bluuebunny/crossref_metadata_2025 huggingface dataset:
Jaw mechanism and dental function in the late cretaceous basal eusuchian Iharkutosuchus
Authors: Ősi Attila , Weishampel David B.
Published: February 2009

Iharkutosuchus makadii is a basal eusuchian crocodylian with multicusped teeth discovered from the Upper Cretaceous of Hungary. Skull and dentition morphology indicates an active food processing for this crocodylian. First among crocodylians, a combination of different analyses, including cranial adductor muscle reconstruction, tooth wear pattern, and enamel microstructure studies, is applied here to support this hypothesis. Data provide unambiguous evidence for significant dental occlusion that was a result of a unique, transverse mandibular movement. Reconstruction of the jaw adductors demonstrates strong muscles responsible for slow but active jaw closure as the motor of transverse jaw movement; nevertheless muscles producing rapid jaw closure were reduced. Macrowear orientations show a dominantly transverse movement of the mandibles completed by a slight anteroposterior component. Along with quadrate morphology, macrowear further indicates that this motion was accomplished by alternate rotation of the mandibles about the quadrate condyles. Dental morphology and wear patterns suggest two types of power stroke: a slicing–crushing stroke associated dominantly with anterior tooth–food–tooth contact (with a low degree of transverse mandibular movement) during in the early stage of mastication, and a grinding stroke with significant posterior tooth–tooth contact and a dynamic transverse movement occurring later. The patterns of microwear show a diverse diet for Iharkutosuchus including both soft and hard items. This is also supported by the microstructure of the thick, wrinkled enamel built up mostly by poorly developed columnar units. Based on wear patterns, ontogenetic variation in feeding habits of Iharkutosuchus is also recognized. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.
Intracellular distribution of the reductive and oxidative pentose phosphate pathways in two diatoms
Authors: Gruber Ansgar , Weber Till , Bártulos Carolina Río , Vugrinec Sascha , Kroth Peter G.
Published: February 2009

Diatoms contribute a large proportion to the worldwide primary production and are particularly effective in fixing carbon dioxide. Possibly because diatom plastids originate from a secondary endocytobiosis, their cellular structure is more complex and metabolic pathways are rearranged within diatom cells compared to cells containing primary plastids. We annotated genes encoding isozymes of the reductive and oxidative pentose phosphate pathways in the genomes of the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum and bioinformatically inferred their intracellular distribution. Prediction results were confirmed by fusion of selected presequences to Green Fluorescent Protein and expression of these constructs in P. tricornutum. Calvin cycle enzymes for the carbon fixation and reduction of 3‐phosphoglycerate are present in single isoforms, while we found multiple isoenzymes involved in the regeneration of ribulose‐1,5‐bisphosphate. We only identified one cytosolic sedoheptulose‐1,7‐bisphosphatase in both investigated diatoms. The oxidative pentose phosphate pathway seems to be restricted to the cytosol in diatoms, since we did not find stromal glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconolactone dehydrogenase isoforms. However, the two species apparently possess a plastidic phosphogluconolactonase. A 6‐phosphogluconolactone dehydrogenase is apparently plastid associated in P. tricornutum and might be active in the periplastidic compartment, suggesting that this compartment might be involved in metabolic processes in diatoms.

Abbreviations: AL: aldolase, ATP: adenosine triphosphate, Chl: Chlorophyll, DIC: Normanski differential interference contrast, ER: endoplasmic reticulum, EST: expressed sequence tag, FBA: fructose‐1,6‐bisphosphate aldolase, FBPase: fructose‐1,6‐bisphosphatase, GAPDH: glycerinaldehyd‐3‐phosphate dehydrogenase, GFP: enhanced Green Fluorescent Protein, GPDH: glucose‐6‐phosphate dehydrogenase, GPI: glucose‐6‐phosphate isomerase, HMM: Hidden Markov Models, JGI: Joint Genome Institute, NADPH: nicotinamide adenine dinucleotide phosphate, NN: Neuronal networks, OPP: oxidative pentose phosphate pathway, PCR: Polymerase Chain Reaction, PGDH: 6‐phosphogluconolactone dehydrogenase, PGK: phosphoglycerate kinase, PGL: phosphogluconolactonase, Phatr2: version 2.0 of the Phaeodactylum tricornutum genome, PRK: phosphoribulokinase, RPE: ribulose‐phosphate epimerase, RPI: ribose‐5‐phosphate isomerase, RuBisCO: ribulose‐1,5‐bisphosphate carboxylase/oxygenase, SBPase: sedoheptulose‐1,7‐bisphosphatase, TAL: transaldolase, Thaps3: version 3.0 of the Thalassiosira pseudonana genome, TKL: transketolase, TPI: triosephosphate isomerase, UGGtransferase: UDP glucose‐starch glycosyl transferase. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
FGF23 is a putative marker for bone healing and regeneration
Authors: Goebel Sascha , Lienau Jasmin , Rammoser Ulrich , Seefried Lothar , Wintgens Karl Florian , Seufert Jochen , Duda Georg , Jakob Franz , Ebert Regina
Published: February 2009

Besides numerous other factors, fibroblast growth factor receptor (FGFR) signaling is involved in fracture healing and bone remodeling. FGF23 is a phosphatonin produced by osteoblastic cells, which signals via FGFR1, thereby exerting effects in bone and kidney. We analyzed if serum FGF23 levels might be an indicator to predict fracture healing and union. FGF23 (C‐Term) was elevated on day 3 postoperatively in 55 patients sustaining an exchange of total hip implants due to aseptic loosening. A prospective study of 40 patients undergoing primary hip arthroplasty also showed elevated FGF23 (C‐Term) but no change in FGF23 (intact) levels on days 1, 4, and 10 postoperatively. Serum phosphate and phosphate clearance stayed within normal ranges. FGF23 mRNA expression in ovine callus was compared between a standard and delayed course of osteotomy healing. In the standard model, a marked increase in FGF23 mRNA expression compared to the delayed healing situation was observed. Immunohistochemical analysis showed FGF23 production of osteoblasts and granulation tissue in the fracture callus during bone healing. In conclusion, FGF23 is involved in bone healing, can be measured by a sensitive assay in peripheral blood, and is a promising candidate as an indicator for healing processes prone to reunion versus nonunion. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Effects of Light‐Induced Regularity on the Physical Properties of Multiphase Polymers
Authors: Murata Kosuke , Murata Tasuku , Nakanishi Hideyuki , Norisuye Tomohisa , Tran‐Cong‐Miyata Qui
Published: February 2009

A method that combines UV irradiation and pausing was developed to manipulate the regularity and the length scales of the morphology generated by phase separation in full‐interpenetrating polymer networks of polystyrene and poly(methyl methacrylate). Upon increasing the pause time of photopolymerization and photo‐crosslink processes, the morphology gradually changes from hexagonal‐like packing to random structures. The width of the loss tan δ obtained for these phase‐separated materials changes with the morphological regularity, suggesting a potential technique for fabrication of mechanical bandgap materials.

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New Strategy Targeting Well‐Defined Polymethylene‐block‐Polystyrene Copolymers: The Combination of Living Polymerization of Ylides and Atom Transfer Radical Polymerization
Authors: Chen Jian‐Zhuang , Cui Kun , Zhang Shu‐Yuan , Xie Peng , Zhao Qiao‐Ling , Huang Jin , Shi Li‐Ping , Li Guang‐Yu , Ma Zhi
Published: February 2009

Well‐defined polymethylene‐block‐polystyrene (PM‐b‐PS) diblock copolymers were synthesized via a combination of living polymerization of ylides and atom transfer radical polymerization (ATRP) of styrene. A series of hydroxyl‐terminated polymethylenes (PM‐OHs) with different molecular weight and narrow molecular weight distribution were prepared using living polymerization of ylides following efficient oxidation in a quantitive functionality. Then, the macroinitiators (PM‐MIs ($\overline M _{\rm n}$ = 1 900–15 000; PDI = 1.12–1.23)) transformed from PM‐OHs in ≈ 100% conversion initiated ATRPs of styrene to construct PM‐b‐PS copolymers. The GPC traces indicated the successful extension of PS segment ($\overline M _{\rm n}$ of PM‐b‐PS = 5 000–41 800; PDI = 1.08–1.23). Such copolymers were characterized by 1H NMR and DSC.

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Efficient Usage of Thiocarbonates for Both the Production and the Biofunctionalization of Polymers
Authors: Boyer Cyrille , Bulmus Volga , Davis Thomas P.
Published: February 2009

End group modification of polymers prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization was accomplished by conversion of trithiocarbonate into reactive functions able to conjugate easily with biomolecules or bioactive functionality. Polymers were prepared by RAFT, and subsequent aminolysis led to sulfhydryl‐terminated polymers that reacted in situ with an excess of dithiopyridyl disulfide to yield pyridyl disulfide‐terminated macromolecules or in the presence of ene to yield functional polymers. In the first route, the pyridyl disulfide end groups allowed coupling with oligonucleotide and peptide. The second approach exploited thiol–ene chemistry to couple polymers and model compounds such as carbohydrate and biotin with high yield.

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Particle Formation in RAFT‐Mediated Emulsion Polymerization: A Calorimetric Study
Authors: Leswin J. S. K. , Meuldijk J. , Gilbert R. G. , van Herk A. M.
Published: February 2009

Ab initio RAFT‐mediated semi‐continuous emulsion polymerizations with controlled monomer feed have been studied by reaction calorimetry. This online monitoring technique provided detailed information about the onset of the nucleation period in semi‐batch processes for emulsion polymerization of styrene and n‐butyl acrylate. The reactions were carried out under controlled radical polymerization conditions by employing amphipathic macro‐RAFT agents of various degrees of surface activity. For n‐butyl acrylate as well as for styrene the more surface active polymeric macro‐RAFT agents led to higher initial polymerization rates. Polymerizations with hydrophobic macro‐RAFT agents also adapted more quickly to an increased monomer feed than polymerizations with hydrophilic macro‐RAFT agents.This points to the presence of more particles when a hydrophobic macro‐RAFT agent is used. Hydrophobic macro‐RAFT agents demonstrated to be promising for producing latex products with a proper control of particle number and particle size distribution.
Entrepreneurial signaling to attract resources: the case of franchising
Authors: Michael Steven C.
Published: February 2009

Why firms and individuals reveal information is the subject of considerable theoretical research, but little empirical work has been possible due to a lack of suitable data. In this paper we examine why entrepreneurs selling business opportunities (franchisors) reveal information regarding potential profits (termed earnings claims). Empirical analysis shows that: first, contrary to theory, only a small percentage of franchisors claim; and, second, the franchisors that do claim have lower costs or are responding to competition. In particular, the prediction of theoretical models from economics that resource providers will not transact if information is not disclosed is not supported; resource providers can and do make significant investments even when entrepreneurs refuse to disclose information. Copyright © 2009 John Wiley & Sons, Ltd.
Magnetic resonance diffusion characteristics of histologically defined prostate cancer in humans
Authors: Xu Junqian , Humphrey Peter A. , Kibel Adam S. , Snyder Abraham Z. , Narra Vamsidhar R. , Ackerman Joseph J.H. , Song Sheng‐Kwei
Published: February 2009

The contrast provided by diffusion‐sensitive magnetic resonance offers the promise of improved tumor localization in organ‐confined human prostate cancer (PCa). Diffusion tensor imaging (DTI) measurements of PCa were performed in vivo, in patients undergoing radical prostatectomy, and later, ex vivo, in the same patients' prostatectomy specimens. The imaging data were coregistered to histological sections of the prostatectomy specimens, thereby enabling unambiguous characterization of diffusion parameters in cancerous and benign tissues. Increased cellularity, and hence decreased luminal spaces, in peripheral zone PCa led to approximately 40% and 50% apparent diffusion policy (ADC) decrease compared with benign peripheral zone tissues in vivo and ex vivo, respectively. In contrast, no significant diffusion anisotropy differences were observed between the cancerous and noncancerous peripheral zone tissues. However, the dense fibromuscular tissues in prostate, such as stromal tissues in benign prostatic hyperplasia in central gland, exhibited high diffusion anisotropy. A tissue classification method is proposed to combine DTI and T2‐weighted image contrasts that may provide improved specificity of PCa detection over T2‐weighted imaging alone. PCa identified in volume rendered MR images qualitatively correlates well with histologically determined PCa foci. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.
High frequency of tumours in Mulibrey nanism
Authors: Karlberg Niklas , Karlberg Susann , Karikoski Riitta , Mikkola Sakari , Lipsanen‐Nyman Marita , Jalanko Hannu
Published: February 2009

Mulibrey nanism (MUL) is a monogenic disorder with prenatal‐onset growth failure, typical clinical characteristics, cardiopathy and tendency for a metabolic syndrome. It is caused by recessive mutations in the TRIM37 gene encoding for the peroxisomal TRIM37 protein with ubiquitin‐ligase activity. In this work, the frequency and pathology of malignant and benign tumours were analysed in a national cohort of 89 Finnish MUL patients aged 0.7–76 years. The subjects had a clinical and radiological evaluation, and histological and immunohistocemical analyses on specimens obtained from biopsy, surgery or autopsy, were performed. The results show that the MUL patients have disturbed architecture with ectopic tissues and a high frequency of both benign and malignant tumours detectable in several internal organs. A total of 210 tumorous lesions were detected in 66/89 patients (74%). Fifteen malignancies occurred in 13 patients (15%), seven of them in the kidney (five Wilms' tumours), three in the thyroid gland, two gynaecological cancers, one gastrointestinal carcinoid tumour, one neuropituitary Langerhans cell histiocytosis and one case of acute lymphoblastic leukaemia (ALL). Tumours detected by radiology in the liver and other organs mainly comprised strongly dilated blood vessels (peliosis), vascularized cysts and nodular lesions. The lesions showed strong expression of the endothelial cell markers CD34 and CD31 as well as the myocyte marker α‐smooth muscle actin (α‐SMA). Our findings show that MUL is associated with frequent malignant tumours and benign adenomatous and vascular lesions, as well as disturbed organ development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Silicon‐containing liquid polymer electrolytes for application in lithium ion batteries
Authors: Rossi Nicholas AA , West Robert
Published: February 2009

Since the initial discovery of poly(ethylene glycol) (PEG) as a good lithium ion conductor at elevated temperatures, efforts have been made to find ways of incorporating this biocompatible polymer into lithium ion batteries. In areas of research involving implantable medical devices, consumer electronics and automotive power sources, there is a need to develop a ‘safe’ alternative to the solvent‐based, flammable and toxic electrolytes currently employed. However, PEG has been shown to be electrochemically unstable and crystalline at room temperature. In order to overcome these problems, polysiloxanes have been conjugated to PEG in order to improve its conductivity and physical properties. Over the last few years, the group at the University of Wisconsin‐Madison has been involved in collaborations with scientists at Argonne National Laboratories, Grinnell College and Quallion LLC to develop commercially viable silicon‐containing polymer electrolytes. This mini‐review discusses the electrochemical, thermal and physical properties of these electrolytes, and highlights the progress from high molecular weight polysiloxane‐based electrolytes to low‐viscosity, highly conducting oligosiloxane and silane electrolytes. Copyright © 2009 Society of Chemical Industry
Chemical actuation in responsive hydrogels
Authors: Swann Joshua MG , Ryan Anthony J
Published: February 2009

Chemically actuated hydrogels share a unique feature with living systems: they are both driven by their ability to convert chemical energy into a mechanical response. However, macroscopic applications of hydrogels are limited due to the slow response rate of these materials. This problem has been overcome by developing materials at micrometre length scales, as the diffusion kinetics are related to the square of the size of the smallest dimension of the gel. Herein, the progress in the field of chemical actuation in hydrogel systems is reviewed, with an emphasis on the ways in which new devices have been designed. Copyright © 2009 Society of Chemical Industry
Sperm cell proteomics
Authors: Oliva Rafael , de Mateo Sara , Estanyol Josep Maria
Published: February 2009

The spermatozoon is an accessible cell which can be easily purified and therefore it is particularly well suited for proteomic analysis. It is also an extremely differentiated cell with very marked genetic, cellular, functional and chromatin changes as compared to other cells, and has profound implications for fertility, embryo development and heredity. The recent developments in MS have boosted the potential for identification and study of the sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. The present article reviews the available scientific publications dealing with the composition and function of the sperm cell using an MS proteomic approach.
CNT templated regioselective enzymatic polymerization of phenol in water and modification of surface of MWNT thereby
Authors: Peng Yun , Liu Hewen , Zhang Xingyuan , Li Yuesheng , Liu Shiyong
Published: February 2009

Carbon nanotubes (CNTs) are used as templates to synthesize regioselective polymers from enzymatic polymerization of phenol in water. About 90% of total polymeric units in the obtained polymers are the highly thermally stable oxyphenylene units. The polymer‐yields are dependent on the quantities of CNTs used. On the basis of MWNT‐templated enzymatic polymerization of phenol, covalent attachment of polyphenol chains to the surface of MWNT by way of a linking molecule, hydroquinone, is achieved. This approach supplies a novel way for producing high‐performance polymers and for functionalization of the surface of CNT. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1627–1635, 2009
Application of Plasma Surface Treatment to Solid‐State Microscopic Particulates
Authors: Sonnenfeld Axel , Spillmann Adrian , Arpagaus Cordin , Rudolf von Rohr Philipp
Published: February 2009

The plasma‐assisted improvement of wettability and flowability of fine powders is described. The plasma down‐stream reactor designed for an adequate treatment of particulates allows efficient surface modification and functionalization of particulates. It is discussed that the resulting rapidity of the processes is due to the homogeneous dispersion of particulates in the reactor. This enables intense particle/plasma interaction presumably due to the formation of floating potential sheaths around each single particle immersed in the plasma. Thus, although the overall energy influx applied is in the range of 10 to 100 J · m−2 of the particulate surfaces, the treatment time in the order of a tenth of a second instead of hours compared to conventional processes.

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Non‐invasive assessment of exercise performance in children with cystic fibrosis (CF) and non‐cystic fibrosis bronchiectasis: Is there a CF specific muscle defect?
Authors: Rosenthal Mark , Narang Indra , Edwards Liz , Bush Andrew
Published: February 2009

Introduction

Peripheral muscle dysfunction is increasingly recognized as complicating respiratory disease, but this is difficult to measure non‐invasively.

Research Question

Can skeletal muscle function and efficiency be measured during exercise non‐invasively using respiratory mass spectrometry (RMS); and is the known exercise dysfunction in cystic fibrosis (CF) children related in part to a disease specific defect of skeletal muscle, or a non‐specific manifestation of chronic airway infection and inflammation.

Methods

Calculations of effective pulmonary blood flow and stroke volume, blood oxygen content and oxygen dispatch from the lungs, skeletal muscle oxygen extraction and consumption, anerobic threshold and capacity, and gross, net and work efficiency in 106 controls and 36 children (18 CF) with bronchiectasis, all aged from 8 to 17 years.

Results

Normal values for control subjects are tabulated. CF and non‐CF bronchiectatic subjects had similar physiology, and skeletal muscle abnormalities could not be detected. Reduced oxygen dispatch from the lungs, due to an inability to raise stroke volume, without an increase in functional residual capacity was the major factor in reduced exercise ability.

Conclusions

Non‐invasive RMS can be used to determine skeletal muscle function in children. The changes observed in CF subjects were very similar to non‐CF bronchiectatic subjects and thus a CF specific defect was not demonstrated. Pediatr Pulmonol. 2009; 44:222–230. © 2009 Wiley‐Liss, Inc.
Recommendations for treatment of intermittent mild persistent asthma in children and adolescents
Authors: Naspitz Charles K. , Cropp Gerd J.
Published: February 2009

Many parents and caretakers of children and adolescents with mild persistent asthma (MPA) do not follow proposed guidelines, namely the daily and continuous administration of inhaled corticosteroids (ICS). Instead, parents and caretakers tend to use ICS and bronchodilators intermittently for short periods and restart such therapy only when symptoms reappear. It is our opinion that intermittent treatment of MPA in children and adolescents might achieve the same level of asthma control as has been achieved in adults. We propose, therefore, that after an initial period of stabilization with age‐appropriate doses of oral glucocorticoids or high‐dose ICS and short‐acting beta‐2 agonists (SABA), caretakers can stop treatment once there are no longer signs or symptoms of asthma. When asthmatic symptoms recur, treatment should be restarted with ICS and SABA, or oral corticosteroids if the exacerbation is severe. The perception of developing asthma symptoms remains an unsolved problem. Based on our clinical experience in children and adolescents with asthma, we list a number of signs and symptoms that precede an exacerbation of asthma, allowing for an early re‐introduction of treatment to prevent an exacerbation. Pediatr Pulmonol. 2009; 44:205–208. © 2009 Wiley‐Liss, Inc.
Airway humidification with a heated wire humidifier during high‐frequency ventilation using babylog 8000 plus® in neonates
Authors: Nagaya Ken , Okamoto Toshio , Nakamura Eiki , Hayashi Tokitsugi , Fujieda Kenji
Published: February 2009

Objective

Little data are available on airway humidity during high‐frequency ventilation (HFV). Our purpose is to evaluate the airway humidification during HFV.

Methods

We examined the airway humidification and temperature in a neonatal HFV system using Babylog 8000 plus®. The absolute humidity (AH), relative humidity (RH), and temperature at different sites and under different HFV conditions were compared with those during conventional intermittent positive pressure ventilation (IPPV).

Results

The mean AH and RH at the patient end of the respiratory circuit under 37°C in the humidification chamber (HC) during HFV were less than 35 mg/L and 65%, respectively, while those during IPPV were 42.3 mg/L and 96.8%, respectively. The humidification at the outlet of the HC was similar results. Moreover, during HFV an increase in the bias‐flow of ventilator led to a further decrease in the humidity at the patient end of respiratory circuit and the outlet of HC. It was necessary to set the temperature in the HC at >39°C to maintain adequate humidity at the HC and the patient end of respiratory circuit during HFV. An increase in the incubator temperature led to an increase in the temperature at the patient end of the respiratory circuit. The temperature at the patient end of the respiratory circuit was about 39–40°C when the incubator temperature was 35–37°C.

Conclusions

The airway humidification at the patient end of respiratory circuit and the outlet of HC in HFV were poorer than those in IPPV. However, the adequacy of humidification and safety in HFV remain to be demonstrated in clinical practice. Pediatr Pulmonol. 2009; 44:260–266. © 2009 Wiley‐Liss, Inc.
Structural basis for chloramphenicol tolerance in Streptomyces venezuelae by chloramphenicol phosphotransferase activity
Authors: Izard Tina
Published: February 2009

Streptomyces venezuelae synthesizes chloramphenicol (Cm), an inhibitor of ribosomal peptidyl transferase activity, thereby inhibiting bacterial growth. The producer escapes autoinhibition by its own secondary metabolite through phosphorylation of Cm by chloramphenicol phosphotransferase (CPT). In addition to active site binding, CPT binds its product 3‐phosphoryl‐Cm, in an alternate product binding site. To address the mechanisms of Cm tolerance of the producer, the crystal structures of CPT were determined in complex with either the nonchlorinated Cm (2‐N‐Ac‐Cm) at 3.1 Å resolution or the antibiotic's immediate precursor, the p‐amino analog p‐NH2‐Cm, at 2.9 Å resolution. Surprisingly, p‐NH2‐Cm binds CPT in a novel fashion. Additionally, neither 2‐N‐Ac‐Cm nor p‐NH2‐Cm binds to the secondary product binding site.
In Memoriam: Professor G.N. Ramachandram (1922–2001)
Authors: Ramakrishnan C.
Published: February 2009

Editor's note: Few scientists contribute an idea of such clarity and power that it appears in all the discipline's textbooks and bears the author's name. For the contribution to be relevant and universally employed almost forty years after it first appeared is even less common. Structural biology lost the author of such an idea with the death of G.N. Ramachandran, whose picture appears on the cover of this issue of Protein Science. His seminal contribution is described in remembrances of Professor Ramachandran's life and career by colleague and co‐author of the 1963 paper, C. Ramakrishnan. A perspective by George D. Rose follows, which articulates the enduring impact of that work.
Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB
Authors: Nagy Maria , Wu Hui‐Chuan , Liu Zhonghua , Kedzierska‐Mieszkowska Sabina , Zolkiewski Michal
Published: February 2009

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide‐binding P‐loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker‐A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T‐to‐N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP‐bound conformation and the high‐affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker‐A Thr in sensing the nucleotide's γ‐phosphate and in maintaining an allosteric linkage between the P‐loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate‐remodeling activity.
Genetic selection system for improving recombinant membrane protein expression in E. coli
Authors: Massey‐Gendel Elizabeth , Zhao Anni , Boulting Gabriella , Kim Hye‐Yeon , Balamotis Michael A. , Seligman Len M. , Nakamoto Robert K. , Bowie James U.
Published: February 2009

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold.
Structure and sequence relationships in the lipocalins and related proteins
Authors: Flower Darren R. , North ANTHONY C.T. , Attwood Teresa K.
Published: February 2009

The lipocalins and fatty acid‐binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger “structural superfamily”; we have christened this overall group the calycins to reflect the cup‐shaped structure of its members.
Reduced representation model of protein structure prediction: Statistical potential and genetic algorithms
Authors: Sun Shaojian
Published: February 2009

A reduced representation model, which has been described in previous reports, was used to predict the folded structures of proteins from their primary sequences and random starting conformations. The molecular structure of each protein has been reduced to its backbone atoms (with ideal fixed bond lengths and valence angles) and each side chain approximated by a single virtual united‐atom. The coordinate variables were the backbone dihedral angles ϕ and Ψ. A statistical potential function, which included local and nonlocal interactions and was computed from known protein structures, was used in the structure minimization. A novel approach, employing the concepts of genetic algorithms, has been developed to simultaneously optimize a population of conformations. With the information of primary sequence and the radius of gyration of the crystal structure only, and starting from randomly generated initial conformations, I have been able to fold melittin, a protein of 26 residues, with high computational convergence. The computed structures have a root mean square error of 1.66 Å (distance matrix error = 0.99 Å) on average to the crystal structure. Similar results for avian pancreatic polypeptide inhibitor, a protein of 36 residues, are obtained. Application of the method to apamin, an 18‐residue polypeptide with two disulfide bonds, shows that it folds apamin to native‐like conformations with the correct disulfide bonds formed.
Energy coupling between DNA binding and subunit association is responsible for the specificity of DNA‐Arc interaction
Authors: Silva Jerson L. , Silveira Cristina F.
Published: February 2009

The effects of several DNA molecules on the free energy of subunit association of Arc repressor were measured. The association studies under equilibrium conditions were performed by the dissociating perturbation of hydrostatic pressure. The magnitude of stabilization of the subunit interaction was determined by the specificity of the protein‐DNA interaction. Operator DNA stabilized the free energy of association by about 2.2 kcal/mol of monomeric unit, whereas poly(dG‐dC) stabilized the subunit interaction by only 0.26 kcal. Measurements of the stabilizing free energy at different DNA concentrations revealed a stoichiometry of two dimers per 21 bp for the operator DNA sequence and for the nonspecific DNA poly(dA‐dT). However, the maximum stabilization was much larger for operator sequence (Δp = 1, 750 bar) as compared for poly(dA‐dT) (Δp = 750 bar). The importance of the free‐energy linkage for the recognition process was corroborated by its absence in a mutant Arc protein (PL8) that binds to operator and nonspecific DNA sequences with equal, low affinity. We conclude that the coupling accounts for the high specificity of the Arc‐operator DNA interaction. We hypothesize a mutual coupling between the protein subunits and the two DNA strands, in which the much higher persistency of the associated form when Arc is bound to operator would stabilize the interactions between the two DNA strands.
Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains
Authors: Powers Vincent M. , Yang Ying R. , Fogli Michael J. , Schachman H. K.
Published: February 2009

Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with α‐chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654‐658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26, 000 and 8, 000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild‐type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N‐(phosphonacetyl)‐l‐aspartate, are increased about 7‐ and 15‐fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino‐terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1‐240 and 241‐310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild‐type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild‐type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re‐associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.
Purification and characterization of Klebsiella aerogenes UreE protein: A nickel‐binding protein that functions in urease metallocenter assembly
Authors: Lee Mann Hyung , Pankratz H. Stuart , Wang Shengke , Scott Robert A. , Finnegan Michael G. , Johnson Michael K. , Ippolito Joseph A. , Christianson David W. , Hausinger Robert P.
Published: February 2009

The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324‐4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic β‐strand and exhibits unusually tight binding to phenyl‐Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (Mr = 35, 000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 + 1.3 μM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X‐ray absorption and variable‐temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo‐octahedral geometry with 3‐5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X‐ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.
Structure, function, and regulation of adrenergic receptors
Authors: Strosberg A. D.
Published: February 2009

Adrenergic receptors for adrenaline and noradrenaline belong to the large multigenic family of receptors coupled to GTP‐binding proteins. Three pharmacologic types have been identified: α1‐, α2‐, and β‐adrenergic receptors. Each of these has three subtypes, characterized by both structural and functional differences. The α2and β receptors are coupled negatively and positively, respectively, to adenylyl cyclase via Gior Gsregulatory proteins, and the α1receptors modulate phospholipase C via the Goprotein. Subtype expression is regulated at the level of the gene, the mRNA, and the protein through various transcriptional and postsynthetic mechanisms. Adrenergic receptors constitute, after rhodopsin, one of the best studied models for the other receptors coupled to G proteins that are likely to display similar structural and functional properties.
Cloning, characterization, and expression of the β subunit of pig heart succinyl‐CoA synthetase
Authors: Bailey Darrin L. , Wolodko William T. , Bridger William A.
Published: February 2009

The form of succinyl‐CoA synthetase found in mammalian mitochondria is known to be an αβ dimer. Both GTP‐and ATP‐specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the β subunit of pig heart succinyl‐CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4‐kb sequence encoding the cytoplasmic precursor to the β subunit comprised of 417 amino acid residues including a 22‐residue mitochondrial targeting sequence. The cDNA encoding the 395‐amino acid, 42,502‐Da mature protein was confirmed to be the succinyl‐CoA synthetase β subunit by agreement with the N‐terminal protein sequence and by high homology to prokaryotic forms of the β subunit that were previously cloned (about 45% identical to β from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429–434), we found no tryptophan residue to be encoded in the sequence for the mature β subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl‐CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart β subunit and its counterpart α subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl‐CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.
Conformational intermediates in the folding of a coiled‐coil model peptide of the N‐terminus of tropomyosin and αα‐tropomyosin
Authors: Greenfield Norma J. , Hitchcock‐Degregori Sarah E.
Published: February 2009

Circular dichroism was used to study the folding of αα‐tropomyosin and AcTM43, a 43‐residue peptide designed to serve as a model for the N‐terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (<25 °C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled‐coil α‐helix of native αα‐tropomyosin. The second curve resembled the spectrum of single‐stranded short α‐helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the α‐helical content increased before most of the coiled coil was formed.

When the CD spectrum of striated muscle αα‐tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 °C, there was a decrease in the coiled coil and an increase in the conventional α‐helix type spectrum without formation of random coil. The major transition, occurring at 40 °C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.
Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution
Authors: Eriksson A. Elisabeth , Matthews Brian W. , Cousens Lawrence S.
Published: February 2009

The three‐dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 Å resolution. The structure has a Kunitz‐type fold and is composed of 12 antiparallel β‐strands, 6 of which form a β‐barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441–3445). Two β‐mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the Sγ atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site.

The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 Å resolution and the structure refined to an R‐factor of 13.8%. The binding of the more electron‐dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 Å from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.
Backbone assignments and secondary structure of the Escherichia coli enzyme‐II mannitol a domain determined by heteronuclear three‐dimensional NMR spectroscopy
Authors: Kroon Gerard J. A. , Dijkstra Klaas , Scheek Ruud M. , Robillard George T. , Grötzinger Joachim
Published: February 2009

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme‐IImtl.

The backbone resonances were partially assigned using three‐dimensional heteronuclear 1H NOE 1H‐15N single‐quantum coherence (15N NOESY‐HSQC) spectroscopy and three‐dimensional heteronuclear 1H total correlation 1H‐15N single‐quantum coherence (15N TOCSY‐HSQC) spectroscopy on uniformly 15N enriched protein. Triple‐resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three‐dimensional 1H‐15N‐13Cα correlation experiments (HNCA and HN(CO)CA), a three‐dimensional 1H‐15N‐13CO correlation experiment (HNCO), and a three‐dimensional 1Hα‐13Cα‐13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances.

Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY‐HSQC and the 1Hα and 13Cα secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X‐ray.
A study of the effects of altering the sites for N‐glycosylation in α‐1‐proteinase inhibitor variants M and S
Authors: Samandari Taraz , Brown Jerry L.
Published: February 2009

α‐1‐Proteinase inhibitor (A1Pi) is a monomeric secreted protein glycosylated at asparagines 46, 83, and 247. For this study cDNAs for M (normal) and S (Glu264 → Val) variants of A1Pi were altered by site‐directed mutagenesis to produce the combinations of single, double, and triple mutants that can be generated by changing the codons normally specifying these Asn residues to encode Gln. The fates of the mutant proteins were followed in transiently transfected COS‐1 cells. All variants with altered glycosylation sites are secreted at reduced rates, are partially degraded, accumulate intracellularly, and some form Nonidet P‐40‐insoluble aggregates. The carbohydrate attached at Asn83 seems to be of particular importance to the export of both A1PiM and A1PiS from the endoplasmic reticulum. All mutations affecting glycosylation of A1PiS notably reduce secretion, cause formation of insoluble aggregates, and influence degradation of the altered proteins. The variant of A1PiS missing all three glycosylation sites is poorly secreted, is incompletely degraded, and accumulates in unusual perinuclear vesicles. These studies show that N‐linked oligosaccharides in A1Pi are vital to its efficient export from the endoplasmic reticulum and that the consequences of changing the normal pattern of glycosylation vary depending upon the sites altered and the variant of A1Pi bearing these alterations.