Indirect observation by 13C NMR spectroscopy of a novel CO2 fixation pathway in methanogens.

High-field carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the isotopic dilution of doubly carbon-13-labeled precursors for 2,3-cyclopyrophosphoglycerate, a novel primary metabolite that occurs in certain methanogens. A unique carbon dioxide fixation pathway that gives rise to asymmetric labeling of acetyl coenzyme A has been demonstrated in Methanobacterium thermoautotrophicum. The effect of selected metabolic inhibitors on the labeled species in the pathway has been examined by NMR. These techniques establish a general, sensitive method for the delineation of convergent biosynthetic pathways.

A tyrosine residue is involved in the allosteric binding of tryptophan to yeast chorismate mutase.

Comparative 1H-NMR studies have been carried out on wild-type chorismate mutase, which is activated by tryptophan and inhibited by tyrosine and phenylalanine, and mutant yeast chorismate mutase, which has a single point mutation (T226I) and is not allosterically regulated but 'locked' in the activated state. Double quantum-filtered COSY spectra show cross-peaks which have been assigned to a tyrosine that are absent in the mutant enzyme and in the wild-type enzyme plus tryptophan when compared with the wild-type enzyme alone. These observations indicate the involvement of a tyrosine at or near the allosteric binding site. The involvement of tyrosine in tryptophan binding was tested by modification of tyrosine in yeast chorismate mutase by nitration with tetranitromethane. All forms of the enzyme exhibited an approx. 50% reduction in specific activity, but it was found that preincubation of the wild-type with the allosteric activator, tryptophan, lead to partial protection against loss in specific activity. Only one tyrosine residue was nitrated in the wild-type enzyme and this tyrosine was identified by tryptic digestion and sequencing, and found to be very close to the site of the single point mutation in the mutant enzyme. It is proposed that Tyr-234 is located at or near the allosteric activation site.

Multinuclear NMR studies of the trp-repressor.

The binding of the corepressor, L-tryptophan, to the Escherichia coli trp-aporepressor in solution has been examined by 13C- and 19F-NMR spectroscopy. The binding of a number of tryptophan analogues have been studied by equilibrium dialysis. Evidence is presented that support the crystallographic studies (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L. and Sigler, P. B. (1985) Nature 317, 782-786) that Val-58 is within the ring currents of the bound tryptophan and also close in space to the indole 5'-position, on the basis of heteronuclear 19F(1H)-NOE experiments. The tryptophan carboxylate is in hydrogen-bonding distance to a highly positively charged residue, probably Arg-54 and this bond strengthens on formation of the trp-repressor-DNA complex.

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme-ligand complexes in solution, we have isolated the 26-kDa N-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate-3-phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N-terminal domain upon S3P binding.

Time-resolved solid-state REDOR NMR studies of UDP N-acetylglucosamine enolpyruvyl transferase.

The new method of time-resolved solid-state rotational echo double resonance (REDOR) NMR spectroscopy introduced recently by this laboratory has been applied to the enzyme uridine N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase (EPT), with the goal of probing the interactions between reactive species and their enzyme active site. The approach has been used in a qualitative fashion with the enzyme-inhibitor and enzyme-intermediate complexes of uniformly 15N-labeled UDP-NAG EPT, trapped under steady-state and pre-steady-state conditions. A different set of intermolecular interactions between the substrates UDP-NAG, UDP-NAG plus 3-Z-fluorophosphoenolpyruvate, covalent O-phosphothioketal, and UDP-NAG plus phosphoenolpyruvate trapped under time-resolved conditions (after 50 ms reaction time), and the EPT enzyme active site were observed, and this is contrasted to a similar study of the interactions in a related enzyme, 5-enolpyruvyl-shikimate-3-phosphate synthase.

Hemodynamic and biochemical characteristics of the aorta in the WKY, SHR, WKHT, and WKHA rat strains.

This study was designed to characterize the hemodynamic and biochemical properties of the abdominal aorta in four genetically related inbred rat strains that express genetic hypertension and hyperactive behavior in varying combinations. These include (1) the spontaneously hypertensive rat (SHR), which is hypertensive, hyperactive, and hyperreactive to stress; (2) Wistar-Kyoto (WKY) rats, which express none of these traits; (3) WKHT rats, which are hypertensive but not hyperactive; and (4) WKHA rats, which are hyperactive and hyperreactive to stress, but normotensive. Together, these four strains allowed us to examine the structural and functional changes in the aorta in the hypertensive SHR, the most widely used animal model of genetic hypertension, while controlling for the variables of hyperactivity and hyperreactivity that are also expressed in the SHR. Four groups of animals of both sexes were studied: (1) WKY, n = 101, (2) WKHA, n = 33, (3) WKHT, n = 91, and (4) SHR, n = 28. Blood pressure (BP) was determined by tail plethysmography as well as direct intraarterial monitoring under anesthesia. Fixed specimens were prepared for histologic analysis and the wall thickness determined morphometrically. Quantification of soluble tissue protein, elastin, and collagen in the aortic tissue was determined by measuring leucine (leu), hydroxyproline (HP/leu), and desmosine (DES/leu). The hypertensive strains (SHR and WKHT) had significantly higher tail BP than the normotensive strains (WKY and WKHA)-WKY: 128.7 +/- 22.3; WKHA: 126.7 +/- 14.6; WKHT: 162.8 +/- 21.2; SHR: 164.2 +/- 36.1 (p < 0.0001). Additionally, intraaortic diastolic BP and mean BP were higher in SHR rats than in WKHT. Morphometric studies showed the media thickness in the SHR rats was significantly greater than in the WKY and WKHA rats and no different than in the WKHT rats. Significantly less of the aortic wall protein was present as elastin in the hypertensive rats (SHR and WKHT), as well as the hyperactive rats (WKHA), compared to rats that had neither trait (WKY). These studies provide new information regarding aortic structure and function in genetic hypertension using inbred strains to control for the hyperactivity/hyperreactivity traits that coexist with hypertension in the SHR. They reveal that hypertensive aortas have altered matrix proteins that cannot be explained simply on the basis of blood pressure alone.

Altered artery mechanics and structure in monocrotaline pulmonary hypertension.

Pulmonary hypertension in rats, induced by an injection of monocrotaline, is associated with changes in the wall structure of the pulmonary arterial bed. We have studied the effects of this remodeling on mechanical properties of cylindrical pulmonary artery segments from rats 21 days after monocrotaline (MCT) injection. Resting and active (KCl induced) circumference-tension relationships were established for segments of extrapulmonary and intrapulmonary arteries isolated from the hilum and the fifth lateral branch from the axial pathway (all preacinar). The thicknesses of the vessel wall, the media, and adventitia were measured at several positions around the circumference of the artery by computerized analysis of histological cross sections of the segments fixed at a standard circumference. Resting and active stress were also calculated. The study shows that active circumferential tension and active stress are reduced in vessels from MCT-treated rats. Based on our findings, it is unlikely that altered contractile function of preacinar arteries contributes significantly to the increased vascular resistance seen in this model.

Comparative studies of phase-cycling schemes for multiple pi-pulse sequences.

Recently, a new phase cycling scheme was introduced by this laboratory for use in biological solid-state NMR experiments involving multiple pi-pulses with characteristics that suggested it may enhance the sensitivity of these kind of experiments (Y. Li and J. N. S. Evans, 1995, Chem. Phys. Lett. 241, 79 and Erratum, 1995, ibid. 246, 527; Y. Li and J. N. S. Evans, 1996, J. Magn. Reson. B 111, 296). The new sequence followed the supercycled concept proposed a decade ago for heteronuclear decoupling experiments. In this paper, more detailed experiments demonstrate that the claim of enhanced sensitivity was unfounded, and in fact the supercycle proposed differs little from the established XY-8 and XY-16 based supercycles.

Reactivity of rabbit basilar artery to alterations in extracellular potassium and calcium after subarachnoid hemorrhage.

Hemolysis of periarterial clots after subarachnoid hemorrhage may liberate large quantities of K+ into the vicinity of cerebral blood vessels and possibly change their sensitivity to endogenous vasoactive agents. The current study examined the influence of subarachnoid hemorrhage on the sensitivity of rabbit basilar arterial segments to K+ and Ca++. An analysis of K+ and Ca++ dose-response curves demonstrated that incubated arterial segments isolated from animals with subarachnoid hemorrhage were substantially more sensitive to these cations than were corresponding controls. We speculate that chronically elevated K+ levels in areas of periarterial clot lysis or brain ischemia might initiate vascular smooth muscle depolarization and vasospasm. Our data provide additional rationale for the use of calcium channel blockers in preventing or treating vasospasm in cases of subarachnoid hemorrhage.

Head and neck manifestations of histiocytosis-X.

Histiocytosis-X is an uncommon disease, but is relevant to the otolaryngologist because patients with this malady present with, or ultimately develop, head and neck manifestations. Sixty-two patients with histologically confirmed histiocytosis-X have been examined at the Hospital for Sick Children in London since 1958. Most patients were under 2 years of age on initial examination and had either osseous or dermatologic disease involvement. Less commonly, otic or oral lesions were found. Histiocytosis-X is a systemic disease and must be treated accordingly. Isolated lesions may mimic common disease processes, therefore it is imperative that if a seemingly simple problem does not resolve with routine therapy, further patient evaluation be undertaken, If diagnosed, histiocytosis-X can generally be controlled with steroid therapy, or in more recalcitrant cases, by the addition of chemotherapy.

Endoscopic repair of supraglottic laryngeal clefts.

We describe the technique of endoscopic diagnosis and endoscopic surgical repair used in the management of supraglottic interarytenoid laryngeal clefts in 11 children seen between 1981 and 1988 at the Hospital for Sick Children, London, England. Six of the children had primary type I clefts that required endoscopic repair. The symptoms included inspiratory stridor, choking during eating, and aspiration. Five of the children had previous transcervical repair of type II clefts that had partial breakdown in the interarytenoid area causing symptoms of aspiration, which required secondary repair endoscopically. All the patients had successful microlaryngoscopic closure; in two children, however, the breakdown of the repair necessitated repeated endoscopic correction. The only complication occurred in a case of postoperative supraglottitis, which was successfully managed with intubation and antibiotics. We conclude that endoscopic repair is a useful and reliable technique and an elegant alternative to the open transcervical approach for the closure of supraglottic laryngeal clefts.

Pediatric vocal fold paralysis: a long-term retrospective study.

OBJECTIVE: To review our experience of pediatric vocal fold paralysis (VFP), with particular emphasis on etiological factors, associated airway pathologic conditions, and treatment and prognostic outcomes. DESIGN: Retrospective case review of a cohort of patients presenting with VFP. SETTING: Tertiary referral center. PATIENTS: A consecutive sample of 102 patients presenting with VFP to Great Ormond Street Hospital for Children, London, England, over a 14-year period from 1980 to 1994. RESULTS: There was an almost equal distribution of unilateral (52% [n = 53]) and bilateral (48% [n = 49]) VFP. Iatrogenic causes (43% [n = 44]) formed the largest group, followed by idiopathic VFP (35% [n = 36]), neurological causes (16% [n = 16]), and finally birth trauma (5% [n = 5]). Associated upper airway pathologic conditions were noted in 66% (n = 23) of patients who underwent tracheotomy. Tracheotomy was necessary in only 57% (n = 28) of children with bilateral VFP. Prognosis was variable depending upon the cause, with neurological VFP having the highest rate of recovery (71% [5/7]) and iatrogenic VFP the lowest rate (46% [12/26]). CONCLUSION: Recovery after an interval of up to 11 years was seen in idiopathic bilateral VFP; this has significant implications when considering lateralization procedures in these patients.

Management of posterior laryngeal and laryngotracheoesophageal clefts.

OBJECTIVE: To review the clinical features, associated congenital abnormalities, management, and morbidity of infants presenting with posterior laryngeal and laryngotracheal clefts. DESIGN: Case series. SETTING: Great Ormond Street Hospital for Sick Children NHS Trust, London, England. PATIENTS: Consecutive sample of 44 patients presenting with posterior laryngeal and laryngotracheal clefts between December 10, 1979, and January 30, 1992. MAIN OUTCOME MEASURES: Clinical features, incidence of surgery, and associated morbidity and mortality related to different types of airway cleft. RESULTS: The main presenting features were stridor and aspiration, which were more evident with the more extensive clefts. Twenty-five patients (56%) had associated congenital abnormalities. Fourteen patients (32%) were treated conservatively. Sixteen patients (36%) underwent primary endoscopic surgical repair. Eight patients (18%) underwent primary repair via an anterior laryngofissure; and six patients (14%) underwent primary repair via a lateral pharyngotomy. Eight patients (18%) required revision surgery, two (4%) of them on more than one occasion. Ten patients (23%) required fundoplication to control gastroesophageal reflux. Six patients (14%) died. CONCLUSIONS: The identification of an airway cleft requires a high index of suspicion. Morbidity and mortality are reduced by securing the airway, controlling gastroesophageal reflux, and using a multidisciplinary pediatric team. We recommend the anterior laryngofissure because of the ease of surgical access.

Tracheal reconstruction in children using cadaveric homograft trachea.

OBJECTIVE: We report the use of cadaveric human tracheal homograft in the treatment of severe long segment congenital tracheal stenosis in children. METHODS: Five children (aged 5 months-8 years) with severe life-threatening airway obstruction due to long segment congenital tracheal stenosis had failed conventional management. All were ventilator dependent or rapidly deteriorating at the time of surgery, two were on extracorporeal membrane oxygenation, and no alternative therapy was available. The stenosed trachea was removed and the posterior trachealis muscle left in situ when possible. Surgical technique involved the use of cardiopulmonary bypass in four of five cases. Stored cadaveric tracheal homograft tissue was shaped and inserted over a silastic intra-luminal stent which was kept in place for up to 3 months. Regular bronchoscopy was necessary postoperatively to clear granulation tissue, which resolved on removal of the stent. RESULTS: Four patients are all now without stents, intubation or tracheostomy. Three are without airway problems 16, 14, and 9 months after surgery and one attends for occasional dilatation of a distal tracheal stenosis, but is now at home despite other severe multiple congenital problems. One patient presented with complete disruption of the trachea and mediastinal sepsis and was supported on extracorporeal membrane oxygenation prior to surgery; this patient eventually died of airway failure and sepsis. CONCLUSIONS: The application of cadaveric human tracheal homograft to congenital tracheal stenosis is novel. Its use in five children who would otherwise have died has provided an extra therapy in an extremely difficult group of patients.

Early markers of lung injury.

The goal of this study was to develop an early marker of lung injury that might change in response to exposure to a mobile source emission. Nitrogen dioxide (NO2)2 was chosen as an example of an atmospheric pollutant that is related to automobile emissions. Since reorganization of the connective tissue matrix of the lung occurs in response to injury, markers of connective tissue metabolism were selected as targets. Hydroxylysine became the marker of choice. It is an amino acid that is virtually exclusive to collagen, although it does occur in minimal amounts in other proteins. Furthermore, it is excreted in the urine, which makes it readily available for analysis using noninvasive techniques. Other markers evaluated as part of the study included angiotensin-converting enzyme as a marker of lung injury, desmosine as a marker of elastin degradation, and hydroxyproline as another marker of collagen metabolism. Male Fischer-344 rats were exposed in whole-body chambers to controlled concentrations of NO2 for various doses and periods of time. The concentrations of NO2 ranged from 0.5 to 30 parts per million (ppm); the rats were exposed for six hours per day for periods of two days to four weeks. Urine and bronchoalveolar lavage samples were collected and analyzed for the appropriate marker. In addition, pulmonary function studies and histologic examinations of the lungs were completed at selected time points. Urinary hydroxylysine concentration increased as a function of NO2 concentration during six-hour-per-day exposures for two days. This short-term exposure required relatively high doses to achieve significant changes in the hydroxylysine output. During one-week exposures to either 25 or 30 ppm NO2, there was an increase in urinary hydroxylysine associated with changes in lavage concentrations of angiotensin-converting enzyme and hydroxylysine. The lungs of these animals demonstrated histologic changes typical of oxidant injury. Four-week exposure protocols using 0.5 and 1 ppm NO2 were most interesting in terms of the sensitivity of the marker. There was minimal damage revealed by the histology and function studies, yet there were significant increases in the excretion of hydroxylysine. It appears that hydroxylysine can be indicative of exposure when other parameters are normal. It will require long-term follow-up of exposed rats to determine whether or not the change in marker concentration is predictive of damage. Hydroxylysine may be an excellent marker of exposure to oxidants in the human population. Controlled studies to establish base-line values are needed, followed by carefully controlled studies in individuals with connective tissue abnormalities of the lung.

Management of the cleft larynx and tracheoesophageal clefts.

The anomaly of posterior laryngeal cleft and the more extensive laryngotracheoesophageal cleft is extremely rare. Twenty-one cases of cleft larynx are reviewed. A new, simpler clinically oriented classification of the clefts is proposed, and the difficulty in establishing the precise diagnosis is stressed. Stridor is the commonest presenting symptom in type 1 clefts, and aspiration on feeding, recurrent pneumonia, and abnormalities of cry suggest type 2 clefts. Type 3 clefts present with severe aspiration cyanosis and incipient cardiorespiratory failure. Aspiration reflux and persistent stridor are extremely common postoperative problems and are responsible for the delays in decannulation in spite of apparently successful operative closure of the clefts.

Laryngotracheal reconstruction in children. Five-year follow-up.

Congenital and acquired subglottic stenosis is a commonly encountered problem in the pediatric population. In acquired cases endotracheal intubation is responsible for its development in the great majority of cases, but high tracheotomy, laryngeal burns, external neck trauma, and tumors, both intrinsic and extrinsic, are occasionally seen. The management of mature subglottic stenosis in children remains a controversial issue. The prevailing attitude of otolaryngologists is to perform a tracheotomy and hope for decannulation after one or two years, due to the expected growth of the larynx. Unfortunately, some of the acquired lesions are so severe that often no lumen is demonstrable. In such cases no amount of growth will allow extubation. A variety of endoscopic methods, such as dilation with or without resection using diathermy or laser, are certainly helpful in the early phases of wound healing while the scar tissue is soft and pliable. To deal with the mature, hard, fibrous, unresponsive scar various authors have proposed differing laryngotracheal reconstructive techniques. The authors discuss a unique experience of laryngotracheal reconstruction in 103 children. They define their indications for the three procedures that are most widely used, and address the issue raised by opponents of laryngotracheal reconstruction in children, namely the consideration that laryngeal growth potential may be adversely affected by such external operations. The authors have evidence that this has not occurred in 35 cases followed for a minimum of five years.

Pediatric airway reconstruction at Great Ormond Street: a ten-year review. III. Decannulation and suprastomal collapse.

One hundred eight consecutive patients with pediatric laryngotracheal stenosis requiring airway reconstruction over a 10-year period were reviewed. Ninety (83%) of the patients were decannulated. Over three quarters of the decannulations took place within 20 months of primary reconstruction. More than half of the patients (47, or 52%) had persistent tracheocutaneous fistulae after decannulation, which required elective closure. The likelihood of a persisting tracheocutaneous fistula is directly related to duration of cannulation.

Pediatric airway reconstruction at Great Ormond Street: a ten-year review. II. Revisional airway reconstruction.

One hundred eight consecutive patients with pediatric laryngotracheal stenosis requiring airway reconstruction over a 10-year period were reviewed. Thirty-two patients required revisional airway reconstruction in an attempt to achieve decannulation. Patients underwent from one to four revisional airway reconstructions, most often laryngotracheal reconstruction with costal cartilage grafting. In the Cotton grading scheme of preoperative stenosis, those patients requiring revisional airway surgery tended to come from the more severely affected categories. Twenty-two patients of 32 (69%) achieved decannulation with revisional airway reconstruction. Thus, revisional airway reconstruction is indicated if the first attempt fails.

Pediatric airway reconstruction at Great Ormond Street: a ten-year review. I. Laryngotracheoplasty and laryngotracheal reconstruction.

One hundred eight consecutive patients with pediatric laryngotracheal stenosis requiring airway reconstruction over a 10-year period were reviewed. One hundred forty-nine operations consisting of 75 laryngotracheoplasties and 74 laryngotracheal reconstructions with costal cartilage grafting were performed. The Cotton grading scheme of preoperative stenosis was useful in predicting likelihood of decannulation. In all, 90 patients (83%) were decannulated.