bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical and Molecular Characterization of AtPAP26, a Vacuolar Purple Acid Phosphatase Up-Regulated in Phosphate-Deprived Arabidopsis Suspension Cells and Seedlings
Authors: Veljanovski Vasko , Vanderbeld Barbara , Knowles Vicki L. , Snedden Wayne A. , Plaxton William C. 
Published: September 2006

Abstract
A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (A max = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a &gt;30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.
Probing protein structure and dynamics by second‐derivative ultraviolet absorption analysis of cation–π interactions
Authors: Lucas Laura H. , Ersoy Baran A. , Kueltzo Lisa A. , Joshi Sangeeta B. , Brandau Duane T. , Thyagarajapuram Nagarajan , Peek Laura J. , Middaugh C. Russell 
Published: September 2006

AbstractWe describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation–π interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second‐derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N‐acetylated C‐ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation–π interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.
Theoretical investigation of the photoinitiated folding of HP‐36
Authors: Jang Soonmin , Sreerama Narasimha , Liao Vivian H.‐C. , Lu S. Hsiu‐Feng , Li Feng‐Yin , Shin Seokmin , Woody Robert W. , Lin Sheng Hsien 
Published: September 2006

AbstractA computational model was developed to examine the phototriggered folding of a caged protein, a protein modified with an organic photolabile cross‐linker. Molecular dynamics simulations of the modified 36‐residue fragment of subdomain B of chicken villin head piece with a photolabile linker were performed, starting from both the caged and the uncaged structures. Construction of a free‐energy landscape, based on principal components as well as on radius of gyration versus root‐mean‐square deviation, and circular dichroism calculations were employed to characterize folding behavior and structures. The folded structures observed in the molecular dynamics trajectories were found to be similar to that of the wild‐type protein, in agreement with the published experimental results. The free‐energy landscapes of the modified and wild‐type proteins have similar topology, suggesting common thermodynamic/kinetic behavior. The existence of small differences in the free‐energy surface of the modified protein from that of the native protein, however, indicates subtle differences in the folding behavior.
Design and NMR conformational study of a β‐sheet peptide based on Betanova and WW domains
Authors: Fernández‐Escamilla Ana M. , Ventura Salvador , Serrano Luis , Jiménez M. Angeles 
Published: September 2006

AbstractA good approach to test our current knowledge on formation of protein β‐sheets is de novo protein design. To obtain a three‐stranded β‐sheet mini‐protein, we have built a series of chimeric peptides by taking as a template a previously designed β‐sheet peptide, Betanova‐LLM, and incorporating N‐ and/or C‐terminal extensions taken from WW domains, the smallest natural β‐sheet domain that is stable in absence of disulfide bridges. Some Betanova‐LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt β‐sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest β‐sheet population according to the CD spectra, named 3SBWW‐2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW‐2 adopts a well defined three‐stranded antiparallel β‐sheet structure with a disordered C‐terminal tail. To discern between the contributions to β‐sheet stability of strand residues and the C‐terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW‐2 but lacking the C‐terminal extension, named Betanova‐LYYL, was also investigated. β‐Sheet stability in these two peptides, in the parent Betanova‐LLM and in WW‐P, a prototype WW domain, decreased in the order WW‐P > 3SBWW‐2 > Betanova‐LYYL > Betanova‐LLM. Conclusions about the contributions to β‐sheet stability were drawn by comparing structural properties of these four peptides.
Oligomerization behavior of the archaeal Sm2‐type protein from Archaeoglobus fulgidus
Authors: Kilic Turgay , Sanglier Sarah , Van Dorsselaer Alain , Suck Dietrich 
Published: September 2006

AbstractAs part of a functional analysis of archaeal Sm‐related proteins, we have studied the oligomerization behavior of the Sm‐2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA‐bound heptamer increases at lower pH.
Localization of binding sites in protein structures by optimization of a composite scoring function
Authors: Rossi Andrea , Marti‐Renom Marc A. , Sali Andrej 
Published: September 2006

AbstractThe rise in the number of functionally uncharacterized protein structures is increasing the demand for structure‐based methods for functional annotation. Here, we describe a method for predicting the location of a binding site of a given type on a target protein structure. The method begins by constructing a scoring function, followed by a Monte Carlo optimization, to find a good scoring patch on the protein surface. The scoring function is a weighted linear combination of the z‐scores of various properties of protein structure and sequence, including amino acid residue conservation, compactness, protrusion, convexity, rigidity, hydrophobicity, and charge density; the weights are calculated from a set of previously identified instances of the binding‐site type on known protein structures. The scoring function can easily incorporate different types of information useful in localization, thus increasing the applicability and accuracy of the approach. To test the method, 1008 known protein structures were split into 20 different groups according to the type of the bound ligand. For nonsugar ligands, such as various nucleotides, binding sites were correctly identified in 55%–73% of the cases. The method is completely automated (http://salilab.org/patcher) and can be applied on a large scale in a structural genomics setting.
Amyloid formation in denatured single‐mutant lysozymes where residual structures are modulated
Authors: Mishima Tomonori , Ohkuri Takatoshi , Monji Akira , Imoto Taiji , Ueda Tadashi 
Published: September 2006

AbstractReduced hen lysozyme has a residual structure involving long‐range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long‐range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid‐fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.
Active TEM‐1 β‐lactamase mutants with random peptides inserted in three contiguous surface loops
Authors: Mathonet Pascale , Deherve Julie , Soumillion Patrice , Fastrez Jacques 
Published: September 2006

AbstractEngineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non‐natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM‐1 β‐lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two β‐strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well‐conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N‐terminal helix and the first β‐strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final β‐strand to the C‐terminal helix, particularly if the wild‐type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium‐sized loops. The combination of these activity‐selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.
Selection of allosteric β‐lactamase mutants featuring an activity regulation by transition metal ions
Authors: Mathonet Pascale , Barrios Humberto , Soumillion Patrice , Fastrez Jacques 
Published: September 2006

AbstractLibraries of phage‐displayed β‐lactamase mutants in which up to three loops have been engineered by genetic introduction of random peptide sequences or by randomization of the wild‐type sequence have been submitted to selection protocols designed to find mutants in which binding of transition metal ions to the engineered secondary binding site leads to significant effects on the enzymatic activity. A double‐selection protocol was applied: The phage‐displayed libraries were first selected for transition metal ions affinity by panning on IMAC support, then a second selection step was applied to isolate mutants that have retained significant catalytic activity. The analysis of the kinetic properties of mutants in the presence of nickel, copper, or zinc ions allowed isolation of a few mutants whose activity was either enhanced or inhibited by factors up to three and >10, respectively, in a metal‐specific manner. A remarkable mutant exhibiting differential allosteric regulation depending on the metal was found. Its activity was activated by nickel ion binding, inhibited by cupric ion binding, and nearly unaffected by zinc ions. These observations point to an interesting potential for up‐ or down‐regulation of activity within a monomeric enzyme by binding to an “allosteric site” relatively remote from the active site.
Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
Authors: Johansson Patrik , Castell Alina , Jones T. Alwyn , Bäckbro Kristina 
Published: September 2006

AbstractA large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β‐sheet on top of a long and a short α‐helix. Two monomers in turn pack to form a double‐hotdog‐folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R‐specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans‐2‐enoyl‐coenzyme A moiety with a k cat of 1.1 × 102 sec−1. The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site‐directed mutagenesis. In contrast to many hotdog‐folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate‐binding channels of long‐chain eukaryotic hydratase 2 enzymes.
NMR structure of the enzyme GatB of the galactitol‐specific phosphoenolpyruvate‐dependent phosphotransferase system and its interaction with GatA
Authors: Volpon Laurent , Young Christopher R. , Matte Allan , Gehring Kalle 
Published: September 2006

AbstractThe phosphoenolpyruvate‐dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate‐specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol‐specific transporter (IIgat) belongs to the glucitol family and is structurally the least well‐characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three‐dimensional structure of the IIB subunit (GatB). GatB consists of a central four‐stranded parallel β‐sheet flanked by α‐helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between β1 and α1 that folds into a P‐loop‐like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA‐interacting residues.
Endocrinology and Dialysis: Management of Lipid Abnormalities Associated with End‐Stage Renal Disease
Authors: Liu Jia , Kalantarinia Kambiz , Rosner Mitchell H. 
Published: September 2006

AbstractThe management of lipid abnormalities in patients with end‐stage renal disease (ESRD) remains controversial. Large, well‐designed studies investigating the effects of dyslipidemia on cardiovascular (CV) morbidity and mortality and the role of cholesterol lowering drugs in reducing mortality in ESRD patients are lacking. While it seems reasonable to suspect that dyslipidemia and its treatment in ESRD patients will affect CV morbidity and mortality similar to that in the general population, recent studies have suggested that this may not be the case. Furthermore, the pharmacokinetics of lipid lowering drugs are altered in patients with ESRD and must be considered when treating this group of patients. This article reviews the major classes of drugs used to treat dyslipidemia, emphasizing their role in patients with ESRD.
Repellency of inseminated Culex pipiens (Diptera: Culicidae) females to various wing‐beat sound frequencies in modulated intensities in a flight chamber
Authors: LEE Dong‐Kyu 
Published: September 2006

AbstractWing‐beat sound frequencies at 370, 440, 493 and 554 Hz were evaluated for female mosquito repellency. One hundred inseminated females of Culex pipiens were introduced into a 30 cm × 30 cm × 3 m screened cage with an electronic device (cellular phone) emitting selected frequencies positioned at one end of the chamber. The mosquitoes were counted in areas of 0.0–0.5, 0.5–1.0, 1.0–2.0 and 2.0–3.0 m from the cellular phone. In a second test, 50 Cx. pipiens were released in the screened cage with a shaved rat as bait placed beside the cellular phone (370 Hz) at the center of the chamber. Statistical analysis (anova) showed that significantly fewer (P < 0.05) mosquitoes rested near the cellular phone at 370 Hz, but not at the other frequencies. Similarly, there was significant reduction (mean 33.0%) in the number of mosquitoes taking a blood meal on the shaved rat when the cellular phone was turned on (P < 0.01).
General biology of Ostrinia palustralis memnialis (Walker), a potential biological control agent of Rumex spp. in Korea
Authors: KWON Ohseok , PARK Jinyoung , LEE In‐Yong , PARK Jae‐Eup 
Published: September 2006

Abstract Ostrinia palustralis memnialis is an oligophagous insect that feeds on the species of the genus Rumex. Continuous rearing of O. palustralis memnialis was carried out from 2004 to 2005, and its general biology was studied. The preliminary host specificity test showed a promising result for O. palustralis memnialis as a potential biological control agent for Rumex spp.
External quality assurance of HER2 fluorescence in situ hybridisation testing: results of a UK NEQAS pilot scheme
Authors: Bartlett John M S , Ibrahim Merdol , Miller Keith , Jasani Bharat , Morgan John M , Ellis Ian , Kay Elaine , Magee Hilary , Barnett Sarah 
Published: September 2006

Background and Aims:Trastuzumab provides clinical benefit for advanced and early breast cancer patients whose tumours over-express or have gene amplification of the HER2 oncogene. The UK National External Quality Assessment Scheme (NEQAS) for immunohistochemical testing was established to assess and improve the quality of HER2 immunohistochemical testing. However, until recently, no provision was available for HER2 fluorescence in situ hybridisation (FISH) testing. A pilot scheme was set up to review the performance of FISH testing in clinical diagnostic laboratories.Methods:FISH was performed in 6 reference and 31 participating laboratories using a cell line panel with known HER2 status.Results:Using results from reference laboratories as a criterion for acceptable performance, 60% of all results returned by participants were appropriate and 78% either appropriate or acceptable. However, 22.4% of results returned were deemed inappropriate, including 13 cases (4.2%) where a misdiagnosis would have been made had these been clinical specimens.Conclusions:The results of three consecutive runs show that both reference laboratories and a proportion of routine clinical diagnostic (about 25%) centres can consistently achieve acceptable quality control of HER2 testing. Data from a significant proportion of participating laboratories show that further steps are required, including those taken via review of performance under schemes such as NEQAS, to improve quality of HER2 testing by FISH in the “real world”.
Diabetes alters vascular mechanotransduction: pressure-induced regulation of mitogen activated protein kinases in the rat inferior vena cava
Authors: Rice Kevin M , Desai Devashish H , Kakarla Sunil K , Katta Anjaiah , Preston Deborah L , Wehner Paulette , Blough Eric R 
Published: September 2006

Abstract

Background
Diabetes mellitus is an important risk factor for increased vein graft failure after bypass surgery. However, the cellular and molecular mechanism(s) underlying vessel attrition in this population remain largely unexplored. Recent reports have suggested that the pathological remodeling of vein grafts may be mediated by mechanically-induced activation of the mitogen activated protein kinase (MAPK) signaling pathways and the MAPK-related induction of caspase-3 activity. On the basis of these findings, we hypothesized that diabetes may be associated with alterations in how veins "sense" and "respond" to altered mechanical loading.

Methods
Inferior venae cavae (IVC) from the non-diabetic lean (LNZ) and the diabetic obese (OSXZ) Zucker rats were isolated and incubated ex vivo under basal or pressurized conditions (120 mmHg). Protein expression, basal activation and the ability of increased pressure to activate MAPK pathways and apoptosis-related signaling was evaluated by immunoblot analysis.

Results
Immunoblot analyses revealed differential expression and activation of extracellular signal-regulated kinase (ERK1/2), p38 and c-Jun NH2-terminal kinase (JNK) MAPKs in the IVCs of diabetic rats as compared to non-diabetic rats. In particular, the expression and basal phosphorylation of p38β- (52.3 ± 11.8%; 45.8 ± 18.2%), JNK 1- (21.5 ± 9.3%; 19.4 ± 11.6%) and JNK3-MAPK (16.8 ± 3.3%; 29.5 ± 17.6%) were significantly higher (P < 0.05) in the diabetic vena cava. An acute increase in IVC intraluminal pressure failed to increase the phosphorylation of ERK1-, JNK-2, or any of the p38-MAPKs in the diabetic obese Zucker rats. Also, IVC loading in the LNZ led to a 276.0 ± 36.0% and 85.8 ± 25.1% (P < 0.05) increase in the cleavage of caspase-3 and caspase-9, respectively, with no effect on these molecules in the OSXZ. No differences were found in the regulation of Bax and Bcl-2 between groups. However, basal expression levels of Akt, phospho-Akt, PTEN, phospho-PTEN and phospho-Bad were higher in the diabetic venae cavae (P < 0.05).

Conclusion
These data suggest that diabetes is associated with significant alteration in the ability of the vena cava to activate MAPK- and apoptosis-related signaling. Whether these changes are associated with the increased vein graft attrition seen in the diabetic population will require further investigation.