bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination
Authors: Liu Yunqing , Liao Jing , Zhu Bin , Wang En-Duo , Ding Jianping 
Published: March 2006

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.
Unique structure of Ascaris suum b5-type cytochrome: an additional α-helix and positively charged residues on the surface domain interact with redox partners
Authors: Yokota Takehiro , Nakajima Yoshitaka , Yamakura Fumiyuki , Sugio Shigetoshi , Hashimoto Muneaki , Takamiya Shinzaburo 
Published: March 2006

Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 Å (1 Å=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six α-helices and five β-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional α-helix, α4A, between helices α4 and α5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80° relative to the axis formed by haem-Fe and Nϵ atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode metmyoglobin can be hypothesized.
L-Cysteate sulpho-lyase, a widespread pyridoxal 5′-phosphate-coupled desulphonative enzyme purified from Silicibacter pomeroyi DSS-3T
Authors: Denger Karin , Smits Theo H. M. , Cook Alasdair M. 
Published: March 2006

Quantitative utilization of L-cysteate (2-amino-3-sulphopropionate) as the sole source of carbon and energy for growth of the aerobic, marine bacterium Silicibacter pomeroyi DSS-3T was observed. The sulphonate moiety was recovered in the medium largely as sulphite, and the appropriate amount of the ammonium ion was also observed. Genes [suyAB (3-sulpholactate sulpho-lyase)] encoding the known desulphonation reaction in cysteate degradation were absent from the genome, but a homologue of a putative sulphate exporter gene (suyZ) was found, and its neighbour, annotated as a D-cysteine desulphhydrase, was postulated to encode pyridoxal 5′-phosphate-coupled L-cysteate sulpho-lyase (CuyA), a novel enzyme. Inducible CuyA was detected in cysteate-grown cells. The enzyme released equimolar pyruvate, sulphite and the ammonium ion from L-cysteate and was purified to homogeneity by anion-exchange, hydrophobic-interaction and gel-filtration chromatography. The N-terminal amino acid sequence of this 39-kDa subunit confirmed the identification of the cuyA gene. The native enzyme was soluble and homomultimeric. The Km-value for L-cysteate was high (11.7 mM) and the enzyme also catalysed the D-cysteine desulphhydrase reaction. The gene cuyZ, encoding the putative sulphite exporter, was co-transcribed with cuyA. Sulphite was exported despite the presence of a ferricyanide-coupled sulphite dehydrogenase. CuyA was found in many bacteria that utilize cysteate.
Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase
Authors: Shiryaev Sergey A. , Ratnikov Boris I. , Chekanov Alexei V. , Sikora Sergey , Rozanov Dmitri V. , Godzik Adam , Wang Jun , Smith Jeffrey W. , Huang Ziwei , Lindberg Iris , Samuel Melanie A. , Diamond Michael S. , Strongin Alex Y. 
Published: March 2006

Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9–12-mer peptides are potent inhibitors of WNV NS3 with Ki values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.
Role of N-terminal residues in the ubiquitin-independent degradation of human thymidylate synthase
Authors: Peña Maria Marjorette O. , Xing Yang Yang , Koli Sangita , Berger Franklin G. 
Published: March 2006

Thymidylate synthase (TS) catalyses the reductive methylation of dUMP to form dTMP, a reaction that is essential for maintenance of nucleotide pools during cell growth. Because the enzyme is indispensable for DNA replication in actively dividing cells, it is an important target for cytotoxic drugs used in cancer chemotherapy, including fluoropyrimidines (e.g. 5-fluorouracil and 5-fluoro-2′-deoxyuridine) and anti-folates (e.g. raltitrexed, LY231514, ZD9331 and BW1843U89). These drugs generate metabolites that bind to the enzyme's active site and inhibit catalytic activity, leading to thymidylate deprivation and cellular apoptosis. Ligand binding to TS results in stabilization of the enzyme and an increase in its intracellular concentration. Previously, we showed that degradation of the TS polypeptide is carried out by the 26 S proteasome in a ubiquitin-independent manner. Such degradation is directed by the disordered N-terminal region of the TS polypeptide, and is abrogated by ligand binding. In the present study, we have verified the ubiquitin-independent nature of TS proteolysis by showing that a ‘lysine-less’ polypeptide, in which all lysine residues were replaced by arginine, is still subject to proteasome-mediated degradation. In addition, we have mapped the structural determinants of intracellular TS degradation in more detail and show that residues at the N-terminal end of the molecule, particularly the penultimate amino acid Pro2, play an important role in governing the half-life of the enzyme. This region is capable on its own of destabilizing an evolutionarily distinct TS molecule that normally lacks this domain, indicating that it functions as a degradation signal. Interestingly, degradation of an intrinsically unstable mutant form of TS, containing a Pro→Leu substitution at residue 303, is directed by C-terminal, rather than N-terminal, sequences. The implications of these findings for the control of TS expression, and for the regulation of protein degradation in general, are discussed.
Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin–26 S proteasome system in the living rat
Authors: Dominy John E. , Hirschberger Lawrence L. , Coloso Relicardo M. , Stipanuk Martha H. 
Published: March 2006

Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin–26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.
A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae
Authors: Fernandez Luisa E. , Aimanova Karlygash G. , Gill Sarjeet S. , Bravo Alejandra , Soberón Mario 
Published: March 2006

A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI–ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI–ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI–ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI–ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.
Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts
Authors: Morty Rory E. , Bulau Patrick , Pellé Roger , Wilk Sherwin , Abe Koji 
Published: March 2006

Peptidases of parasitic protozoans are emerging as novel virulence factors and therapeutic targets in parasitic infections. A trypanosome-derived aminopeptidase that exclusively hydrolysed substrates with Glp (pyroglutamic acid) in P1 was purified 9248-fold from the plasma of rats infected with Trypanosoma brucei brucei. The enzyme responsible was cloned from a T. brucei brucei genomic DNA library and identified as type I PGP (pyroglutamyl peptidase), belonging to the C15 family of cysteine peptidases. We showed that PGP is expressed in all life cycle stages of T. brucei brucei and is expressed in four other blood-stream-form African trypanosomes. Trypanosome PGP was optimally active and stable at bloodstream pH, and was insensitive to host plasma cysteine peptidase inhibitors. Native purified and recombinant hyper-expressed trypanosome PGP removed the N-terminal Glp blocking groups from TRH (thyrotrophin-releasing hormone) and GnRH (gonadotropin-releasing hormone) with a kcat/Km value of 0.5 and 0.1 s−1·μM−1 respectively. The half-life of TRH and GnRH was dramatically reduced in the plasma of trypanosome-infected rats, both in vitro and in vivo. Employing an activity-neutralizing anti-trypanosome PGP antibody, and pyroglutamyl diazomethyl ketone, a specific inhibitor of type I PGP, we demonstrated that trypanosome PGP is entirely responsible for the reduced plasma half-life of TRH, and partially responsible for the reduced plasma half-life of GnRH in a rodent model of African trypanosomiasis. The abnormal degradation of TRH and GnRH, and perhaps other neuropeptides N-terminally blocked with a pyroglutamyl moiety, by trypanosome PGP, may contribute to some of the endocrine lesions observed in African trypanosomiasis.
Oxidative folding of hirudin in human serum
Authors: Chang Jui-Yoa , Lu Bao-Yun , Lai Por-Hsiung 
Published: March 2006

Human serum contains factors that promote oxidative folding of disulphide proteins. We demonstrate this here using hirudin as a model. Hirudin is a leech-derived thrombin-specific inhibitor containing 65 amino acids and three disulphide bonds. Oxidative folding of hirudin in human serum is shown to involve an initial phase of rapid disulphide formation (oxidation) to form the scrambled isomers as intermediates. This is followed by the stage of slow disulphide shuffling of scrambled isomers to attain the native hirudin. The kinetics of regenerating the native hirudin depend on the concentrations of both hirudin and human serum. Quantitative regeneration of native hirudin in undiluted human serum can be completed within 48 h, without any redox supplement. These results cannot be adequately explained by the existing oxidized thiol agents in human serum or the macromolecular crowding effect, and therefore indicate that human serum may contain yet to be identified potent oxidase(s) for assisting protein folding.
Evaluation of a Safety Resheathable Winged Steel Needle for Prevention of Percutaneous Injuries Associated With Intravascular-Access Procedures Among Healthcare Workers
Authors: Mendelson Meryl H. , Lin-Chen Bao Ying , Solomon Robin , Bailey Eileen , Kogan Gene , Goldbold James 
Published: March 2006

AbstractObjective:To compare the percutaneous injury rate associated with a standard versus a safety resheathable winged steel (butterfly) needle.Design:Before-after trial of winged steel needle injuries during a 33-month period (19-month baseline, 3-month training, and 11-month study intervention), followed by a 31-month poststudy period.Setting:A 1,190-bed acute care referral hospital with inpatient and outpatient services in New York City.Participants:All healthcare workers performing intravascular-access procedures with winged steel needles.Intervention:Safety resheathable winged steel needle.Results:The injury rate associated with winged steel needles declined from 13.41 to 6.41 per 100,000 (relative risk [RR], 0.48; 95% confidence interval [CI95], 0.31 to 0.73) following implementation of the safety device. Injuries occurring during or after disposal were reduced most substantially (RR 0.15; CI95, 0.06 to 0.43 Safety winged steel needle injuries occurred most often before activation of the safety mechanism was appropriate (39%); 32% were due to the user choosing not to activate the device, 21% occurred during activation, and 4% were due to improper activation. Preference for the safety winged steel needle over the standard device was 63%. The safety feature was activated in 83% of the samples examined during audits of disposal containers. Following completion of the study, the safety winged steel needle injury rate (7.29 per 100,000) did not differ significantly from the winged steel needle injury rate during the study period.Conclusion:Implementation of a safety resheathable winged steel needle substantially reduced injuries among healthcare workers performing vascular-access procedures. The residual risk of injury associated with this device can be reduced further with increased compliance with proper activation procedures.
Nosocomial Transmission of Hepatitis C Virus Associated With the Use of Multidose Saline Vials
Authors: Krause Gérard , Trepka Mary Jo , Whisenhunt Robert S. , Katz Dolly , Nainan Omana , Wiersma Steven T. , Hopkins Richard S. 
Published: March 2006

AbstractObjective:To identify the source of an outbreak of acute hepatitis C virus (HCV) infection among 3 patients occurring within 8 weeks of hospitalization in the same ward of a Florida hospital during November 1998.Design:A retrospective cohort study was conducted among 41 patients hospitalized between November 11 and 19, 1998. Patients' blood was tested for antibodies to HCV, and HCV RNA-positive samples were genotyped and sequenced.Results:Of the 41 patients, 24 (59%) participated in the study. HCV genotype 1b infections were found in 5 patients. Three of 4 patients who received saline flushes from a multidose saline vial on November 16 had acute HCV infection, whereas none of the 9 patients who did not receive saline flushes had HCV infection (P= .01). No other significant exposures were identified. The HCV sequence was available for 1 case of acute HCV and differed by a single nucleotide (0.3%) from that of the indeterminate case.Conclusion:This outbreak of HCV probably occurred when a multidose saline vial was contaminated with blood from an HCV-infected patient. Hospitals should emphasize adherence to standard procedures to prevent blood-borne infections. In addition, the use of single-dose vials or prefilled saline syringes might further reduce the risk for nosocomial transmission of blood-borne pathogens.
Occupationally Acquired Human Immunodeficiency Virus (HIV) Infection: National Case Surveillance Data During 20 Years of the HIV Epidemic in the United States
Authors: Do Ann N. , Ciesielski Carol A. , Metler Russ P. , Hammett Teresa A. , Li Jianmin , Fleming Patricia L. 
Published: March 2006

AbstractObjective:To characterize occupationally acquired human immunodeficiency virus (HIV) infection detected through case surveillance efforts in the United States.Design:National surveillance systems, based on voluntary case reporting.Setting:Healthcare or laboratory (clinical or research) settings.Patients:Healthcare workers, defined as individuals employed in healthcare or laboratory settings (including students and trainees), who are infected with HIV.Methods:Review of data reported through December 2001 in the HIV/AIDS Reporting System and the National Surveillance for Occupationally Acquired HIV Infection.Results:Of 57 healthcare workers with documented occupationally acquired HIV infection, most (86%) were exposed to blood, and most (88%) had percutaneous injuries. The circumstances varied among 51 percutaneous injuries, with the largest proportion (41%) occurring after a procedure, 35% occurring during a procedure, and 20% occurring during disposal of sharp objects. Unexpected circumstances difficult to anticipate during or after procedures accounted for 20% of all injuries. Of 55 known source patients, most (69%) had acquired immunodeficiency syndrome (AIDS) at the time of occupational exposure, but some (11%) had asymptomatic HIV infection. Eight (14%) of the healthcare workers were infected despite receiving postexposure prophylaxis (PEP).Conclusions:Prevention strategies for occupationally acquired HIV infection should continue to emphasize avoiding blood exposures. Healthcare workers should be educated about both the benefits and the limitations of PEP, which does not always prevent HIV infection following an exposure. Technologic advances (eg, safety-engineered devices) may further enhance safety in the healthcare workplace
Monsel's Solution: A Potential Vector for Nosocomial Infection?
Authors: Rupp Mark E. , Medcalf Sharon J. , Fey Paul D. , Handke Luke D. , Marion Nedra D. 
Published: March 2006

AbstractMonsel's solution is a common topically applied hemostatic agent used in minor dermatologic and gynecologic surgery. Clinically, because it is often stored for long periods and dispensed from a common source for multiple patients, Monsel's solution is a potential vector for transmission of infection. However, microbiologic inoculation studies and contamination surveys indicate that Monsel's solution has properties that prohibit microbial growth, making it an unlikely vector for nosocomial infection.