bluuebunny/crossref_metadata_2025 huggingface dataset:
Prion protein expression in different species: Analysis with a panel of new mAbs
Authors: Zanusso Gianluigi , Liu Dacai , Ferrari Sergio , Hegyi Ivan , Yin Xinghua , Aguzzi Adriano , Hornemann Simone , Liemann Susanne , Glockshuber Rudi , Manson Jean C. , Brown Paul , Petersen Robert B. , Gambetti Pierluigi , Sy Man-Sun
Published: July 2002

By immunizing prion knockout mice (
Prnp
−/−) with recombinant murine prion protein (PrP
c
), we obtained a panel of mAbs specific for murine PrP
c
. These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrP
c
) and protease-resistant (PrP
res
) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrP
c
. These newly generated PrP
c
antibodies will help to explore the biology of PrP
c
and to establish the diagnosis of prion diseases in both humans and animals.
A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G
β5
subunits
Authors: Snow Bryan E. , Krumins Andrejs M. , Brothers Greg M. , Lee Sheu-Fen , Wall Mark A. , Chung Stephen , Mangion Joan , Arya Sudha , Gilman Alfred G. , Siderovski David P.
Published: July 2002

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the α subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein γ subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the
Caenorhabditis elegans
protein EGL-10. Coexpression of RGS11 with different G
β
subunits reveals specific interaction between RGS11 and G
β5
. The expression of mRNA for RGS11 and G
β5
in human tissues overlaps. The G
β5
/RGS11 heterodimer acts as a GAP on G
αo
, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G
α
proteins and form complexes with specific G
β
subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.
Prions
Authors: Prusiner Stanley B.
Published: July 2002

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt–Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPScthrough a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPScacts as a template upon which PrPCis refolded into a nascent PrPScmolecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.
SopB, a protein required for virulence of
Salmonella dublin
, is an inositol phosphate phosphatase
Authors: Norris F. Anderson , Wilson Monita P. , Wallis Timothy S. , Galyov Edouard E. , Majerus Philip W.
Published: July 2002

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by
Salmonella dublin
, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity
in vitro
. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca
2+
-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5,6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F.,
et al.
(1997)
Proc. Natl. Acad. Sci. USA
94, 14456–14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain,
S. dublin
SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with
S. dublin
SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type
S. dublin
-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.
The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins
Authors: Hummer Gerhard , Garde Shekhar , García Angel E. , Paulaitis Michael E. , Pratt Lawrence R.
Published: July 2002

Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.
Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency
Authors: Xie Weiqiao , Li Limin , Cohen Stanley N.
Published: July 2002

TSG101 is a recently discovered tumor susceptibility gene whose functional inactivation in mouse fibroblasts results in cell transformation and the ability to form metastatic tumors in nude mice. Although restoration of
TSG101
activity reverses tumorigenesis, neoplasia is irreversible in some cells, suggesting that permanent genetic alteration can occur during TSG101 inactivation. Here we describe studies that support this notion. We find that localization of TSG101 is cell cycle-dependent, occurring in the nucleus and Golgi complex during interphase, and in mitotic spindles and centrosomes during mitosis; cells made neoplastic by a deficiency in TSG101 expression show a series of mitosis-related abnormalities, including multiple microtubule organizing centers, aberrant mitotic spindles, abnormal distribution of metaphase chromatin, aneuploidy, and nuclear anomalies. Our findings suggest that TSG101 deficiency may lead to genome instability in addition to previously reported reversible neoplastic transformation.
Modulation of
Neisseria gonorrhoeae
susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family
Authors: Shafer W. M. , Qu X.-D. , Waring A. J. , Lehrer R. I.
Published: July 2002

We have previously described the antibacterial capacity of protegrin-1 (PG-1), a cysteine-rich, cationic peptide from porcine leukocytes, against
Neisseria gonorrhoeae
. We now report genetic and biochemical evidence that gonococcal susceptibility to the lethal action of PG-1 and other structurally unrelated antibacterial peptides, including a peptide (LL-37) that is expressed constitutively by human granulocytes and testis and inducibly by keratinocytes, is modulated by an energy-dependent efflux system termed
mtr
. These results indicate that such efflux systems may enable mucosal pathogens like gonococci to resist endogenous antimicrobial peptides that are thought to act during infection.
Germacrene C synthase fromLycopersicon esculentumcv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase
Authors: Colby Sheila M. , Crock John , Dowdle-Rizzo Barbara , Lemaux Peggy G. , Croteau Rodney
Published: July 2002

Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil ofLycopersicon esculentumcv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity inEscherichia coliby using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr= 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity).
Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition
Authors: Gudi Sivaramaprasad , Nolan John P. , Frangos John A.
Published: July 2002

Mechanical forces arising from strain, pressure, and fluid shear stress are sensed by cells through an unidentified mechanoreceptor(s) coupled to intracellular signaling pathways. In vascular endothelial cells, fluid shear stress is transduced via pathway(s) involving heterotrimeric guanine nucleotide-binding proteins (G proteins) by molecular mechanisms that are unknown. In the present study, we investigated the activation of purified G proteins reconstituted into phospholipid vesicles. Vesicles containing G proteins were loaded with [γ-
32
P]GTP and subjected to physiological levels of fluid shear stress in a cone-and-plate viscometer. Steady-state GTP hydrolysis was measured as an index of G protein function. Shear stress (0–30 dynes/cm
2
) activated G proteins in dose-dependent manner (0.48–4.6 pmol/min per μg of protein). Liposomes containing lysophosphatidylcholine (30 mol %) or treated with benzyl alcohol (40 mM), conditions that increase bilayer fluidity, exhibited 3- to 5-fold enhancement of basal GTPase activity. Conversely, incorporation of cholesterol (24 mol %) into liposomes reduced the activation of G proteins by shear. These results demonstrate the ability of the phospholipid bilayer to mediate the shear stress-induced activation of membrane-bound G proteins in the absence of protein receptors and that bilayer physical properties modulate this response.
Rational design of a triple helix-specific intercalating ligand
Authors: Escudé Christophe , Nguyen Chi Hung , Kukreti Shrikant , Janin Yves , Sun Jian-Sheng , Bisagni Emile , Garestier Thérèse , Hélène Claude
Published: July 2002

DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3,4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson–Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.
Determination of the rate of the glutamate/glutamine cycle in the human brain by
in vivo
13
C NMR
Authors: Shen Jun , Petersen Kitt F. , Behar Kevin L. , Brown Peter , Nixon Terrence W. , Mason Graeme F. , Petroff Ognen A. C. , Shulman Gerald I. , Shulman Robert G. , Rothman Douglas L.
Published: July 2002

Recent
13
C NMR studies in rat models have shown that the glutamate/glutamine cycle is highly active in the cerebral cortex and is coupled to incremental glucose oxidation in an ≈1:1 stoichiometry. To determine whether a high level of glutamatergic activity is present in human cortex, the rates of the tricarboxylic acid cycle, glutamine synthesis, and the glutamate/glutamine cycle were determined in the human occipital/parietal lobe at rest. During an infusion of [1-
13
C]-glucose,
in vivo
13
C NMR spectra were obtained of the time courses of label incorporation into [4-
13
C]-glutamate and [4-
13
C]-glutamine. Using a metabolic model we have validated in the rat, we calculated a total tricarboxylic acid cycle rate of 0.77 ± 0.07 μmol/min/g (mean ± SD,
n
= 6), a glucose oxidation rate of 0.39 ± 0.04 μmol/min/g, and a glutamate/glutamine cycle rate of 0.32 ± 0.05 μmol/min/g (mean ± SD,
n
= 6). In agreement with studies in rat cerebral cortex, the glutamate/glutamine cycle is a major metabolic flux in the resting human brain with a rate ≈80% of glucose oxidation.
Long-distance charge transport in duplex DNA: The phonon-assisted polaron-like hopping mechanism
Authors: Henderson Paul T. , Jones Denise , Hampikian Gregory , Kan Yongzhi , Schuster Gary B.
Published: July 2002

An anthraquinone-linked duplex DNA oligomer containing 60 base pairs was synthesized by PCR. The strand complementary to the quinone-containing strand has four isolated GG steps, which serve as traps for a migrating radical cation. Irradiation of the quinone leads to electron transfer from the DNA to the quinone forming the anthraquinone radical anion and a base radical cation. The radical cation migrates through the DNA, causing reaction at GG steps revealed as strand breaks. The efficiency of strand cleavage falls off exponentially with distance from the quinone (slope = −0.02 Å
−1
). This finding necessitates reinterpretation of mechanisms proposed for radical cation migration in DNA. We propose that radical cations form self-trapped polarons that migrate by thermally activated hopping.
The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance
Authors: Austin D. J. , Kristinsson K. G. , Anderson R. M.
Published: July 2002

The threat to human health posed by antibiotic resistance is of growing concern. Many commensal and pathogenic organisms have developed resistance to well established and newer antibiotics. The major selection pressure driving changes in the frequency of antibiotic resistance is the volume of drug use. However, establishing a quantitative relationship between the frequency of resistance and volume of drug use has proved difficult. Using population genetic methods and epidemiological observations, we report an analysis of the influence of the selective pressure imposed by the volume of drug use on temporal changes in resistance. Analytical expressions are derived to delineate key relationships between resistance and drug consumption. The analyses indicate that the time scale for emergence of resistance under a constant selective pressure is typically much shorter than the decay time after cessation or decline in the volume of drug use and that significant reductions in resistance require equally significant reductions in drug consumption. These results highlight the need for early intervention once resistance is detected.
Metamorphosis is inhibited in transgenic
Xenopus laevis
tadpoles that overexpress type III deiodinase
Authors: Huang Haochu , Marsh-Armstrong Nicholas , Brown Donald D.
Published: July 2002

One of the genes that is up-regulated by thyroid hormone (TH) during
Xenopus laevis
metamorphosis encodes a type III deiodinase (D3) that inactivates TH. Transgenic
X. laevis
tadpoles overexpressing a GFP-D3 fusion protein were produced. These transgenic tadpoles had high levels of deiodinase activity and were resistant to exogenous TH added 1 week after fertilization. They developed normally throughout embryogenesis and premetamorphic stages but became retarded in their development late in prometamorphosis when endogenous TH reaches its highest level. Gill and tail resorption were delayed and most of the animals arrested and died. One tadpole completed its metamorphosis without resorbing its tail. These results demonstrate that D3 can modulate the action of TH
in vivo
, and document the value of the new transgenic method for functional analysis of genes involved in metamorphosis.
Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold
Authors: Beste Gerald , Schmidt Frank S. , Stibora Thomas , Skerra Arne
Published: July 2002

We demonstrate that the ligand pocket of a lipocalin from
Pieris brassicae
, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (
i
) that the molecular recognition of fluorescein is specific and (
ii
) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.
Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides
Authors: Suzuki Koichi , Mori Atsumi , Ishii Ken J. , Saito Jun , Singer Dinah S. , Klinman Dennis M. , Krause Philip R. , Kohn Leonard D.
Published: July 2002

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.
Insulin increases near-membrane but not global Ca
2+
in isolated skeletal muscle
Authors: Bruton Joseph D. , Katz Abram , Westerblad Håkan
Published: July 2002

It has long been debated whether changes in Ca
2+
are involved in insulin-stimulated glucose uptake in skeletal muscle. We have now investigated the effect of insulin on the global free myoplasmic Ca
2+
concentration and the near-membrane free Ca
2+
concentration ([Ca
2+
]
mem
) in intact, single skeletal muscle fibers from mice by using fluorescent Ca
2+
indicators. Insulin has no effect on the global free myoplasmic Ca
2+
concentration. However, insulin increases [Ca
2+
]
mem
by ≈70% and the half-maximal increase in [Ca
2+
]
mem
occurs at an insulin concentration of 110 microunits per ml. The increase in [Ca
2+
]
mem
by insulin persists when sarcoplasmic reticulum Ca
2+
release is inhibited but is lost by perfusing the fiber with a low Ca
2+
medium or by addition of L-type Ca
2+
channel inhibitors. Thus, insulin appears to stimulate Ca
2+
entry into muscle cells via L-type Ca
2+
channels. Wortmannin, which inhibits insulin-mediated activation of glucose transport in isolated skeletal muscle, also inhibits the insulin-mediated increase in [Ca
2+
]
mem
. These data demonstrate a new facet of insulin signaling and indicate that insulin-mediated increases in [Ca
2+
]
mem
in skeletal muscle may underlie important actions of the hormone.
EDS1
, an essential component of
R
gene-mediated disease resistance in
Arabidopsis
has homology to eukaryotic lipases
Authors: Falk Anders , Feys Bart J. , Frost Louise N. , Jones Jonathan D. G. , Daniels Michael J. , Parker Jane E.
Published: July 2002

A major class of plant disease resistance (
R
) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the
Drosophila
Toll and human IL-1 receptors. In
Arabidopsis thaliana
,
EDS1
is indispensable for the function of these
R
genes. The
EDS1
gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective
eds1
alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of
EDS1
mRNA and marker mRNAs (
PR1
and
PDF1.2
, respectively) for these two pathways in wild-type and
eds1
mutant plants after different challenges. The results suggest that EDS1 functions upstream of salicylic acid-dependent
PR1
mRNA accumulation and is not required for jasmonic acid-induced
PDF1.2
mRNA expression.
Enantiomer discrimination illustrated by high-resolution crystal structures of the human nuclear receptor hRARγ
Authors: Klaholz Bruno P. , Mitschler André , Belema Makonen , Zusi C. , Moras Dino
Published: July 2002

The human retinoic acid receptor (hRAR) is a member of the nuclear receptor superfamily that regulates the transcription of target genes in a ligand-dependent manner. The three hRAR isotypes are targets for retinoids that are used in the treatment of various diseases, including breast cancer and skin diseases. Drug efficiency and safety depend on the pharmacological activity of enantiomers, which can differ because of the chiral environment generated by the target. We report the crystal structures of the hRARγ ligand-binding domain bound to two enantiomers, the active BMS270394 and the inactive BMS270395, solved at 1.6 Å and 1.7 Å resolution, respectively. The crystal structures reveal that in both enantiomers, the hydroxyl moiety attached to the chiral center forms a hydrogen bond to the Met-272 sulfur atom, thus imposing a conformation of BMS270395 that differs significantly from that observed for BMS270394 and other known retinoids. BMS270395 adopts an energetically unfavorable conformation, accounting for its inactivity; in contrast, the conformation of BMS270394 is close to an energy minimum. Our high-resolution data allow rationalization of enantiomer discrimination by the receptor and provide a model system for the pharmacological properties of enantiomeric pairs.
Both early and delayed anti-CD40L antibody treatment induces a stable plaque phenotype
Authors: Lutgens Esther , Cleutjens Kitty B. J. M. , Heeneman Sylvia , Koteliansky Victor E. , Burkly Linda C. , Daemen Mat J. A. P.
Published: July 2002

In the present study, we investigated the role of the CD40L-CD40 pathway in a model of progressive atherosclerosis. ApoE−/− mice were treated with an anti-CD40L antibody or a control antibody for 12 wk. Antibody treatment started early (age 5 wk) or was delayed until after the establishment of atherosclerosis (age 17 wk). In both the early and delayed treatment groups, anti-CD40L antibody did not decrease plaque area or inhibit lesion initiation or age-related increase in lesion area. The morphology of initial lesions was not affected, except for a decrease in T-lymphocyte content. Effects of anti-CD40L antibody treatment on the morphology of advanced lesions were pronounced. In both the early and delayed treatment groups, T-lymphocyte content was significantly decreased. Furthermore, a pronounced increase in collagen content, vascular smooth muscle cell/myofibroblast content, and fibrous cap thickness was observed. In the delayed treatment group, a decrease in lipid core and macrophage content occurred. Interestingly, advanced lesions of anti-CD40L antibody-treated mice exhibited an increased transforming growth factor β1 immunoreactivity, especially in macrophages. In conclusion, both early and delayed treatment with an anti-CD40L antibody do not affect atherosclerotic lesion initiation but do result in the development of a lipid-poor collagen-rich stable plaque phenotype. Furthermore, delayed treatment with anti-CD40L antibody can transform the lesion profile from a lipid-rich to a lipid-poor collagen-rich phenotype. Postulated mechanisms of this effect on plaque phenotype are the down-regulation of proinflammatory pathways and up-regulation of collagen-promoting factors like transforming growth factor β.
Dissociation of response conflict, attentional selection, and expectancy with functional magnetic resonance imaging
Authors: Casey B. J. , Thomas Kathleen M. , Welsh Tomihisa F. , Badgaiyan Rajendra D. , Eccard Clayton H. , Jennings J. Richard , Crone Eveline A.
Published: July 2002

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.
The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters
Authors: Wender Paul A. , Mitchell Dennis J. , Pattabiraman Kanaka , Pelkey Erin T. , Steinman Lawrence , Rothbard Jonathan B.
Published: July 2002

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat49–57(RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49–57were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49–57play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat49–57in cellular uptake. In contrast, a 9-mer ofl-arginine (R9) was 20-fold more efficient than Tat49–57at cellular uptake as determined by Michaelis–Menton kinetic analysis. Thed-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat49–57play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat49–57and even to r9. Overall, a transporter has been developed that is superior to Tat49–57, protease resistent, and more readily and economically prepared.
Climate and infectious disease: Use of remote sensing for detection of
Vibrio cholerae
by indirect measurement
Authors: Lobitz Brad , Beck Louisa , Huq Anwar , Wood Byron , Fuchs George , Faruque A. S. G. , Colwell Rita
Published: July 2002

It has long been known that cholera outbreaks can be initiated when
Vibrio cholerae
, the bacterium that causes cholera, is present in drinking water in sufficient numbers to constitute an infective dose, if ingested by humans. Outbreaks associated with drinking or bathing in unpurified river or brackish water may directly or indirectly depend on such conditions as water temperature, nutrient concentration, and plankton production that may be favorable for growth and reproduction of the bacterium. Although these environmental parameters have routinely been measured by using water samples collected aboard research ships, the available data sets are sparse and infrequent. Furthermore, shipboard data acquisition is both expensive and time-consuming. Interpolation to regional scales can also be problematic. Although the bacterium,
V. cholerae
, cannot be sensed directly, remotely sensed data can be used to infer its presence. In the study reported here, satellite data were used to monitor the timing and spread of cholera. Public domain remote sensing data for the Bay of Bengal were compared directly with cholera case data collected in Bangladesh from 1992–1995. The remote sensing data included sea surface temperature and sea surface height. It was discovered that sea surface temperature shows an annual cycle similar to the cholera case data. Sea surface height may be an indicator of incursion of plankton-laden water inland, e.g., tidal rivers, because it was also found to be correlated with cholera outbreaks. The extensive studies accomplished during the past 25 years, confirming the hypothesis that
V. cholerae
is autochthonous to the aquatic environment and is a commensal of zooplankton, i.e., copepods, when combined with the findings of the satellite data analyses, provide strong evidence that cholera epidemics are climate-linked.
Nonlinearity in genetic decoding: Homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting
Authors: Larsen Bente , Wills Norma M. , Nelson Chad , Atkins John F. , Gesteland Raymond F.
Published: July 2002

The τ and γ subunits of DNA polymerase III are both encoded by a single gene in
Escherichia coli
and
Thermus thermophilus
. γ is two-thirds the size of τ and shares virtually all its amino acid sequence with τ.
E. coli
and
T. thermophilus
have evolved very different mechanisms for setting the approximate 1:1 ratio between τ and γ. Both mechanisms put ribosomes into alternate reading frames so that stop codons in the new frame serve to make the smaller γ protein. In
E. coli
, ≈50% of initiating ribosomes translate the
dnaX
mRNA conventionally to give τ, but the other 50% shift into the −1 reading frame at a specific site (A AAA AAG) in the mRNA to produce γ. In
T. thermophilus
ribosomal frameshifting is not required: the
dnaX
mRNA is a heterogeneous population of molecules with different numbers of A residues arising from transcriptional slippage on a run of nine T residues in the DNA template. Translation of the subpopulation containing nine As (or +/− multiples of three As) yields τ. The rest of the population of mRNAs (containing nine +/− nonmultiples of three As) puts ribosomes into the alternate reading frames to produce the γ protein(s). It is surprising that two rather similar
dnaX
sequences in
E. coli
and
T. thermophilus
lead to very different mechanisms of expression.
Accurate protein crystallography at ultra-high resolution: Valence electron distribution in crambin
Authors: Jelsch Christian , Teeter Martha M. , Lamzin Victor , Pichon-Pesme Virginie , Blessing Robert H. , Lecomte Claude
Published: July 2002

The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 Å) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.
Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco
Authors: Yang Kwang-Yeol , Liu Yidong , Zhang Shuqun
Published: July 2002

Hypersensitive response (HR), a form of programmed cell death, is
frequently associated with plant disease resistance. It has been
proposed that mitogen-activated protein kinase (MAPK) cascades regulate
HR cell death based on pharmacological studies by using kinase
inhibitors. However, direct evidence is lacking. Here, we demonstrate
that NtMEK2, a MAPK kinase, is upstream of salicylic acid-induced
protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two
tobacco MAPKs that are activated by various pathogens or
pathogen-derived elicitors. Expression of a constitutively active
mutant of
NtMEK2
induces HR-like cell death in tobacco,
which is preceded by the activation of endogenous SIPK and
WIPK. In addition, NtMEK2-SIPK/WIPK cascade appears to control the
expression of 3-hydroxy-3-methylglutaryl CoA reductase
(
HMGR
) and
l
-phenylalanine ammonia lyase
(
PAL
), two defense genes encoding key enzymes in the
phytoalexin and salicylic acid biosynthesis pathways. These results
demonstrate that a plant MAPK cascade controls multiple defense
responses against pathogen invasion.
The Meaning of Lexical Classes
Authors: Josefsson Gunlög
Published: July 2002

Following the spirit of Relativized Extreme Functionalism, I argue that a set of grammatical features, traditionally thought of as devoid of semantics, lexical class (declension and conjugation), in fact has semantic content. Taking Josefsson (1995. 1997. 1998) as a point of departure, I suggest that the lexical class determines the word class of a word, hence relating the word to a major ontological category such as THING and EVENT. A certain lexical class may correspond to a semantic subclass of a major ontological category, but this does not need to be the case. The approach taken explains certain morphological phenomena in Swedish, such as stem vowels, and explains why the non-head part of a compound is undetermined for major ontological category.
Th1/Th2 cytokines and ICAM–1 levels post‐liver transplant do not predict early rejection
Authors: Granot E. , Tarcsafalvi A. , Emre S. , Sheiner P. , Guy S. , Schwartz M. E. , Boros P. , Miller C. M.
Published: July 2002

Th1 derived cytokines IFN‐γ and IL–2, Th2 cytokine IL–4, and ICAM–1 have been implicated in liver allograft rejection. In order to determine whether monitoring of cytokine profiles during the first days post‐liver transplant can predict early rejection we measured IFN‐gg, IL–2, sIL–2 receptor, IL–4 and ICAM–1 in 22 patients, in plasma samples obtained within 4h after liver perfusion (baseline) and between postoperative days (POD) 3–6. ICAM–1 and sIL–2R levels at POD 3–6 were significantly higher than at baseline but did not differ in presence or absence of rejection. Mean percentage increase of ICAM–1 levels was significantly lower in patients with Muromonab‐C3 Orthoclone OKT3 (J.C. Health Care) (OKT3) whereas percentage increase of sIL–2R levels was higher in OKT3‐treated patients. IFN‐γ levels at POD 3–6 increased from baseline while IL–4 levels were unchanged. Levels of IFN‐γ, IL–4 and their ratios did not correlate with rejection or immunosuppressive therapy. Thus, Th1/Th2 cytokine monitoring during the first week post‐transplant does not predict early rejection and immunosuppressive therapy is the predominant factor affecting ICAM and sIL–2R levels after liver transplantation.
Anti‐inflammatory effect of glucose—mannose binding lectins isolated from Brazilian beans
Authors: Assreuy A. M. S. , Shibuya M. D. , Martins G. J. , De Souza M. L. P. , Cavada B. S. , Moreira R. A. , Oliveira J. T. A. , Ribeiro R. A. , Flores C. A.
Published: July 2002

Selectins are essential for leukocyte recruitment in inflammation. Because of a lectin domain present in the selectin structure, we investigated the anti‐inflammtory activity of six mannose–glucose binding lectins from brazilian beans: Dioclea guianensis‐DguiL; D. grandiflora‐DgL; Cratylia floribunda‐CfL;
D. violacea‐D.vL; D. virgata‐DvirL and Canavalia brasiliensis‐ConBr. The lectins were injected intravenously (i.v.) into rats (0.1 and 1.0 mg/kg; 30 min before irritants) and its activities compared to
E. coli endotoxin (LPS,30 μg/kg i.v.). Three lectins (DvL, CfL and DguiL), although less intense than LPS, inhibited the neutrophil migration induced by carrageenan (Cg, 300 μg) in a dose‐dependent manner (0.1 and 1.0 mg/kg). DvL activity was reversed by 0.1 M α‐D‐methyl‐mannoside (α‐CH3), but not by 0.1 M α‐D‐galactose. The fMLP (44 ng)‐induced neutrophil migration was also reduced by these lectins. Endotoxin contamination of lectin samples could be excluded since α‐CH3 treatment reversed the DvL effect, but did not modify LPS inhibitory activity. Carrageenan (300 μg)‐induced paw oedema was also reduced by LPS or lectin treatments. Conversely, none of the tested lectins inhibited dextran (Dex, 300 μg)‐induced paw oedema, a classical leukocyte independent model, or zymosan (Zy, 1.0 mg)‐induced peritonitis and paw oedema. LPS showed no effect upon Dex‐induced paw oedema and barely reduced (25%) the oedematogenic effects of zymosan. As proposed for LPS, the lectin inhibitory activity was better observed on neutrophil‐mediated inflammatory reactions. We speculate that the plant lectin antiinflammatory activity is probably due to a competitive blockage of a common leukocyte and/or endothelial selectin carbohydrate ligand.
Endocrine-Modulating Substances in the Environment: Estrogenic Effects of Pharmaceutical Products
Authors: Arcand-Hoy L. D. , Nimrod A. C. , Benson W. H.
Published: July 2002

Several classes of anthropogenic substances modulate endocrine pathways. To date, most of the research has focused on chemical classes such as chlorinated hydrocarbon contaminants, pesticides, or degradation products of high-use industrial surfactants. However, little is known about the environmental impact of another important class of chem icals: hormonally active pharmaceutical products. This deficit of information is surprising, given the biological potencies of these widely used substances and their potential to enter the environment through sewage treatment effluents and runoff from agricultural applications. Estrogenic pharmaceutical products are used extensively in human estrogen-replacement therapy and oral contraceptive formulations. Estrogenic substances are also used as growth enhancement products in veterinary medicine. This report describes the potencies, pharm acokinetics, and metabolic pathways of such estrogenic products. The amounts of pharmaceutical-derived estrogens that reach the environment from doses and usage are estimated. Factors that affect environmental concentrations including metabolism and biodegradation are also considered. Finally, we present both chemical and analytical data, as well as bioassay evidence, that suggest that biologically active concentrations of estrogenic compounds are being released into the aquatic environment.
Chemical and analytical aspects of areca nut
Authors: Lord G. A. , Lim C. K. , Warnakulasuriya S. , Peters T. J.
Published: July 2002

Abstract A capillary zone electrophoretic (CZE) method for the rapid analysis of the major alkaloids (arecoline and guavacoline) in areca nut extract is described. Areca nuts were pulverized and then extracted with water by sonication in a water bath. After centrifugation, the supernatant was analysed on a fused‐silica capillary with 100 mM ammonium acetat–acetic acid (pH 4.6) as the running buffer at a voltage of 20 kV and temperature of 30#176;C. The method is applicable to the analysis of alkaloids in the nut, commercial preparations (pan masala) and in the saliva of areca nut chewers.
Diffuse Intramuscular Lipomatosis of a Lower Limb
Authors: Morris Alfred D. , Jane Mike J. , Ritchie David , Helliwell Tim
Published: July 2002

Patient. A 40‐year‐old man presented with a swelling of the left thigh which had been increasing in size over 10 months. Surgery confirmed a diagnosis of lipoma. After 6 months, another swelling appeared, this time in the left calf. Ultrasound‐guided biopsies revealed that the tissue showed appearances consistent with intramuscular lipoma. No further surgery was performed and the man is to be reviewed regularly, with possible debulking if necessary.

Discussion. This case presents an atypical case of lipomatosis. Magnetic resonance imaging is useful for assessing the extent of the lesion.
Brush Border Myosin–I Structure and ADP-dependent Conformational Changes Revealed by Cryoelectron Microscopy and Image Analysis
Authors: Jontes James D. , Milligan Ronald A.
Published: July 2002

Brush border myosin–I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751–753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain–binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The ∼31° swing corresponds to ∼63 Å at the end of the rigid light chain–binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.
Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles
Authors: Su Tin Tin , O'Farrell Patrick H.
Published: July 2002

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.