bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in platelet count in older, poor‐risk patients with acute myeloid leukemia or myelodysplastic syndrome treated with valproic acid and all‐trans retinoic acid
Authors: Pilatrino Chiara , Cilloni Daniela , Messa Emanuela , Morotti Alessandro , Giugliano Emilia , Pautasso Marisa , Familiari Ubaldo , Cappia Susanna , Pelicci Pier Giuseppe , Lo Coco Francesco , Saglio Giuseppe , Guerrasio Angelo
Published: May 2005

BACKGROUND

The authors investigated the efficacy and safety of the histone deacetylase inhibitors valproic acid (VPA) and all‐trans retinoic acid (ATRA) as differentiation agents in a cohort of older, poor‐risk patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

METHODS

Twenty older patients with recurrent or refractory AML or MDS were treated in a Phase II protocol with sequential VPA and ATRA therapy. VPA was started at a dose of 10 mg/kg per day and then escalated to achieve the serum concentration of 45–100 μg/mL. ATRA was added at 45 mg/square meters (sm) per day when VPA reached the target serum concentration. Only patients treated continuously for ≥ 2 months were considered evaluable.

RESULTS

Hematologic improvement, according to World Health Organization criteria, was observed in 6 of 20 patients enrolled in the protocol but in 6 of 11 considered evaluable. In five patients, a major platelet response was observed, achieving platelet transfusion independence. Three of these five patients also exhibited a minor erythroid response. A sixth patient showed both a minor erythroid response and a platelet response. The median duration of response was 189 days (range, 63–550 days). No significant reduction in the blast count was observed. Grade 3 neurocortical toxicity was observed in four patients. Severe bone pain was experienced by 4 patients (2 Grade 4 and 2 Grade 3) and was associated with an increase in the peripheral blast cell count. Treatment with ATRA did not modify the response observed with VPA alone.

CONCLUSIONS

Differentiation therapy with VPA was of clinical benefit in approximately 30% of elderly patients with AML and MDS of the refractory anemia with excess of blast type with unfavorable prognostic features. A striking platelet transfusion independence lasting several months may be obtained in some patients, reducing the burden of palliative care and improving the quality of life. Cancer 2005;. © 2005 American Cancer Society.
CpG methylation at promoter site −140 inactivates TGFβ2 receptor gene in prostate cancer
Authors: Zhao Hong , Shiina Hiroaki , Greene Kirsten L. , Li Long‐Cheng , Tanaka Yuichiro , Kishi Hirofumi , Igawa Mikio , Kane Christopher J. , Carroll Peter , Dahiya Rajvir
Published: May 2005

BACKGROUND

The action of transforming growth factor β (TGF‐β) is mediated through type 1 (TβRI) and type 2 (TβRII) receptors. Prostate cancer cells are often resistant to TGF‐β signaling due to loss of TβRII expression. The authors of the current study hypothesized that CpG methylation of the TβRII promoter at the Sp1 binding site −140 mediates this loss of TβRII expression in prostate cancer.

METHODS

Sixty‐seven prostate cancer (PC) samples, 8 benign prostatic hyperplasia (BPH) samples, and 4 prostate cancer cell lines (DUPro, LNCaP, ND‐1 and PC‐3) were analyzed for 1) TβRII mRNA expression by semiquantitative RT‐PCR, 2) TβRII protein expression by immunohistochemistry, and 3) TGFβRII promoter methylation at CpG site −140 by methylation specific PCR and bisulfite DNA sequencing. Prostate cancer cell lines were treated with the demethylating agent 5aza2′deoxycytidine to determine if TβRII gene expression could be increased by blocking promoter methylation.

RESULTS

mRNA and protein expression of TβRII was lower in the PC samples than in the BPH samples. CpG methylation at site −140 was higher in PC than in BPH (P < 0.01). Promoter methylation was inversely correlated with TβRII mRNA expression in the PC and BPH samples (P < 0.0001). PC3, ND1, and DUPro TβRII mRNA expression increased following treatment of cells with 5‐aza‐2′‐deoxycytidine.

CONCLUSION

CpG methylation of the TβRII promoter at CPG site −140 leads to functional loss of the TβRII gene in prostate cancer. Treatment with 5‐aza‐2′ deoxycytidine can restore gene expression. The current study results report the first association between prostate cancer and loss of the TGF‐ β signaling pathway by TβRII DNA promoter methylation. Cancer 2005;. © 2005 American Cancer Society.
Outcomes and prognostic factors after recurrence in children and adolescents with nonmetastatic rhabdomyosarcoma
Authors: Mazzoleni Stefano , Bisogno Gianni , Garaventa Alberto , Cecchetto Giovanni , Ferrari Andrea , Sotti Guido , Donfrancesco Alberto , Madon Enrico , Casula Letizia , Carli Modesto ,   Associazione Italiana di Ematologia e Oncologia Pediatrica Soft Tissue Sarcoma Committee
Published: May 2005

BACKGROUND

Although > 90% of children with nonmetastatic rhabdomyosarcoma (RMS) achieve complete remission with current treatment, up to one‐third of them experience a recurrence. Survival rates are not always poor in patients who develop recurrences; thus, prognostic factors are needed to tailor salvage treatment.

METHODS

The current analysis included 125 children who were affected by localized RMS and were enrolled in 3 consecutive Italian protocols (RMS79, RMS88, and RMS96) who developed recurrences after complete remission. Patient, tumor, and treatment characteristics were studied in univariate and multivariate analyses to determine the independent significance of different factors.

RESULTS

The median time from diagnosis to recurrence was 17.8 months. Most patients had local recurrences (72%). The 5‐year overall survival (OS) rate was 28.3% ± 8.7%. Multivariate analysis identified 4 factors that were associated with poor survival: 1) alveolar subtype (relative risk [RR], 2.0), 2) parameningeal or “other” sites (RR, 2.6), 3) systemic recurrence (RR, 3.1), and 4) recurrence on therapy (RR, 2.3). The absence of any of these risk factors identified a “favorable risk” group (12% of patients) with a 5‐year OS rate of 71.8% ± 23.5%. Patients with a single risk factor (32%) had an OS rate of 37.5% ± 17.2%. Combining patients with 0 or 1 risk factor, the OS rate was 66.5% in the subgroup who had not received radiotherapy compared with an OS rate of 30.3% in the subgroup who had received radiotherapy; this difference was significant (P= 0.03).

CONCLUSIONS

The results of the current analysis demonstrated that groups with a different prognosis can be identified among patients with recurrent RMS. Patients with a nonalveolar histology, a primary site other than the parameningeal or “other” sites, local recurrence, and recurrence off therapy had a better prognosis. First‐line treatment may have an impact on prognostic variables. In fact, patients who had no or only one risk factor and patients who had tumors with a nonalveolar histology benefited more from salvage therapy if they had not received radiotherapy for their initial treatment. These data may be useful in planning risk‐adapted salvage protocols. Cancer 2005;. © 2005 American Cancer Society.
Protein phosphatase 4 – from obscurity to vital functions
Authors: Cohen Patricia T.W. , Philp Amanda , Vázquez-Martin Cristina
Published: May 2005

Protein phosphatase 4 (Ppp4) is a ubiquitous serine/threonine phosphatase in the PPP family that is now recognised to regulate a variety of cellular functions independently of protein phosphatase 2A (PP2A). Regulatory subunits (R1 and R2) have been identified in mammals that interact with the catalytic subunit of Ppp4 (Ppp4c) and control its activity. Ppp4c–R2 complexes play roles in organelle assembly; not only are they essential for maturation of the centrosome, but they are also involved in spliceosomal assembly via interaction with the survival of motor neurons (SMNs) complex. Several cellular signalling routes, including NF‐κB and the target of rapamycin (TOR) pathways appear to be regulated by Ppp4. Emerging evidence indicates that Ppp4 may play a role in the DNA damage response and that Ppp4c–R1 complexes decrease the activity of a histone deacetylase, implicating Ppp4 in the regulation of chromatin activities. Antitumour agents, cantharidin and fostriecin, potently inhibit the activity of Ppp4. Orthologues of mammalian Ppp4 subunits in Saccharomyces cerevisiae confer resistance to the anticancer, DNA‐binding drugs, cisplatin and oxaliplatin.
Peroxynitrite‐mediated matrix metalloproteinase‐2 activation in human hepatic stellate cells
Authors: Migita Kiyoshi , Maeda Yumi , Abiru Seigo , Komori Atsumasa , Yokoyama Terufumi , Takii Yasushi , Nakamura Minoru , Yatsuhashi Hiroshi , Eguchi Katsumi , Ishibashi Hiromi
Published: May 2005

To investigate whether hepatic stellate cells (HSCs) alter their expression of MMPs after exposure to nitrogen oxide intermediate (NOI), a human hepatic stellate cell line, LI90 cells, was stimulated with an NO donor, SNAP, or a peroxynitrite donor, SIN‐1, and the culture supernatants were analyzed by gelatin zymography or anti‐MMPs immunoblot. Although SIN‐1 did not enhance the secretions of MMP‐1 and 13, SIN‐1 induced the NF‐κB activation, MT1‐MMP expression and the secretion of activated MMP‐2 in LI90 cells. These results suggest that peroxynitrite may contribute to the remodeling of the extracellular matrix in liver by activating pro‐MMP‐2.
Folding transition into a loosely collapsed state in plasmid DNA as revealed by single‐molecule observation
Authors: Sato Yuko T. , Hamada Tsutomu , Kubo Koji , Yamada Ayako , Kishida Tsunao , Mazda Osam , Yoshikawa Kenichi
Published: May 2005

The conformational transition of a plasmid DNA, pGEG.GL3 (12.5 kbp, circular), induced by spermine(4+) was studied through the observation of individual DNA by fluorescence microscopy. We deduced the change in the hydrodynamic radius R
H from an analysis of the Brownian motion of single DNA molecules. R
H decreases in a continuous manner with an increase in spermine(4+), in contrast to the large discrete on/off change for long linear DNA. Just after the transition to the collapsed state, a small number of DNA molecules tend to form an assembly, which disperses in the bulk solution without precipitation.
Using fingerprint image quality to improve the identification performance of the U.S. Visitor and Immigrant Status Indicator Technology Program
Authors: Wein Lawrence M. , Baveja Manas
Published: May 2005

Motivated by the difficulty of biometric systems to correctly match fingerprints with poor image quality, we formulate and solve a game-theoretic formulation of the identification problem in two settings: U.S. visa applicants are checked against a list of visa holders to detect visa fraud, and visitors entering the U.S. are checked against a watchlist of criminals and suspected terrorists. For three types of biometric strategies, we solve the game in which the U.S. Government chooses the strategy's optimal parameter values to maximize the detection probability subject to a constraint on the mean biometric processing time per legal visitor, and then the terrorist chooses the image quality to minimize the detection probability. At current inspector staffing levels at ports of entry, our model predicts that a quality-dependent two-finger strategy achieves a detection probability of 0.733, compared to 0.526 under the quality-independent two-finger strategy that is currently implemented at the U.S. border. Increasing the staffing level of inspectors offers only minor increases in the detection probability for these two strategies. Using more than two fingers to match visitors with poor image quality allows a detection probability of 0.949 under current staffing levels, but may require major changes to the current U.S. biometric program. The detection probabilities during visa application are ≈11–22% smaller than at ports of entry for all three strategies, but the same qualitative conclusions hold.
Sexually dimorphic expression of trkB, a Z-linked gene, in early posthatch zebra finch brain
Authors: Chen Xuqi , Agate Robert J. , Itoh Yuichiro , Arnold Arthur P.
Published: May 2005

Sexual differentiation of the zebra finch (
Taeniopygia guttata
) neural song circuit is thought to be initiated by sex differences in sex chromosome gene expression in brain cells. One theory is that Z-linked genes, present in the male's ZZ genome at double the dose of females' (ZW), are expressed at higher levels and trigger masculine patterns of development. We report here that trkB (tyrosine kinase receptor B) is Z-linked in zebra finches. trkB is the receptor for neurotrophic factors BDNF and neurotrophin 4, and mediates their influence on neuronal survival, migration, and specification. trkB mRNA is expressed at a higher level in the male telencephalon or whole brain than in corresponding regions of the female in adulthood, and at posthatch day (P) 6, when the song circuit is undergoing sexual differentiation. Moreover, this expression is higher in the song nucleus high vocal center (HVC) than in the surrounding telencephalon at P6, and in males relative to females. In addition, trkB protein is expressed more highly in male than female whole brain at P6. These results establish trkB as a candidate factor that contributes to masculine differentiation of HVC because of its Z-linkage, which leads to sex differences in expression. BDNF is known to be stimulated by estrogen and to be expressed at higher levels in males than females at later ages in HVC. Thus, the trkB-BDNF system may be a focal point for convergent masculinizing influences of Z-linked factors and hormones.
Subsecond reorganization of the actin network in cell motility and chemotaxis
Authors: Diez Stefan , Gerisch Günther , Anderson Kurt , Müller-Taubenberger Annette , Bretschneider Till
Published: May 2005

Actin networks are continuously reorganized in cells that rapidly change their shape. Applying total internal reflection fluorescence microscopy at acquisition rates of 10–20 Hz, we measured an average growth rate of 3 μm·sec
–1
for filamentous actin structures throughout the entire substrate-attached cortex of
Dictyostelium
cells. New filaments often proceed along preexisting ones, resulting in bundle formation concurrent with filament growth. In cells that orientate in a gradient of chemoattractant, prominent assemblies of actin enriched in the Arp2/3 complex are inserted into the network, primarily at the base of filopods that point into the direction of the gradient. We propose that high turnover rates of actin filaments confer the plasticity to the cell cortex that is required for rapid accommodation to external stimuli.
Dissecting coherent vibrational spectra of small proteins into secondary structural elements by sensitivity analysis
Authors: Zhuang Wei , Abramavicius Darius , Mukamel Shaul
Published: May 2005

The response of proteins to sequences of femtosecond infrared pulses provides a multidimensional view into their equilibrium distribution of structures and snapshot pictures of fast-triggered dynamical events. Analyzing these experiments requires advanced computational tools for assigning regions in the resulting multi-dimensional correlation plots to specific secondary-structure elements and their couplings. A differential sensitivity analysis technique based on a perturbation of the local (real space) Hamiltonian is developed to achieve that goal. Application to the amide I region of a small globular protein reveals regions associated with the α-helix, β-sheet, and their coupling. Comparison of signals generated in different directions shows that the double-quantum-coherence signal has a higher sensitivity to the couplings compared with the single-quantum-coherence (photon echo) technique.
Virulence and competitive ability in genetically diverse malaria infections
Authors: de Roode Jacobus C. , Pansini Riccardo , Cheesman Sandra J. , Helinski Michelle E. H. , Huijben Silvie , Wargo Andrew R. , Bell Andrew S. , Chan Brian H. K. , Walliker David , Read Andrew F.
Published: May 2005

Explaining parasite virulence is a great challenge for evolutionary biology. Intuitively, parasites that depend on their hosts for their survival should be benign to their hosts, yet many parasites cause harm. One explanation for this is that within-host competition favors virulence, with more virulent strains having a competitive advantage in genetically diverse infections. This idea, which is well supported in theory, remains untested empirically. Here we provide evidence that within-host competition does indeed select for high parasite virulence. We examine the rodent malaria
Plasmodium chabaudi
in laboratory mice, a parasite–host system in which virulence can be easily monitored and competing strains quantified by using strain-specific real-time PCR. As predicted, we found a strong relationship between parasite virulence and competitive ability, so that more virulent strains have a competitive advantage in mixed-strain infections. In transmission experiments, we found that the strain composition of the parasite populations in mosquitoes was directly correlated with the composition of the blood-stage parasite population. Thus, the outcome of within-host competition determined relative transmission success. Our results imply that within-host competition is a major factor driving the evolution of virulence and can explain why many parasites harm their hosts.
Diacylglycerol kinase ι regulates Ras guanyl-releasing protein 3 and inhibits Rap1 signaling
Authors: Regier Debra S. , Higbee Jared , Lund Katrina M. , Sakane Fumio , Prescott Stephen M. , Topham Matthew K.
Published: May 2005

To study the physiological function of diacylglycerol (DAG) kinase ι (DGKι), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGKι. DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGKι inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGKι bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGKι by crossing wild-type or DGKι-deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGKι-deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGKι regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGKζ, which is structurally similar to DGKι, inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK.
The
Arabidopsis
SUMO E3 ligase SIZ1 controls phosphate deficiency responses
Authors: Miura Kenji , Rus Ana , Sharkhuu Altanbadralt , Yokoi Shuji , Karthikeyan Athikkattuvalasu S. , Raghothama Kashchandra G. , Baek Dongwon , Koo Yoon Duck , Jin Jing Bo , Bressan Ray A. , Yun Dae-Jin , Hasegawa Paul M.
Published: May 2005

Plants sense phosphate (Pi) deficiency and initiate signaling that controls adaptive responses necessary for Pi acquisition. Herein, evidence establishes that AtSIZ1 is a plant small ubiquitin-like modifier (SUMO) E3 ligase and is a focal controller of Pi starvation-dependent responses. T-DNA insertional mutated alleles of
AtSIZ1
(At5g60410) cause
Arabidopsis
to exhibit exaggerated prototypical Pi starvation responses, including cessation of primary root growth, extensive lateral root and root hair development, increase in root/shoot mass ratio, and greater anthocyanin accumulation, even though intracellular Pi levels in
siz1
plants were similar to wild type. AtSIZ1 has SUMO E3 ligase activity
in vitro
, and immunoblot analysis revealed that the protein sumoylation profile is impaired in
siz1
plants. AtSIZ1-GFP was localized to nuclear foci. Steadystate transcript abundances of Pi starvation-responsive genes
AtPT2
,
AtPS2
, and
AtPS3
were moderate but clearly greater in
siz1
seedlings than in wild type, where Pi is sufficient. Pi starvation induced the expression of these genes to the same extent in
siz1
and wild-type seedlings. However, two other Pi starvation-responsive genes,
AtIPS1
and
AtRNS1
, are induced more slowly in
siz1
seedlings by Pi limitation. PHR1, a MYB transcriptional activator of
AtIPS1
and
AtRNS1
, is an AtSIZ1 sumoylation target. These results indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a control mechanism that acts both negatively and positively on different Pi deficiency responses.
Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain
Authors: Nakanishi Kotaro , Fukai Shuya , Ikeuchi Yoshiho , Soma Akiko , Sekine Yasuhiko , Suzuki Tsutomu , Nureki Osamu
Published: May 2005

Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA
Ile
2
and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from
Aquifex aeolicus
at 2.42-Å resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long α-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA
Ile
2
is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear.
Escherichia coli
TilS (called MesJ) has an additional CTD, which may recognize the tRNA
Ile
2
acceptor stem. In contrast, a mutational study revealed that
A. aeolicus
TilS does not recognize the tRNA acceptor stem but recognizes the C29·G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA
Ile
2
from tRNA
Met
by strategies similar to that used by IleRS, but in distinct manners.
Autophagy promotes MHC class II presentation of peptides from intracellular source proteins
Authors: Dengjel Jörn , Schoor Oliver , Fischer Rainer , Reich Michael , Kraus Marianne , Müller Margret , Kreymborg Katharina , Altenberend Florian , Brandenburg Jens , Kalbacher Hubert , Brock Roland , Driessen Christoph , Rammensee Hans-Georg , Stevanovic Stefan
Published: May 2005

MHC–peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4+T cell-mediated processes.
The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure
Authors: Garrity David , Call Matthew E. , Feng Jianwen , Wucherpfennig Kai W.
Published: May 2005

The activating NKG2D receptor plays a critical role in innate and adaptive immune responses by natural killer cells and subpopulations of T cells. The human receptor assembles with the DAP10 signaling dimer, and it is thought that one NKG2D homodimer pairs with a single DAP10 dimer by formation of two salt bridges between charged transmembrane (TM) residues. However, direct stoichiometry measurements demonstrated that one NKG2D homodimer assembles with four DAP10 chains. Selective mutation of one of the basic TM residues of NKG2D resulted in loss of two DAP10 chains, indicating that each TM arginine serves as an interaction site for a DAP10 dimer. Assembly of the hexameric structure was cooperative because this mutation also significantly reduced NKG2D dimerization. A monomeric NKG2D TM peptide was sufficient for assembly with a DAP10 dimer, indicating that the interaction between these proteins occurs in the membrane environment. Formation of a three-helix interface among the TM domains involved ionizable residues from all three chains, the TM arginine of NKG2D and both TM aspartic acids of the DAP10 dimer. The organization of the TM domains thus shows similarities to the T cell antigen receptor–CD3 complex, in particular to the six-chain assembly intermediate between T cell antigen receptor and the CD3δε and CD3γε dimers. Binding of a single ligand can thus result in phosphorylation of four DAP10 chains, which may be relevant for the sensitivity of NKG2D receptor signaling, in particular in situations of low ligand density.
High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein
Authors: Chiu Thang K. , Kubelka Jan , Herbst-Irmer Regine , Eaton William A. , Hofrichter James , Davies David R.
Published: May 2005

The 35-residue subdomain of the villin headpiece (HP35) is a small ultrafast folding protein that is being intensely studied by experiments, theory, and simulations. We have solved the x-ray structures of HP35 and its fastest folding mutant [K24 norleucine (nL)] to atomic resolution and compared their experimentally measured folding kinetics by using laser temperature jump. The structures, which are in different space groups, are almost identical to each other but differ significantly from previously solved NMR structures. Hence, the differences between the x-ray and NMR structures are probably not caused by lattice contacts or crystal/solution differences, but reflect the higher accuracy of the x-ray structures. The x-ray structures reveal important details of packing of the hydrophobic core and some additional features, such as cross-helical H bonds. Comparison of the x-ray structures indicates that the nL substitution produces only local perturbations. Consequently, the finding that the small stabilization by the mutation is completely reflected in an increased folding rate suggests that this region of the protein is as structured in the transition state as in the folded structure. It is therefore a target for engineering to increase the folding rate of the subdomain from ≈0.5 μs–1for the nL mutant to the estimated theoretical speed limit of ≈3 μs–1.
The
elongata
mutants identify a functional Elongator complex in plants with a role in cell proliferation during organ growth
Authors: Nelissen Hilde , Fleury Delphine , Bruno Leonardo , Robles Pedro , De Veylder Lieven , Traas Jan , Micol José Luis , Van Montagu Marc , Inzé Dirk , Van Lijsebettens Mieke
Published: May 2005

The key enzyme for transcription of protein-encoding genes in eukaryotes is RNA polymerase II (RNAPII). The recruitment of this enzyme during transcription initiation and its passage along the template during transcription elongation is regulated through the association and dissociation of several complexes. Elongator is a histone acetyl transferase complex, consisting of six subunits (ELP1–ELP6), that copurifies with the elongating RNAPII in yeast and humans. We demonstrate that point mutations in three
Arabidopsis thaliana
genes, encoding homologs of the yeast Elongator subunits
ELP1
,
ELP3
(histone acetyl transferase), and
ELP4
are responsible for the phenotypes of the
elongata2
(
elo2
),
elo3
, and
elo1
mutants, respectively. The
elo
mutants are characterized by narrow leaves and reduced root growth that results from a decreased cell division rate. Morphological and molecular phenotypes show that the
ELONGATA
(
ELO
) genes function in the same biological process and the epistatic interactions between the
ELO
genes can be explained by the model of complex formation in yeast. Furthermore, the plant Elongator complex is genetically positioned in the process of RNAPII-mediated transcription downstream of Mediator. Our data indicate that the Elongator complex is evolutionarily conserved in structure and function but reveal that the mechanism by which it stimulates cell proliferation is different in yeast and plants.
Physics-based protein-structure prediction using a hierarchical protocol based on the UNRES force field: Assessment in two blind tests
Authors: Ołdziej S. , Czaplewski C. , Liwo A. , Chinchio M. , Nanias M. , Vila J. A. , Khalili M. , Arnautova Y. A. , Jagielska A. , Makowski M. , Schafroth H. D. , Kaźmierkiewicz R. , Ripoll D. R. , Pillardy J. , Saunders J. A. , Kang Y. K. , Gibson K. D. , Scheraga H. A.
Published: May 2005

Recent improvements in the protein-structure prediction method developed in our laboratory, based on the thermodynamic hypothesis, are described. The conformational space is searched extensively at the united-residue level by using our physics-based UNRES energy function and the conformational space annealing method of global optimization. The lowest-energy coarse-grained structures are then converted to an all-atom representation and energy-minimized with the ECEPP/3 force field. The procedure was assessed in two recent blind tests of protein-structure prediction. During the first blind test, we predicted large fragments of α and α+β proteins [60–70 residues with C
α
rms deviation (rmsd) <6 Å]. However, for α+β proteins, significant topological errors occurred despite low rmsd values. In the second exercise, we predicted whole structures of five proteins (two α and three α+β, with sizes of 53–235 residues) with remarkably good accuracy. In particular, for the genomic target TM0487 (a 102-residue α+β protein from
Thermotoga maritima
), we predicted the complete, topologically correct structure with 7.3-Å C
α
rmsd. So far this protein is the largest α+β protein predicted based solely on the amino acid sequence and a physics-based potential-energy function and search procedure. For target T0198, a phosphate transport system regulator PhoU from
T. maritima
(a 235-residue mainly α-helical protein), we predicted the topology of the whole six-helix bundle correctly within 8 Å rmsd, except the 32 C-terminal residues, most of which form a β-hairpin. These and other examples described in this work demonstrate significant progress in physics-based protein-structure prediction.
On the orientation of the backbone dipoles in native folds
Authors: Ripoll Daniel R. , Vila Jorge A. , Scheraga Harold A.
Published: May 2005

The role of electrostatic interactions in determining the native fold of proteins has been investigated by analyzing the alignment of peptide bond dipole moments with the local electrostatic field generated by the rest of the molecule with and without solvent effects. This alignment was calculated for a set of 112 native proteins by using charges from a gas phase potential. Most of the peptide dipoles in this set of proteins are on average aligned with the electrostatic field. The dipole moments associated with α-helical conformations show the best alignment with the electrostatic field, followed by residues in β-strand conformations. The dipole moments associated with other secondary structure elements are on average better aligned than in randomly generated conformations. The alignment of a dipole with the local electrostatic field depends on both the topology of the native fold and the charge distribution assumed for all of the residues. The influences of (
i
) solvent effects, (
ii
) different sets of charges, and (
iii
) the charge distribution assumed for the whole molecule were examined with a subset of 22 proteins each of which contains <30 ionizable groups. The results show that alternative charge
distribution
models lead to significant differences among the associated electrostatic fields, whereas the electrostatic field is less sensitive to the particular set of the adopted charges themselves (empirical conformational energy program for peptides or parameters for solvation energy).
Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia
Authors: Zheng Pei-Zheng , Wang Kan-Kan , Zhang Qun-Ye , Huang Qiu-Hua , Du Yan-Zhi , Zhang Qing-Hua , Xiao Da-Kai , Shen Shu-Hong , Imbeaud Sandrine , Eveno Eric , Zhao Chun-Jun , Chen Yu-Long , Fan Hui-Yong , Waxman Samuel , Auffray Charles , Jin Gang , Chen Sai-Juan , Chen Zhu , Zhang Ji
Published: May 2005

Understanding the complexity and dynamics of cancer cells in response to effective therapy requires hypothesis-driven, quantitative, and high-throughput measurement of genes and proteins at both spatial and temporal levels. This study was designed to gain insights into molecular networks underlying the clinical synergy between retinoic acid (RA) and arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which results in a high-quality disease-free survival in most patients after consolidation with conventional chemotherapy. We have applied an approach integrating cDNA microarray, 2D gel electrophoresis with MS, and methods of computational biology to study the effects on APL cell line NB4 treated with RA, ATO, and the combination of the two agents and collected in a time series. Numerous features were revealed that indicated the coordinated regulation of molecular networks from various aspects of granulocytic differentiation and apoptosis at the transcriptome and proteome levels. These features include an array of transcription factors and cofactors, activation of calcium signaling, stimulation of the IFN pathway, activation of the proteasome system, degradation of the PML–RARα oncoprotein, restoration of the nuclear body, cell-cycle arrest, and gain of apoptotic potential. Hence, this investigation has provided not only a detailed understanding of the combined therapeutic effects of RA/ATO in APL but also a road map to approach hematopoietic malignancies at the systems level.
Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog
Authors: Premkumar Lakshmanane , Greenblatt Harry M. , Bageshwar Umesh K. , Savchenko Tatyana , Gokhman Irena , Sussman Joel L. , Zamir Ada
Published: May 2005

Protein molecular adaptation to drastically shifting salinities was studied in dCA II, an α-type carbonic anhydrase (EC 4.2.1.1) from the exceptionally salt-tolerant unicellular green alga
Dunaliella salina
. The salt-inducible, extracellular dCA II is highly salt-tolerant and thus differs from its mesophilic homologs. The crystal structure of dCA II, determined at 1.86-Å resolution, is globally similar to other α-type carbonic anhydrases except for two extended α-helices and an added Na-binding loop. Its unusual electrostatic properties include a uniformly negative surface electrostatic potential of lower magnitude than that observed in the highly acidic halophilic proteins and an exceptionally low positive potential at a site adjoining the catalytic Zn
2+
compared with mesophilic homologs. The halotolerant dCA II also differs from typical halophilic proteins in retaining conformational stability and solubility in low to high salt concentrations. The crucial role of electrostatic features in dCA II halotolerance is strongly supported by the ability to predict the unanticipated halotolerance of the murine CA XIV isozyme, which was confirmed biochemically. A proposal for the functional significance of the halotolerance of CA XIV in the kidney is presented.
Starch-Related α-Glucan/Water Dikinase Is Involved in the Cold-Induced Development of Freezing Tolerance in Arabidopsis
Authors: Yano Ryoichi , Nakamura Masanobu , Yoneyama Tadakatsu , Nishida Ikuo
Published: May 2005

Cold-induced soluble sugar accumulation enhances the degree of freezing tolerance in various cold-hardy plants including Arabidopsis (Arabidopsis thaliana), where soluble sugars accumulate in only a few hours at 2°C. Hence, along with photosynthesis, starch degradation might play a significant role in cold-induced sugar accumulation and enhanced freezing tolerance. Starch-related α-glucan/water dikinase (EC 2.7.9.4), encoded by Arabidopsis STARCH EXCESS 1 (SEX1), is hypothesized to regulate starch degradation in plastids by phosphorylating starch, thereby ensuring better accessibility by starch-degrading enzymes. Here, we show that Arabidopsis sex1 mutants, when incubated at 2°C for 1 d, were unable to accumulate maltooligosaccharides or normal glucose and fructose levels. In addition, they displayed impaired freezing tolerance. After 7 d at 2°C, sex1 mutants did not show any of the above abnormal phenotypes but displayed slightly higher leaf starch contents. The impaired freezing tolerance of sex1 mutants was restored by overexpression of wild-type SEX1 cDNA using the cauliflower mosaic virus 35S promoter. The results demonstrate a genetic link between the SEX1 locus and plant freezing tolerance, and show that starch degradation is important for enhanced freezing tolerance during an early phase of cold acclimation. However, induction of starch degradation was not accompanied by significant changes in α-glucan/water dikinase activity in leaf extracts and preceded cold-induced augmentation of SEX1 transcripts. Therefore, we conclude that augmentation of SEX1 transcripts might be a homeostatic response to low temperature, and that starch degradation during an early phase of cold acclimation could be regulated by a component(s) of a starch degradation pathway(s) downstream of SEX1.
Genome-Wide Identification of Genes Expressed in Arabidopsis Pistils Specifically along the Path of Pollen Tube Growth
Authors: Tung Chih-Wei , Dwyer Kathleen G. , Nasrallah Mikhail E. , Nasrallah June B.
Published: May 2005

Plant reproductive development is dependent on successful pollen-pistil interactions. In crucifers, the pollen tube must breach the stigma surface and burrow through the extracellular matrix of the stigma epidermal cells and transmitting tract cells before reaching its ovule targets. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen/pollen tubes and cells of the pistil that line their path. However, with few exceptions, little is known about the genes that control these interactions. The specialized functions of stigma epidermal cells and transmitting tract cells are likely to depend on the activity of genes expressed specifically in these cells. In order to identify these genes, we used the Arabidopsis (Arabidopsis thaliana) ATH1 microarray to compare the whole-genome transcriptional profiles of stigmas and ovaries isolated from wild-type Arabidopsis and from transgenic plants in which cells of the stigma epidermis and transmitting tract were specifically ablated by expression of a cellular toxin. Among the 23,000 genes represented on the array, we identified 115 and 34 genes predicted to be expressed specifically in the stigma epidermis and transmitting tract, respectively. Both gene sets were significantly enriched in predicted secreted proteins, including potential signaling components and proteins that might contribute to reinforcing, modifying, or remodeling the structure of the extracellular matrix during pollination. The possible role of these genes in compatible and incompatible pollen-pistil interactions is discussed.
Pigment Deficiency in Nightshade/Tobacco Cybrids Is Caused by the Failure to Edit the Plastid ATPase α-Subunit mRNA
Authors: Schmitz-Linneweber Christian , Kushnir Sergei , Babiychuk Elena , Poltnigg Peter , Herrmann Reinhold G. , Maier Rainer M.
Published: May 2005

The subgenomes of the plant cell, the nuclear genome, the plastome, and the chondriome are known to interact through various types of coevolving macromolecules. The combination of the organellar genome from one species with the nuclear genome of another species often leads to plants with deleterious phenotypes, demonstrating that plant subgenomes coevolve. The molecular mechanisms behind this nuclear–organellar incompatibility have been elusive, even though the phenomenon is widespread and has been known for >70 years. Here, we show by direct and reverse genetic approaches that the albino phenotype of a flowering plant with the nuclear genome of Atropa belladonna (deadly nightshade) and the plastome of Nicotiana tabacum (tobacco) develops as a result of a defect in RNA editing of a tobacco-specific editing site in the plastid ATPase α-subunit transcript. A plastome-wide analysis of RNA editing in these cytoplasmic hybrids and in plants with a tobacco nucleus and nightshade chloroplasts revealed additional defects in the editing of species-specific editing sites, suggesting that differences in RNA editing patterns in general contribute to the pigment deficiencies observed in interspecific nuclear–plastidial incompatibilities.
Brand power revisited: measuring brand equity in cyber‐space
Authors: Na WoonBong , Marshall Roger
Published: May 2005

Purpose

Cyber‐brands are regularly valued but, to date, there has been no reported research designed to understand the derivation of cyber brands’ equity, so that attempts can be made to increase their brands’ equity efficiently. This research aims to address the issue.

Design/methodology/approach

To measure the strength of cyber brands, prior off‐line research is replicated, taking a customer focus to identify determinate variables. These customer‐focused variables are tested against cyber consumers’ stated satisfaction and site‐visit intention behavior, using regression analysis.

Findings

The results satisfactorily justify the suggested model, which has a predictive power ranging from 62 to 72 per cent.

Practical implications

The study shows that practitioners intent on raising the power of their cyber brand can be guided by the same model as used off‐line.

Originality/value

The major theoretical contribution of the study is to show that there is little difference between measuring and increasing brand power on or off‐line.
A New View of Lesbian Subtypes: Stable Versus Fluid Identity Trajectories over an 8-Year Period
Authors: Diamond Lisa M.
Published: May 2005

Research has increasingly documented that the distinction between lesbian and bisexual women is one of degree rather than kind, and some researchers have therefore argued for an end to sexual categorization altogether. To the contrary, I maintain that researchers should explore alternative criteria for sexual categorization that might allow us to discern novel and meaningful subtypes of same-sex sexuality Toward this end, I explore the usefulness of a typology that focuses on change in lesbian identification over time, using a sample of young sexual-minority women that has been observed longitudinally for a period of 8 years. Specifically, I contrast women who have maintained consistent lesbian identifications over this time period (stable lesbians) to women who have alternated between lesbian and nonlesbian labels (fluid lesbians) and women who never adopted lesbian labels (stable nonlesbians). The pattern of similarities and differences among the groups changes as a function of the specific phenomenon being assessed (e.g., sexual and emotional attractions, sexual contact, developmental histories), suggesting the value of using multiple, alternative sexual typologies as heuristics to guide future research into life span development of sexual orientation and identity.
A Dual-Identity Framework for Understanding Lesbian Experience
Authors: Fingerhut Adam W. , Peplau Letitia Anne , Ghavami Negin
Published: May 2005

The diverse life experiences of contemporary lesbians are shaped by women's differing ties to two social worlds, the majority heterosexual society and the minority subculture of the lesbian or sexual-minority world. This article presents a detailed conceptual analysis of a dual-identity framework that emphasizes lesbians' simultaneous affiliations with both lesbian and mainstream/heterosexual communities. The usefulness of this approach is discussed, with emphasis on implications for understanding individual differences in exposure to gay-related stress and mental health. Results from a survey of 116 lesbians showed that scores on measures of Lesbian Identity and Mainstream Identity were not significantly correlated with each other. Both lesbian and mainstream identities were significantly related to lesbians' reported experiences of discrimination, feelings of internalized homophobia, and life satisfaction. Limitations of the dual-identity framework and suggestions for future research are considered.
Identity Support, Identity Devaluation, and Well-Being Among Lesbians
Authors: Beals Kristin P. , Peplau Letitia Anne
Published: May 2005

This research tested predictions about the association of identity support and identity devaluation with psychological well-being (self-esteem, life satisfaction, and depression). Lesbian women completed baseline surveys ( N = 42), then provided daily experience reports during a 2-week period ( n = 38), and completed a 2-month follow-up survey ( n = 34). Several types of evidence consistently demonstrated the importance of identity support for well-being. Lesbians who reported more identity support scored higher on measures of well-being at initial assessment, during the daily phase of the study, and at the follow-up. Comparable results were found for the negative association between identity devaluation and well-being. Identity measures proved to be significant predictors of lesbians' well-being even after controlling for other types of support and social stress. Both identity support and identity devaluation contributed independently to the prediction of daily self-esteem and life satisfaction. Strengths and limitations of this study are discussed.
Similarities and Differences in Relational Boundaries among Heterosexuals, Gay Men, and Lesbians
Authors: Harkless Lynne E. , Fowers Blaine J.
Published: May 2005

This study investigated the relative contributions of gender and sexual orientation as factors associated with the formation of boundaries in dyadic intimate relating in both same- and opposite-sex couples. The study examined a relational pattern previously not empirically investigated but widely accepted as an actuality unique to lesbians; specifically, that lesbians tend to remain connected to ex-serious-relationship partners after breakup. The study utilized a research design approach emphasizing the methodological utility and heuristic value of including sexual orientation as an independent variable in studies of gender dynamics. Two general classes of theoretical frameworks, those emphasizing gender role socialization influences and those emphasizing systems influences, were discussed in terms of their relative goodness of fit as conceptual bases for the data. Questionnaires were completed by 60 lesbians, 37 gay men, 45 heterosexual women, and 39 heterosexual men. Lesbians and gay men reported higher levels of connection to ex-serious-relationship partners than heterosexuals. The data reflect how inclusion of sexual orientation can broaden understandings of gender differentiated phenomena beyond more traditional gender-only based accounts.