bluuebunny/crossref_metadata_2025 huggingface dataset:
Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs
Authors: Chalasani Geetha , Dai Zhenhua , Konieczny Bogumila T. , Baddoura Fady K. , Lakkis Fadi G.
Published: July 2002

The allospecifc T cell population responding to a transplanted organ consists of both naïve and memory lymphocytes. Although it is established that naive T cells are activated by antigen within the organized structures of secondary lymphoid organs (the spleen, lymph nodes, and mucosal lympoid tissues), it is not clear whether memory T cell activation and propagation depend on homing to these organs. To answer this question, we investigated whether allospecific naïve or memory T cells can mediate acute cardiac allograft rejection in mutant mice that lack all of their secondary lymphoid tissues. The results of our experiments demonstrated that antigen-experienced memory T cells have two advantages over naïve T cells: (i) memory T cells mount a vigorous immune response that leads to allograft rejection independent of secondary lymphoid organs; and (ii) memory T cells generate more memory T cells without homing to secondary lymphoid organs. These unique properties of memory T cells were further confirmed by showing that memory-like T cells that arise from the homeostatic proliferation of naive T cells in the absence of antigenic stimulation are suboptimal at rejecting allografts and do not generate memory T cells in mice devoid of secondary lymphoid tissues.
Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE
Authors: Hu Zonglin , Gogol Edward P. , Lutkenhaus Joe
Published: July 2002

Selection of the division site in
Escherichia coli
is regulated by the
min
system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE. In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles. We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicles into tubes. Stimulation of the MinD ATPase by addition of MinE led to disassembly of the tubes and the release of MinD from the vesicles. It is proposed that this MinE-regulated dynamic assembly of MinD underlies MinD oscillation.
The pK
a
of His-24 in the folding transition state of apomyoglobin
Authors: Jamin Marc , Geierstanger Bernhard , Baldwin Robert L.
Published: July 2002

In native apomyoglobin, His-24 cannot be protonated, although at pH
4 the native protein forms a molten globule folding intermediate in
which the histidine residues are readily protonated. The inability to
protonate His-24 in the native protein dramatically affects the
unfolding/refolding kinetics, as demonstrated by simulations for a
simple model. Kinetic data for wild type and for a mutant lacking
His-24 are analyzed. The pK
a
values of histidine residues
in native apomyoglobin are known from earlier studies, and the average
histidine pK
a
in the molten globule is determined from the
pH dependence of the equilibrium between the native and molten globule
forms. Analysis of the pH-dependent unfolding/refolding kinetics
reveals that the average pK
a
of the histidine residues,
including His-24, is closely similar in the folding transition state to
the value found in the molten globule intermediate. Consequently,
protonation of His-24 is not a barrier to refolding of the molten
globule to the native protein. Instead, the normal pK
a
of
His-24 in the transition state, coupled with its inaccessibility in the
native state, promotes fast unfolding at low pH. The analysis of the
wild-type results is confirmed and extended by using the wild-type
parameters to fit the unfolding kinetics of a mutant lacking His-24.
Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin
Authors: Neufang Gitta , Fürstenberger Gerhard , Heidt Markus , Marks Friedrich , Müller-Decker Karin
Published: July 2002

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.
Rapid genetic analysis of
Helicobacter pylori
gastric mucosal colonization in suckling mice
Authors: Guo Betty P. , Mekalanos John J.
Published: July 2002

Previously described animal models for
Helicobacter pylori
infection have been limited by cumbersome host requirements (e.g., germ-free conditions or unusual species) or are applicable to only special subsets of
H. pylori
strains (e.g., fresh clinical isolates or animal-adapted derivatives). Here, we report that 5- to 6-day-old outbred CD-1 (ICR) suckling mice support 24-h colonization of all
H. pylori
strains tested (SS1, 26695 SmR-1, 43504 SmR-1, and G27 SmR-1), including lab-passaged strains that cannot be adapted for colonization of adult animals. Total colony-forming units (cfu) recovered from infection with lab-passaged strains did not differ from those with mouse-adapted SS1. We also tested this model's ability to detect colonization defects in strains carrying mutations in known virulence genes by coinfecting with wild-type
H. pylori
and measuring differential recovery. This competition assay identified colonization defects in several classes of known attenuated mutants, including those defective in acid resistance (
ureA
), metabolism (
frdA
), motility (
motB
), and chemotaxis (
cheY
). A mutant defective in
copA
(copper transporting P-type ATPase) is nonattenuated in adult and infant mice. Possibly because of the limited duration of infection, our model did not identify defects in vacuolating cytotoxin (
vacA
) or γ-glutamyltranspeptidase (
ggt
) as attenuating, in contrast to results from other animal models. We also identified a new virulence gene (HP0507) encoding a conserved hypothetical protein, which is important for colonization in our model. The suckling mouse model offers a rapid method to identify colonization defects in any
H. pylori
strain and may have utility as a new tool for studying immunity to primary infection.
A gene network model accounting for development and evolution of mammalian teeth
Authors: Salazar-Ciudad Isaac , Jernvall Jukka
Published: July 2002

Generation of morphological diversity remains a challenge for evolutionary biologists because it is unclear how an ultimately finite number of genes involved in initial pattern formation integrates with morphogenesis. Ideally, models used to search for the simplest developmental principles on how genes produce form should account for both developmental process and evolutionary change. Here we present a model reproducing the morphology of mammalian teeth by integrating experimental data on gene interactions and growth into a morphodynamic mechanism in which developing morphology has a causal role in patterning. The model predicts the course of tooth-shape development in different mammalian species and also reproduces key transitions in evolution. Furthermore, we reproduce the known expression patterns of several genes involved in tooth development and their dynamics over developmental time. Large morphological effects frequently can be achieved by small changes, according to this model, and similar morphologies can be produced by different changes. This finding may be consistent with why predicting the morphological outcomes of molecular experiments is challenging. Nevertheless, models incorporating morphology and gene activity show promise for linking genotypes to phenotypes.
Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the F
O
sector of
Escherichia coli
ATP synthase
Authors: Hutcheon Marcus L. , Duncan Thomas M. , Ngai Helen , Cross Richard L.
Published: July 2002

Subunit rotation within the F
1
catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded F
O
sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. F
O
F
1
was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998)
Proc. Natl. Acad. Sci. USA
95, 6607–6612]. Membranes were treated with
N
,
N
′-dicyclohexyl-[
14
C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little
14
C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/P
i
. Furthermore, preincubation with MgADP and azide to inhibit F
1
or with high concentrations of
N
,
N
′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.
The complete sequence of the 1,683-kb pSymB megaplasmid from the N
2
-fixing endosymbiont
Sinorhizobium meliloti
Authors: Finan Turlough M. , Weidner Stefan , Wong Kim , Buhrmester Jens , Chain Patrick , Vorhölter Frank J. , Hernandez-Lucas Ismael , Becker Anke , Cowie Alison , Gouzy Jérôme , Golding Brian , Pühler Alfred
Published: July 2002

Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from
the symbiotic N
2
-fixing bacterium
Sinorhizobium
meliloti
revealed that the replicon has a high gene density
with a total of 1,570 protein-coding regions, with few insertion
elements and regions duplicated elsewhere in the genome. The only
copies of an essential arg-tRNA gene and the
minCDE
genes are located on pSymB. Almost 20% of the pSymB sequence carries
genes encoding solute uptake systems, most of which were of the
ATP-binding cassette family. Many previously unsuspected genes involved
in polysaccharide biosynthesis were identified and these, together with
the two known distinct exopolysaccharide synthesis gene clusters, show
that 14% of the pSymB sequence is dedicated to polysaccharide
synthesis. Other recognizable gene clusters include many involved in
catabolic activities such as protocatechuate utilization and
phosphonate degradation. The functions of these genes are consistent
with the notion that pSymB plays a major role in the saprophytic
competence of the bacteria in the soil environment.
FEZ1/LZTS1
gene at 8p22 suppresses cancer cell growth and regulates mitosis
Authors: Ishii Hideshi , Vecchione Andrea , Murakumo Yoshiki , Baldassarre Gustavo , Numata Shinichiro , Trapasso Francesco , Alder Hansjuerg , Baffa Raffaele , Croce Carlo M.
Published: July 2002

The
FEZ1
/
LZTS1
gene maps to chromosome 8p22, a region that is frequently deleted in human tumors. Alterations in
FEZ1
/
LZTS1
expression have been observed in esophageal, breast, and prostate cancers. Here, we show that introduction of
FEZ1
/
LZTS1
into Fez1/Lzts1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S–G
2
/M stage of the cell cycle. Fez1/Lzts1 protein is hyperphosphorylated by cAMP-dependent kinase during cell-cycle progression. We found that Fez1/Lzts1 is associated with microtubule components and interacts with p34
cdc2
at late S–G
2
/M stage
in vivo
. Present data show that
FEZ1
/
LZTS1
inhibits cancer cell growth through regulation of mitosis, and that its alterations result in abnormal cell growth.
Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids
Authors: Schmidt Edward E. , Taylor Deborah S. , Prigge Justin R. , Barnett Sheila , Capecchi Mario R.
Published: July 2002

The bacteriophage P1 Cre/
lox
P system has
become a powerful tool for
in vivo
manipulation of the
genomes of transgenic mice. Although
in vitro
studies
have shown that Cre can catalyze recombination between cryptic
“pseudo-
lox
P” sites in mammalian genomes, to date
there have been no reports of
lox
P-site infidelity in
transgenic animals. We produced lines of transgenic mice that use the
mouse
Protamine 1
(
Prm1
) gene promoter to
express Cre recombinase in postmeiotic spermatids. All male founders
and all Cre-bearing male descendents of female founders were sterile;
females were unaffected. Sperm counts, sperm motility, and sperm
morphology were normal, as was the mating behavior of the transgenic
males and the production of two-celled embryos after mating. Mice that
expressed similar levels of a derivative transgene that carries an
inactive Cre exhibited normal male fertility. Analyses of embryos from
matings between sterile Cre-expressing males and wild-type females
indicated that Cre-catalyzed chromosome rearrangements in the
spermatids that lead to abortive pregnancies with 100% penetrance.
Similar Cre-mediated, but
lox
P-independent, genomic
alterations may also occur in somatic tissues that express Cre, but,
because of the greater difficulty of assessing deleterious effects of
somatic mutations, these may go undetected. This study indicates that,
following the use of the Cre/
lox
P site-specific
recombination systems
in vivo
, it is prudent to
eliminate or inactivate the
Cre
recombinase gene as
rapidly as possible.
Ancestral major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms.
Authors: Gaur L K , Nepom G T
Published: July 2002

Genes within the major histocompatibility complex (MHC) are characterized by extensive polymorphism within species and also by a remarkable conservation of contemporary human allelic sequences in evolutionarily distant primates. Mechanisms proposed to account for strict nucleotide conservation in the context of highly variable genes include the suggestion that intergenic exchange generates repeated sets of MHC DRB polymorphisms [Gyllensten, U. B., Sundvall, M. & Erlich, H. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3686-3690; Lundberg, A. S. & McDevitt, H. 0. (1992) Proc. Natl. Acad. Sci. USA 89, 6545-6549]. We analyzed over 50 primate MHC DRB sequences, and identified nucleotide elements within macaque and baboon DRB6-like sequences with deletions corresponding to specific exon 2 hypervariable regions, which encode a discrete alpha helical segment of the MHC antigen combining site. This precisely localized deletion provides direct evidence implicating segmental exchange of MHC-encoded DRB gene fragments as one of the evolutionary mechanisms both generating and maintaining MHC diversity. Intergenic exchange at this site may be fundamental to the diversification of immune protection in populations by permitting alteration in the specificity of the MHC that determines the repertoire of antigens bound.
Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserve.
Authors: Gross W L , Bak M I , Ingwall J S , Arstall M A , Smith T W , Balligand J L , Kelly R A
Published: July 2002

Cardiac myocytes express both constitutive and cytokine-inducible nitric oxide syntheses (NOS). NO and its congeners have been implicated in the regulation of cardiac contractile function. To determine whether NO could affect myocardial energetics, 31P NMR spectroscopy was used to evaluate high-energy phosphate metabolism in isolated rat hearts perfused with the NO donor S-nitrosoacetylcysteine (SNAC). All hearts were exposed to an initial high Ca2+ (3.5 mM) challenge followed by a recovery period, and then, either in the presence or absence of SNAC, to a second high Ca2+ challenge. This protocol allowed us to monitor simultaneously the effect of SNAC infusion on both contractile reserve (i.e., baseline versus high workload contractile function) and high-energy phosphate metabolism. The initial high Ca2+ challenge caused the rate-pressure product to increase by 74 +/- 5% in all hearts. As expected, ATP was maintained as phosphocreatine (PCr) content briefly dropped and then returned to baseline during the subsequent recovery period. Control hearts responded similarLy to the second high Ca2+ challenge, but SNAC-treated hearts did not demonstrate the expected increase in rate-pressure product. In these hearts, ATP declined significantly during the second high Ca2+ challenge, whereas phosphocreatine did not differ from controls, suggesting that phosphoryl transfer by creatine kinase (CK) was inhibited. CK activity, measured biochemically, was decreased by 61 +/- 13% in SNAC-treated hearts compared to controls. Purified CK in solution was also inhibited by SNAC, and reversal could be accomplished with DTT, a sulfhydryl reducing agent. Thus, NO can regulate contractile reserve, possibly by reversible nitrosothiol modification of CK.
Centromere mapping and orientation of the molecular linkage map of rice (Oryza sativa L.).
Authors: Singh K , Ishii T , Parco A , Huang N , Brar D S , Khush G S
Published: July 2002

Rice has become a model cereal plant for molecular genetic research. Rice has the most comprehensive molecular linkage maps with more than 2000 DNA markers and shows synteny and colinearity with the maps of other cereal crops. Until now, however, no information was available about the positions of centromeres and arm locations of markers on the molecular linkage map. Secondary and telotrisomics were used to assign restriction fragment length polymorphism markers to specific chromosome arms and thereby to map the positions of centromeres. More than 170 restriction fragment length polymorphism markers were assigned to specific chromosome arms through gene dosage analysis using the secondary and telotrisomics and the centromere positions were mapped on all 12 linkage groups. The orientations of seven linkage groups were reversed to fit the "short arm on top" convention and the corrected map is presented.
Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.
Authors: Garcia R A , Bustamante C J , Reich N O
Published: July 2002

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.
The recombination signals for adeno-associated virus site-specific integration.
Authors: Linden R M , Winocour E , Berns K I
Published: July 2002

The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.
Recruitment and replacement of hippocampal neurons in young and adult chickadees: an addition to the theory of hippocampal learning.
Authors: Barnea A , Nottebohm F
Published: July 2002

We used [3H]thymidine to document the birth of neurons and their recruitment into the hippocampal complex (HC) of juvenile (4.5 months old) and adult blackcapped chickadees (Parus atricapillus) living in their natural surroundings. Birds received a single dose of [3H]thymidine in August and were recaptured and killed 6 weeks later, in early October. All brains were stained with Cresyl violet, a Nissl stain. The boundaries of the HC were defined by reference to the ventricular wall, the brain surface, or differences in neuronal packing density. The HC of juveniles was as large as or larger than that of adults and packing density of HC neurons was 31% higher in juveniles than in adults. Almost all of the 3H-labeled HC neurons were found in a 350-m-wide layer of tissue adjacent to the lateral ventricle. Within this layer the fraction of 3H-labeled neurons was 50% higher in juveniles than in adults. We conclude that the HC of juvenile chickadees recruits more neurons and has more neurons than that of adults. We speculate that juveniles encounter greater environmental novelty than adults and that the greater number of HC neurons found in juveniles allows them to learn more than adults. At a more general level, we suggest that (i) long-term learning alters HC neurons irreversibly; (ii) sustained hippocampal learning requires the periodic replacement of HC neurons; (iii) memories coded by hippocampal neurons are transferred elsewhere before the neurons are replaced.
A unique human gene that spans over 230 kb in the human chromosome 8p11-12 and codes multiple family proteins sharing RNA-binding motifs.
Authors: Shimamoto A , Kitao S , Ichikawa K , Suzuki N , Yamabe Y , Imamura O , Tokutake Y , Satoh M , Matsumoto T , Kuromitsu J , Kataoka H , Sugawara K , Sugawara M , Sugimoto M , Goto M , Furuichi Y
Published: July 2002

A unique gene, RBP-MS, spanning over 230 kb in the human chromosome 8p11-12 near the Werner syndrome gene locus is described. The single-copy RBP-MS gene is alternatively spliced, resulting in a family of at least 12 transcripts (average length of 1.5 kb). Nine different types of cDNAs that encode an RNa-binding motif at the N terminus and helix-rich sequences at the C terminus have been identified thus far. Among the 16 exons identified, four 5'-proximal exons contained sequences homologous to the RNA-binding domain of Drosophila couch potato gene. Northern blot analysis showed that the RBP-MS gene was expressed strongly in the heart, prostate, intestine, and ovary, and poorly in the skeletal muscle, spleen, thymus, brain, and peripheral leukocytes. The possible role of this gene in RNA metabolism is discussed.
A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in
Mycobacterium tuberculosis
Authors: Yuan Ying , Barry Clifton E.
Published: July 2002

Mycobacterium tuberculosis
produces three classes of
mycolic acids that differ primarily in the presence and nature of
oxygen-containing substituents in the distal portion of the
meromycolate branch. The methoxymycolate series has a methoxy group
adjacent to a methyl branch, in addition to a cyclopropane in the
proximal position. Using the gene for the enzyme that introduces the
distal cyclopropane (
cma
1) as a probe, we have cloned
and sequenced a cluster of genes coding for four highly homologous
methyl transferases (
mma
1–4). When introduced into
Mycobacterium smegmatis
, this gene cluster conferred the
ability to synthesize methoxymycolates. By determining the structure of
the mycolic acids produced following expression of each of these genes
individually and in combination, we have elucidated the biosynthetic
steps responsible for the production of the major series of
methoxymycolates. The
mma
4 gene product (MMAS-4)
catalyzes an unusual
S
-adenosyl-
l
-methionine-dependent
transformation of the distal
cis
-olefin into a secondary
alcohol with an adjacent methyl branch. MMAS-3
O
-methylates this secondary alcohol to form the
corresponding methyl ether, and MMAS-2 introduces a
cis
-cyclopropane in the proximal position of the methoxy
series. The similarity of these reactions and the enzymes that catalyze
them suggests that some of the structural diversity of mycolic acids
results from different chemical fates of a common cationic
intermediate, which in turn results from methyl group addition to an
olefinic mycolate precursor.
Symmetries in geology and geophysics
Authors: Turcotte Donald L. , Newman William I.
Published: July 2002

Symmetries have played an important role in a variety of problems
in geology and geophysics. A large fraction of studies in mineralogy
are devoted to the symmetry properties of crystals. In this paper,
however, the emphasis will be on scale-invariant (fractal) symmetries.
The earth’s topography is an example of both statistically
self-similar and self-affine fractals. Landforms are also associated
with drainage networks, which are statistical fractal trees. A
universal feature of drainage networks and other growth networks is
side branching. Deterministic space-filling networks with
side-branching symmetries are illustrated. It is shown that naturally
occurring drainage networks have symmetries similar to
diffusion-limited aggregation clusters.
Equilibration rates in lipid monolayers
Authors: McConnell Harden M.
Published: July 2002

A theoretical analysis is given for the rate of change of domain
sizes in lipid monolayers at the air–water interface. The calculation
is applicable to liquid domains formed from binary mixtures of lipids
that form two coexisting liquid phases. Under conditions where the two
lipid molecules have approximately equal areas, the equilibration rate
does not involve macroscopic hydrodynamic flow in the subphase but
rather depends on the diffusion coefficient of the lipid molecules. The
calculation shows that the equilibration rate in binary mixtures of
cholesterol and phosphatidylcholine is remarkably slow, the radius of a
typical 20-μm diameter domain changing by as little as a part in a
million per second. Under these circumstances, equilibration times of
the order of days or weeks are expected. Even with such long times, the
final state reached by the monolayer will in general be a state of
metastable equilibrium, rather than true equilibrium.
Newborn primate infants are entrained by low intensity lighting
Authors: Rivkees Scott A. , Hofman Paul L. , Fortman Jeffrey
Published: July 2002

At the present time we do not know when the circadian timing system of human infants becomes responsive to light. Because of human study limitations, it is not currently possible to address this issue in clinical studies. Therefore, to provide insights into when the circadian system of humans becomes responsive to light, baboons were studied. We first assessed if the biological clock located in suprachiasmatic nuclei (SCN) is responsive to light at birth. When term newborn infants were exposed to bright light at night (5000 lux), SCN metabolic activity and c-
fos
mRNA expression increased, indicating the presence of photic responsiveness. When photic entrainment of developing rhythmicity was examined in infants, low intensity (200 lux) cycled lighting was sufficient to entrain circadian phase. However, low intensity lighting was not sufficient to induce changes in SCN metabolic activity or c-
fos
mRNA expression. Phase–response studies indicated that light exposure (200 lux) before the onset of activity most effectively shifted circadian phase. These data provide direct evidence that the SCN are responsive to visually mediated light information in a primate at birth. Further consideration of lighting conditions that infants are exposed to is therefore warranted.
Resistance to apoptosis caused byPIG-Agene mutations in paroxysmal nocturnal hemoglobinuria
Authors: Brodsky Robert A. , Vala Milada S. , Barber James P. , Medof M. Edward , Jones Richard J.
Published: July 2002

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder resulting from mutations in an X-linked gene,PIG-A, that encodes an enzyme required for the first step in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors.PIG-Amutations result in absent or decreased cell surface expression of all GPI-anchored proteins. Although many of the clinical manifestations (e.g., hemolytic anemia) of the disease can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 and CD55, it is unclear why the PNH clone dominates hematopoiesis and why it is prone to evolve into acute leukemia. We found thatPIG-Amutations confer a survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34+(CD59−) cells from PNH patients survived longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normalPIG-Agene in PNH cell lines reversed the cellular resistance to apoptosis. Inhibited apoptosis resulting fromPIG-Amutations appears to be the principle mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. These data also suggest that GPI anchors are important in regulating apoptosis.
VMAT2 knockout mice: Heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity
Authors: Takahashi Nobuyuki , Miner Lucinda L. , Sora Ichiro , Ujike Hiroshi , Revay Randal S. , Kostic Vladimir , Jackson-Lewis Vernice , Przedborski Serge , Uhl George R.
Published: July 2002

The brain vesicular monoamine transporter (VMAT2) pumps monoamine neurotransmitters and Parkinsonism-inducing dopamine neurotoxins such as 1-methyl-4-phenyl-phenypyridinium (MPP
+
) from neuronal cytoplasm into synaptic vesicles, from which amphetamines cause their release. Amphetamines and MPP
+
each also act at nonvesicular sites, providing current uncertainties about the contributions of vesicular actions to their
in vivo
effects. To assess vesicular contributions to amphetamine-induced locomotion, amphetamine-induced reward, and sequestration and resistance to dopaminergic neurotoxins, we have constructed transgenic VMAT2 knockout mice. Heterozygous VMAT2 knockouts are viable into adult life and display VMAT2 levels one-half that of wild-type values, accompanied by smaller changes in monoaminergic markers, heart rate, and blood pressure. Weight gain, fertility, habituation, passive avoidance, and locomotor activities are similar to wild-type littermates. In these heterozygotes, amphetamine produces enhanced locomotion but diminished behavioral reward, as measured by conditioned place preference. Administration of the MPP
+
precursor
N
-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to heterozygotes produces more than twice the dopamine cell losses found in wild-type mice. These mice provide novel information about the contributions of synaptic vesicular actions of monoaminergic drugs and neurotoxins and suggest that intact synaptic vesicle function may contribute more to amphetamine-conditioned reward than to amphetamine-induced locomotion.
Interferon-α/β inhibition of interleukin 12 and interferon-γ productionin vitroand endogenously during viral infection
Authors: Cousens Leslie P. , Orange Jordan S. , Su Helen C. , Biron Christine A.
Published: July 2002

Interferon (IFN)-α/β-mediated negative regulation of interleukin 12 (IL-12) and IFN-γ proteins is reported here. Both IFN-α and IFN-β inhibited fixedStaphylococcus aureusCowan strain induction of IL-12 and IFN-γ production by mouse splenic leukocytes in culture. Extended studies with IFN-α demonstrated that inhibition was at the level of biologically active IL-12 p70. Effects were selective, as induction of tumor necrosis factor was unaffected and induction of IL-6 was enhanced. Neutralization of IFN-α/β expressed endogenously during infections with murine cytomegalovirus (MCMV) enhanced early IL-12 and IFN-γ protein production. Furthermore, during infections of mice with lymphocytic choriomeningitis virus (LCMV), this treatment revealed a previously undetected early IL-12 and IFN-γ protein expression, and mice deficient in IFN-α/β receptor function, but not control mice, also expressed endogenous LCMV-induced IL-12. The effects of IFN-α/β neutralization on production of IL-12 and IFN-γ during the viral infections were detected in both serum samples and medium conditioned with splenic leukocytes isolated from infected animals.In vitrostudies demonstrated that splenic leukocytes isolated from LCMV-infected mice were primed to produce IL-12 in response to stimulation withStaphylococcus aureusCowan strain, but that this responsiveness was sensitive to added IFN-α. Moreover, endogenous IFN-α/β induced by LCMV inhibitedin vivolipopolysaccharide stimulation of IL-12 production. These results demonstrate a new pathway for regulating cytokine responses, and suggest a mechanism for inhibition of IL-12-dependent immune responses during viral infections.
Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase
Authors: Sasaki Yukiko , Kozaki Akiko , Hatano Mika
Published: July 2002

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined
in vitro
for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated
in vitro
more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2–5 mM Mg
2+
, indicating that light-produced changes in stromal pH and Mg
2+
concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg
2+
concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.
Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein
Authors: Zhong Guangming , e Sousa Caetano Reis , Germain Ronald N.
Published: July 2002

Several unanswered questions in T cell immunobiology relating to intracellular processing orin vivoantigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Akas a model system. mAbs specific for either HEL-(46–61)–Akor HEL-(116–129)–Akhave been isolated. They cross-react with a small subset of I-Akmolecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Akspecific mAbs to visualize intracellular compartments where I-Akis loaded with HEL-derived peptides early after antigen administration. Other uses, especially forin vivotracking of specific ligand-bearing antigen-presenting cells, are discussed.
DNA topoisomerase targets of the fluoroquinolones: A strategy for avoiding bacterial resistance
Authors: Zhao Xilin , Xu Chen , Domagala John , Drlica Karl
Published: July 2002

Fluoroquinolones are antibacterial agents that attack DNA gyrase and topoisomerase IV on chromosomal DNA. The existence of two fluoroquinolone targets and stepwise accumulation of resistance suggested that new quinolones could be found that would require cells to obtain two topoisomerase mutations to display resistance. For wild-type cells to become resistant, the two mutations must be acquired concomitantly. That is expected to occur infrequently. To identify such compounds, fluoroquinolones were tested for the ability to kill a moderately resistant gyrase mutant. Compounds containing a C8-methoxyl group were particularly lethal, and incubation of wild-type cultures on agar containing C8-methoxyl fluoroquinolones produced no resistant mutant, whereas thousands arose during comparable treatment with control compounds lacking the C8 substituent. When the test strain contained a preexisting topoisomerase IV mutation, which by itself conferred no resistance, equally high numbers of resistant mutants were obtained for C8-methoxyl and control compounds. Thus C8-methoxyl fluoroquinolones required two mutations for expression of resistance. Although highly lethal, C8-methoxyl fluoroquinolones were not more effective than C8-H controls at blocking bacterial growth. Consequently, quinolone action involves two events, which we envision as formation of drug–enzyme–DNA complexes followed by release of lethal double-strand DNA breaks. Release of DNA breaks, which must occur less frequently than complex formation, is probably the process stimulated by the C8-methoxyl group. Understanding this stimulation should provide insight into intracellular quinolone action and contribute to development of fluoroquinolones that prevent selection of resistant bacteria.
Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops
Authors: Cai Kewen , Langen Ralf , Hubbell Wayne L. , Khorana H. Gobind
Published: July 2002

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335–T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4′-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240–V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.
A novel 3′-end repair mechanism in an RNA virus
Authors: Nagy Peter D. , Carpenter Clifford D. , Simon Anne E.
Published: July 2002

Many positive-stranded RNA viruses contain short, single-stranded 3′ ends that are vulnerable to degradation by host cellular RNases. Therefore, the existence of a 3′-end repair mechanism (analogous to cellular telomerases) must be required and/or advantageous for RNA viruses. Accordingly, we provide evidence suggesting that deletions of up to 6 nt from the 3′ end of satellite (sat-) RNA C (a small parasitic RNA associated with turnip crinkle carmovirus) are repaired to the wild-type sequence
in vivo
and
in vitro
. The novel 3′-end repair mechanism involves the production of 4–8 nt oligoribonucleotides by abortive synthesis by the viral replicase using the 3′ end of the viral genomic RNA as template. Based on our
in vitro
results, we postulate that the oligoribonucleotides are able to prime synthesis of wild-type negative-strand sat-RNA C in a reaction that does not require base pairing of the oligoribonucleotides to the mutant, positive-strand RNA template. The discovery of a 3′-end repair mechanism opens up new strategies for interfering with viral infections.
Signaling in plants
Authors: Mulligan R. Michael , Chory Joanne , Ecker Joseph R.
Published: July 2002

Higher plants are sessile organisms that perceive environmental
cues such as light and chemical signals and respond by changing their
morphologies. Signaling pathways utilize a complex network of
interactions to orchestrate biochemical and physiological responses
such as flowering, fruit ripening, germination, photosynthetic
regulation, and shoot or root development. In this session, the
mechanisms of signaling systems that trigger plant responses to light
and to the gaseous hormone, ethylene, were discussed. These signals are
first sensed by a receptor and transmitted to the nucleus by a complex
network. A signal may be transmitted to the nucleus by any of several
systems including GTP binding proteins (G proteins), which change
activity upon GTP binding; protein kinase cascades, which sequentially
phosphorylate and activate a series of proteins; and membrane ion
channels, which change ionic characteristics of the cells. The signal
is manifested in the nucleus as a change in the activity of DNA-binding
proteins, which are transcription factors that specifically interact
and modulate the regulatory regions of genes. Thus, detection of an
environmental signal is transmitted through a transduction pathway, and
changes in transcription factor activity may coordinate changes in the
expression of a portfolio of genes to direct new developmental
programs.
The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from
Escherichia coli
Authors: Arscott L. David , Gromer Stephan , Schirmer R. Heiner , Becker Katja , Williams Charles H.
Published: July 2002

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide–disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit
M
r
≈55,000). Although the mechanism and structure of thioredoxin reductase from
Escherichia coli
are distinct (
M
r
≈35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a
M
r
of ≈55,000 [Luthman, M. & Holmgren, A. (1982)
Biochemistry
21, 6628–6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995)
FEBS Lett
373, 5–9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996)
Proc. Natl. Acad. Sci. USA
93, 6146–6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate–flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate seleno-cysteine [Tamura, T. & Stadtman, T.C. (1996)
Proc. Natl. Acad. Sci. USA
93, 1006–1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of
E. coli
thioredoxin reductase.