BrAIST-Calc: Prediction of Individualized Benefit From Bracing for Adolescent Idiopathic Scoliosis.

STUDY DESIGN: Prospective multicenter study data were used for model derivation and externally validated using retrospective cohort data. OBJECTIVE: Derive and validate a prognostic model of benefit from bracing for adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: The Bracing in Adolescent Idiopathic Scoliosis Trial (BrAIST) demonstrated the superiority of bracing over observation to prevent curve progression to the surgical threshold; 42% of untreated subjects had a good outcome, and 28% progressed to the surgical threshold despite bracing, likely due to poor adherence. To avoid over-treatment and to promote patient goal setting and adherence, bracing decisions (who and how much) should be based on physician and patient discussions informed by individual-level data from high-quality predictive models. MATERIALS AND METHODS: Logistic regression was used to predict curve progression to <45° at skeletal maturity (good prognosis) in 269 BrAIST subjects who were observed or braced. Predictors included age, sex, body mass index, Risser stage, Cobb angle, curve pattern, and treatment characteristics (hours of brace wear and in-brace correction). Internal and external validity were evaluated using jackknifed samples of the BrAIST data set and an independent cohort (n=299) through estimates of discrimination and calibration. RESULTS: The final model included age, sex, body mass index, Risser stage, Cobb angle, and hours of brace wear per day. The model demonstrated strong discrimination ( c -statistics 0.83-0.87) and calibration in all data sets. Classifying patients as low risk (high probability of a good prognosis) at the probability cut point of 70% resulted in a specificity of 92% and a positive predictive value of 89%. CONCLUSION: This externally validated model can be used by clinicians and families to make informed, individualized decisions about when and how much to brace to avoid progression to surgery. If widely adopted, this model could decrease overbracing of AIS, improve adherence, and, most importantly, decrease the likelihood of spinal fusion in this population.

Surgeon and SterEOS agreement of spinal sagittal plane parameters in patients with adolescent idiopathic scoliosis.

PURPOSE: To compare agreement between surgeons and sterEOS sagittal plane measurements. METHODS: EOS radiographs of 74 patients with adolescent idiopathic scoliosis were reviewed. The measurements were generated by two surgeons and compared to sterEOS. Intraclass correlations (ICC) were calculated. Agreement was also analyzed for the following subgroups: Cobb angle < 70° vs ≥ 70°, lumbar modifier A vs B/C, and BMI of < 24.5 kg/m2 vs ≥ 24.5 kg/m2. Agreement was poor if the ICC was < 0.5, moderate if 0.5-0.75, good if 0.75-0.9, and excellent if > 0.9. Paired t tests were performed to compare the surgeon's and sterEOS means. RESULTS: For the surgeons, agreement was good (0.75-0.89) except for pelvic tilt (PT) and sacral slope (SS), which were excellent (0.91-0.92). Agreement between the surgeons and sterEOS were good (0.78-0.9) except PT and SS, which were excellent (0.91-0.93). Agreement was negatively affected for T4-T12 kyphosis, PI, and SS in the ≥ 70°group, LL when BMI was ≥ 24.5 kg/m2, and LL, PI, and SS in the lumbar modifier B/C group. The ICCs overlapped with the 95% confidence intervals (95% CI). Paired t-test showed a significant difference for T4-T12 kyphosis (p < 0.001). This was also true in the < 70° group (p < 0.001), the ≥ 70° group (p = 0.04), and the BMI < 24.5 kg/m2 group. PT was significantly different for the ≥ 70° group. CONCLUSIONS: There was good to excellent agreement between the surgeons and surgeons and sterEOS. Some variables may affect agreement. The surgeons overestimated T4-T12 kyphosis.

The classification of scoliosis braces developed by SOSORT with SRS, ISPO, and POSNA and approved by ESPRM.

PURPOSE: Studies have shown that bracing is an effective treatment for patients with idiopathic scoliosis. According to the current classification, almost all braces fall in the thoracolumbosacral orthosis (TLSO) category. Consequently, the generalization of scientific results is either impossible or misleading. This study aims to produce a classification of the brace types. METHODS: Four scientific societies (SOSORT, SRS, ISPO, and POSNA) invited all their members to be part of the study. Six level 1 experts developed the initial classifications. At a consensus meeting with 26 other experts and societies' officials, thematic analysis and general discussion allowed to define the classification (minimum 80% agreement). The classification was applied to the braces published in the literature and officially approved by the 4 scientific societies and by ESPRM. RESULTS: The classification is based on the following classificatory items: anatomy (CTLSO, TLSO, LSO), rigidity (very rigid, rigid, elastic), primary corrective plane (frontal, sagittal, transverse, frontal & sagittal, frontal & transverse, sagittal & transverse, three-dimensional), construction-valves (monocot, bivalve, multisegmented), construction-closure (dorsal, lateral, ventral), and primary action (bending, detorsion, elongation, movement, push-up, three points). The experts developed a definition for each item and were able to classify the 15 published braces into nine groups. CONCLUSION: The classification is based on the best current expertise (the lowest level of evidence). Experts recognize that this is the first edition and will change with future understanding and research. The broad application of this classification could have value for brace research, education, clinical practice, and growth in this field.

Iliac screw instrumentation to the pelvis in children with neuromuscular and syndromic scoliosis. No lateral connectors and respect sagittal balance.

STUDY DESIGN: One-center retrospective cohort study. BACKGROUND: Compared to the traditional iliac screw technique, the modified iliac screw technique has a lower rate of distal implant failure in the treatment of neuromuscular scoliosis patients with pelvic obliquity. However, the reasons for decreased failure with the modified iliac screw technique are controversial. QUESTIONS/PURPOSES: (1) Is distal implant failure, as evident by implant breakage or disconnection, more likely to occur in patients receiving the traditional iliac screw technique (PSIS) compared to the modified S2AI (MODS2) technique? (2) After controlling for relevant confounding variables, are there other identifiable risk factors for distal implant failure? METHODS: We identified patients who underwent pelvic screw fixation by three pediatric spine surgeons from January 2007 to July 2017. Based on the starting point of the iliac screws, patients were divided into two groups. Group 1 consisted of PSIS fixation with an offset connector. Group 2 consisted of modified S2AI fixation without an offset connector. Demographic, operative, and radiographic data were obtained. RESULTS: Cobb angle, lumbar lordosis, and pelvic obliquity were not significantly different between the two groups. Overall distal implant failure was 40/100 (40%) and significant between Group 1 PSIS 29/53 (55%) and Group 2 MODS2 11/47 (23%) (p = 0.002). No other complications were significant. Three risk factors were identified with implant failure: high pelvic incidence (17-fold increase, 95% confidence interval [CI] = 5.5 to 53.1, p < 0.001), high angle rod contour (3.8-fold increase, 95% CI = 1.2 to 11.9, p = 0.023), and use of an offset connector (3.2-fold increase, 95% CI = 1.0 to 10.3, p = 0.049). Failure did not correlate with the use of a cross-link, iliac screw diameter, or screw density. Revision surgery related to distal implant failure did not significantly differ between the two groups. CONCLUSIONS: Compared to the use of an offset connector with PSIS fixation, MODS2 fixation had a lower rate of implant failure. Sagittal balance parameters, namely pelvic incidence and angle of rod bend, were the major risk factors for implant failure. LEVEL OF EVIDENCE: III.

Low-Dose Versus High-Dose Postoperative Naloxone Infusion Combined With Patient-Controlled Analgesia for Adolescent Idiopathic Scoliosis Surgery: A Randomized Controlled Trial.

STUDY DESIGN: Randomized controlled trial. OBJECTIVES: The aim of this prospective randomized clinical trial was to compare low (0.5 µg/kg/h) and high (2.5 µg/kg/h) dose naloxone infusion on the time to tolerate liquids and meals after surgery, patient-controlled analgesia (PCA) opioid requirements, nausea and pruritus ratings, and hospital length of stay. SUMMARY OF BACKGROUND DATA: Adolescents undergoing posterior spinal fusion often receive PCA after surgery and may experience common opioid-associated side effects, including nausea and pruritus. Low-dose naloxone infusion has been shown to reduce the incidence of pruritus and nausea while preserving analgesia, although an ideal dose has not been determined. Less is known about the potential for naloxone to improve bowel function after surgery. METHODS: Eighty-four patients (age 10-21 years) were randomly allocated to receive low- or high-dose naloxone infusion postoperatively. Surgical anesthetic consisted of propofol and opioid infusion with intrathecal morphine (10-15 µg/kg) at the conclusion of surgery. A visual analog scale (VAS) was used to rate nausea and pruritus. RESULTS: The groups had similar time to oral liquid intake after surgery and transition from PCA to oral pain medication. The VAS scores for pruritus and nausea were also similar, as was the need to treat these side effects. Morphine equivalents were similar between groups on postoperative day (POD) 0 and 1. On POD 2, the high-dose infusion group had significantly greater PCA bolus use (1.41±0.9 vs. 1.04±0.6; p<.05), although pain scores did not differ significantly. Hospital length of stay was similar for the two groups. CONCLUSION: High-dose naloxone infusion was associated with similar rates of opioid side effects as low-dose. Increased PCA use noted on POD 2 may represent partial reversal of opioid analgesia in the high-dose naloxone group. LEVEL OF EVIDENCE: Level 1.

2016 SOSORT guidelines: orthopaedic and rehabilitation treatment of idiopathic scoliosis during growth.

BACKGROUND: The International Scientific Society on Scoliosis Orthopaedic and Rehabilitation Treatment (SOSORT) produced its first guidelines in 2005 and renewed them in 2011. Recently published high-quality clinical trials on the effect of conservative treatment approaches (braces and exercises) for idiopathic scoliosis prompted us to update the last guidelines' version. The objective was to align the guidelines with the new scientific evidence to assure faster knowledge transfer into clinical practice of conservative treatment for idiopathic scoliosis (CTIS). METHODS: Physicians, researchers and allied health practitioners working in the area of CTIS were involved in the development of the 2016 guidelines. Multiple literature reviews reviewing the evidence on CTIS (assessment, bracing, physiotherapy, physiotherapeutic scoliosis-specific exercises (PSSE) and other CTIS) were conducted. Documents, recommendations and practical approach flow charts were developed using a Delphi procedure. The process was completed with the Consensus Session held during the first combined SOSORT/IRSSD Meeting held in Banff, Canada, in May 2016. RESULTS: The contents of the new 2016 guidelines include the following: background on idiopathic scoliosis, description of CTIS approaches for various populations with flow-charts for clinical practice, as well as literature reviews and recommendations on assessment, bracing, PSSE and other CTIS. The present guidelines include a total of 68 recommendations divided into following topics: bracing (n = 25), PSSE to prevent scoliosis progression during growth (n = 12), PSSE during brace treatment and surgical therapy (n = 6), other conservative treatments (n = 2), respiratory function and exercises (n = 3), general sport activities (n = 6); and assessment (n = 14). According to the agreed strength and level of evidence rating scale, there were 2 recommendations on bracing and 1 recommendation on PSSE that reached level of recommendation "I" and level of evidence "II". Three recommendations reached strength of recommendation A based on the level of evidence I (2 for bracing and one for assessment); 39 recommendations reached strength of recommendation B (20 for bracing, 13 for PSSE, and 6 for assessment).The number of paper for each level of evidence for each treatment is shown in Table 8. CONCLUSION: The 2016 SOSORT guidelines were developed based on the current evidence on CTIS. Over the last 5 years, high-quality evidence has started to emerge, particularly in the areas of efficacy of bracing (one large multicentre trial) and PSSE (three single-centre randomized controlled trials). Several grade A recommendations were presented. Despite the growing high-quality evidence, the heterogeneity of the study protocols limits generalizability of the recommendations. There is a need for standardization of research methods of conservative treatment effectiveness, as recognized by SOSORT and the Scoliosis Research Society (SRS) non-operative management Committee.

The effect of scoliotic deformity on spine kinematics in adolescents.

BACKGROUND: While adolescent idiopathic scoliosis (AIS) produces well characterized deformation in spinal form, the effect on spinal function, namely mobility, is not well known. Better understanding of scoliotic spinal mobility could yield better treatment targets and diagnoses. The purpose of this study was to characterize the spinal mobility differences due to AIS. It was hypothesized that the AIS group would exhibit reduced mobility compared to the typical adolescent (TA) group. METHODS: Eleven adolescents with right thoracic AIS, apices T6-T10, and eleven age- and gender-matched TAs moved to their maximum bent position in sagittal and coronal plane bending tasks. A Trakstar (Ascension Technologies Burlington, VT) was used to collect position data. The study was approved by the local IRB. Using MATLAB (MathWorks, Natick, MA) normalized segmental angles were calculated for upper thoracic (UT) from T1-T3, mid thoracic (MT) from T3-T6, lower thoracic (LT) from T6-T10, thoracolumbar (TL) from T10-L1, upper lumbar (UL) from L1-L3, and thoracic from T1-L1 by subtracting the standing position from the maximum bent position and dividing by number of motion units in each segment. Mann Whitney tests (α = 0.05) were used to determine mobility differences. RESULTS: The findings indicated that the AIS group had comparatively increased mobility in the periapical regions of the spine. The AIS group had an increase of 1.2° in the mid thoracic region (p = 0.01) during flexion, an increase of 1.0° in the mid thoracic region (p = 0.01), 1.5° in the thoracolumbar region (p = 0.02), and 0.7° in thoracic region (p = 0.04) during left anterior-lateral flexion, an increase of 6.0° in the upper lumbar region (p = 0.02) during right anterior-lateral flexion, and an increase of 2.2° in the upper lumbar region during left lateral bending (p < 0.01). CONCLUSIONS: Participants with AIS did not have reduced mobility in sagittal or coronal motion. Contrarily, the AIS group often had a greater mobility, especially in segments directly above and below the apex. This indicates the scoliotic spine is flexible and may compensate near the apex.

Recommendations for research studies on treatment of idiopathic scoliosis: Consensus 2014 between SOSORT and SRS non-operative management committee.

The two main societies clinically dealing with idiopathic scoliosis are the Scoliosis Research Society (SRS), founded in 1966, and the international Society on Scoliosis Orthopedic and Rehabilitation Treatment (SOSORT), started in 2004. Inside the SRS, the Non-Operative Management Committee (SRS-NOC) has the same clinical interest of SOSORT, that is the Orthopaedic and Rehabilitation (or Non-Operative, or conservative) Management of idiopathic scoliosis patients. The aim of this paper is to present the results of a Consensus among the best experts of non-operative treatment of Idiopathic Scoliosis, as represented by SOSORT and SRS, on the recommendation for research studies on treatment of Idiopathic Scoliosis. The goal of the consensus statement is to establish a framework for research with clearly delineated inclusion criteria, methodologies, and outcome measures so that future meta- analysis or comparative studies could occur. A Delphi method was used to generate a consensus to develop a set of recommendations for clinical studies on treatment of Idiopathic Scoliosis. It included the development of a reference scheme, which was judged during two Delphi Rounds; after this first phase, it was decided to develop the recommendations and 4 other Delphi Rounds followed. The process finished with a Consensus Meeting, that was held during the SOSORT Meeting in Wiesbaden, 8-10 May 2014, moderated by the Presidents of SOSORT (JP O'Brien) and SRS (SD Glassman) and by the Chairs of the involved Committees (SOSORT Consensus Committee: S Negrini; SRS Non-Operative Committee: MT Hresko). The Boards of the SRS and SOSORT formally accepted the final recommendations. The 18 Recommendations focused: Research needs (3), Clinically significant outcomes (4), Radiographic outcomes (3), Other key outcomes (Quality of Life, adherence to treatment) (2), Standardization of methods of non-operative research (6).

Predictors of postoperative pain trajectories in adolescent idiopathic scoliosis.

STUDY DESIGN: Prospective observational study. OBJECTIVE: To determine the typical trajectory of pain during the first 6 months after spinal fusion surgery for adolescent idiopathic scoliosis (AIS) and the extent to which certain demographic, medical, and psychological variables modify this trajectory. SUMMARY OF BACKGROUND DATA: Pain after spinal fusion surgery for AIS may not improve predictably with elapsed healing time, and limited data exist on predictors of the course of pain during the initial months after surgery. METHODS: Fifty patients ages 11 to 17 (mean = 14.5, standard deviation = 1.9) with AIS and undergoing posterior spinal fusion surgery comprised the study sample. Pain outcomes were assessed at 4 time points after surgery (2-week, 6-week, 3-month, and 6-month postsurgery). Preoperative predictor variables comprising demographics, baseline Cobb angle, body mass index, baseline pain, and psychological variables (anxiety, negative mood, and confidence in ability to control pain) were assessed 2 weeks before surgery. Perioperative predictor variables comprising pain, pain coping efficacy, negative mood, surgery length, length and lowest level of fusion, and analgesic use were assessed by self-report or record review. Multilevel growth models were used to evaluate hypotheses pertaining to predictors of pain trajectories. RESULTS: Pain level on average declined predictably with days since surgery (b = -0.14 to -0.19, P < 0.01). For 22% of adolescents, pain was at or above baseline levels through 6 months after surgery. Greater baseline pain and anxiety predicted slower improvement in pain, whereas greater confidence in ability to control pain predicted more rapid declines in pain. None of the demographic or medical variables reliably modified postsurgical pain trajectories. CONCLUSION: Although pain typically declines predictably with healing time from spinal fusion surgery for AIS, higher preoperative levels of pain and anxiety may be risk factors for chronic postsurgical pain whereas greater pain coping efficacy may help optimize postsurgical pain outcomes. LEVEL OF EVIDENCE: 3.

The pendulum swings back to scoliosis screening: screening policies for early detection and treatment of idiopathic scoliosis - current concepts and recommendations.

This editorial article initiates the school scoliosis screening thematic series of the Scoliosis journal. The various issues on screening policies are discussed; clinical and practical recommendations of setting up school screening programs are also described.

Vectored cranial-cervical traction limits facial contact pressure from prone positioning during posterior spinal deformity surgery.

STUDY DESIGN: Prospective, two-way complete block design analyzing facial contact pressures during prone positioning with the use of cervical traction for spinal surgery. Level 2 evidence. OBJECTIVE: To assess the effect of varying traction angle and traction weight to limit facial contact pressure. SUMMARY OF BACKGROUND DATA: Posterior spine surgery has known hazards related to the prone positioning. Cervical traction is used to limit downward pressure exerted to the face to stabilize the head and neck and to aide in deformity correction. The effects of the traction angle and force on facial contact pressure have not been studied. METHODS: Facial contact pressure was measured for 10 patients undergoing posterior spine surgery in the prone position with Gardner-Wells tongs applied for cervical traction. The facial contact pressure was measured with a force transducer at each of three angles from horizontal (0°, 30°, 45°) and each of four traction weights (0, 5, 10,15 lb), a total of 12 measurement parameters for each patient. An in-line tensiometer provided consistent application of force throughout the traction system. RESULTS: Ten patients, average age 15 ± 0.6 years, six female, BMI 21.3 ± 1.7, underwent facial pressure monitoring. Post hoc analysis showed that both higher traction weights and angles significantly limited facial pressure (P = 0.0001). The lowest overall average facial pressure of 0.51 lb (95% CI = 0.28-0.73) occurred with 15 lb of traction applied at 45° above the horizontal. This was significantly less facial pressure than found when traction was applied at all weights tested using the commonly employed 0° in-line traction angle (P < 0.0001). CONCLUSION: A combination of upward vectored 45° traction angle and 15 lb of weight significantly decreased facial contact pressure. The use of an "in-line tensiometer" assured an accurate force application.

Vertical expandable prosthetic titanium rib as treatment of thoracic insufficiency syndrome in spondylocostal dysplasia.

BACKGROUND: Spondylocostal dysplasia (SCD) constitutes a heterogeneous patient group with multiple vertebral formations and segmentation defects of the entire spine, with asymmetric rib malformations. Respiratory failure has been reported in spondylocostal dysplasia secondary to thoracic insufficiency syndrome. The vertical expandable prosthetic titanium rib (VEPTR) reconstructs the chest wall to address the thoracic insufficiency seen in this patient population. The purpose of this study is to evaluate spinal deformity correction and respiratory function outcomes in a spondylocostal dysplasia population treated with VEPTR. METHODS: A cohort of 20 patients with spondylocostal dysplasia and 2-year follow-up were evaluated from a multicenter IDE study of 214 patients who had surgery with the VEPTR device. Data collected included gender, nonskeletal malformations, age at surgery, number of procedures, estimated blood loss, length of stay, and surgical time. Clinical and radiographic parameters were collected, and respiratory function was assessed. RESULTS: In 14 of 20 patients (70%), spinal deformity was controlled evidenced by a decrease of the initial Cobb coronal angle at last follow-up. Fourteen patients (70%) maintained their oxygen level throughout treatment. At preoperative and last evaluation, assisted ventilation rating (AVR) scores showed that 5 patients improved their level of ventilation and 14 patients maintained their AVR level at room air. One patient decreased his level from supplemental oxygen to night ventilation. Mean thoracic spinal length (growth) by year was 0.82 cm. No mortality occurred in this group of patients. CONCLUSIONS: VEPTR implantation in SCD allows continued thoracic spine growth while controlling progressive spine deformity. The improved AVR ratings after surgery suggest a beneficial effect on the natural history of TIS in this population. Mortality and complication rate seem acceptable in this high-risk population of SCD patients. LEVEL OF EVIDENCE: Therapeutic study, Level IV, (case series, no comparison group).

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme: differential expression of alternatively spliced variants in Drosophila tissues.

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A. We performed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1 mRNA were found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K(0.5)) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects.

Rat liver carnitine palmitoyltransferase 1 forms an oligomeric complex within the outer mitochondrial membrane.

Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1-82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling.

The mitochondrial intermembrane loop region of rat carnitine palmitoyltransferase 1A is a major determinant of its malonyl-CoA sensitivity.

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.

Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines.

Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol.2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased k(cat)/K(m) values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-alpha-methylbenzylamine (N-CalphaMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CalphaMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.

Structure and regulation of acetyl-CoA carboxylase genes of metazoa.

Acetyl-CoA carboxylase (ACC) plays a fundamental role in fatty acid metabolism. The reaction product, malonyl-CoA, is both an intermediate in the de novo synthesis of long-chain fatty acids and also a substrate for distinct fatty acyl-CoA elongation enzymes. In metazoans, which have evolved energy storage tissues to fuel locomotion and to survive periods of starvation, energy charge sensing at the level of the individual cell plays a role in fuel selection and metabolic orchestration between tissues. In mammals, and probably other metazoans, ACC forms a component of an energy sensor with malonyl-CoA, acting as a signal to reciprocally control the mitochondrial transport step of long-chain fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I (CPT I). To reflect this pivotal role in cell function, ACC is subject to complex regulation. Higher metazoan evolution is associated with the duplication of an ancestral ACC gene, and with organismal complexity, there is an increasing diversity of transcripts from the ACC paraloges with the potential for the existence of several isozymes. This review focuses on the structure of ACC genes and the putative individual roles of their gene products in fatty acid metabolism, taking an evolutionary viewpoint provided by data in genome databases.

Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity.

We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N-C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N-C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N-C interactions. This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N-C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24-->Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40-47) as being involved in the chemical N-C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.