridoid glycoside, which is stably kept in a compartment separate from activating enzymes. When the leaf tissue is destroyed by herbivores, enzymes localized in organelles start to activate oleuropein into a very strong protein denaturant that has protein-crosslinking and lysine-decreasing activities. These activities are stronger than ever reported from plant systems and have adverse effects against herbivores by decreasing the nutritive value of dietary protein completely. We report here that strong oleuropein-specific β-glucosidase in organelles activates oleuropein by converting the secoiridoid glucoside moiety of oleuropein into a glutaraldehyde-like structure, which is also an α,β-unsaturated aldehyde. Oleuropein activated by β-glucosidase had very strong protein-denaturing, protein-crosslinking, and lysine-alkylating activities that are very similar to, but stronger than, those of glutaraldehyde. Aucubin, another iridoid glycoside, had similar activities after β-glucosidase treatment. We also detected polyphenol oxidase activity in organelles that activate the dihydroxyphenolic moiety to have protein-crosslinking activities. These data suggest that the privet tree has developed an effective defense mechanism with oleuropein, a unique multivalent alkylator ideal as a protein-crosslinker. Our results that iridoid glycosides are precursors of alkylators may elucidate the chemical bases that underlie various bioactivities and ecological roles of iridoid glycosides.
Digital PCR
Authors: Vogelstein Bert , Kinzler Kenneth W.
Published: July 2002

The identification of predefined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. Here, we describe an approach for transforming the exponential, analog nature of the PCR into a linear, digital signal suitable for this purpose. Single molecules are isolated by dilution and individually amplified by PCR; each product is then analyzed separately for the presence of mutations by using fluorescent probes. The feasibility of the approach is demonstrated through the detection of a mutant
ras
oncogene in the stool of patients with colorectal cancer. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
Two major forms of DNA (cytosine-5) methyltransferase in human somatic tissues
Authors: Hsu Duen-Wei , Lin Meng-Jau , Lee Tai-Lin , Wen Shau-Ching , Chen Xin , Shen C.-K. James
Published: July 2002

Thus far, only one major form of vertebrate DNA (cytosine-5) methyltransferase (CpG MTase, EC
2.1.1.37
) has been identified, cloned, and extensively studied. This enzyme, dnmt1, has been hypothesized to be responsible for most of the maintenance as well as the
de novo
methylation activities occurring in the somatic cells of vertebrates. We now report the discovery of another abundant species of CpG MTase in various types of human cell lines and somatic tissues. Interestingly, the mRNA encoding this CpG MTase results from alternative splicing of the primary transcript from the
Dnmt1
gene, which incorporates in-frame an additional 48 nt between exons 4 and 5. Furthermore, this 48-nt exon sequence is derived from the first, or the most upstream, copy of a set of seven different Alu repeats located in intron 4. The ratios of expression of this mRNA to the expression of the previously known, shorter Dnmt1 mRNA species, as estimated by semiquantitative reverse transcription–PCR analysis, range from two-thirds to three-sevenths. This alternative splicing scheme of the Dnmt1 transcript seems to be conserved in the higher primates. We suggest that the originally described and the recently discovered forms of CpG MTase be named dnmt1-a and dnmt1-b, respectively. The evolutionary and biological implications of this finding are discussed in relation to the cellular functions of the CpG residues and the CpG MTases.
A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem
Authors: Hooper Lora V. , Xu Jian , Falk Per G. , Midtvedt Tore , Gordon Jeffrey I.
Published: July 2002

Little is known about how members of the indigenous microflora interact with their mammalian hosts to establish mutually beneficial relationships. We have used a gnotobiotic mouse model to show that
Bacteroides thetaiotaomicron
, a component of the intestinal microflora of mice and humans, uses a repressor, FucR, as a molecular sensor of
l
-fucose availability. FucR coordinates expression of an operon encoding enzymes in the
l
-fucose metabolic pathway with expression of another locus that regulates production of fucosylated glycans in intestinal enterocytes. Genetic and biochemical studies indicate that FucR does this by using fucose as an inducer at one locus and as a corepressor at the other locus. Coordinating this commensal’s immediate nutritional requirements with production of a host-derived energy source is consistent with its need to enter and persist within a competitive ecosystem.
Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry
Authors: Walker Matthew , Zhang Xue-Zhong , Jiang Wei , Trinick John , White Howard D.
Published: July 2002

The mechanism of binding of myosin subfragment-1 (S1) to actin in the absence of nucleotides was studied by a combination of stopped-flow fluorescence and ms time resolution electron microscopy. The fluorescence data were obtained by using pyrene-labeled actin and exhibit a lag phase. This demonstrates the presence of a transient intermediate after the collision complex and before the formation of the stable “rigor” complex. The transient intermediate predominates 2–15 ms after mixing, whereas the rigor complex predominates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixing revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed the “arrowhead” appearance characteristic of rigor. The most likely explanation of the disorder of the transient intermediate is that the binding is through one or more flexible loops on the surfaces of the proteins. The transition from disordered to ordered binding is likely to be part of the force-generating step in muscle.
Functional neuroanatomical double dissociation of mnemonic and executive control processes contributing to working memory performance
Authors: Postle Bradley R. , Berger Jeffrey S. , D’Esposito Mark
Published: July 2002

We used event-related functional MRI to investigate the neural bases of two categories of mental processes believed to contribute to performance of an alphabetization working memory task: memory storage and memory manipulation. Our delayed-response tasks required memory for the identity and position-in-the-display of items in two- or five-letter memory sets (to identify load-sensitive regions) or memory for the identity and relative position-in-the-alphabet of items in five-letter memory sets (to identify manipulation-sensitive regions). Results revealed voxels in the left perisylvian cortex of five of five subjects showing load sensitivity (as contrasted with alphabetization-sensitive voxels in this region in only one subject) and voxels of dorsolateral prefrontal cortex in all subjects showing alphabetization sensitivity (as contrasted with load-sensitive voxels in this region in two subjects). This double dissociation was reliable at the group level. These data are consistent with the hypothesis that the nonmnemonic executive control processes that can contribute to working memory function are primarily prefrontal cortex-mediated whereas mnemonic processes necessary for working memory storage are primarily posteriorly mediated. More broadly, they support the view that working memory is a faculty that arises from the coordinated interaction of computationally and neuroanatomically dissociable processes.
Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence
Authors: Ismail Abdelbagi M. , Hall Anthony E. , Close Timothy J.
Published: July 2002

Dehydrins (DHNs, LEA D-11) are plant proteins present during environmental stresses associated with dehydration or low temperatures and during seed maturation. Functions of DHNs have not yet been defined. Earlier, we hypothesized that a ≈35-kDa DHN and membrane properties that reduce electrolyte leakage from seeds confer chilling tolerance during seedling emergence of cowpea (
Vigna unguiculata
L. Walp.) in an additive and independent manner. Evidence for this hypothesis was not rigorous because it was based on correlations of presence/absence of the DHN and slow electrolyte leakage with chilling tolerance in closely related cowpea lines that have some other genetic differences. Here, we provide more compelling genetic evidence for involvement of the DHN in chilling tolerance of cowpea. We developed near-isogenic lines by backcrossing. We isolated and determined the sequence of a cDNA corresponding to the ≈35-kDa DHN and used gene-specific oligonucleotides derived from it to test the genetic linkage between the DHN presence/absence trait and the DHN structural gene. We tested for association between the DHN presence/absence trait and both low-temperature seed emergence and electrolyte leakage. We show that allelic differences in the
Dhn
structural gene map to the same position as the DHN protein presence/absence trait and that the presence of the ≈35-kDa DHN is indeed associated with chilling tolerance during seedling emergence, independent of electrolyte leakage effects. Two types of allelic variation in the
Dhn
gene were identified in the protein-coding region, deletion of one Φ-segment from the DHN-negative lines and two single amino acid substitutions.
Regulation of mitochondrial ATP synthesis by calcium: Evidence for a long-term metabolic priming
Authors: Jouaville Laurence S. , Pinton Paolo , Bastianutto Carlo , Rutter Guy A. , Rizzuto Rosario
Published: July 2002

In recent years, mitochondria have emerged as important targets of agonist-dependent increases in cytosolic Ca
2+
concentration. Here, we analyzed the significance of Ca
2+
signals for the modulation of organelle function by directly measuring mitochondrial and cytosolic ATP levels ([ATP]
m
and [ATP]
c
, respectively) with specifically targeted chimeras of the ATP-dependent photoprotein luciferase. In both HeLa cells and primary cultures of skeletal myotubes, stimulation with agonists evoking cytosolic and mitochondrial Ca
2+
signals caused increases in [ATP]
m
and [ATP]
c
that depended on two parameters: (
i
) the amplitude of the Ca
2+
rise in the mitochondrial matrix, and (
ii
) the availability of mitochondrial substrates. Moreover, the Ca
2+
elevation induced a long-lasting priming that persisted long after agonist washout and caused a major increase in [ATP]
m
upon addition of oxidative substrates. These results demonstrate a direct role of mitochondrial Ca
2+
in driving ATP production and unravel a form of cellular memory that allows a prolonged metabolic activation in stimulated cells.
Biodiversity and ecosystem productivity in a fluctuating environment: The insurance hypothesis
Authors: Yachi Shigeo , Loreau Michel
Published: July 2002

Although the effect of biodiversity on ecosystem functioning has
become a major focus in ecology, its significance in a fluctuating
environment is still poorly understood. According to the insurance
hypothesis, biodiversity insures ecosystems against declines in their
functioning because many species provide greater guarantees that some
will maintain functioning even if others fail. Here we examine this
hypothesis theoretically. We develop a general stochastic dynamic model
to assess the effects of species richness on the expected temporal mean
and variance of ecosystem processes such as productivity, based on
individual species’ productivity responses to environmental
fluctuations. Our model shows two major insurance effects of species
richness on ecosystem productivity: (
i
) a buffering
effect, i.e., a reduction in the temporal variance of productivity, and
(
ii
) a performance-enhancing effect, i.e., an increase
in the temporal mean of productivity. The strength of these insurance
effects is determined by three factors: (
i
) the way
ecosystem productivity is determined by individual species responses to
environmental fluctuations, (
ii
) the degree of
asynchronicity of these responses, and (
iii
) the
detailed form of these responses. In particular, the greater the
variance of the species responses, the lower the species richness at
which the temporal mean of the ecosystem process saturates and the
ecosystem becomes redundant. These results provide a strong theoretical
foundation for the insurance hypothesis, which proves to be a
fundamental principle for understanding the long-term effects of
biodiversity on ecosystem processes.
Expression pattern and, surprisingly, gene length shape codon usage inCaenorhabditis,Drosophila, andArabidopsis
Authors: Duret Laurent , Mouchiroud Dominique
Published: July 2002

We measured the expression pattern and analyzed codon usage in 8,133, 1,550, and 2,917 genes, respectively, fromCaenorhabditis elegans,Drosophila melanogaster, andArabidopsis thaliana. In those three species, we observed a clear correlation between codon usage and gene expression levels and showed that this correlation is not due to a mutational bias. This provides direct evidence for selection on silent sites in those three distantly related multicellular eukaryotes. Surprisingly, there is a strong negative correlation between codon usage and protein length. This effect is not due to a smaller size of highly expressed proteins. Thus, for a same-expression pattern, the selective pressure on codon usage appears to be lower in genes encoding long rather than short proteins. This puzzling observation is not predicted by any of the current models of selection on codon usage and thus raises the question of how translation efficiency affects fitness in multicellular organisms.
Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates
Authors: Palmer Jeffrey D. , Adams Keith L. , Cho Yangrae , Parkinson Christopher L. , Qiu Yin-Long , Song Keming
Published: July 2002

We summarize our recent studies showing that angiosperm
mitochondrial (mt) genomes have experienced remarkably high rates of
gene loss and concomitant transfer to the nucleus and of intron
acquisition by horizontal transfer. Moreover, we find substantial
lineage-specific variation in rates of these structural mutations and
also point mutations. These findings mostly arise from a Southern blot
survey of gene and intron distribution in 281 diverse angiosperms.
These blots reveal numerous losses of mt ribosomal protein genes but,
with one exception, only rare loss of respiratory genes. Some lineages
of angiosperms have kept all of their mt ribosomal protein genes
whereas others have lost most of them. These many losses appear to
reflect remarkably high (and variable) rates of functional transfer of
mt ribosomal protein genes to the nucleus in angiosperms. The recent
transfer of
cox2
to the nucleus in legumes provides both
an example of interorganellar gene transfer in action and a starting
point for discussion of the roles of mechanistic and selective forces
in determining the distribution of genetic labor between organellar and
nuclear genomes. Plant mt genomes also acquire sequences by horizontal
transfer. A striking example of this is a homing group I intron in the
mt
cox1
gene. This extraordinarily invasive mobile
element has probably been acquired over 1,000 times separately during
angiosperm evolution via a recent wave of cross-species horizontal
transfers. Finally, whereas all previously examined angiosperm mtDNAs
have low rates of synonymous substitutions, mtDNAs of two distantly
related angiosperms have highly accelerated substitution rates.
Increased baseline occupancy of D
2
receptors by dopamine in schizophrenia
Authors: Abi-Dargham Anissa , Rodenhiser Janine , Printz David , Zea-Ponce Yolanda , Gil Roberto , Kegeles Lawrence S. , Weiss Richard , Cooper Thomas B. , Mann J. John , Van Heertum Ronald L. , Gorman Jack M. , Laruelle Marc
Published: July 2002

The classical dopamine hypothesis of schizophrenia postulates a hyperactivity of dopaminergic transmission at the D
2
receptor. We measured
in vivo
occupancy of striatal D
2
receptors by dopamine in 18 untreated patients with schizophrenia and 18 matched controls, by comparing D
2
receptor availability before and during pharmacologically induced acute dopamine depletion. Acute depletion of intrasynaptic dopamine resulted in a larger increase in D
2
receptor availability in patients with schizophrenia (19% ± 11%) compared with control subjects (9% ± 7%,
P
= 0.003). The increased occupancy of D
2
receptors by dopamine occurred both in first-episode neuroleptic-naive patients and in previously treated chronic patients experiencing an episode of illness exacerbation. In addition, elevated synaptic dopamine was predictive of good treatment response of positive symptoms to antipsychotic drugs. This finding provides direct evidence of increased stimulation of D
2
receptors by dopamine in schizophrenia, consistent with increased phasic activity of dopaminergic neurons.