Radical effects on mutation spectra in lambda phage.

Mutations in the lambda repressor gene cI (710 bp) were induced by 60Co-gamma radiation in dissolved lambda phage DNA. After in vitro DNA packaging to lambda phage particles (pack phage) and phenotypic expression of the mutants, DNA was sequenced directly. Two-thirds of mutations were located in the amino terminus region of the gene without any signs of hotspots. Changes consisted of (+1) insertions (25%) and base substitutions (75%). Transitions were exclusively G/C to A/T. Transversions were mostly G/C to C/G and few G/C to T/A. We did not find A/T to T/A transversions, A/T to G/C transitions, deletions and gross rearrangements. In most of the base substitutions a pre-existing base pair had been replaced by an A/T pair; this might come from 'non-instructional sites' like abasic sites. Several mechanisms for base substitutions are considered.     

Functional abnormalities of experimental autogenous vein graft neoendothelium.

When a vein is grafted into the arterial circulation, the endothelium of the graft is damaged. Regeneration of an intact neoendothelium occurs, but the functional properties of this surface have not been clarified. In this study, the functional integrity of the neoendothelium of veins grafted into the carotid artery of the rabbit was assessed through the use of acetylcholine and histamine to stimulate the production of the important endothelium-derived relaxing factor (EDRF). Control veins, precontracted with norepinephrine [10(-5) M], relaxed after exposure to acetylcholine [( 10(-7) M], 42.4% +/- 6.4%, p = 0.008) and histamine [( 10(-6) M], 30.6% +/- 4.3%, p = 0.03). This relaxation response was abolished after mechanical removal of the endothelium. By contrast, neither acetylcholine nor histamine caused an endothelium-dependent relaxation in the vein grafts, even though scanning electron microscopy demonstrated the presence of a morphologically intact endothelium. However, addition of stabilized EDRF purified from cultured endothelial cells induced relaxation of the vein grafts (35.8% +/- 3.6%, p = 0.002). These data indicate that vein graft endothelium is unable to produce EDRF in response to exposure to acetylcholine or histamine. The inability to produce this potent smooth muscle cell relaxing factor and anti-aggregatory substance may be a predisposition to vein graft failure.     

Evaluation of postsurgical crestal bone levels adjacent to non‐submerged dental implants

In most of the studies on long-term radiographic evaluations of crestal bone levels adjacent to dental implants, no baseline radiographs taken immediately post-surgically had been obtained.The aim of this study was to test the reproducibility of a simple radiographic method for linear measurements of changes in bone levels and to evaluate changes in crestal bone levels adjacent co non-submerged ITI® implants 1 year following the surgical procedure. From 128 patients enrolled in a clinical and radiographic longitudinal study 40 patients also had radiographs taken immediately postsurgically. They were, however, not obtained as “identical” images. The radiographs were mounted onto slides and projected on a screen. Mesially and distally from 57 implants triplicate linear measurements of the distance implant shoulder to bone crest were taken, using known dimensions of the implants as internal reference distances. The median difference of 213 (out of 228 possible) duplicate measurements was 0.00 mm (ranging from ?1.72 mm to +1.47 mm when comparing the second co the third reading). Some 81% of the double measurements were within ±0.5 mm and the precision was 0.30 mm. In the immediate postoperative radiographs the median mesial bone level was located at 2.07 mm (distally 2.19 mm) from the implant shoulder. A statistically significant amount of bone loss in the first year was observed mesially (median=?0.78 mm) and distally (0.85 mm)(Wilcoxon matched pairs signed rank test ±0.001). No statistically significant influence of the implant location, the implant length, type of the implant (screw; cylinder) was observed (Kruskal-Wallis P>0.05).The age of the patients was not correlated significantly to the amount of bone loss observed. In conclusion, methodological limitations existed when evaluating linear bone changes in non-identical radiographs using reference dimensions of the implants. The amount of postsurgical bone loss estimated in other studies was confirmed when using an immediate postoperative radiograph as a baseline.     

Increased cell death after therapy with an Arg-Gly-Asp-linked somatostatin analog.

Receptor-targeted scintigraphy and radionuclide therapy with radiolabeled somatostatin analogs are successfully applied for somatostatin receptor-positive tumors. The synergistic effects of an apoptosis-inducing factor, for example, the Arg-Gly-Asp (RGD) motif, can increase the radiotherapeutic efficacy of these peptides. Hence, the tumoricidal effects of the hybrid peptide RGD-diethylaminetriaminepentaacetic acid (DTPA)-Tyr3-octreotate (cyclic[c](Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr), hereafter referred to as RGD-DTPA-octreotate, were evaluated in comparison with those of RGD (c(Arg-Gly-Asp-D-Tyr-Asp)) and Tyr3-octreotate (D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). METHODS: The therapeutic effects of RGD-111In-DTPA-octreotate, 111In-DTPA-RGD, and 111In-DTPA-Tyr3-octreotate were investigated with various cell lines by use of a colony-forming assay, and caspase-3 activity was also determined. RESULTS: Tumoricidal effects were found with 111In-DTPA-RGD, 111In-DTPA-Tyr3-octreotate, and RGD-111In-DTPA-octreotate, in order from least effective to most effective. Also, the largest increase in caspase-3 levels was found with RGD-111In-DTPA-octreotate. CONCLUSION: RGD-111In-DTPA-octreotate has more pronounced tumoricidal effects than 111In-DTPA-RGD and 111In-DTPA-Tyr3-octreotate, because of increased apoptosis, as indicated by increased caspase-3 activity.     

Synthetic and computer-assisted analyses of the pharmacophore for the benzodiazepine receptor inverse agonist site

The structural requirements for ligand binding to the benzodiazepine receptor (BzR) inverse agonist site were probed through the synthesis and in vitro evaluation of 3-substituted beta-carbolines 6, 7, 11, 12, gamma-carboline 13, and diindoles 18-21, 23-25, 27, 28, and 34. On the basis of the apparent binding affinities of these and other analogues, a hydrogen bond acceptor site (A2) on the receptor is proposed to interact with the N(9) hydrogen atom of the beta-carbolines or the N(7) hydrogen nuclei of the diindoles. Likewise, a proposed hydrogen bond donating site (H1) interacts with the N(2) nitrogen atom of the beta-carbolines or the N(5) nitrogen atom of the diindoles. It appears that interaction with both sites is a prerequisite for high affinity since analogues which have either one or both of these positions blocked exhibit substantial reduction in affinity. Moreover, H1 appears to be capable of engaging in a three-centered hydrogen bond with appropriately functionalized ligands, which explains the increase in potency observed in the following series of 3-substituted beta-carbolines: the n-butyl (12, IC50 = 245 nM), n-propoxy (9, IC50 = 11 nM), and propyl ketone (11, IC50 = 2.8 nM) congeners. In addition to H1 and A2, there appears to be a relatively narrow hydrophobic pocket in the binding cleft that can accommodate substituents at the 3-position of the beta-carbolines which have chain lengths less than or equal to C5. There is a 1 order of magnitude decrease in affinity between n-propoxy analogue 9 (IC50 = 11 nM, chain length = 4) and n-butoxy derivative 7 (IC50 = 98 nM, chain length = 5). Furthermore, alpha- and gamma-branching [e.g. ethoxycarbonyl (2), IC50 = 5 nM and tert-butoxycarbonyl (31) IC50 = 10 nM] but not beta- and delta-branching [e.g. isopropoxy (6), IC50 = 500 nM and (neopentyloxy) carbonyl (48), IC50 = 750 nM] at position 3 are tolerated. Occupation of this hydrophobic pocket is clearly important for high affinity as evidenced by the relatively low affinity of 30, a beta-carboline which possesses a hydrogen atom at the 3-position. This same hydrophobic pocket is partially filled by the D and E rings of the diindoles, which accounts for the high affinity of several members of this series. An excluded volume analysis using selected 3-substituted beta-carbolines and ring-E substituted pyridodiindoles is consistent with the presence of this hydrophobic pocket (see Figure 1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Aortic aneurysm in a 74-year-old man with coronary disease and obstructive lung disease: is double jeopardy enough?

A previous decision analysis examined a patient with severe CAD, diminished ventricular function, and an abdominal aortic aneurysm and also concluded that CABG followed by aneurysm repair was optimal. This patient, who had well-preserved cardiac function but severely compromised pulmonary status, stood to gain less from CABG than would a patient with more severe coronary disease, thus accounting for the "close-call" between the CABG-AAA and AAA only strategies. Nevertheless, the analysis did emphasize the benefit of aneurysm repair, whether done alone or after CABG. The analysis also highlighted the significant risk of aneurysm rupture the patient is exposed to while recovering from CABG surgery. The operative mortality risks of the two procedures are similar; thus, the patient's total operative risk is approximately doubled if he undergoes both procedures rather than aneurysm repair alone. The key question raised by the analysis is whether this double jeopardy is more than compensated by the degree to which prior CABG reduces both short-term cardiac risk at subsequent aneurysm repair and long-term cardiac mortality. For this patient, who had good cardiac function, the gains appeared sufficient to offset the interval risk of aneurysm rupture and the additional risk associated with a surgical procedures. THE REAL WORLD The patient indeed underwent and tolerated CABG, although he had a stormy prolonged postoperative course due to pulmonary failure. After discharge from the hospital, he declined readmission for repair of the aneurysm. We did not model that possibility, clearly an inadequacy in our tree. Some six months later, the patient was still alive and was, reluctantly, readmitted for aneurysmorrhaphy. At that time, however, his pulmonary function had deteriorated and both the anesthesiologist and the pulmonary consultant stated unequivocally that further surgery was now impossible. In retrospect, the expected utility of CABG without aneurysm repair (thus providing only a decrease in the long-term mortality risk from his CAD) would have been 1.95 (DEALE) or 2.06 (Markov) years. Sensitivity analysis revealed that, even if long-term cardiac risk were completely eliminated by CABG, immediate aneurysm repair would have been a better approach had the patient's physicians known he would be likely to refuse or not be a candidate for the second operation. In summary, although the patient's comorbidities did indeed place him at significant operative risk for either aneurysmorrhaphy alone or two sequential procedures, the benefits to be gained were shown to far outweigh the risks when compared with expectant observation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alteration by burn injury of the pharmacokinetics and pharmacodynamics of cimetidine in children

Summary We have studied the mechanisms of the increased dosage requirements of the H₂-receptor antagonist cimetidine in paediatric burned patients in a pharmacokinetic and pharmacodynamic study.Cimetidine (10–15 mg·kg^–1) was given to 21 burned children and multiple blood samples were obtained for determination of plasma cimetidine concentrations and pharmacokinetic analysis.The relation of gastric pH to plasma cimetidine concentrations was studied in five of these children who had nasogastric tubes. In an additional four patients the effects of cimetidine on gastric pH were studied during a continuous infusion of cimetidine, which maintained steady-state plasma cimetidine concentrations above 0.5 µg·ml^–1.The mean (SEM) clearance of cimetidine in burned children was 16.22 ml·kg^–1 and cimetidine half-life was 1.06 h. The cimetidine clearance and half-life values were significantly higher in burned children compared with our previously reported values for normal adult patients, 8.2 ml·min·kg^–1 and 2.21 h respectively.Endogenous creatinine clearance normalized to 70 kg in burned children was 190 ml·min^–1. In burned children 41% of the dose of intact cimetidine was excreted during 8 h of the study compared with 45% excretion during 24 h in healthy adult controls previously reported. The correlation coefficient between creatinine and cimetidine clearances was 0.93 (r ²=0.85).The plasma concentration of cimetidine needed to increase gastric pH to 4.0 was 1.0 µg·ml^–1, which contrasts with the value of >0.5 µg·ml^–1 required for adult burned patients.These findings support the hypothesis that the higher dosage requirements of cimetidine in burned children is due both to enhanced elimination kinetics and to alterations in target organ sensitivity, requiring higher than normal plasma concentrations for the desired effect. In burned children Cimetidine should be given in higher doses and/or more frequently.     

Urinary lactate excretion to monitor the efficacy of treatment of type I glycogen storage disease

The purpose of this study was to investigate the usefulness of urinary lactate measurements to assess the adequacy of dietary treatment in patients with type I glycogen storage disease (GSD-I). We determined the correlation of urine and blood lactate concentrations in 21 GSD-I patients during 24-h admissions to the General Clinical Research Center (GCRC) during which hourly blood samples and aliquots of every void were obtained. In all but 1 patient, we found a good correlation between blood lactate concentrations and urinary lactate excretion. One patient did not excrete lactate in significant amounts despite elevated blood lactate concentrations. In 17 patients, the highest blood lactate concentrations occurred during the night. Markedly elevated nighttime average blood lactate concentrations above 3.5 mmol/l resulted in a urinary lactate concentration above the normal limit of 0.067 mmol/mmol creatinine in the first morning urine specimen. Mildly elevated nighttime blood lactate concentrations (between 2.2 and 3.5 mmol/l) led to urinary lactate concentrations that were either normal or moderately elevated. All patients with normal blood lactate concentrations during the night also had normal first morning urinary lactate concentrations. The degree of urinary lactate excretion in relation to blood lactate concentrations varied by individual. Urinary filter paper specimens, collected at home during the night and in the morning and mailed to the laboratory, were used to monitor the dietary compliance of 5 GSD-I patients at home over a period of 6 to 9 weeks prior to their GCRC admissions. These data suggested variable degrees of dietary control. In conclusion, the urinary lactate concentration is a useful parameter to monitor therapy of GSD-I patients at home. To be interpretable, the baseline urinary lactate concentration in relation to the blood lactate concentration has to be determined.