bluuebunny/crossref_metadata_2025 huggingface dataset:
Transmission electron microscopic observations of amorphous NiZr alloy formation by solid-state reaction
Authors: Newcomb S. B. , Tu K. N.
Published: July 2002

The interdiffusion of nickel and zirconium by a solid-state reaction to form an amorphous NiZr alloy has been followed by transmission electron microscopy. Cross-sectional examination of NiZr bilayers annealed at 300 °C has shown the formation of amorphous NiZr and the development of large voids in the nickel neighboring the Ni/NiZr interface. Our results indicate that nickel is the dominant diffusing species in the amorphous alloy and that the diffusion requires a defect mechanism. Prolonged annealing at 300 °C results in the formation of a crystalline NiZr phase, which co-exists with the amorphous alloy.
Observation of shock waves and cooling waves in the laser ablation of Kapton films in air
Authors: Koren G.
Published: July 2002

Deflections of a He-Ne laser beam in excimer and CO2 laser-produced plumes from Kapton films were measured. They show that in addition to the existence of a shock wave observed earlier, a slower cooling wave is also developed. A model calculation is presented which explains the present experimental data as well as previous results regarding the decrease of etching efficiency with increasing ambient gas pressure.
Current fluctuations and silicon oxide wear-out in metal-oxide-semiconductor tunnel diodes
Authors: Farmer K. R. , Saletti R. , Buhrman R. A.
Published: July 2002

We have studied the behavior of very thin oxide (∼20 Å) metal-oxide-semiconductor tunnel diodes under high electrical field bias. These devices do not usually experience catastrophic breakdown, but can be worn out at high fields through the creation of a low barrier tunneling path. The effective area of the path increases during stress, while the barrier height remains essentially constant at ∼1 eV. The formation of the path is correlated to the presence of multilevel switching fluctuations in the diode current. The same complex fluctuations and excess currents are seen in oxides up to 70 Å where the fluctuations show up as noisy precursors to catastrophic breakdown.
Cytoplasmic–nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation
Authors: Kim Jae Eun , Chen Jie
Published: July 2002

Translation initiation is one of the key events regulated in response to mitogenic stimulation and nutrient availability, tightly coupled to mammalian cell cycle progression and growth. FKBP12-rapamycin-associated protein (FRAP; also named mTOR or RAFT1), a member of theataxia telangiectasiamutated (ATM)-related kinase family, governs a rapamycin-sensitive membrane-to-cytoplasm signaling cascade that modulates translation initiation via p70 S6 kinase (p70s6k) and eIF-4E binding protein 1 (4E-BP1). Our studies reported here reveal a surprising regulatory mechanism of this signaling, which involves cytoplasmic–nuclear shuttling of FRAP. By using leptomycin B (LMB), a specific inhibitor of nuclear export receptor Crm1, we show that FRAP is a cytoplasmic–nuclear shuttling protein. Inhibition of FRAP nuclear export by LMB coincides with diminished p70s6kactivation and 4E-BP1 phosphorylation. Further investigation by altering FRAP's nuclear shuttling activity with exogenous nuclear import and export signals has yielded results that are consistent with a direct link between nuclear shuttling of FRAP and mitogenic stimulation of p70s6kactivation and 4E-BP1 phosphorylation. Furthermore, by using a reporter system, we provide evidence suggesting that nuclear shuttling of FRAP regulates mitogen-stimulated rapamycin-sensitive translation initiation. These findings uncover a function for the nucleus in the direct regulation of the protein synthesis machinery via extracellular signals.
Translocation of Cockayne syndrome group A protein to the nuclear matrix: Possible relevance to transcription-coupled DNA repair
Authors: Kamiuchi Shinya , Saijo Masafumi , Citterio Elisabetta , de Jager Martijn , Hoeijmakers Jan H. J. , Tanaka Kiyoji
Published: July 2002

Transcription-coupled repair (TCR) efficiently removes a variety of
lesions from the transcribed strand of active genes. By allowing rapid
resumption of RNA synthesis, the process is of major importance for
cellular resistance to transcription-blocking genotoxic damage.
Mutations in the Cockayne syndrome group A or B (CSA or CSB) gene
result in defective TCR. However, the exact mechanism of TCR in
mammalian cells remains to be elucidated. We found that CSA protein is
rapidly translocated to the nuclear matrix after UV irradiation. The
translocation of CSA was independent of Xeroderma pigmentosum group
C, which is specific to the global genome repair subpathway of
nucleotide excision repair (NER) and of the core NER factor Xeroderma
pigmentosum group A but required the CSB protein. In
UV-irradiated cells, CSA protein colocalized with the
hyperphosphorylated form of RNA polymerase II, engaged in transcription
elongation. The translocation of CSA was also induced by treatment
of the cells with cisplatin or hydrogen peroxide, both of which produce
damage that is subjected to TCR but not induced by treatment with
dimethyl sulfate, which produces damage that is not subjected to TCR.
The hydrogen peroxide-induced translocation of CSA was also CSB
dependent. These findings establish a link between TCR and the nuclear
matrix mediated by CSA.
The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice
Authors: Joyce Charles W. , Amar Marcelo J. A. , Lambert Gilles , Vaisman Boris L. , Paigen Beverly , Najib-Fruchart Jamila , Hoyt Robert F. , Neufeld Edward D. , Remaley Alan T. , Fredrickson Donald S. , Brewer H. Bryan , Santamarina-Fojo Silvia
Published: July 2002

Identification of mutations in the ABCA1 transporter (ABCA1)
as the genetic defect in Tangier disease has generated interest in
modulating atherogenic risk by enhancing ABCA1 gene expression. To
investigate the role of ABCA1 in atherogenesis, we analyzed
diet-induced atherosclerosis in transgenic mice overexpressing human
ABCA1 (hABCA1-Tg) and spontaneous lesion formation in
hABCA1-Tg × apoE-knockout (KO) mice. Overexpression of hABCA1 in
C57BL/6 mice resulted in a unique anti-atherogenic profile
characterized by decreased plasma cholesterol (63%), cholesteryl ester
(63%), free cholesterol (67%), non-high density lipoprotein
(HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but
markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and
apoE (2.8-fold) levels. These beneficial changes in the lipid profile
led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg
mice. In marked contrast, ABCA1 overexpression had a minimal effect on
the plasma lipid profile of apoE-KO mice and resulted in a 2- to
2.6-fold increase in aortic lesion area. These combined results
indicate that overexpression of ABCA1 in C57BL/6 mice on a high
cholesterol diet results in an atheroprotective lipoprotein profile and
decreased atherosclerosis, and thus provide previously
undocumented
in vivo
evidence of an
anti-atherogenic role for the ABCA1 transporter. In contrast,
overexpression of ABCA1 in an apoE-KO background led to increased
atherosclerosis, further substantiating the important role of apoE in
macrophage cholesterol metabolism and atherogenesis. In summary, these
results establish that, in the presence of apoE, overexpression of
ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism
and reduces atherosclerosis
in vivo
, and indicate that
pharmacological agents that will increase ABCA1 expression may reduce
atherogenic risk in humans.
A stilbene synthase from Japanese red pine (
Pinus densiflora
): Implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis
Authors: Kodan Atsushi , Kuroda Hiroyuki , Sakai Fukumi
Published: July 2002

Stilbene synthase (STS) and chalcone synthase (CHS) are plant-specific polyketide synthases that play key roles in the stilbenoid and flavonoid biosyntheses, respectively. We have recently isolated from
Pinus densiflora
three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) and one CHS cDNA (PDCHSX). We then heterologously expressed these cDNAs in
Escherichia coli
and characterized their properties. An unusual STS isozyme, PDSTS3, lacks the common C-terminal extension of STS because of a frame-shift mutation and shows the highest pinosylvin-forming activity among the STSs tested. Pinosylvin was shown to be a potent inhibitor of PDCHSX (
K
i
= 6 μM) as well as PDSTS2 (
K
i
= 13 μM), which presumably maintains the balance between the stilbenoid and flavonoid biosyntheses. PDSTS3 was insensitive to product inhibition. We identified PDSTS3 in the pine seedlings as well as full-length STS. The data provide evidence that PDSTS3 is involved in the potential regulation of the stilbenoid and flavonoid biosynthetic pathways in pine trees.
Circularly permuted green fluorescent proteins engineered to sense Ca
2+
Authors: Nagai Takeharu , Sawano Asako , Park Eun Sun , Miyawaki Atsushi
Published: July 2002

To visualize Ca
2+
-dependent protein–protein
interactions in living cells by fluorescence readouts, we used a
circularly permuted green fluorescent protein (cpGFP), in which the
amino and carboxyl portions had been interchanged and reconnected by a
short spacer between the original termini. The cpGFP was fused to
calmodulin and its target peptide, M13. The chimeric
protein, which we have named “pericam,” was fluorescent and its
spectral properties changed reversibly with the amount of
Ca
2+
, probably because of the interaction between
calmodulin and M13 leading to an alteration of the
environment surrounding the chromophore. Three types of pericam were
obtained by mutating several amino acids adjacent to the chromophore.
Of these, “flash-pericam” became brighter with Ca
2+
,
whereas “inverse-pericam” dimmed. On the other hand,
“ratiometric-pericam” had an excitation wavelength changing in a
Ca
2+
-dependent manner. All of the pericams expressed in
HeLa cells were able to monitor free Ca
2+
dynamics, such as
Ca
2+
oscillations in the cytosol and the nucleus.
Ca
2+
imaging using high-speed confocal line-scanning
microscopy and a flash-pericam allowed to detect the free propagation
of Ca
2+
ions across the nuclear envelope. Then, free
Ca
2+
concentrations in the nucleus and mitochondria were
simultaneously measured by using ratiometric-pericams having
appropriate localization signals, revealing that extra-mitochondrial
Ca
2+
transients caused rapid changes in the concentration
of mitochondrial Ca
2+
. Finally, a “split-pericam” was
made by deleting the linker in the flash-pericam. The
Ca
2+
-dependent interaction between calmodulin
and M13 in HeLa cells was monitored by the association of the two
halves of GFP, neither of which was fluorescent by itself.
CD4+T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production
Authors: Zeng Gang , Wang Xiang , Robbins Paul F. , Rosenberg Steven A. , Wang Rong-Fu
Published: July 2002

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8+T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4+T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43–70% of Caucasians. The ESO p157–170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4+T cells as well as HLA-A2-restricted CD8+T cells. Thus, the ESO p157–170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4+and CD8+T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4+T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4+T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.
F-spondin is a contact-repellent molecule for embryonic motor neurons
Authors: Tzarfati-Majar Vered , Burstyn-Cohen Tal , Klar Avihu
Published: July 2002

The floor plate plays a key role in patterning axonal trajectory in
the embryonic spinal cord by providing both long-range and local
guidance cues that promote or inhibit axonal growth toward and across
the ventral midline of the spinal cord, thus acting as an intermediate
target for a number of crossing (commissural) and noncrossing (motor)
axons. F-spondin, a secreted adhesion molecule expressed in the
embryonic floor plate and the caudal somite of birds, plays a dual role
in patterning the nervous system. It promotes adhesion and outgrowth of
commissural axons and inhibits adhesion of neural crest cells. In the
current study, we demonstrate that outgrowth of embryonic motor axons
also is inhibited by F-spondin protein in a contact-repulsion fashion.
Three independent lines of evidence support our hypothesis:
substrate-attached F-spondin inhibits outgrowth of dissociated motor
neurons in an outgrowth assay; F-spondin elicits acute growth cone
collapse when applied to cultured motor neurons; and challenging
ventral spinal cord explants with aggregates of HEK 293 cells
expressing F-spondin, causes contact-repulsion of motor neurites.
Structural–functional studies demonstrate that the processed
carboxyl-half protein that contains the thrombospondin type 1
repeats is more prominent in inhibiting outgrowth, suggesting that the
processing of F-spondin is important for enhancing its inhibitory
activity.
Desorption/ionization on silicon (DIOS): A diverse mass spectrometry platform for protein characterization
Authors: Thomas John J. , Shen Zhouxin , Crowell John E. , Finn M. G. , Siuzdak Gary
Published: July 2002

Since the advent of matrix-assisted laser desorption/ionization
and electrospray ionization, mass spectrometry has played an
increasingly important role in protein functional characterization,
identification, and structural analysis. Expanding this role,
desorption/ionization on silicon (DIOS) is a new approach that allows
for the analysis of proteins and related small molecules. Despite the
absence of matrix, DIOS-MS yields little or no fragmentation and is
relatively tolerant of moderate amounts of contaminants commonly found
in biological samples. Here, functional assays were performed on an
esterase, a glycosidase, a lipase, as well as exo- and endoproteases by
using enzyme-specific substrates. Enzyme activity also was monitored in
the presence of inhibitors, successfully demonstrating the ability of
DIOS to be used as an inhibitor screen. Because DIOS is a matrix-free
desorption technique, it also can be used as a platform for multiple
analyses to be performed on the same protein. This unique advantage was
demonstrated with acetylcholine esterase for qualitative and
quantitative characterization and also by its subsequent identification
directly from the DIOS platform.
Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions
Authors: Vasquez Karen M. , Christensen Jesper , Li Lei , Finch Rick A. , Glazer Peter M.
Published: July 2002

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.
Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression
Authors: Iwashima Makio , Takamatsu Masako , Yamagishi Hiroko , Hatanaka Yasue , Huang Yi-Ying , McGinty Courtnie , Yamasaki Sho , Koike Toru
Published: July 2002

Shc, a prototypic adapter molecule, has been implicated in T cell receptor (TCR) signal transduction, but its role has not been identified clearly. Here we report that Shc is essential for TCR-induced IL-2 production but is dispensable for CD69 or CD25 expression. Engagement of TCR in mutant Jurkat T cells lacking Shc fails to produce IL-2 because of impaired mitogen-activated protein kinase activation. Activation of c-Rel, a transcription factor essential for IL-2 expression, was impaired also. In contrast, activation of nuclear factor of activated T cell and expression of CD69/CD25 were comparable between the mutant and wild-type Jurkat cells. These defects were rescued by expression of exogenous Shc. Activation of c-Rel using the estrogen receptor fusion protein restored the activation of the IL-2 promoter in an estrogen-dependent manner. These results show that Shc plays an essential role in the TCR-induced activation of c-Rel and the IL-2 promoter.
Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy
Authors: Ulrich Andreas , Schmitz Arndt A. P. , Braun Thomas , Yuan Tao , Vogel Hans J. , Vergères Guy
Published: July 2002

MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic segment that mediates binding of MRP to CaM. We show that the effector domain is in a polar environment in free MRP, suggesting exposure to water, in agreement with a rod-shaped structure of the protein. The effector domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties of the myristoylated and unmyristoylated forms of a construct in which a tryptophan residue was introduced at position 4 close to the myristoylated N terminus of MRP suggests that the lipid moiety is also involved in the interaction of MRP with CaM.
Modulation of tRNA
Ala
identity by inorganic pyrophosphatase
Authors: Wolfson Alexey D. , Uhlenbeck Olke C.
Published: July 2002

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3′-
32
P-labeled tRNA. When applied to
Escherichia coli
alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA
Ala
. The effect of tRNA
Ala
identity mutations on both aminoacylation efficiency (
k
cat
/
K
M
) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the
k
cat
/
K
M
value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between
in vivo
and
in vitro
analysis of tRNA
Ala
identity.
Selective adsorption ofl- andd-amino acids on calcite: Implications for biochemical homochirality
Authors: Hazen Robert M. , Filley Timothy R. , Goodfriend Glenn A.
Published: July 2002

The emergence of biochemical homochirality was a key step in the origin of life, yet prebiotic mechanisms for chiral separation are not well constrained. Here we demonstrate a geochemically plausible scenario for chiral separation of amino acids by adsorption on mineral surfaces. Crystals of the common rock-forming mineral calcite (CaCO3), when immersed in a racemic aspartic acid solution, display significant adsorption and chiral selectivity ofd- andl-enantiomers on pairs of mirror-related crystal-growth surfaces. This selective adsorption is greater on crystals with terraced surface textures, which indicates thatd- andl-aspartic acid concentrate along step-like linear growth features. Thus, selective adsorption of linear arrays ofd- andl-amino acids on calcite, with subsequent condensation polymerization, represents a plausible geochemical mechanism for the production of homochiral polypeptides on the prebiotic Earth.
Simple neural networks for the amplification and utilization of small changes in neuron firing rates
Authors: Adair Robert K.
Published: July 2002

I describe physiologically plausible “voter-coincidence” neural networks such that secondary “coincidence” neurons fire on the simultaneous receipt of sufficiently large sets of input pulses from primary sets of neurons. The networks operate such that the firing rate of the secondary, output neurons increases (or decreases) sharply when the mean firing rate of primary neurons increases (or decreases) to a much smaller degree. In certain sensory systems, signals that are generally smaller than the noise levels of individual primary detectors, are manifest in very small increases in the firing rates of sets of afferent neurons. For such systems, this kind of network can act to generate relatively large changes in the firing rate of secondary “coincidence” neurons. These differential amplification systems can be cascaded to generate sharp, “yes–no” spike signals that can direct behavioral responses.
Evolutionary EST analysis identifies rapidly evolving male reproductive proteins inDrosophila
Authors: Swanson Willie J. , Clark Andrew G. , Waldrip-Dail Heidi M. , Wolfner Mariana F. , Aquadro Charles F.
Published: July 2002

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland ofDrosophila simulansto their orthologs in its close relativeDrosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen (∼11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that ∼90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.
Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster
Authors: Chiu Hsien-Tai , Hubbard Brian K. , Shah Aseema N. , Eide Jonathan , Fredenburg Ross A. , Walsh Christopher T. , Khosla Chaitan
Published: July 2002

Streptomyces lavendulae
produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein–protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an α-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning
ca.
50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or α-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.
The evolution of methicillin resistance in
Staphylococcus aureus
: Similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones
Authors: Crisóstomo M. Inês , Westh Henrik , Tomasz Alexander , Chung Marilyn , Oliveira Duarte C. , de Lencastre Hermínia
Published: July 2002

The key genetic component of methicillin resistance, the
mecA
determinant, is not native to
Staphylococcus
aureus
. Thus, the evolution of methicillin-resistant
S.
aureus
(MRSA) must have begun with the acquisition of the
mecA
determinant from an unknown heterologous source
some time before the first reported appearance of MRSA isolates in
clinical specimens in the U.K. and Denmark (in the early 1960s). We
compared the genetic backgrounds and phenotypes of a group of
methicillin-susceptible
S. aureus
(MSSA) isolates to the
properties of MRSA strains isolated in Denmark and the U.K. during the
same time period, and also to the genetic profiles of contemporary
epidemic clones of MRSA. All early MRSA isolates resembled a large
group of the early MSSA blood isolates in phenotypic and genetic
properties, including phage group, antibiotype (resistance to
penicillin, streptomycin, and tetracycline), pulsed-field gel
electrophoresis pattern, and
spaA
type and multilocus
sequence type, strongly suggesting that the early MSSA examined here
represented the progeny of a strain that served as one of the first
S. aureus
recipients of the methicillin-resistance
determinant in Europe. The genetic background of this group of early
MSSA isolates was also very similar to that of the widely disseminated
contemporary “Iberian clone” of MRSA, suggesting that genetic
determinants present in early MSSA and essential for some aspects of
the epidemicity and/or virulence of these strains may have been
retained by this highly successful contemporary MRSA lineage.
Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ
Authors: Pohl Peter , Saparov Sapar M. , Borgnia Mario J. , Agre Peter
Published: July 2002

Aquaporins are membrane channels selectively permeated by
water or water plus glycerol. Conflicting reports have described ion
conductance associated with some water channels, raising the question
of whether ion conductance is a general property of the aquaporin
family. To clarify this question, a defined system was developed to
simultaneously measure water permeability and ion conductance. The
Escherichia coli
water channel aquaporin-Z (AqpZ) was
studied, because it is a highly stable tetramer. Planar lipid bilayers
were formed from unilamellar vesicles containing purified AqpZ. The
hydraulic conductivity of bilayers made from the total extract of
E. coli
lipids increased 3-fold if reconstituted with
AqpZ, but electric conductance was unchanged. No channel activity was
detected under voltage-clamp conditions, indicating that less than one
in 10
9
transport events is electrogenic. Microelectrode
measurements were simultaneously undertaken adjacent to the membrane.
Changes in sodium concentration profiles accompanying transmembrane
water flow permitted calculation of the activation energies: 14
kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid
bilayers containing AqpZ. Neither the water permeability nor the
electric conductivity exhibited voltage dependence. This sensitive
system demonstrated that AqpZ is permeated by water but not charged
ions and should permit direct analyses of putative electrogenic
properties of other aquaporins.
Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll
Authors: Matysik Jörg , Alia  , Gast Peter , van Gorkom Hans J. , Hoff Arnold J. , de Groot Huub J. M.
Published: July 2002

We report13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance13C provide information on the electronic structure of the primary electron donor P680(chlorophyllamolecules absorbing around 680 nm) and on the pzspin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophylla(Chla) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chlaradical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chlamacroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.
The
Arabidopsis
male-sterile mutant,
opr3
, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis
Authors: Stintzi Annick , Browse John
Published: July 2002

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of
Arabidopsis
,
opr3
. The
opr3
mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in
opr3
are alleviated by transformation of the mutant with an
OPR3
cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.
Cattle lack vascular receptors for
Escherichia coli
O157:H7 Shiga toxins
Authors: Pruimboom-Brees Ingrid M. , Morgan Tim W. , Ackermann Mark R. , Nystrom Evelyn D. , Samuel James E. , Cornick Nancy A. , Moon Harley W.
Published: July 2002

Escherichia coli
O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of
E. coli
O157:H7. We were interested in knowing why cattle are tolerant to infection with
E. coli
O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb
3
), for receptivity to Stx binding
in vitro,
and for susceptibility to the enterotoxic effects of Stx
in vivo
. TLC was used to detect Gb
3
in tissues from a newborn calf. Gb
3
was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (
n
= 3) and adult cattle (
n
= 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb
3
and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable
E. coli
O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for
E. coli
O157:H7.
Cysteinyl-tRNA synthetase is not essential for viability of the archaeon
Methanococcus maripaludis
Authors: Stathopoulos Constantinos , Kim Wonduck , Li Tong , Anderson Iain , Deutsch Britta , Palioura Sotiria , Whitman William , Söll Dieter
Published: July 2002

The methanogenic archaea
Methanocaldococcus
jannaschii
and
Methanothermobacter
thermautotrophicus
contain a dual-specificity prolyl-tRNA
synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA)
and cysteinyl-tRNA (Cys-tRNA) suitable for
in vivo
translation. This intriguing enzyme may even perform its dual role in
organisms that possess a canonical single-specificity cysteinyl-tRNA
synthetase (CysRS), raising the question as to whether this latter
aminoacyl-tRNA synthetase is indeed required for cell viability. To
test the postulate that all synthetase genes are essential, we
disrupted the
cysS
gene (encoding CysRS) of
Methanococcus maripaludis
. The knockout strain was
viable under normal growth conditions. Biochemical analysis showed that
the pure
M. maripaludis
ProCysRS was capable of forming
Cys-tRNA, implying that the dual-specificity enzyme compensates
in vivo
for the loss of CysRS. The canonical CysRS has a
higher affinity for cysteine than ProCysRS, a reason why
M.
maripaludis
may have acquired
cysS
by a late
lateral gene transfer. These data challenge the notion that all twenty
aminoacyl-tRNA synthetases are essential for the viability of a cell.
Increased anxiety and synaptic plasticity in estrogen receptor β-deficient mice
Authors: Krȩżel Wojciech , Dupont Sonia , Krust Andrée , Chambon Pierre , Chapman Paul F.
Published: July 2002

Estrogens are powerful modulators of neuronal physiology and in humans may affect a broad range of functions, including reproductive, emotional, and cognitive behaviors. We studied the contribution of estrogen receptors (ERs) in modulation of emotional processes and analyzed the effects of deleting ERα or ERβ in mice. Behavior consistent with increased anxiety was observed principally in ERβ mutant females and was associated with a reduced threshold for the induction of synaptic plasticity in the basolateral amygdala. Local increase of 5-hydroxytryptamine 1a receptor expression in medial amygdala may contribute to these changes. Our data show that, particularly in females, there is an important role for ERβ-mediated estrogen signaling in the processing of emotional behavior.
The genome sequence of the facultative intracellular pathogen
Brucella melitensis
Authors: DelVecchio Vito G. , Kapatral Vinayak , Redkar Rajendra J. , Patra Guy , Mujer Cesar , Los Tamara , Ivanova Natalia , Anderson Iain , Bhattacharyya Anamitra , Lykidis Athanasios , Reznik Gary , Jablonski Lynn , Larsen Niels , D'Souza Mark , Bernal Axel , Mazur Mikhail , Goltsman Eugene , Selkov Eugene , Elzer Philip H. , Hagius Sue , O'Callaghan David , Letesson Jean-Jacques , Haselkorn Robert , Kyrpides Nikos , Overbeek Ross
Published: July 2002

Brucella melitensis
is a facultative intracellular
bacterial pathogen that causes abortion in goats and sheep and Malta
fever in humans. The genome of
B. melitensis
strain 16M
was sequenced and found to contain 3,294,935 bp distributed over two
circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197
ORFs. By using the bioinformatics suite ERGO,
2,487 (78%) ORFs were assigned functions. The origins of replication
of the two chromosomes are similar to those of other
α-proteobacteria. Housekeeping genes, including those involved in DNA
replication, transcription, translation, core metabolism, and cell wall
biosynthesis, are distributed on both chromosomes. Type I, II, and III
secretion systems are absent, but genes encoding
sec
-dependent,
sec
-independent, and
flagella-specific type III, type IV, and type V secretion systems as
well as adhesins, invasins, and hemolysins were identified. Several
features of the
B. melitensis
genome are similar to
those of the symbiotic
Sinorhizobium meliloti
.
The plant oncogene
rolD
encodes a functional ornithine cyclodeaminase
Authors: Trovato Maurizio , Maras Bruno , Linhares Francisco , Costantino Paolo
Published: July 2002

The plant oncogene
rolD
stimulates the reproductive phase transition in plants. We define here the function of its gene product. We show that the RolD protein bears sequence homology with ornithine cyclodeaminase, an uncommon enzyme of specialized-niche eubacteria and archaea that catalyzes the unusual NAD
+
-dependent conversion of ornithine to proline. To confirm the prediction of the bioinformatic analysis, the RolD protein was expressed in
Escherichia coli
and purified. An ornithine-dependent NAD
+
reduction that can be ascribed only to ornithine cyclodeaminase (OCD) activity was detected both in bacterial extracts containing RolD and in assays on the purified RolD protein. Furthermore, OCD activity was observed in soluble extracts from plants overexpressing
rolD
. The role of
rolD
in plant pathogenesis and its effect on plant reproductive development are discussed in light of the newly demonstrated enzymatic activity of its gene product.
Diversity of T cell repertoire shaped by a single peptide ligand is critically affected by its amino acid residue at a T cell receptor contact
Authors: Fukui Yoshinori , Oono Takamasa , Cabaniols Jean-Pierre , Nakao Kazuki , Hirokawa Katsuiku , Inayoshi Ayumi , Sanui Terukazu , Kanellopoulos Jean , Iwata Eiko , Noda Mayuko , Katsuki Motoya , Kourilsky Philippe , Sasazuki Takehiko
Published: July 2002

T cell differentiation in the thymus is driven by positive selection through the interaction of αβ T cell receptors (TCRs) with self-peptides bound to self-major histocompatibility complex molecules, yet the influence of the peptide sequence on this process remains unknown. To address this issue, we have compared CD4+T cell differentiation between two sets of mouse lines in which MHC class II I-Abmolecules are occupied with either Eα chain-derived peptide (pEα) or its variant,p60K, with one amino acid substitution from leucine to lysine at P5 residue of TCR contacts. Here, we show that despite the comparable expression of I-Ab–peptide complex in the thymus, this substitution from leucine to lysine affects efficiency of positive selection, resulting in extremely small numbers of CD4+T cells to be selected to mature on I-Ab–p60K complex. Furthermore, we show that, although I-Ab–pEα complex selects diverse T cells, T cell repertoire shaped by I-Ab–p60K complex is markedly constrained. Our findings thus suggest that positive selection is both specific and degenerate, depending on the amino acid residues at TCR contacts of the selecting self-peptides.
Association between divergence and interspersed repeats in mammalian noncoding genomic DNA
Authors: Chiaromonte Francesca , Yang Shan , Elnitski Laura , Yap Von Bing , Miller Webb , Hardison Ross C.
Published: July 2002

The amount of noncoding genomic DNA sequence that aligns
between human and mouse varies substantially in different regions of
their genomes, and the amount of repetitive DNA also varies. In this
report, we show that divergence in noncoding nonrepetitive DNA is
strongly correlated with the amount of repetitive DNA in a
region. We investigated aligned DNA in four large genomic
regions with finished human sequence and almost or completely finished
mouse sequence. These regions, totaling 5.89 Mb of DNA, are on
different chromosomes and vary in their base composition. An analysis
based on sliding windows of 10 kb shows that the fraction of aligned
noncoding nonrepetitive DNA and the fraction of repetitive DNA are
negatively correlated, both at the level of an entire region and
locally within it. This conclusion is strongly supported by a
randomization study, in which repetitive elements are removed and
randomly relocated along the sequences. Thus, regions of noncoding
genomic DNA that accumulated fewer point mutations since the
primate–rodent divergence also suffered fewer retrotransposition
events. These results indicate that some regions of the genome are more
“flexible” over the time scale of mammalian evolution, being able
to accommodate many point mutations and insertions, whereas other
regions are more “rigid” and accumulate fewer changes. Stronger
conservation is generally interpreted as indicating more extensive or
more important function. The evidence presented here of correlated
variation in the rates of different evolutionary processes across
noncoding DNA must be considered in assessing such conservation for
evidence of selection.
Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.
Authors: Sell D R , Lane M A , Johnson W A , Masoro E J , Mock O B , Reiser K M , Fogarty J F , Cutler R G , Ingram D K , Roth G S , Monnier V M
Published: July 2002

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.
Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.
Authors: Nielsen S , Terris J , Smith C P , Hediger M A , Ecelbarger C A , Knepper M A
Published: July 2002

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.