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S S Ashraf

Publications and source records attributed to S S Ashraf.

At least 19 recordsLinked to original sources
PubMed2000

Role of modified nucleosides of yeast tRNA(Phe) in ribosomal binding.

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.

Nucleosides↗
PubMed1999

Routine sternal closure using interlocking multitwisted wires.

We describe a method of sternal closure that enhances sternal stabilization and minimizes bleeding from sternal fractures caused by retraction. With the technique of interlocking multitwisted wires the initial placement of the wire sutures is the same as in traditional sternal closure, however the twisting technique is improved, with multiple twisting including four twisted strands. Our method of closure is effective, simple and quick to perform and has several advantages over conventional or figure-of-eight closure. This closure is also biomechanically more rigid than conventional or figure-of-eight closure. We therefore recommend routine sternal closure using interlocking multitwisted wires.

Bone Wires↗
PubMed1999

Single atom modification (O-->S) of tRNA confers ribosome binding.

Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.

Aldehydes↗
PubMed1999

The uridine in "U-turn": contributions to tRNA-ribosomal binding.

"U-turns" represent an important class of structural motifs in the RNA world, wherein a uridine is involved in an abrupt change in the direction of the polynucleotide backbone. In the crystal structure of yeast tRNAPhe, the invariant uridine at position 33 (U33), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson-Crick interactions: hydrogen bonding of the 2'-OH to N7 of A35 and the N3-H to A36-phosphate, and stacking between C32 and A35-phosphate. The functional importance of each noncanonical interaction was determined by assaying the ribosomal binding affinities of tRNAPhe anticodon stem and loop domains (ASLs) with substitutions at U33. An unsubstituted ASL bound 30S ribosomal subunits with an affinity (Kd = 140+/-50 nM) comparable to that of native yeast tRNAPhe (Kd = 100+/-20 nM). However, the binding affinities of ASLs with dU-33 (no 2'-OH) and C-33 (no N3-H) were significantly reduced (2,930+/-140 nM and 2,190+/-300 nM, respectively). Surprisingly, the ASL with N3-methyluridine-33 (no N3-H) bound ribosomes with a high affinity (Kd = 220+/-20 nM). In contrast, ASLs constructed with position 33 uridine analogs in nonstacking, nonnative, and constrained conformations, dihydrouridine (C2'-endo), 6-methyluridine (syn) and 2'O-methyluridine (C3'-endo) had almost undetectable binding. The inability of ASLs with 6-methyluridine-33 and 2'O-methyluridine-33 to bind ribosomes was not attributable to any thermal instability of the RNAs. These results demonstrate that proton donations by the N3-H and 2'OH groups of U33 are not absolutely required for ribosomal binding. Rather, the results suggest that the overall uridine conformation, including a dynamic (C3'-endo > C2'-endo) sugar pucker, anti conformation, and ability of uracil to stack between C32 and A35-phosphate, are the contributing factors to a functional U-turn.

Anticodon↗
PubMed1999

Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains.

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.

Aldehydes↗
PubMed1997

Effects of cardiopulmonary bypass on neonatal and paediatric inflammatory profiles.

OBJECTIVE: Cardiopulmonary bypass (CPB) causes significant morbidity in paediatric patients, yet the mechanisms involved in the related inflammatory processes (resulting in capillary leak and edema) are poorly understood. Moreover, earlier palliative and corrective intervention in neonates and infants has provided the cohorts of patients about whom little is known of their proinflammatory response. METHODS: In the present two group study, 14 neonates (age 1-28 days, 2.5-4.5 kg) and 13 infants (2-12 months, 3-7 kg), undergoing CPB for congenital heart disease were consecutively recruited. The two cohorts were well matched in terms of CPB and aortic cross-clamp times (P > 0.1). Blood samples were collected on induction of anaesthesia, 5 min following onset of CPB, at the end of CPB, and 30 min, 2 and 24 h post-protamine (PP) administration. Plasma concentration of cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), terminal complement complex (C5b-9) neutrophil counts and leucocyte elastase were measured. RESULTS: Plasma levels of all inflammatory markers significantly increased in both groups during and following CPB as compared to baseline. During and following CPB the change in IL-8 level was more pronounced in neonates (peak 30 min PP, median(range): 1062 (182-3872) pg/ml) than in infants 568 (172-1368) pg/ml), P = 0.01. Changes in IL-6 level were indistinguishable between groups intraoperatively, but remained significantly higher at 24 h in neonates (P = 0.02). Peri and postoperative levels of C5b-9 were significantly higher in infants than in neonates (peak 30 min PP, median (range): 984 (118-1142) ng/ml vs 458 (22 1340) ng/ml in neonates respectively, P = 0.01) but were similar at 24 h. Despite this, leucocyte elastase profiles did not differ significantly between the respective cohorts. CONCLUSION: These results indicate that there may be differences between neonates and infants with regard to the inflammatory response to CPB and neonatal patients merit further investigation in order to elucidate whether the pathophysiology of their CPB related inflammatory response and its clinical sequelae differs from their older counterparts.

Cardiopulmonary Bypass↗
PubMed1997

Proinflammatory cytokine release during pediatric cardiopulmonary bypass: influence of centrifugal and roller pumps.

OBJECTIVE: It has been proposed that nonocclusive centrifugal pumps may elicit less blood cell trauma and hence a reduced inflammatory response than standard roller pumps. However, there have been no reports describing the impact of such pumps on proinflammatory cytokine release in pediatric cohorts. DESIGN: A prospective randomized study was undertaken. SETTING: A regional cardiothoracic center of a university hospital. PARTICIPANTS: Thirty-four pediatric patients undergoing cardiopulmonary bypass (CPB) for the correction of complex congenital heart defects were recruited. INTERVENTIONS: Either standard twin roller (n = 17), or centrifugal vortex (Biopump, Medtronic Biomedicus Inc, MN) (n = 17) blood pumping. MEASUREMENTS AND MAIN RESULTS: Venous blood was drawn (1) on induction of anesthesia, (2) 5 minutes on bypass, (3) end of CPB, (4) 30 minutes post-protamine, (5) 2 hours and (6) 24 hours postoperation. Neutrophil count, level of plasma leukocyte elastase, terminal complement complex (C5b-9); interleukin-6 (IL-6) and interleukin-8 (IL-8) were increased during and after CPB compared with the postinduction baseline. C5b-9 levels in both groups peaked at the end of CPB before returning to baseline at 24 hours: (median [range]), 564 (16 to 1,136) ng/mL in centrifugal group versus 508 (0 to 1,128) ng/mL in the roller group. IL-6 in both groups reached its peak level at 2 hours postprotamine (208 [98 to 411] pg/mL in centrifugal versus 205 [60-327] pg/mL in the roller group), before coming back to baseline at 24 hours. Plasma leukocyte elastase and IL-8 reached their maximum level 15 minutes after protamine administration: 215 (64 to 375) pg/mL in centrifugal versus 235 (87 to 410) pg/mL in roller group; and 700 (90 to 5,925) ng/mL versus 362 (120 to 3,400) ng/mL, respectively. CONCLUSIONS: The current study confirms the proinflammatory nature of pediatric CPB surgery, but failed to show a significant advantage of centrifugal pumping over roller perfusion in terms of the inflammatory response.

Cardiopulmonary Bypass↗
PubMed1997

Ribosomal binding of modified tRNA anticodons related to thermal stability.

The physicochemical contributions of modified nucleosides to tRNA functions are not well understood. In order to determine the contributions of specific modifications to tRNA stability as well as to ribosomal binding, ten variously modified yeast tRNA(Phe) anticodon stems and loops (tRNA(Phe)AC) were synthesized. Thermal denaturation studies on these synthetic 17mers show dramatic stabilization (or destabilization) by the presence of the various naturally occurring nucleoside modifications. Adapting a novel molecular biology approach (initially pioneered by Moazed and Noller), the interactions of these variously modified anticodons with the E. coli 16S rRNA "P-site" residues are being quantitated. The binding (affinity) constant (kD) of the tRNA(Phe)AC to the 8 of the ten 16S rRNA nucleosides that interact with tRNA and synthetic anticodons are being examined. We postulate that the "stabilizing" modifications (m1G37, psi 39, and m5C40) in the presence of an "open loop" will dramatically increase the binding affinity of the tRNA(Phe)AC to the 30S E. coli ribosomal subunit when compared to unmodified tRNA(Phe)AC. On the other hand, "destabilizing" modifications are expected to reduce the binding affinity of the tRNA(Phe)AC to the E. coli 30S ribosomal subunit. The results from these experiments have demonstrated the importance of nucleoside modifications to tRNA stability and ribosomal binding affinity, and will relate the structural contributions of nucleoside modifications to tRNA function.

Anticodon↗
PubMed1996

The management of stab wounds to the chest: sixteen years' experience.

In a retrospective review of 16 years of admissions to a regional cardiothoracic unit, 76 patients were admitted with stab wounds to the chest. Of these wounds, 75% were managed conservatively with tube drainage and observation and 25% required surgical intervention. 5.2% were managed as emergency room thoracotomies, 15.8% as urgent procedures in the operating theatre and 4% as late procedures. The total mortality was 1.3% and was seen in the emergency room thoracotomy group (mortality 25%). The risk if sub-diaphragmatic injury in the presence of chest injury is highlighted.

Adolescent↗
PubMed1995

Determinants of early and late mortality in patients with end-stage renal disease undergoing cardiac surgery.

To determine factors influencing early and late mortality associated with cardiovascular surgery in end-stage renal disease, 48 consecutive patients (mean age 56.3 years) were reviewed: 30 underwent coronary and 18 valvular surgery. There were eight early deaths, three in the former and five in the latter group. Factors significantly related to early mortality in univariate testing included infective valvular disease, emergency vs elective surgery (both p = 0.02) poor left ventricular function and prolonged clamping and bypass times (all p = 0.001). When these factors were included in a stepwise logistic regression analysis, infective valvular disease (p = 0.02), poor left ventricular function (p = 0.01) and long cross-clamping (p = 0.01) were independently associated with early mortality. There were six late deaths. Survival for the whole cohort at 1, 5 and 7 years was 95%, 60.4% and 42.6%, respectively. Related to late mortality at univariate testing were age (p = 0.03), smoking (p = 0.04), diabetes (p = 0.03) and poor left ventricular function (p = 0.02), and stepwise logistic regression analysis showed independent association with age, diabetes and impaired left ventricular function. Mortality associated with cardiac surgery in patients with end-stage nephropathy can be reduced by better patient selection, early operation in patients with infective endocarditis, and minimized cross-clamping and bypass times.

Adult↗
PubMed1994

S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: evidence against a role for glutathione disulfide.

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neutrophils stimulated with phorbol diester to produce superoxide anion. Rapid S-thiolation of several proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thio by HPLC chromatography, contributing considerably more than 85% of the total. Minor amounts of homocysteine and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione became protein bound (2 nmol/mg of protein). There was no increase in glutathione disulfide suggesting that S-thiolation of the proteins did not occur by thiol/disulfide exchange. Approximately 10 mol% of one heavily modified band (29 kDa) was S-thiolated after 30 min. A second major band of 42 kDa was identified as actin. It contained 1/10th of the total protein-bound glutathione and approximately 5 mol% was S-thiolated after 30 min. These experiments identify a subset of S-thiolated neutrophil proteins, including actin, whose modification is related to the phorbol diester stimulation of superoxide anion production in human neutrophils. Ten percent of the total glutathione pool became protein-bound without an appreciable change in non-bound concentration of glutathione or glutathione disulfide. These results suggest that glutathione was synthesized during initial phases of the respiratory burst, compensating for the amount of glutathione that became protein-bound. Since there was no significant increase in glutathione disulfide, it was probably not important in the observed protein S-thiolation.

Actins↗
PubMed1994

Early and late results following combined coronary bypass surgery and mitral valve replacement.

To study the effect of various perioperative and operative variables, we analysed the results of 66 consecutive patients undergoing mitral valve replacement (MVR) and coronary artery bypass grafting (CABG). The mean age was 61.2 years (34 males and 32 females), the mean follow-up 54.71 +/- 7.8 months. The hospital mortality rate was 7.6% (5/66). New York Heart Association (NYHA) functional class (P < 0.01), left ventricular global wall motion score (increased scores indicating impaired function, P = 0.005) and cross-clamp time (P < 0.05) were associated with hospital mortality. There was no significant relationship of age (certainly up to the age of 70), cause of mitral valve disease, severity of mitral regurgitation, number of grafts, presence of angina, or previous myocardial infarction with hospital mortality. There were eight late deaths, survival at 1, 3 and 5 years was 92.4%, 83.2% and 80.2%, respectively. Although there was a trend for pulmonary vascular resistance (P = 0.15), NYHA class (P = 0.18) and aortic cross-clamp time (P = 0.09) to be associated with late survival, the only factor significantly related to late survival was global wall motion score (P = 0.001), i.e. those with scores of more than 10. Severity of mitral regurgitation and cause of mitral valve disease have been reported as being related to late survival in patients undergoing combined CABG and MVR, but we have found no such relationship. Our results indicate that both hospital and late mortality after this operation are strongly correlated with left ventricular function.

Adult↗
PubMed1994

Bicoronary-pulmonary fistulae with coexistent mitral valve prolapse: a case report and literature review of coronary-pulmonary fistula.

Bicoronary-pulmonary artery fistulae are rare congenital coronary artery fistulae. We report the case of a 57-year-old man who presented with biventricular failure and angina pectoris. At angiography the coronary arteries were normal, but bicoronary-pulmonary fistulae were noted; severe mitral regurgitation secondary to mitral valve prolapse was also present. At right heart catheterization the calculated left to right shunt was 2.1. Fistula ligation and repair of both mitral and tricuspid valves was undertaken. The literature regarding coronary-pulmonary fistula is also reviewed.

Arterio-Arterial Fistula↗
PubMed1994

A comparison of early mortality and morbidity after single and bilateral internal mammary artery grafting with the free right internal mammary artery.

OBJECTIVE: To compare differences in early mortality and morbidity in patients receiving a single internal mammary artery graft (SIMA) with those receiving bilateral internal mammary artery grafts (BIMA) with a free right internal mammary artery (RIMA). DESIGN: Retrospective analysis of 150 patients undergoing BIMA grafting between 1989-1992 who were carefully matched with 150 patients undergoing SIMA grafting between 1987-1992 for known cardiovascular risk factors, extent of coronary disease, left ventricular function, and number of coronary grafts. Operative variables noted included aortic cross clamp time and bypass time. Postoperative cardiac, respiratory, and wound complications were also noted. RESULTS: Operative mortality was 2% in the SIMA group and 1.3% in the BIMA group (NS). Other than the prevalence of ventricular arrhythmias (P = 0.025), which were more common in the BIMA group, there were no significant differences between the two groups in terms of postoperative morbidity. At median (interquartile range) follow up of 27.94(0.86) and 23.94(0.74) months for the SIMA and BIMA groups respectively there were no deaths. 87% of the SIMA group and 91% of the BIMA group were free of symptoms at follow up. CONCLUSIONS: The earlier fears regarding increased early mortality and morbidity after BIMA surgery were not confirmed by this study. All patients receiving both mammary arteries had a free rather than pedicle right internal mammary graft. The early mortality and morbidity reported here compares favourably with previous reports on the use of a pedicle graft.

Adult↗
PubMed1994

A prospective study of serum lipoproteins after coronary artery bypass surgery.

We examined the acute and long-term effects of coronary artery bypass (CABG) surgery on serum lipid, lipoprotein and apolipoprotein levels. One series of 34 patients having CABG surgery was studied pre-operatively and for six weeks afterwards, and another 22 patients were investigated before and two years after CABG surgery. None of the patients studied received any lipid-lowering drug therapy or specific dietary advice. In both groups, pre-operative serum lipoprotein (a) (Lp(a)) and serum triglyceride concentrations were raised and serum high-density lipoprotein (HDL) cholesterol and apolipoprotein AI (apo AI) were low compared to healthy people. Acutely, there were profound decreases of 40-60% in the serum levels of cholesterol (p < 0.001), low-density lipoprotein cholesterol (p < 0.05), triglycerides (p < 0.01), Lp(a) (p < 0.05) and apolipoprotein B (apo B) (p < 0.05). There was a small decrease in serum apo A1 (p < 0.05), and serum HDL cholesterol showed no change. All these variables regained their pre-operative values within six weeks. Two years postoperatively, serum Lpa was 40% less than its pre-operative concentration (p < 0.001) and HDL cholesterol had increased (p < 0.001). Triglyceride levels decreased (p < 0.02) when beta-blockade was withdrawn. The long-term decrease in Lp(a) following surgery is unlikely to be due either to stopping beta-blockers or to life-style changes. Myocardial ischaemia relieved by the operation may have been partially responsible for its previously raised concentration.

Adult↗