bluuebunny/crossref_metadata_2025 huggingface dataset:
Transition state structure of arginine kinase: Implications for catalysis of bimolecular reactions
Authors: Zhou Genfa , Somasundaram Thayumanasamy , Blanc Eric , Parthasarathy Golapakrishnan , Ellington W. Ross , Chapman Michael S.
Published: July 2002

Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with fluctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-Å resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transition-state analog complex studied here, a nitrate mimics the planar γ-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.
Proposed mechanism for stability of proteins to evolutionary mutations
Authors: Nelson Erik David , Onuchic José Nelson
Published: July 2002

It is shown that the sequence-ordering tendencies induced by design into different fast-folding, thermally stable native structures interfere. This interference results in a type of quasiorthogonality between optimal native structures, which divides sequence space into fast-folding, thermally stable families surrounded by slow-folding, low stability shells. A concrete example of this effect is provided by using a simple α carbon type model in which a complete correspondence is established between sequence and structure. It is speculated that gaps can occur in the space of protein-like sequences separating the sequence families and resulting in a mechanism for stability and diversity of protein sequence information.
Identification of the binding site for Gqα on its effector Bruton’s tyrosine kinase
Authors: Ma Yong-Chao , Huang Xin-Yun
Published: July 2002

Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqα binds directly to the nonreceptor Bruton’s tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both
in vitro
and
in vivo
. Only active GTP-bound Gqα, not inactive GDP-bound Gqα, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqα also eliminate Btk stimulation by Gqα, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqα to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.
A novel amplicon at 8p22–23 results in overexpression of cathepsin B in esophageal adenocarcinoma
Authors: Hughes Steven J. , Glover Thomas W. , Zhu Xiao-Xiang , Kuick Rork , Thoraval Didier , Orringer Mark B. , Beer David G. , Hanash Samir
Published: July 2002

Cathepsin B (
CTSB
) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the
CTSB
gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22–23 that results in
CTSB
overexpression in esophageal adenocarcinoma. Amplified genomic
Not
I–
Hin
fI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent
in situ
hybridization defined a single region of amplification involving chromosome bands 8p22–23. We investigated the candidate cancer-related gene
CTSB
, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (
FDFT1
), arylamine
N-
acetyltransferase (
NAT-1
), lipoprotein lipase (
LPL
), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only
CTSB
and
FDFT1
were consistently amplified in eight (12.1%) of the tumors. Neither
NAT-1
nor
LPL
were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72.5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22–23 resulting in
CTSB
gene amplification and overexpression supports an important role for
CTSB
in esophageal adenocarcinoma and possibly in other tumors.
Neural basis of an inherited speech and language disorder
Authors: Vargha-Khadem F. , Watkins K. E. , Price C. J. , Ashburner J. , Alcock K. J. , Connelly A. , Frackowiak R. S. J. , Friston K. J. , Pembrey M. E. , Mishkin M. , Gadian D. G. , Passingham R. E.
Published: July 2002

Investigation of the three-generation KE family, half of whose members are affected by a pronounced verbal dyspraxia, has led to identification of their core deficit as one involving sequential articulation and orofacial praxis. A positron emission tomography activation study revealed functional abnormalities in both cortical and subcortical motor-related areas of the frontal lobe, while quantitative analyses of magnetic resonance imaging scans revealed structural abnormalities in several of these same areas, particularly the caudate nucleus, which was found to be abnormally small bilaterally. A recent linkage study [Fisher, S., Vargha-Khadem, F., Watkins, K. E., Monaco, A. P. & Pembry, M. E. (1998)Nat. Genet.18, 168–170] localized the abnormal gene (SPCH1) to a 5.6-centiMorgan interval in the chromosomal band 7q31. The genetic mutation or deletion in this region has resulted in the abnormal development of several brain areas that appear to be critical for both orofacial movements and sequential articulation, leading to marked disruption of speech and expressive language.
Characterization of two patched receptors for the vertebrate hedgehog protein family
Authors: Carpenter David , Stone Donna M. , Brush Jennifer , Ryan Anne , Armanini Mark , Frantz Gretchen , Rosenthal Arnon , de Sauvage Frederic J.
Published: July 2002

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human
PTCH2
gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity
in vivo
. Chromosomal localization of
PTCH2
places it on chromosome 1p33–34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.
Estrogen alters behavior and forebrain c-
fos
expression in ovariectomized rats subjected to the forced swim test
Authors: Rachman Ilya M. , Unnerstall James R. , Pfaff Donald W. , Cohen Rochelle S.
Published: July 2002

Estrogen has been implicated in brain functions related to affective state, including hormone-related affective disorders in women. Although some reports suggest that estrogen appears to decrease vulnerability to affective disorders in certain cases, the mechanisms involved are unknown. We used the forced swim test (FST), a paradigm used to test the efficacy of antidepressants, and addressed the hypotheses that estrogen alters behavior of ovariectomized rats in the FST and the FST-induced expression of c-
fos
, a marker for neuronal activity, in the rat forebrain. The behaviors displayed included struggling, swimming, and immobility. One hour after the beginning of the test on day 2, the animals were perfused, and the brains were processed for c-
fos
immunocytochemistry. On day 1, the estradiol benzoate-treated animals spent significantly less time struggling and virtually no time in immobility and spent most of the time swimming. Control rats spent significantly more time struggling or being immobile during a comparable period. On day 2, similar behavioral patterns with still more pronounced differences were observed between estradiol benzoate and ovariectomized control groups in struggling, immobility, and swimming. Analysis of the mean number of c-
fos
immunoreactive cell nuclei showed a significant reduction in the estradiol benzoate versus control groups in areas of the forebrain relating to sensory, contextual, and integrative processing. Our results suggest that estrogen-induced neurochemical changes in forebrain neurons may translate into an altered behavioral output in the affective domain.
Ribosome display efficiently selects and evolves high-affinity antibodiesin vitrofrom immune libraries
Authors: Hanes Jozef , Jermutus Lutz , Weber-Bornhauser Susanne , Bosshard Hans Rudolf , Plückthun Andreas
Published: July 2002

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 ± 1) × 10−11M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome displayin vitro, and that this constitutes an affinity maturation inherent in this method. Thein vitro-selected scFvs could be functionally expressed in theEscherichia coliperiplasm with good yields or prepared byin vitrorefolding. Thus, ribosome display can be a powerful methodology forin vitrolibrary screening and simultaneous sequence evolution.
Genetic control of social organization in an ant
Authors: Ross Kenneth G. , Keller Laurent
Published: July 2002

A central issue in evolutionary biology is the extent to which complex social organization is under genetic control. We have found that a single genomic element marked by the protein-encoding geneGp-9is responsible for the existence of two distinct forms of social organization in the fire antSolenopsis invicta. This genetic factor influences the reproductive phenotypes and behavioral strategies of queens and determines whether workers tolerate a single fertile queen or multiple queens per colony. Furthermore, this factor affects worker tolerance of queens with alternate genotypes, thus explaining the dramatic differences inGp-9allele frequencies observed between the two social forms in the wild. These findings reveal how a single genetic factor can have major effects on complex social behavior and influence the nature of social organization.
Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of
Glossina morsitans morsitans
Authors: Cappello Michael , Li Song , Chen Xiaoai , Li Chang-Ben , Harrison Lisa , Narashimhan Sukanya , Beard Charles B. , Aksoy Serap
Published: July 2002

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of
Glossina morsitans morsitans
(Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in
Escherichia coli
and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.
A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families
Authors: Gaffney Patrick M. , Kearns Grainne M. , Shark Katherine B. , Ortmann Ward A. , Selby Scott A. , Malmgren Michelle L. , Rohlf Kristine E. , Ockenden Theresa C. , Messner Ronald P. , King Richard A. , Rich Stephen S. , Behrens Timothy W.
Published: July 2002

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [
D6S257
, logarithm of odds (lod) = 3.90,
P
= 0.000011] and at three additional regions: 16q13 (
D16S415
, lod = 3.64,
P
= 0.000022), 14q21–23 (
D14S276
, lod = 2.81,
P
= 0.00016), and 20p12 (
D20S186
, lod = 2.62,
P
= 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21–33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores ≥1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.
Nuclear localization of prostaglandin E
2
receptors
Authors: Bhattacharya Mousumi , Peri Krishna G. , Almazan Guillermina , Ribeiro-da-Silva Alfredo , Shichi Hitoshi , Durocher Yves , Abramovitz Mark , Hou Xin , Varma Daya R. , Chemtob Sylvain
Published: July 2002

Prostaglandin E
2
receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP
1
was also found in fibroblast Swiss 3T3 cells stably overexpressing EP
1
and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP
1
fused to green fluorescent protein. High-resolution immunostaining of EP
1
revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and
in situ
in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.
Investigating neural correlates of conscious perception by frequency-tagged neuromagnetic responses
Authors: Tononi Giulio , Srinivasan Ramesh , Russell D. Patrick , Edelman Gerald M.
Published: July 2002

In binocular rivalry, a subject views two incongruent stimuli through each eye but consciously perceives only one stimulus at a time, with a switch in perceptual dominance every few seconds. To investigate the neural correlates of perceptual dominance in humans, seven subjects were recorded with a 148-channel magnetoencephalography array while experiencing binocular rivalry. A red vertical grating flickering at one frequency was presented to one eye through a red filter and a blue horizontal grating flickering at a different frequency was presented to the other eye through a blue filter. Steady-state neuromagnetic responses at the two frequencies were used as tags for the two stimuli and analyzed with high-resolution power spectra. It was found that a large number of channels showed peaks at both frequencies, arranged in a horseshoe pattern from posterior to anterior regions, whether or not the subject was consciously perceiving the corresponding stimulus. However, the amount of power at the stimulus frequency was modulated in relation to perceptual dominance, being lower in many channels by 50–85% when the subject was not conscious of that stimulus. Such modulation by perceptual dominance, although not global, was distributed to a large subset of regions showing stimulus-related responses, including regions outside visual cortex. The results demonstrate a correlation between the conscious perception of a visual stimulus and the synchronous activity of large populations of neurons as reflected by steady-state neuromagnetic responses.
The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplicesin vitro
Authors: Laggerbauer Bernhard , Achsel Tilmann , Lührmann Reinhard
Published: July 2002

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplicesin vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.
Unique molecular surface features ofin vivotolerized T cells
Authors: Maier Curtis C. , Bhandoola Avinash , Borden William , Yui Katsuyuki , Hayakawa Kyoko , Greene Mark I.
Published: July 2002

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergicin vivoby superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vβ8.1 T cell antigen receptor (TCR) transgenic mice with a Vβ8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1a) induces tolerance to Mls-1aby clonal anergy. CD4 lymph node T cells from Mls-1ainoculated transgenic mice enriched for the 6C10−phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10+CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10−T cells remain 3G11hibut express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.
Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT
2
) angiotensin receptor
Authors: Siragy Helmy M. , Inagami Tadashi , Ichiki Toshihiro , Carey Robert M.
Published: July 2002

The vast majority of the known biological effects of the renin–angiotensin system are mediated by the type-1 (AT
1
) receptor, and the functions of the type-2 (AT
2
) receptor are largely unknown. We investigated the role of the AT
2
receptor in the vascular and renal responses to physiological increases in angiotensin II (ANG II) in mice with targeted deletion of the AT
2
receptor gene. Mice lacking the AT
2
receptor (AT
2
-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (
P
< 0.0001). In AT
2
-null mice, infusion of ANG II (4 pmol/kg/min) for 7 days produced a marked and sustained increase in SBP [from 116 ± 0.5 to 208 ± 1 mmHg (
P
< 0.0001) (1 mmHg = 133 Pa)] and reduction in urinary sodium excretion (U
Na
V) [from 0.6 ± 0.01 to 0.05 ± 0.002 mM/day (
P
< 0.0001)] whereas neither SBP nor U
Na
V changed in WT mice. AT
2
-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3′,5′-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3′,5′-monophosphate by ≈4-fold (
P
< 0.0001) whereas no changes were observed in AT
2
-null mice. These results demonstrate that the AT
2
receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT
2
receptor leads to vascular and renal hypersensitivity to ANG II, including sustained antinatriuresis and hypertension. These results strongly suggest that the AT
2
receptor plays a counterregulatory protective role mediated via BK and nitric oxide against the antinatriuretic and pressor actions of ANG II.
The roles of prefrontal brain regions in components of working memory: Effects of memory load and individual differences
Authors: Rypma Bart , D’Esposito Mark
Published: July 2002

Using an event-related functional MRI design, we explored the relative roles of dorsal and ventral prefrontal cortex (PFC) regions during specific components (Encoding, Delay, Response) of a working memory task under different memory-load conditions. In a group analysis, effects of increased memory load were observed only in dorsal PFC in the encoding period. Activity was lateralized to the right hemisphere in the high but not the low memory-load condition. Individual analyses revealed variability in activation patterns across subjects. Regression analyses indicated that one source of variability was subjects’ memory retrieval rate. It was observed that dorsal PFC plays a differentially greater role in information retrieval for slower subjects, possibly because of inefficient retrieval processes or a reduced quality of mnemonic representations. This study supports the idea that dorsal and ventral PFC play different roles in component processes of working memory.
Ultraviolet A radiation induces immediate release of iron in human primary skin fibroblasts: The role of ferritin
Authors: Pourzand Charareh , Watkin Richard D. , Brown Jonathan E. , Tyrrell Rex M.
Published: July 2002

In mammalian cells, the level of the iron-storage protein ferritin (Ft) is tightly controlled by the iron-regulatory protein-1 (IRP-1) at the posttranscriptional level. This regulation prevents iron acting as a catalyst in reactions between reactive oxygen species and biomolecules. The ultraviolet A (UVA) radiation component of sunlight (320–400 nm) has been shown to be a source of oxidative stress to skin via generation of reactive oxygen species. We report here that the exposure of human primary skin fibroblasts, FEK4, to UVA radiation causes an immediate release of “free” iron in the cells via proteolysis of Ft. Within minutes of exposure to a range of doses of UVA at natural exposure levels, the binding activity of IRP-1, as well as Ft levels, decreases in a dose-dependent manner. This decrease coincides with a significant leakage of the lysosomal components into the cytosol. Stabilization of Ft molecules occurs only when cells are pretreated with lysosomal protease inhibitors after UVA treatment. We propose that the oxidative damage to lysosomes that leads to Ft degradation and the consequent rapid release of potentially harmful “free” iron to the cytosol might be a major factor in UVA-induced damage to the skin.
The intracellular parasiteTheileria parvaprotects infected T cells from apoptosis
Authors: Heussler Volker T. , Machado Joel , Fernandez Paula C. , Botteron Catherine , Chen Chao-Guang , Pearse Martin J. , Dobbelaere Dirk A. E.
Published: July 2002

Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasiteTheileria parvasecures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-κB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-κB activation inT. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-κB activation plays a crucial role in the survival ofT. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-κB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-κB activation pathway, such as IκBα or p65, prompt rapid apoptosis ofT. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-κB activation is an essential step in maintaining the transformed phenotype of the infected cells.
Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D
3
-1α-hydroxylase gene in normocalcemic rats
Authors: Shinki Toshimasa , Ueno Yutaka , DeLuca Hector F. , Suda Tatsuo
Published: July 2002

Regulation of vitamin D metabolism has long been examined by using vitamin D-deficient hypocalcemic animals. We previously reported that, in a rat model of chronic hyperparathyroidism, expression of 25-hydroxyvitamin D
3
-1α-hydroxylase (CYP27B1) mRNA was markedly increased in renal proximal convoluted tubules. It is believed that the major regulator for the expression of renal CYP27B1 is parathyroid hormone (PTH). However, in the normocalcemic state, the mechanism to regulate the renal CYP27B1 gene could be different, since plasma levels of PTH are very low. In the present study, the effect of PTH and calcitonin (CT) on the expression of renal CYP27B1 mRNA was investigated in normocalcemic sham-operated rats and normocalcemic thyroparathyroidectomized (TPTX) rats generated by either PTH or CaCl
2
infusion. A single injection of CT dose-dependently decreased the expression of vitamin D receptor mRNA in the kidney of normocalcemic sham-TPTX rats. Concomitantly, CT greatly increased the expression of CYP27B1 mRNA in the kidney of normocalcemic sham-TPTX rats. CT also increased the expression of CYP27B1 mRNA in the kidney of normocalcemic TPTX rats. Conversion of serum [
3
H]1α,25(OH)
2
D
3
from 25-hydroxy[
3
H]vitamin D
3
in vivo
was also greatly increased by the injection of CT into sham-TPTX rats and normocalcemic TPTX rats, but not into hypocalcemic TPTX rats. In contrast, administration of PTH did not induce the expression of CYP27B1 mRNA in the kidney of vitamin D-replete sham-TPTX rats and hypocalcemic TPTX rats. PTH increased the expression of renal CYP27B1 mRNA only in vitamin D-deficient hypocalcemic TPTX rats. These results suggest that CT plays an important role in the maintenance of serum 1α,25(OH)
2
D
3
under normocalcemic physiological conditions, at least in rats.
The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties
Authors: Moosmann Bernd , Behl Christian
Published: July 2002

Among the family of steroidal molecules, only estrogens have the capability of preventing neuronal cell death caused by increased oxidative burden. Employing neuronal cell lines, brain membrane, and low density lipoprotein oxidation assays, we show that the antioxidant and neuroprotective effects of estrogens are dependent not on their genomic properties as hormones but rather on their basic chemical properties as hydrophobic phenolic molecules. Concentrations of 17β-estradiol of 0.1–500 nM, which confer maximum estrogen receptor-dependent gene transcription
in vitro
as well as maximum estrogen receptor binding, respectively, do not show antioxidant or neuroprotective effects. In contrast, phenolic compounds such as 2,4,6-trimethylphenol,
N
-acetylserotonin, and 5-hydroxyindole exhibit neuroprotective effects without any estrogenicity. Comparing various natural and synthetic mono- and polyphenolic compounds, no correlation between their antioxidant cytoprotective effect and their estrogenic potency can be seen. These results call into question the idea of a general correlation between the intended pharmacological effects of estrogens and phenolic compounds and their effect on estrogen receptor-dependent pathways. Furthermore, they may open the door toward the rational design of neuroprotective antioxidants with decreased hormonal side effects.
Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3
Authors: Woetmann Anders , Nielsen Mette , Christensen Søren T. , Brockdorff Johannes , Kaltoft Keld , Engel Anne-Marie , Skov Søren , Brender Christine , Geisler Carsten , Svejgaard Arne , Rygaard Jørgen , Leick Vagn , Ødum Niels
Published: July 2002

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4
+
T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (
i
) phosphorylation of STAT3 on serine and threonine residues, (
ii
) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (
iii
) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.
Proton transfer from the bulk to the bound ubiquinone Q
B
of the reaction center in chromatophores of
Rhodobacter sphaeroides
: Retarded conveyance by neutral water
Authors: Gopta Oksana A. , Cherepanov Dmitry A. , Junge Wolfgang , Mulkidjanian Armen Y.
Published: July 2002

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule Q
B
by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (
i
) the electron transfer to Q
B
(at 450 nm), (
ii
) the electrogenic proton delivery from the surface to the Q
B
site (by electrochromic carotenoid response at 524 nm), and (
iii
) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to Q
B
−
and the proton-related electrogenesis proceeded with the same time constant of ≈100 μs (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (τ ≈ 400 μs). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (
i
) proton transfer from the surface-located acidic groups into the Q
B
site followed the reduction of Q
B
without measurable delay; (
ii
) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (
iii
) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.
Potent inhibition of tumor survivalin vivoby β-lapachone plus taxol: Combining drugs imposes different artificial checkpoints
Authors: Li Chiang J. , Li You-Zhi , Pinto Antonio Ventura , Pardee Arthur B.
Published: July 2002

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of β-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after β-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. β-Lapachone causes cell-cycle delays in late G1and S phase, and taxol arrests cells at G2/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone “collisions” at cell-cycle checkpoints.
Recurrent invasion and extinction of a selfish gene
Authors: Goddard Matthew R. , Burt Austin
Published: July 2002

Homing endonuclease genes show super-Mendelian inheritance, which allows them to spread in populations even when they are of no benefit to the host organism. To test the idea that regular horizontal transmission is necessary for the long-term persistence of these genes, we surveyed 20 species of yeasts for the ω-homing endonuclease gene and associated group I intron. The status of ω could be categorized into three states (functional, nonfunctional, or absent), and status was not clustered on the host phylogeny. Moreover, the phylogeny of ω differed significantly from that of the host, strong evidence of horizontal transmission. Further analyses indicate that horizontal transmission is more common than transposition, and that it occurs preferentially between closely related species. Parsimony analysis and coalescent theory suggest that there have been 15 horizontal transmission events in the ancestry of our yeast species, through simulations indicate that this value is probably an underestimate. Overall, the data support a cyclical model of invasion, degeneration, and loss, followed by reinvasion, and each of these transitions is estimated to occur about once every 2 million years. The data are thus consistent with the idea that frequent horizontal transmission is necessary for the long-term persistence of homing endonuclease genes, and further, that this requirement limits these genes to organisms with easily accessible germ lines. The data also show that mitochondrial DNA sequences are transferred intact between yeast species; if other genes do not show such high levels of horizontal transmission, it would be due to lack of selection, rather than lack of opportunity.
Head-specific gene expression in
Hydra
: Complexity of DNA– protein interactions at the promoter of
ks1
is inversely correlated to the head activation potential
Authors: Endl Ingrid , Lohmann Jan U. , Bosch Thomas C. G.
Published: July 2002

To gain insight into the molecular mechanisms that direct
position-dependent gene expression in the simple and evolutionarily old
metazoan
Hydra
, we have examined DNA–protein interactions
in the 1.5-kb cis regulatory region of the head-specific gene
ks1
.
In vitro
footprinting and gel-retardation
techniques have been used to map the location of all protein-binding
sites. To our surprise, we found substantially more proteins binding to
ks1
promoter elements in nuclear extract from basal
(gastric) than from apical (head- and tentacle-formation zone) cells.
One of these proteins is the homeobox protein Cnox-2. In the head
regeneration-deficient mutant reg-16, an increased level of nuclear
protein binds to
ks1
promoter elements. Treatment of polyps
with the
ks1
-inducing phorbol ester
12-
O
-tetradecanoylphorbol 13-acetate (TPA) resulted in
reduced binding of nuclear proteins to the
ks1
cis
regulatory region. As activation of
ks1
transcription is
correlated with the absence of nuclear proteins binding to the
ks1
promoter, we propose that the majority of these
proteins act as transcriptional repressors. In this view, the gradient
of head activation along the
Hydra
body axis is caused by a
decreasing amount of inhibitory factors, rather than an increasing
amount of activators, toward the head. Thus, inhibitory mechanisms
might have played a crucial role in regulating position-dependent gene
activation during early metazoan evolution.
Protrusive growth from giant liposomes driven by actin polymerization
Authors: Miyata Hidetake , Nishiyama Shuji , Akashi Ken-ichirou , Kinosita Kazuhiko
Published: July 2002

Development of protrusions in the cell is indispensable in the process of cell motility. Membrane protrusion has long been suggested to occur as a result of actin polymerization immediately beneath the cell membrane at the leading edge, but elucidation of the mechanism is insufficient because of the complexity of the cell. To study the mechanism, we prepared giant liposomes containing monomeric actin (100 or 200 μM) and introduced KCl into individual liposomes by an electroporation technique. On the electroporation, the giant liposomes deformed. Most importantly, protrusive structure grew from the liposomes containing 200 μM actin at rates (ranging from 0.3 to 0.7 μm/s) similar to those obtained in the cell. The deformation occurred in a time range (30 ∼ 100 s) similar to that of actin polymerization monitored in a cuvette (ca. 50 s). Concomitant with deformation, Brownian motion of micron-sized particles entrapped in the liposomes almost ceased. From these observations, we conclude that actin polymerization in the liposomes caused the protrusive formation.
Prebiotic cytosine synthesis: A critical analysis and implications for the origin of life
Authors: Shapiro Robert
Published: July 2002

A number of theories propose that RNA, or an RNA-like substance, played a role in the origin of life. Usually, such hypotheses presume that the Watson–Crick bases were readily available on prebiotic Earth, for spontaneous incorporation into a replicator. Cytosine, however, has not been reported in analyses of meteorites nor is it among the products of electric spark discharge experiments. The reported prebiotic syntheses of cytosine involve the reaction of cyanoacetylene (or its hydrolysis product, cyanoacetaldehyde), with cyanate, cyanogen, or urea. These substances undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation. To favor cytosine formation, reactant concentrations are required that are implausible in a natural setting. Furthermore, cytosine is consumed by deamination (the half-life for deamination at 25°C is ≈340 yr) and other reactions. No reactions have been described thus far that would produce cytosine, even in a specialized local setting, at a rate sufficient to compensate for its decomposition. On the basis of this evidence, it appears quite unlikely that cytosine played a role in the origin of life. Theories that involve replicators that function without the Watson–Crick pairs, or no replicator at all, remain as viable alternatives.
Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide fromShigella dysenteriaetype 1
Authors: Pozsgay Vince , Chu Chiayung , Pannell Lewis , Wolfe Jennifer , Robbins John B. , Schneerson Rachel
Published: July 2002

Our development of vaccines to prevent shigellosis is based on the hypothesis that a critical (protective) level of serum IgG to the O-specific polysaccharide (O-SP) domain ofShigellalipopolysaccharide (LPS) confers immunity. The O-SP is a hapten and must be conjugated to a protein to induce serum antibodies. The O-SP ofShigella dysenteriaetype 1 (≈27 tetrasaccharide repeat units), prepared by acid hydrolysis of the LPS, was bound to human serum albumin (HSA) by multiple point attachment (O-SP-HSA): The molar ratio of HSA to O-SP was 1.0. Synthetic saccharides, composed of one or multiples of the O-SP tetrasaccharide, equipped with a spacer at their reducing end, were bound to HSA by a single point attachment: The average molar ratios of the saccharides to HSA ranged from 4 to 24. Serum IgG anti-LPS, elicited in mice by O-SP-HSA or synthetic tetra-, octa-, dodeca-, and hexadecasaccharide fragments, was measured by ELISA. Outbred 6-week-old female mice were injected s.c. three times at biweekly intervals with 2.5 μg of saccharide as a conjugate and were bled 7 days after the second and third injections. Excepting the tetramer, conjugates of the octamer, dodecamer and hexadecamer elicited IgG LPS antibodies after the second injection, a statistically significant rise (booster) after the third injection, and higher levels than those vaccinated with O-SP-HSA (P= 0.0001). The highest geometric mean levels of IgG anti-LPS were elicited by the hexadecamer with 9 chains or 9 moles of saccharide/HSA (15.5 ELISA units) followed by the octamer with 20 chains (11.1 ELISA units) and the dodecamer with 10 chains (9.52 ELISA units). Clinical evaluation of these synthetic saccharides bound to a medically useful carrier is planned.
Synthetic receptors as models for alkali metal cation-π binding sites in proteins
Authors: De Wall Stephen L. , Meadows Eric S. , Barbour Leonard J. , Gokel George W.
Published: July 2002

The alkali metal cations Na
+
and K
+
have several important physiological roles, including modulating enzyme activity. Recent work has suggested that alkali metal cations may be coordinated by π systems, such as the aromatic amino acid side chains. The ability of K
+
to interact with an aromatic ring has been assessed by preparing a family of synthetic receptors that incorporate the aromatic side chains of phenylalanine, tyrosine, and tryptophan. These receptors are constructed around a diaza-18-crown-6 scaffold, which serves as the primary binding site for an alkali metal cation. The ability of the aromatic rings to coordinate a cation was determined by crystallizing each of the receptors in the presence of K
+
and by solving the solid state structures. In all cases, complexation of K
+
by the π system was observed. When possible, the structures of the unbound receptors also were determined for comparison. Further proof that the aromatic ring makes an energetically favorable interaction with the cation was obtained by preparing a receptor in which the arene was perfluorinated. Fluorination of the arene reverses the electrostatics, but the aromaticity is maintained. The fluorinated arene rings do not coordinate the cation in the solid state structure of the K
+
complex. Thus, the results of the predicted electrostatic reversal were confirmed. Finally, the biological implications of the alkali metal cation-π interaction are addressed.