Syndesmosis injuries in the pediatric and adolescent athlete: an analysis of risk factors related to operative intervention.

PURPOSE: To review all paediatric ankle syndesmotic injuries occurring at our institution and identify risk factors associated with operative intervention. METHODS: Among 22 873 evaluations for ankle trauma, we found 220 children suffering from syndesmotic injuries (incidence: 0.96%). We recorded demographic data, details of the injury, features on examination and treatment variables. Univariable and multivariable logistic regression modelling was performed to identify risk factors associated with operative intervention. RESULTS: The mean age at injury was 15.8 years (8.9 to 19.0) with a median follow-up of 13 weeks (IQR 5 to 30 weeks). A sports-related injury was most common (168/220, 76%). A total of 82 of 220 (37%) patients underwent operative fixation, of which 76 (93%) had an associated fibular fracture. Patients undergoing surgery had a higher incidence of swelling and inability to weight bear (p < 0.001). Statistically significant differences were recorded in tibiofibular (TF) clear space, TF overlap and medial clear space (MCS) between the operative and non-operative cohorts (6.0 vs 4.6 mm (p = 0.002), 5.4 vs 6.9 mm (p = 0.004) and 6.4 vs 3.5 mm (p < 0.001)). Multivariable analysis revealed patients with a fracture of the ankle had 44 times the odds of surgical intervention, patients with a closed physis had over five times the odds of surgical intervention and patients with a medial clear space greater than 5 mm had nearly eight times the odds of requiring surgical intervention. CONCLUSIONS: Operative ankle syndesmotic injuries in the paediatric population are often associated with a closed distal tibial physis and concomitant fibular fracture.

Outcomes following hip arthroscopy for femoroacetabular impingement with associated chondrolabral dysfunction: minimum two-year follow-up.

Over an eight-month period we prospectively enrolled 122 patients who underwent arthroscopic surgery of the hip for femoroacetabular impingement and met the inclusion criteria for this study. Patients with bilateral hip arthroscopy, avascular necrosis and previous hip surgery were excluded. Ten patients refused to participate leaving 112 in the study. There were 62 women and 50 men. The mean age of the patients was 40.6 yrs (95% confidence interval (CI) 37.7 to 43.5). At arthroscopy, 23 patients underwent osteoplasty only for cam impingement, three underwent rim trimming only for pincer impingement, and 86 underwent both procedures for mixed-type impingement. The mean follow-up was 2.3 years (2.0 to 2.9). The mean modified Harris hip score (HHS) improved from 58 to 84 (mean difference = 24 (95% CI 19 to 28)) and the median patient satisfaction was 9 (1 to 10). Ten patients underwent total hip replacement at a mean of 16 months (8 to 26) after arthroscopy. The predictors of a better outcome were the pre-operative modified HHS (p = 0.018), joint space narrowing >or= 2 mm (p = 0.005), and repair of labral pathology instead of debridement (p = 0.032). Hip arthroscopy for femoroacetabular impingement, accompanied by suitable rehabilitation, gives a good short-term outcome and high patient satisfaction.

Nuclear localization of the Saccharomyces cerevisiae HMG protein NHP6A occurs by a Ran-independent nonclassical pathway.

The Saccharomyces cerevisiae non-histone protein 6-A (NHP6A) is a member of the high-mobility group 1/2 protein family that bind and bend DNA of mixed sequence. NHP6A has only one high-mobility group 1/2 DNA binding domain and also requires a 16-amino-acid basic tail at its N-terminus for DNA binding. We show in this report that nuclear accumulation of NHP6A is strictly correlated with its DNA binding properties since only nonhistone protein 6 A-green fluorescent protein chimeras that were competent for DNA binding were localized to the nucleus. Despite the requirement for basic residues within the N-terminal segment for DNA binding and nuclear accumulation, this region does not appear to contain a nuclear localization signal. Moreover, NHP6A does not bind to the yeast nuclear localization signal receptor SRP1 and nuclear targeting of NHP6A does not require the function of the 14 different importins. Unlike histone H2B1 which contains a classical nuclear localization signal, entry of NHP6A into the nucleus was found to be independent of Ran as judged by coexpression of Ran GTPase mutants and was shown to occur at 0 degrees C after a 15-min induction. These unusual properties lead us to suggest that NHP6A entry into the nucleus proceeds by a nonclassical Ran-independent pathway.

Mechanism for specificity by HMG-1 in enhanceosome assembly.

Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding.

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine.

The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Deltanhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain. Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Deltanhp6a/b mutants in vivo.

Steady-state regulation of whole-body thyroid hormone pool sizes and interconversion rates in hypothyroid and moderately T3-stimulated rats.

Steady-state regulation of whole-body T3 and T4 distribution and metabolism were directly evaluated and compared in hypothyroid, euthyroid, and in euthyroid rats moderately T3-stimulated by continuous infusion of 0.15 microg/day L-T3 per 100 g BW, thereby supplementing euthyroid T3 sources by two thirds. Our goal was to develop deeper insights into the hierarchy, quantitative adequacy, and sensitivity of this regulatory system, in response to these hormone production challenges in constant steady state. We used a novel whole-body steady-state experiment design model and data analysis approach, which entails nonexclusive whole-body homogenate extracts and blood collected after 7-day infusions of tracer T3 (T3*) or T4*, quantitatively analyzed chromatographically for T3*, T4* and metabolite* concentrations. Hormone regulation implications across the 3 groups were assessed by comparing (per 100g BW) total body T4 to T3 and T3 from T4 conversion rates (CR(4-3) and CR(3-4)), total body pool sizes (Qtot) and distribution volumes (V(D)), total body production (PR), or plasma appearance rates (PAR), plasma clearance rates (PCR), and elimination rates (k). In the hypothyroid rats, absolute production of T3 from T4 was only a fourth of that in euthyroids: CR(3-4) = 1.55 vs. 6.77 ng/h, but the percent (efficiency) of whole-body T4 converted to T3 was more than double that in euthyroids: %CR(4-3) = 45.4% vs. 21.0%, reflecting an effective doubling of type I and/or type II 5'-deiodinase activity on a whole-body basis in response to severe curtailment of thyroidal production. Whole-body T3 pools and T3 production and clearance rates were all about 2 to 3 times lower in hypo- than in euthyroids: minimum Qtot = 36.8 vs. 100 ng, V(D3) = 148 vs. 236 ml, PAR3 = 3.44 vs. 9.09 ng/h, PCR3 = 13.8 vs. 21.3 ml/h; and nearly all T4 pool size, production, clearance and elimination rates also were very substantially reduced: PCR4 = 0.540 vs. 0.941 ml/h, PR4 = 4.11 vs. 38.3 ng/h, Qtot4 = 128 vs. 702 ng, k4 = 0.0322 vs. 0.0530 h(-1). In moderately T3-stimulated rats, presumed central feedback effects of the added T3 on T4 production and total body pool size also were quite pronounced: PR4 = 21.4 ng/h and Qtot4 = 346 ng were reduced to about half that in euthyroids, but T4 elimination indices were virtually unchanged, and T3 production and elimination were minimally affected. Thus, overall, stabilizing negative feedback regulation of TH functioning at different hierarchical levels is quite bidirectionally sensitive. We found very tight (inhibitory) control over thyroidal T4 secretion, possibly also T3 secretion, and probably also absolute T3 production from T4, in response to moderate (+68%) supplements in T3 production; and the efficiency of total body T3 production from available T4 was amplified substantially in the severe primary hypothyroid state, although not nearly enough to compensate for the malady. Finally, the blood to total body pool fractions (Qb/Qtot) of both T3 and T4, but not the plasma or blood hormone levels, remained remarkably constant in response to these oppositely directed hormone production challenges, suggesting this ratio as an actively regulated, homeostatically-maintained entity.

Direct measurement of whole body thyroid hormone pool sizes and interconversion rates in fasted rats: hormone regulation implications.

Food deprivation markedly reduces thyroid hormone levels in mammalian plasma, but existing data are incomplete and equivocal regards extrathyroidal hormone production and other indices of overall hormone economy. We have used a novel experiment design and analysis to directly measure the whole-body rate of conversion of T4 into T3 and several other steady-state whole organism parameters, in 4-day fasted and fed control rats. Trace amounts of 125I-labeled T3 (T3) or T4 (T*4) were infused for 7 days from osmotic minipumps implanted sc. On day 7, rats were anesthetized, bled, and killed and carcasses were frozen in liquid N2, pulverized, homogenized, and extracted. Extracts and plasma samples were chromatographed on both Sephadex and HPLC. Tracer infusion rates, whole rat tissue weights, and steady state tissue, blood, and plasma T*3, T*4, and total radioactivity concentrations provided all kinetic parameters of interest from simple steady state computations. T4 secretion (SR4) and whole body pool sizes were reduced 49-55% in fasted rats. But the most notable results were that the percent of available extrathyroidal T4 converted to T3 in fasted [41.6 +/- 7.9% (SD)] was 87% greater than that in the fed (22.3 +/- 7.69%) rats and this, in turn, generated an absolute rate of production of T3 from T4 not significantly different in fasted vs. fed controls (7.17 +/- 2.40 vs. 7.54 +/- 3.10 ng/h.100 g BW). The surprisingly high 42% conversion ratio in fasting is explained in part by larger T3 blood pools (which are not sites of T3 production from T4) relative to tissue T3 pools in fasted rats, not accounted for in earlier whole-body studies. In contrast with this finding of an increased T4 to T3 conversion ratio in fasted rats, based on whole body measurements, T3 plasma concentrations (Cp3), clearance rates (PCR3), appearance rates (PAR3 = PCR3Cp3), and more conventional indirect estimates of the T4 to T3 conversion ratio (100 PAR3/SR4) were all substantially reduced, consistent with reports in fasting humans limited to measurements of T3 and T3 turnover in plasma and interpreted as indicative of reduced whole body T4 or T3 conversion. Directly measured total T3 extrathyroidal distribution volumes, reduced 55% in the fasted group from 241 +/- 19.5 to 109 +/- 8.14 ml/100 g BW, are also of interest because fed rat values are 27-61% greater than virtually all previous estimates of this index of total body T3.(ABSTRACT TRUNCATED AT 400 WORDS)

Steady state organ distribution and metabolism of thyroxine and 3,5,3'-triiodothyronine in intestines, liver, kidneys, blood, and residual carcass of the rat in vivo.

We have directly measured the relative distribution and steady state pool sizes of T4 and T3 in blood and extrathyroidal tissues of the whole euthyroid male rat, plus several steady state T3 and T4 metabolism indices in the whole animal, in eight rats infused sc for 7 days with labeled 125I-T3 (T3*) or 125I-T4 (T4*) via implanted minipumps. Liver, kidneys, complete intestine, and residual carcass homogenate extracts and sera were chromatographed on both Sephadex and HPLC. Total tissue T3* and T4* in steady state were assessed and corrected for hormone trapped in residual blood in tissues using radioactive albumin as a vascular marker. Labeled Triac* and/or Tetrac* were prominent among steady state metabolites of T3* or T4* in intestines, feces, and residual carcass, but not in liver or kidneys. Extrathyroidal steady state T3 and T4 pools in residual carcass were 52.8 +/- 5.74 (SD) % of total T3 and 41.2 +/- 4.57% of total T4. The largest extrathyroidal organ pools containing exchangeable T3 and T4 were intestines, with 33.1 +/- 5.46% of total (extrathyroidal) T3 and 18.1 +/- 2.80% of total T4, whereas liver and kidney pools, previously reported as the largest measured, had only 8.0 +/- 1.45% and 2.30 +/- 0.26% of total T3 and 8.72 +/- 1.56% and 1.01 +/- 0.111% of total T4, respectively. Whole blood contained 3.6 +/- 0.385% of total T3 and 31.2 +/- 1.70% of total T4. Also, 21.3 +/- 8.2% of total body T4 was converted to T3 in the whole rat (residual carcass+intestines+liver+kidneys), the production rate of T4 was PR4 = 37.1 +/- 5.28 ng/h.100 g BW and the plasma appearance rate of T3 (PR3min) was 10.6 +/- 2.83 ng/h.100 g BW. T3 production from extrathyroidal T4 was CR3-4 = 6.63 +/- 0.94 ng/h and the minimum T3 secretion rate (SR3min) was 4.02 +/- 0.94 ng/h, each per 100 g BW, indicating that the thyroid secretes more than 38% of whole body T3 in euthyroid steady state. T4 and T3 plasma clearance rates (PCR) were 1.01 +/- 0.29 (T4) and 23.7 +/- 4.36 (T3) ml/h.100 g BW.(ABSTRACT TRUNCATED AT 400 WORDS)

Transfer kinetics of 3,5,3'-triiodothyronine and thyroxine from rat blood to large and small intestines, liver, and kidneys in vivo.

Enterohepatic circulation of thyroid hormone in the rat involves transfer of hormone to intestines in bile from the liver and absorption of some intestinal hormone, via portal blood to liver. Transfer of hormone to intestines from mesenteric arterial blood has generally been considered minimal, although this alternate pathway has received little attention. We have measured uptake kinetics of iv trace doses of radioactivity labeled T3 (T3*) and T4* from blood to intestines, longitudinally in 14 segments of total intestines in situ, with bowel contents included and bile duct ligated, and also to liver and kidneys, for comparison. Both T3* and T4* entered the entire length of intestines from blood, and into contents as well as tissue. Tissue uptake of T3* and T4* were fairly uniform longitudinally, but uptake into contents and thus into total intestines (tissue + contents) decreased linearly from pylorus to anus. Unidirectional uptake rate constants (hours-1) or clearance rates (milliliters per h) of T3* were 13 times greater than corresponding T4* fractional uptake rates to intestines. In contrast, T4 mass fluxes (congruent to 38 ng/h.100 g body wt) exceed T3 fluxes (congruent to 6.3 ng/h.100 g body wt) about 6-fold, because plasma contains far more T4 than T3. Comparing these results with published biliary and fecal T3 and T4 flux data, it appears that mesenteric arterial mass influxes to intestines are 3-5 times greater than both biliary secretion and fecal excretion of T3 and T4. Intestinal kinetics, in the absence of biliary influx, are characterized by a moderately rapid uptake followed by a very slow washout phase, which fit into neither the fast nor slow compartment paradigms of a 3-compartment mammillary model, in contrast to liver and kidney kinetics, which fit well into a single fast compartment. Intestinal dynamics are consistent with an organ in which storage and exchange of thyroid hormone dominate over metabolic and excretory processes.

Sites and patterns of absorption of 3,5,3'-triiodothyronine and thyroxine along rat small and large intestines.

It is clear that some thyroid hormone is absorbed from mammalian intestines, but numerous aspects of this process remain unresolved, including elucidation of the locations, extent and mechanisms of absorption, and the role of absorption in thyroid hormone economy. Our goal was to identify the sites and patterns and estimate rates of absorption of both tracer T4* and T3* comprehensively along the entire length of rat intestines, with normal contents included, to gain further insight into the role of this organ in whole-body thyroid hormone regulation. We measured absorption directly in situ in fed rats, using a variant of the classic intestinal loop technique used by others demonstrating absorption of T4 or of T3 from portions of rat intestines under various conditions. Rats were anesthetized, bile ducts were ligated, and absorption was measured from pylorus to anus, in 14 loops of intestines previously injected with T3* or with T4*, or with T3* and T4* injected in adjacent loops. Rats were maintained under otherwise approximately normal conditions, with intestines in situ and abdomen closed, until killing at 2 h. Excised loop radioactivity was measured and loops were homogenized, extracted, and chromatographed quantitatively to evaluate remaining and absorbed T3* and T4*. Absorption of both T3* and T4* were clearly present and were approximately uniform from all small and large intestinal sections, all containing normal intestinal contents, indicating that the entire organ is involved in whole-body thyroid hormone regulation. Furthermore, T3* and T4* were absorbed at approximately the same rate, adding to evidence reported by others for a simple diffusion absorption mechanism.