Assessment of cervical ligamentous injury in trauma patients using MRI.

During a 7-year period, a consecutive retrospective series of 89 trauma patients at a Level One trauma center who had negative standard radiographs with suspected occult cervical injury were administered a cervical magnetic resonance imaging (MRI) scan. The MRI studies were fully able to assess the ligamentous status of the cervical spine in all patients and were the final step in determining the treatment of the spine. Of the total 89 patients, 82 had no ligamentous injury, and 7 had ligamentous injury. Two patients underwent surgery because of the findings on the MRI study. MRI studies of patients with negative standard radiographs but with suspected occult cervical injury are excellent and safe studies for the evaluation of cervical spinal stability because of their ability to detect ligamentous injuries that are not evident on plain radiographs.

Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase.

The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma-carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C(4)' of the cofactor to yield alpha-keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C(alpha)-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha, gamma-elimination from enzyme-bound SAM is likewise shown to occur after C(alpha)-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.

Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins.

Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx-dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V(max) but similar S(0.5). Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V(max) enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. Inclusive of the present study, activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs.

Congenital radius deficiency: radiographic outcome and survivorship analysis.

The experience with congenital radius deficiency, or radial hemimelia, at the Shriners' Hospital for Children, Los Angeles Unit, was reviewed. A cohort of 29 limbs in 23 patients was identified with an average follow-up period of 50 months. Radiographic parameters were assessed using the hand-forearm angle, hand-forearm position, and ulnar bow. We compared radialization to modified centralization, assessed the efficacy of ulnar osteotomy, and assessed the effect of age, preoperative deformity, ulnar osteotomy, and Bayne's type on the final result. Revisions were noted and a survivorship analysis performed. The cohort had statistically significant correction of hand-forearm angle and hand-forearm position. Radialization was similar to modified centralization in the final outcome. Ulnar osteotomy was an efficacious way to correct ulnar deformity. Age, preoperative deformity, performance of an ulnar osteotomy, and Bayne's type did not affect the final wrist position. Survivorship analysis was performed using revision as the end point, with a survivorship rate at 5 years of 67%. Significant risk factors for revision included radial or positive hand-forearm angle and young age at the time of the index procedure. There was a suggestion that small postoperative hand-forearm position, or radial translation, increased the risk of revision. Preoperative deformity, performance of an ulnar osteotomy, and Bayne's type did not affect the risk of revision. These data offer support for the hypothesis that a more ulnar translation and an ulnar angulation of the wrist is a means of reducing the radial lever arm and thus the incidence of deformity recurrence and need for revision.

A novel, definitive test for substrate channeling illustrated with the aspartate aminotransferase/malate dehydrogenase system.

A novel method is presented that establishes definitively the existence or nonexistence of direct metabolite transfer between consecutive enzymes in a metabolic sequence. The procedure is developed with the specific example of channeling of oxaloacetate between Escherichia coli aspartate aminotransferase (AATase) and malate dehydrogenase (MDH). The assay is carried out in the presence of a large excess of inactive variants of AATase. These mutants would outcompete the much smaller quantities of wild-type AATase for any docking sites on MDH and thus decrease the rate of the coupled L-aspartate to oxaloacetate to malate sequence only if the direct metabolite transfer mechanism is operative. The results show that oxaloacetate is not transferred directly from AATase to MDH because no decrease in rate was observed in the presence of approximately 100 microM inactive mutants. This concentration is 10 times the physiological AATase concentration, which was determined in this work. The methodology can be applied generally.

[Mitoxantrone-induced acute left heart failure after intrapleural administration]. / Mitoxantron-induzierte akute Linksherzinsuffizienz nach intrapleuraler Applikation.

A 79-year-old female was admitted to our hospital because of a malignant pleural effusion following mastectomy 4 years ago. In the patient's history arterial hypertension and previous inferior myocardial infarction have been known. Two doses of 20 mg mitoxantrone were installed intrapleurally at an interval of 4 weeks. Six hours after the second mitoxantrone application and the patient had increasing dyspnea with consecutive left heart failure, pulmonary congestion, and a drop of blood pressure. The white-cell count was 14800/mm3. The levels of creatinine phosphokinase (CPK), lactate dehydrogenase (LDH) and serum aspartate aminotransferase (SGOT) were in the normal range. Transthoracic echocardiography showed concentric left ventricular hypertrophy and a markedly decreased fractional shortening, but no left ventricular dilatation. The electrocardiogram showed newly appeared down-sloping ST-segments and inverted T-waves. Clinical recovery was achieved after 6 days by application of oxygen, dobutamine and furosemide followed by angiotensin converting enzyme inhibition and digitalis. In the echocardiographic control examination 14 days later left ventricular function had normalized. The changes of electrocardiogram normalized 4 weeks later.

Identification of residues of spinach thioredoxin f that influence interactions with target enzymes.

The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood. To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes. Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77. Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared. All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax. In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins. The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f. Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity.

Direct identification of the primary nucleophile of thioredoxin f.

Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.

Cumulative effect of double-site mutations of human epidermal growth factor on receptor binding.

Using site-directed mutagenesis, it was previously found that mutation of the individual residues Tyr13, Tyr22, Ile23, or Leu26 in the amino-terminal domain or of the highly conserved Leu47 in the carboxyl-terminal domain of human epidermal growth factor (hEGF), resulted in significantly decreased receptor binding affinity. In the present study, the single-site hEGF mutants Tyr13-->His, Tyr22-->Asp, Ile23-->Thr, and Leu26-->Gly were genetically combined with the Leu47-->Ala hEGF mutant to produce a series of double-site mutant hEGF gene products having alterations simultaneously at two sites, in separate domains, within the same hEGF molecule. Similarly, the combination of the single-site hEGF mutants Tyr13-->His and Ile23-->Thr generated a double-site mutant having two mutations within the same domain. Finally, combination of the hEGF mutation Ile23-->Ala with Leu23-->Ala altered two side chains located in close proximity within the large beta-sheet region of the molecule. Analysis of the relative receptor binding affinities, determined by radioreceptor competition assays of the various single- and double-site hEGF mutants, demonstrated that mutation at any one site does not substantially alter the effect of mutation at the second site in the molecule. The cumulative effect of simultaneous mutations on relative receptor binding affinity confirms the importance of residues, including those in the large amino-terminal beta-sheet, in receptor binding, and indicates that each of the separate sites functions essentially independently in the interaction of the hEGF molecule with its receptor.

[Relevance of ocular pressure tonometry and classical tonometry with reference to prognosis in suspected glaucoma and ocular hypertension]. / Relevanz von Okulopressionstonometrie und klassischer Tonometrie hinsichtlich der Prognose bei Glaukomverdacht und okulärer Hypertension.

A total of 44 eyes of patients with suspected glaucoma or ocular hypertension were examined both by ocular pressure tonometry (OPT) according to Ulrich and by tonography according to Leydhecker. The visual fields of these eyes were prospectively followed up for 3 years to compare the prognostic value of the two methods. In 30 of the 44 eyes a worsening visual field loss was observed, while in 14 the visual fields showed no change. Much better correlation was noted between values obtained by OPT and changes in visual field than between values obtained by tonography and visual field loss. Conventional tonography yielded 39% of false-negative values, but only 11% were recorded with OPT. Therefore, according to results of these and further investigations, OPT appears to be a more helpful and reliable method for assessment and decisions on therapy in patients with suspected glaucoma or ocular hypertension.