Biomedical subjects

S Cusack

Publications and source records attributed to S Cusack.

At least 37 records · Page 2Linked to original sources

PubMed1995

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and its complex with tRNAHis.

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNAHis. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2(1)2(1)2 with cell parameters a = 171.3 A, b = 214.7 A, c = 49.3 A, alpha = beta = gamma = 90 degrees. A complete data set to a resolution of 2.7A with an Rmerge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNAHis shows that the crystals are tetragonal with cell parameters a = b = 232 A, c = 559 A, alpha = beta = gamma = 90 degrees and diffract to about 4.5 A resolution.

Crystallography, X-Ray↗

PubMed1995

Adenovirus fiber.

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Adenoviridae↗

PubMed1995

Structure, function and evolution of seryl-tRNA synthetases: implications for the evolution of aminoacyl-tRNA synthetases and the genetic code.

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA(ser) in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 aminoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3' end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence.

Acylation↗

PubMed1995

Evaluation of outcome following cardiac arrest in patients presenting to two Scottish emergency departments.

OBJECTIVES: To compare and contrast outcomes following cardiac arrest managed in two Accident and Emergency departments, and to identify factors which might account for such differences. DESIGN: Prospective 1-year evaluation of patients sustaining an out-of-hospital cardiac arrest. SETTING: The Accident and Emergency departments of the Edinburgh (ERI) and Glasgow (GRI) Royal Infirmaries which serve two large urban municipalities. PATIENTS: All patients sustaining a prehospital cardiac arrest and brought to ERI or GRI were included. Children (< 13 years), those declared dead on arrival at the scene, and events related to poisoning, near drowning, trauma and pregnancy were excluded. MEASUREMENTS AND MAIN RESULTS: There were 297 prehospital arrests from ERI, and 158 from GRI. Eighty-two (27.6%) were admitted as 'in-patients' to ERI and 23 (14.6%) to GRI (P < 0.01). Thirty-nine (13.1%) survived to hospital discharge from ERI; 13 (8.2%) survived to discharge from GRI (NS). The proportion of VF/VT:Asystole observed was significantly different between the two centres--162:98 from ERI, 54:73 from GRI (P < 0.001). Significantly more prehospital arrests were witnessed and received bystander CPR in those brought to ERI (P < 0.02). For the combined VF/VT/Asystole groups the ERI ambulance response times were significantly shorter (P < 0.01). However, there was no significant difference in the collapse to EMS arrival at the scene times between ERI and GRI. Two survivors from ERI had asystole as their initial observed rhythm. From GRI, one survivor had asystole, one had electromechanical dissociation and in another the initial rhythm was unknown. No survivor to discharge had severe neurological disability. CONCLUSIONS: Patients suffering out-of-hospital cardiac arrests in Edinburgh have a significantly better chance of being admitted to a ward. There is a trend favouring better survival to discharge in Edinburgh, but with the numbers investigated this does not achieve statistical significance. Amongst those factors which contribute to survival there are fewer witnessed arrests, less bystander CPR and slower ambulance response times in those brought to GRI. There is a need to investigate the environment in which patients collapse, to train the public in CPR, and to review the efficiency and resourcing of the ambulance service.

Adolescent↗

PubMed1995

Eleven down and nine to go.

A series of new crystal structures of aminoacyl-tRNA synthetases sheds light on the evolution of specificity in this ancient family of enzymes.

Amino Acyl-tRNA Synthetases↗

PubMed1994

A new additive for protein crystallization.

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 A. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 A. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

Crystallization↗

PubMed1994

Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent.

The influenza virus genome consists of eight segments of negative-sense RNA, i.e. the viral (v) RNA forms the template for the mRNA. Each segment is encapsidated by the viral nucleoprotein to form a ribonucleoprotein (RNP) particle and each RNP carries its own polymerase complex. We studied the interaction of purified nucleoprotein with RNA in vitro, by using a variety of enzymatic and chemical probes for RNA conformation. Our results suggest that the nucleoprotein binds to the vRNA backbone without apparent sequence specificity, exposing the bases to the outside and melting all secondary structure. In this way, the viral polymerase may transcribe the RNA without the need for dissociating the nucleoprotein and without being stopped by RNA secondary structure, and the viral RNPs are ready to start transcription as soon as they enter the host cell.

Base Composition↗

PubMed1994

The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser).

The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.

Adenosine Triphosphate↗

PubMed1994

A small percentage of influenza virus M1 protein contains zinc but zinc does not influence in vitro M1-RNA interaction.

A peptide containing the CCHH motif, the putative zinc-binding sequence of influenza virus M1 protein, was found to bind zinc in a one-to-one complex with the characteristics of a typical zinc-binding peptide. Intact influenza virus also contained zinc and we show that this zinc is bound to the M1 protein in the virus. However, only a small proportion of M1 contained zinc: 4% in virus and 6 to 9% in isolated protein. One strain, B/Yamagata/16/88, consistently contained more zinc: 15 to 20% both in virus and in isolated protein. We also determined the RNA binding and transcription inhibition activities of various M1 proteins and found that the zinc content of M1 had no influence on either activity. We suggest that the zinc in M1 has a structural role in the virion other than nucleic acid binding.

Amino Acid Sequence↗

PubMed1994

Calcium is needed for the thermostability of influenza B virus neuraminidase.

The activity and stability of influenza virus neuraminidase is known to depend on the presence of calcium ions. The atomic structure of the tetrameric neuraminidase head shows two distinct Ca2+ binding sites, one with low affinity on the molecular fourfold symmetry axis and one with high affinity close to the active site in each of the monomers. Here we show that Ca is essential for the thermostability of the isolated neuraminidase tetramer. Inactivation of Ca-free neuraminidase at high temperatures is accompanied by changes in protein structure leading to protease sensitivity. More than one Ca ion per tetramer is involved in stabilization, suggesting a role for the high affinity Ca binding site and the cooperative stabilization of the subunits. Sites which are located close to the fourfold axis of the neuraminidase tetramer and which are able to bind a variety of different metal ions are also described.

Calcium↗

PubMed1993

Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution.

The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.

Amino Acid Sequence↗

PubMed1993

Influenza B virus neuraminidase can synthesize its own inhibitor.

BACKGROUND: Neuraminidase, one of the two surface glycoproteins of influenza virus, cleaves terminal sialic acid residues from glycolipids or glycoproteins. Its crystal structure is known at high resolution, but the mechanism of glycosyl hydrolysis remains unclear. RESULTS: We have determined the crystal structure at 1.8 A resolution of two complexes of influenza B/Beijing neuraminidase containing either the reaction product, sialic acid, or the transition state analogue inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). The sialic acid is bound in a distorted 'boat' conformation closely resembling that of bound DANA, stabilized by a conserved tyrosine residue (Tyr408). This distortion also gives rise to a suicidal side reaction that converts sialic acid to DANA at a low rate. CONCLUSIONS: The mechanism of neuraminidase action is distinct from that of other known glycosyl hydrolases. Substrate distortion appears to be the driving force in glycosyl bond hydrolysis and the proton required for catalysis can probably be donated by water, rather than by residues in the active site, thus allowing the enzyme to operate at high pH. The side reaction converting sialic acid to DANA appears reasonably favourable, and it is unclear how this is minimized by the enzyme.

Crystallography, X-Ray↗

PubMed1993

Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli.

Crystals of the complex between seryl-tRNA synthetase and tRNA(2ser) from Escherichia coli have been obtained from ammonium sulphate solutions. The crystals are of the 1:2 enzyme:tRNA complex, belong to the space group C222(1), have cell dimensions of a = 128.9 A, b = 164.9 A, c = 127.3 A and diffract anisotropically from 3.5 to 4.5 A. An X-ray diffraction data set to 4 A has been collected. The combination of molecular replacement using the refined structure of the catalytic domain of the native enzyme, data from a heavy atom derivative and solvent flattening was used to produce a map at 4 A resolution. This shows that a tRNA molecule binds across the dimer, the anticodon stem and loop do not contact the protein and the helical arm of the enzyme contacts the T psi C loop and the long extra arm of the tRNA.

Crystallization↗

PubMed1993

Adenovirus type 40 virions contain two distinct fibers.

Human subgroup F adenoviruses, types 40 and 41, have two genes that could specify different fiber proteins, a long fiber with 21 or 22 pseudorepeat motifs in the shaft, and a short fiber with 12 motifs. We show that for adenovirus type 40, both proteins are expressed from separate late regions of the genome. They are incorporated into the virion with only one fiber per penton base, unlike avian adenoviruses which have two fibers extending from each penton. Comparison of known adenovirus fiber sequences suggests that the two fibers in adenovirus 40 and 41 arose through an external recombination event rather than having evolved from each other directly. Two alternative fibers on the virion may imply the existence of two distinct receptors.

Adenoviruses, Human↗

PubMed1993

Comparison of structure and sequence of influenza B/Yamagata and B/Beijing neuraminidases shows a conserved "head" but much greater variability in the "stalk" and NB protein.

In 1988 the B/Yamagata/16/88 influenza virus appeared, which was antigenically quite different from the other B viruses circulating at that time. Neuraminidase heads of this strain were purified, crystallized, and analyzed by X-ray crystallography and compared with the structure of B/Beijing/1/87 neuraminidase. Only two point mutations could be detected in the "head" of the molecule leading to local rearrangement of amino acid side-chains. The nucleotide sequence, however, revealed significant differences in the stalk region and in the NB protein.

Amino Acid Sequence↗

PubMed1993

Sequence, structure and evolutionary relationships between class 2 aminoacyl-tRNA synthetases: an update.

The seven class 2 aminoacyl-tRNA synthetases that are alpha 2 dimers have previously been divided by sequence homology into class 2a (seryl-, threonyl-, prolyl- and histidyl-) and class 2b (aspartyl-, asparaginyl- and lysyl-). It has been more difficult to classify the glycyl-, phenylalanyl- and alanyl-tRNA synthetases which have different subunit stoichiometries and which did not apparently contain all three canonical class 2 motifs. New sequence and structural information relating to the three problematic synthetases will be discussed permitting a step forward to be taken in the understanding of the evolutionary relationships between the class 2 synthetases.

Amino Acid Sequence↗

PubMed1993

Survival from cardiac arrest in an accident and emergency department: the impact of out of hospital advisory defibrillation.

A prospective 1-year audit of cardiac arrests treated in the Accident and Emergency department of the Royal Infirmary, Edinburgh is presented. During the period January 1st, 1991 to December 31st, 1991, 325 patients with cardiac arrest were treated. Two-hundred ninety-seven of these were 'out-of-hospital' and 28 were 'in-department' arrests. Of patients with 'out-of-hospital' ventricular fibrillation/pulseless ventricular tachycardia 22.8% were discharged. Survival rates for patients with asystole or electromechanical dissociation were very poor. The impact of semiautomatic out-of-hospital defibrillation upon the survival and number of patients presenting to the department is discussed.

Aged↗

PubMed1993

The introduction of automatic blood pressure monitoring to an accident & emergency resuscitation room.

Machines for automatic non-invasive blood pressure (BP) monitoring are increasingly available in British accident and emergency departments. Our department recently acquired two machines with this capability for use in the resuscitation room. This provided us with an opportunity to compare the speed and frequency of automatic BP recording with the previously used manual method. We found no significant difference in either the median time to the first recording of BP or in the median number of documented recordings in the first hour. However, the overall frequency of BP recording did show a statistically significant increase. We conclude that automation alone does not improve standard practice in this area greatly.

Automation↗