Crystallization and preliminary X-ray diffraction analysis of E. coli arginyl-tRNA synthetase in complex form with a tRNAArg.

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA(Arg) (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 A and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 A, c = 463.2 A, and alpha = beta = 90 degrees , gamma = 120 degrees , being different from a = b = 110.8 A, c = 377.8 A for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.

Venous aneurysms of the wrist.

We describe 2 cases of primary atraumatic venous aneurysm affecting the wrist. Both aneurysms were in branches of the cephalic vein in close proximity to the radial artery. The definitive treatment for these venous aneurysms was surgical excision. There was no recurrence after 9 years in case 1 and after 11 years in case 2. Modern diagnostic modalities were used, including physical examination, Doppler ultrasonography, aspiration, magnetic resonance imaging, and venography. The pathologic analysis was consistent with those venous aneurysms reported in other parts of the body. The hand surgeon should be aware of this rare condition when formulating a differential diagnosis for soft tissue masses of the wrist.

Ventilator-associated pneumonia with circuit changes every 2 days versus every week.

BACKGROUND: Frequent ventilator circuit changes are expensive and sometimes unnecessary. Following the worldwide trend to lengthen the intervals for ventilator circuit change from 2 days to 1 week, this study aims to assure that low rate of ventilator-associated pneumonia (VAP) can be maintained with cost containment. METHODS: Ventilator circuits were routinely changed every 7 days in the study period for 2 years and every 2 days during the historical control period of another 2 years. Pediatric patients (age less than 15 years) were not included. Nosocomial pneumonia was diagnosed by the criteria of the Centers of Disease Control and Prevention (CDC) of the United States (US). VAP was identified by combining and comparing 2 databases from the Respiratory Therapy Department and the Infection Control Unit of our hospital. RESULTS: In the study group, 225 episodes of pneumonias were observed in 7,068 patients and 87,338 ventilator days. The rate of VAP was 2.58 per 1,000 ventilator days. There were 174 episodes of pneumonia in 6,213 patients and 65,467 ventilator days of the control group. The rate of VAP was 2.66 per 1,000 ventilator days. The difference between both groups was not significant (p = 0.803). Yet, the cost curbed was around 80,000 US dollars per year. CONCLUSIONS: Extending ventilator circuit change interval from 2 days to 7 days do not increase the risk for VAP, but the cost savings for labor and supply are substantial.

Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase.

Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNA(Leu). Several mutants in the CP1 domain of Escherichia coli LeuRS were obtained by introduction of restriction endonuclease sites into its gene, leuS. Of these mutants, only LeuRS-A293F had decreased activity (46%) compared to the native enzyme. To investigate the effect of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D. The mutants were impaired in activity and editing function to varying extents. The decrease in K(m) values for three substrates showed that the binding of ATP to these mutants became much stronger. The inhibition of ATP binding to most of the mutants was also stronger. In particular, LeuRS-A293D had the lowest activity, the strongest ATP binding, and the most impaired editing function. A red shift of the fluorescence emission maximum of LeuRS-A293D indicated a less hydrophobic chromophore environment and a relatively more flexible dynamic conformation. The change in T(m) of LeuRS-A293D was higher than that of all other substitutions. Evidence from sequence alignment and crystal structure of LeuRS from Thermus thermophilus shows that A293 was conserved as R (K) or A and is located at a small helix in the editing domain of the enzyme facing the active site. Hence, any amino acid substitution of A293 may affect the stability of the helix, which may lead to impaired editing function and aminoacylation activity and may be indirectly involved in ATP binding.

A Negative Element Located in the Upstream Flanking Region of the Gene Encoding Arginyl-tRNA Synthetase (argS) from Escherichia coli.

The gene, argS, encoding the arginyl-tRNA synthetase (ArgRS) from Escherichia coli ( E.coli ) was overexpressed 1 000 fold in the transformant when E. coli TG1 was transformed with the recombinant plasmid containing argS and pUC18. In order to investigate the regulation of expression of E. coli argS, a series of deletion mutations was constructed. The results of SDS-PAGE showed that deletions of the whole 5' flanking region (argSdelta1) or the region in front of Shine-Dalgarno Sequence (argSdelta2) or the -10 region of promoter (argSdelta3), caused no overexpression of argS. If argS was deleted from 3' end of the flanking region (-189 nt) to the upstream of -10 region of promoter (argSdelta4), the -35 region (argSdelta5), -52 nt (argSdelta6), -70 nt (argSdelta7) and -122 nt (argSdelta8), respectively, the mutant gene was overexpressed to a level similar to that of argS bringing the full length 5' flanking region. However, in the expression of argSdelta4, argSdelta5, argSdelta6, some of ArgRS formed an inclusion body. By determination of RNA dot hybridization, the amount of mRNA produced in the transcription of argSdelta4, argSdelta5 and argSdelta6 was about 2--3 times than that of the wild type argS, argS delta7 and argS delta8. This indicated that the deletion of a 19 nt sequence (AATAGTGAAAACGGCAATA) located between -52 nt and -70 nt of the gene increased the transcription of argS. The 19 nt sequence is a negative region that represses transcription of argS. Deletion of the negative element may result in a faster production of ArgRS and the accumulation of some unfolding protein intermediates aggregating to form the inclusion body. The result by analysis of gel retardation shows that a factor binds to the negative element. Arginine induced specifically the transcription of argS and its effect correlated with the above negative element.

Mutagenesis of N-terminal Amino Acid Residues in beta-subunit of Glutaryl-7-amino-cephalosporanic Acid Acylase C130.

In many GL-7ACA acylases, the first Ser residue at the N-terminal of beta-subunit is the catalytic center. In order to investigate relationship between the N-terminal structure and catalytic activities, peptide replacement and site-directed mutagenesis were performed at the N-terminal of beta-subunit of GL-7ACA acylase C130. When the N-terminal 8 amino acid residues of C130 were replaced by the corresponding sequence of penicillin acylases PAC and PGA, respectively, the first mutant B8PAC lost the activity of the acylase, and the second mutant B8PGA had lower activity with the K(m) value increasing from 0.44x10(-3)mol.L(-1) to 0.55x10(-3) mol.L(-1), and the k(cat) decreasing from 4.92 s(-1) to 1.64 s(-1). Although the substitution of Trp (beta4) by Tyr did not change the K(m) value, the k (cat) decreased to 2.29 s(-1). When the Trp was substitued by Leu, both the K ( m ) and k ( cat ) values decreased. Compared with the wild type, mutations of Ser (beta3) to Met, Ala and Cys caused decrease of K(m) values by 52.27%, 43.18% and 38.64%, respectively. Mutation of Asn (beta2) to Gln caused the K ( m ) value being increased by 5-fold, and k ( cat ) decreased by 10-fold. These results suggested that the N-terminal amino acid residues of beta-subunit in GL-7ACA acylase C130 are important for enzyme function.

Arm pain as an unusual presentation of postdural puncture intracranial hypotension.

IMPLICATIONS: We report a case of a patient experiencing severe arm pain after dural puncture. This complication has not been reported previously. The patient was successfully treated with an epidural patch.

CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function.

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.

A silent antifungal protein (AFP)-like gene lacking two introns in the mould Trichoderma viride.

In cells of the mould Trichoderma viride, the existence of an antifungal protein (AFP)-like gene consisting of 285 bp was confirmed by Southern analysis that genomic DNA of T. viride could hybridize with the cDNA of mature AFP of Aspergillus giganteus MDH 18894. Except for the absence of two introns, the nucleotide sequence of the AFP-like gene was identical to that of the AFP gene of A. giganteus in positions 336-479, 568-649, and 706-765. The AFP-like gene could not be transcribed into its mRNA in T. viride cells as examined by RT-PCR using total RNAs of T. viride as template. Furthermore, AFP could not be detected either directly from the culture medium of T. viride or by Western analysis. However, the AFP-like gene could be actively expressed like the cDNA of AFP in Escherichia coli cell. Recombinant AFP exhibited similar antifungal activity as native AFP.

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.

Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase.

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher Km values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.

Purification of alpha-sarcin and an antifungal protein from Aspergillus giganteus by blue sepharose CL-6B affinity chromatography.

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from the mold Aspergillus giganteus MDH 18894 has been developed. alpha-Sarcin and AFP were purified simultaneously by carboxymethylcellulose 52 cation-exchange chromatography and Blue Sepharose CL-6B affinity chromatography. By this method, 4.2 mg of alpha-sarcin and 6.8 mg of AFP were obtained from 2 liters of medium. Compared with other purification methods such as gel-filtration chromatography, this procedure was simple and specific. The purified alpha-sarcin and AFP were homogeneous characterized on SDS-polyacrylamide gel. The enzymatic activity of several ribosome-inactivating proteins such as alpha-sarcin, trichosanthin, and cinnamomin was significantly inhibited by the dye Cibacron blue F3GA. In 50 microliter of reaction mixture, 10 microM of the dye could inhibit 50% activity of cinnamomin (7 x 10(-9) M), whereas 50% inhibition of the enzymatic activity of trichosanthin (7 x 10(-9) M) and alpha-sarcin (1 x 10(-7) M) required 100 and 50 microM of the dye, respectively.

The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase.

The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied. The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity. When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one. The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent. These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity. Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure. Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat/Km for arginine. The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol. Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization.

Tendon repair.

Recent studies of tendon repair are reviewed in order to help hand therapists make the most effective decisions regarding treatment of tendon injuries. Flexor tendons are emphasized by inclusion of the most current discussions and research on the different phases of intrinsic and extrinsic healing, the zones of injury, mechanical and chemical influences on tendon healing, the techniques of tendon suturing, and the timing of repair and rehabilitation. The purpose of the article is to update hand therapists on the latest research efforts concerning tendon healing in order to achieve better functional outcomes following repair of these structures.

Scoliosis in patients treated with growth hormone.

Two hundred fifty children being treated with growth hormone were screened for scoliosis by using the Adams and Bunnell techniques. If indicated, an anteroposterior radiograph was done and measured by the Cobb and Risser methods. Scoliosis was defined as a frontal curve of > or = 10 degrees; progression, as a sustained increase of > or = 5 degrees, and a progressive curve as one > or = 25 degrees and meeting our criteria for orthotic management. In 10 of the 250 patients, scoliosis developed. Six curves were double major thoracic and lumbar; three thoraco-lumbar; and one single thoracic. Six of the 10 patients had progressive curves and required an orthosis. Their average annualized rate of progression was 26 degrees. Progression was associated with double major curves and an earlier Risser stage. Despite bracing, progression continued to fusion in three patients. We conclude that growth hormone may increase the risk of progression of scoliosis. Furthermore, the progression is frequently rapid and requires special vigilance by the treating physician.

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.

Avulsion fracture of the supraglenoid tubercle: A variation of the SLAP lesion.

Tears of the anterior and posterior/superior labrum (SLAP) are often associated with overhead throwing sports. This lesion may be present in the absence of glenohumeral instability. Fracture of the supraglenoid tubercle associated with a SLAP lesion has not been previously reported. Two cases of supraglenoid tubercle fracture associated with the SLAP lesion in overhead throwing activities are presented. The presence of a supraglenoid fracture on a plain radiograph is uncommon, but such a finding may allow early noninvasive diagnosis of the SLAP lesion.

Effect on yeast LEU2 expression of upstream activation sequence from yeast ENO2 gene coding for enolase.

The upstream activation sequence from ENO2, one of two genes coding for yeast enolase, was inserted into the upstream 405 HpaI site of LEU2, which codes for beta-isopropylmalate dehydrogenase (E.C. 1.1.1.85), utilizing shuttle plasmid YEp13. The effect of the ENO2 upstream activation sequence on expression of yeast LEU2 was studied. Our results revealed a fourfold increase in expression for LEU2 in both orientations after activation by the ENO2 upstream activation sequence. Leucine repressed LEU2 expression. Glucose did not induce the ENO2 upstream activation sequence effect on LEU2 expression. It is possible to construct a high-level expression system in yeast by using the ENO2 upstream activation sequence.