Fresh Osteochondral Allograft Transplantation of the Capitellum for the Treatment of Osteochondritis Dissecans.

BACKGROUND: Osteochondritis dissecans (OCD) of the humeral capitellum is a rare and challenging condition to treat. Several surgical options exist, but in the last few years, the pendulum has swung from debridement and microfracture to restoration of the articular surface. Osteochondral autografts from the rib and knee have been described, but donor-site morbidity is a concern. PURPOSE: To expand the results of fresh osteochondral allograft transplantation (FOCAT) in a previously published report with inclusion of additional patients and a longer follow-up period. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: After institutional review board approval, the charts of patients who underwent FOCAT for OCD of the capitellum between 2006 and 2022 by a single surgeon were reviewed. The majority of cases (94%) had unstable lesions (Minami grades 2 and 3). A trial of nonoperative treatment had failed in all. All patients underwent diagnostic arthroscopy, followed by a mini-open, ligament-sparing approach with grafting using commercially available guides and instruments. RESULTS: A total of 35 patients were identified, of whom 25 were male. The mean age was 16 ± 3.9 years (range, 11-32 years). There were 24 baseball players (19 pitchers and 5 position players), 5 gymnasts, 3 cheerleaders/tumblers, 1 tennis player, 1 student (who did not participate in athletics), and 1 patient with avascular necrosis from chemotherapy. Eighteen patients had a mean flexion contracture of 14.1°± 11.9°. A single osteochondral allograft plug was used in 23 patients (mean diameter, 11.3 ± 2.8 mm), and 12 patients required 2 plugs (Mastercard technique). The mean follow-up was 92.6 ± 54.5 months (range, 24-204 months). There was significant improvement in Oxford (from 25.5 ± 4.9 to 46.7 ± 3.5; P < .00001) and visual analog scale for pain (from 7.5 ± 2 to 0.3 ± 1.0; P < .0001) scores. The mean Single Assessment Numeric Evaluation score at the time of follow-up was 90.6 ± 10.8 (range, 60-100). In overhead athletes, there was significant improvement in the Kerlan-Jobe Orthopaedic Clinic score (from 40.8 ± 11.8 to 90.6 ± 10.8; P < .00001). A postoperative magnetic resonance imaging scan was obtained in 16 (46%) patients at a mean of 32.6 months. In all cases, the graft was incorporated. All overhead athletes were able to return to their sport and perform at the same level or higher for >2 years. Two elbows required a subsequent arthroscopy for loose-body removal; otherwise, there were no other complications. CONCLUSION: FOCAT is an excellent option for treating OCD lesions of the humeral capitellum. Excellent outcomes and high return-to-sport rates were observed, with midterm follow-up showing no graft failures. FOCAT eliminates donor-site morbidity.

Detection of autism spectrum disorder-related pathogenic trio variants by a novel structure-based approach.

BACKGROUND: Glutamatergic synapse dysfunction is believed to underlie the development of Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in many individuals. However, identification of genetic markers that contribute to synaptic dysfunction in these individuals is notoriously difficult. Based on genomic analysis, structural modeling, and functional data, we recently established the involvement of the TRIO-RAC1 pathway in ASD and ID. Furthermore, we identified a pathological de novo missense mutation hotspot in TRIO's GEF1 domain. ASD/ID-related missense mutations within this domain compromise glutamatergic synapse function and likely contribute to the development of ASD/ID. The number of ASD/ID cases with mutations identified within TRIO's GEF1 domain is increasing. However, tools for accurately predicting whether such mutations are detrimental to protein function are lacking. METHODS: Here we deployed advanced protein structural modeling techniques to predict potential de novo pathogenic and benign mutations within TRIO's GEF1 domain. Mutant TRIO-9 constructs were generated and expressed in CA1 pyramidal neurons of organotypic cultured hippocampal slices. AMPA receptor-mediated postsynaptic currents were examined in these neurons using dual whole-cell patch clamp electrophysiology. We also validated these findings using orthogonal co-immunoprecipitation and fluorescence lifetime imaging (FLIM-FRET) experiments to assay TRIO mutant overexpression effects on TRIO-RAC1 binding and on RAC1 activity in HEK293/T cells. RESULTS: Missense mutations in TRIO's GEF1 domain that were predicted to disrupt TRIO-RAC1 binding or stability were tested experimentally and found to greatly impair TRIO-9's influence on glutamatergic synapse function. In contrast, missense mutations in TRIO's GEF1 domain that were predicted to have minimal effect on TRIO-RAC1 binding or stability did not impair TRIO-9's influence on glutamatergic synapse function in our experimental assays. In orthogonal assays, we find most of the mutations predicted to disrupt binding display loss of function but mutants predicted to disrupt stability do not reflect our results from neuronal electrophysiological data. LIMITATIONS: We present a method to predict missense mutations in TRIO's GEF1 domain that may compromise TRIO function and test for effects in a limited number of assays. Possible limitations arising from the model systems employed here can be addressed in future studies. Our method does not provide evidence for whether these mutations confer ASD/ID risk or the likelihood that such mutations will result in the development of ASD/ID. CONCLUSIONS: Here we show that a combination of structure-based computational predictions and experimental validation can be employed to reliably predict whether missense mutations in the human TRIO gene impede TRIO protein function and compromise TRIO's role in glutamatergic synapse regulation. With the growing accessibility of genome sequencing, the use of such tools in the accurate identification of pathological mutations will be instrumental in diagnostics of ASD/ID.

The "Empty Merchant Sign" in acute patellar tendon ruptures.

INTRODUCTION: We introduce a new sign on a Merchant view present in acute patellar tendon ruptures (APTR). We aim to determine the accuracy, sensitivity, and specificity of this new radiographic sign and measure the effect of a tutorial on these measures between trainees and non-trainees. METHODS: Lateral and Merchant radiographs (22 images) of knees with four conditions (patellar instability, APTR, quadriceps tendon rupture, and controls) were randomly shown to 50 trainees and non-trainees who were asked to make a diagnosis based on radiographs. A brief tutorial was administered describing the "Empty Merchant Sign" and the same 22 images were randomly shown after the tutorial. Accuracy, sensitivity, specificity, and positive predictive value were calculated between the two image types and the effect of tutorial on these measures was assessed. RESULTS: After the tutorial: 1. the "Empty Merchant Sign" had a higher specificity (100%) and positive predictive value (99%) compared to the lateral radiograph (81% and 64% respectively, P < 0.001), 2. There was significant improvement (from 56% to 95.3%; P < 0.001) in the accuracy of the Merchant view, making it as accurate as the lateral view (95.3% vs. 90.7%, respectively; P = 0.113). There was no difference in the accuracy of the Merchant view between trainees (97.2%) and non-trainees (90.5%) (P = 0.079). CONCLUSION: The "Empty Merchant Sign" is a highly sensitive and specific diagnostic sign in cases of APTR. With very little training, physicians can identify this sign to diagnose APTRs on a Merchant view.

Operative Treatment of Acute Patellar Tendon Ruptures.

BACKGROUND: The gold standard for patellar tendon repair is a transosseous technique. Suture anchor repair has gained popularity, with recent biomechanical studies demonstrating significantly less gap formation during cyclic loading and significantly higher ultimate failure loads as compared with transosseous repair. These findings have not been substantiated in a large clinical cohort. PURPOSE: To report demographic and epidemiologic data, clinical and surgical findings, and outcomes and complications of anchor and transosseous suture repairs of acute patellar tendon ruptures. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: All patients who underwent a primary repair of a traumatic patellar tendon rupture within 45 days of injury between 2006 and 2016 were retrospectively reviewed. Surgery was performed at a multisurgeon (120 surgeons) multicenter (14 centers) community-based integrated health care system. Patient demographic information, repair type, complications, and time from surgery to release from medical care were recorded. RESULTS: A total of 361 patients (374 knees) met our inclusion criteria. The mean age was 39.8 years (range, 9-86 years), and 91.7% were male. There were 321 transosseous and 53 anchor repairs. There was no significant difference in the mean age (P = .27), sex (P = .79), tourniquet time (P = .93), or body mass index (P = .78) between the groups. There was a significant difference in rerupture rate between transosseous and anchor repairs (7.5% vs 0%, respectively; P = .034). Based on logistic regression, transosseous repair had 3.24 times the odds of reoperation verseus anchor repair (95% CI, 0.757-13.895; P = .1129). The infection rate was 1.6% for transosseous repair and 7.5% for anchor repair (P = .160). There was no difference in time to release from medical care: 18.4 weeks for anchor and 17.1 weeks for transosseous repairs (P = .92). CONCLUSION: Anchor repair demonstrated a significant decrease in rerupture rate when compared with transosseous repair. There was no difference in reoperation rate, infection rate, or time to release from medical care.

Functional outcomes of ulnar collateral ligament reconstruction with a novel double suspensory fixation.

BACKGROUND: Ulnar collateral ligament reconstruction (UCLR) has allowed the return of overhead athletes to throwing sports. We describe a new double suspensory (DS) technique using a single tunnel in the ulna and humerus, achieving fixation with adjustable loop buttons. METHODS: Inclusion criteria included skeletally mature baseball players with clinical and magnetic resonance imaging diagnosis of UCL insufficiency who failed a trial of structured nonoperative treatment. A total of 36 baseball players underwent DS UCLR, between 2011 and 2017, by 1 surgeon with minimum 2-year follow-up. The graft was fixated with an adjustable button loop on the humeral side and a tension slide technique with a button on the ulnar side. Pre- and postoperative Kerlan-Jobe Orthopaedic Clinic and Single Assessment Numerical Evaluation and postoperative Conway scores were obtained. RESULTS: The mean age was 19.8 ± 4.6 years (range, 14-35 years). All were male. Mean years played before surgery was 14.3 ± 4.6 years (range, 8-28 years). There were 32 (89%) pitchers and 4 (11%) position players. There were 13 (36%) high school, 20 (55%) college, 2 (6%) minor league, and 1 (3%) adult league athletes. The mean follow-up was 55.3 ± 23.7 months (range, 26-97 months). There was significant improvement in Kerlan-Jobe Orthopaedic Clinic (33.2 ± 19.9 to 89.7 ± 15.1, P < .0001) and Single Assessment Numerical Evaluation (20.7 ± 16.7 to 93.6 ± 11.9, P < .0001) scores. Using Conway scoring, 25 (69%) had excellent, 5 (14%) good, 3 (8%) fair, and 3 (8%) poor scores. Mean return to play was 9 ± 1.5 months (range, 6-16 months). Only 1 (3%) athlete required a revision surgery and ultimately returned to play and 1 (3%) hardware removal. None developed ulnar nerve symptoms. CONCLUSION: DS fixation for UCLR in baseball players can lead to excellent clinical results and early return to play.

Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities.

Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders.

Bipolar Osteochondral Allograft Transplantation of the Patella and Trochlea.

OBJECTIVE: To evaluate clinical, functional, and radiographic outcomes of patients who underwent bipolar osteochondral allograft transplantation (OCAT) of the patellofemoral joint (PFJ). DESIGN: Prospectively collected data on 18 knees who underwent fresh osteochondral allograft transplantation of the patella and trochlea by a single surgeon were reviewed. Inclusion criteria were: high-grade chondral lesions of PFJ (5 knees), or recurrent patella dislocations with trochlear dysplasia and chondral injury to the patella and/or trochlea (13 knees). Functional scores were obtained preoperatively and at follow-up appointments included Knee injury and Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee (IKDC), Oxford, Cincinnati, Tenger-Lysholm, visual analogue scale (VAS)-pain, and Single Assessment Numeric Evaluation (SANE). Grafts were also evaluated using Osteochondral Allograft MRI Scoring System (OCAMRISS). RESULTS: Three patients were lost to follow-up, leaving 4 knees in group 1, and 11 knees in group 2. Average age was 28.9 years (range 16-52 years). The average follow-up was 33.2 months (range 12-64 months). There was significant improvement of KOOS (from 38.7 to 83.1), IKDC (from 28.2 to 76.6), Tegner-Lysholm (from 38.3 to 88.3), Oxford (from 22.7 to 42.9), Cincinnati (from 35.1 to 83.6), VAS (from 71 to 17.9.), and SANE (from 43.3 to 83) (P < 0.0001). The OCAMRISS score for patella was 2.23 and for trochlea 4.69. There were no revisions or conversions to arthroplasty. CONCLUSION: Bipolar OCAT of the patella and trochlea provide significant improvement in functional outcomes, relief from pain, activity level, and prevent recurrent instability.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower ß-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases ß-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of ß-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and ß-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that ß-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 × 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.

In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.

Core binding factor beta plays a critical role by facilitating the assembly of the Vif-cullin 5 E3 ubiquitin ligase.

UNLABELLED: The HIV-1 virion infectivity factor (Vif) targets the cellular cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) for degradation via the host ubiquitin-proteasome pathway. Vif recruits a cellular E3 ubiquitin ligase to polyubiquitinate A3G/F. The activity of Vif critically depends on the cellular core binding factor beta (CBFß). In this study, we investigated the Vif-CBFß interaction and the role of CBFß in the E3 ligase assembly. Vif-CBFß interaction requires an extensive region of Vif spanning most of its amino terminus and zinc finger region, and cullin 5 (Cul5) binding enhances the stability of the Vif-CBFß interaction. Our results further demonstrate that CBFß plays a critical role in facilitating Cul5 binding to the Vif/elongin B/elongin C complex. Vif, with or without bound substrate, is unable to bind Cul5 in the absence of CBFß. These studies support the notion that CBFß serves as a molecular chaperone to facilitate Vif-E3 ligase assembly. IMPORTANCE: The host antiviral restriction factors A3G/F inhibit viral replication. The HIV-1 protein Vif targets A3G/F for degradation. This immune evasion activity of Vif is dependent on the cellular factor CBFß. Multiple regions of Vif are known to be important for Vif function, but the mechanisms are unclear. The studies described here provide important information about the Vif-CBFß interaction interface and the function of CBFß in E3 ligase assembly. In particular, our comprehensive Vif-CBFß interface mapping results help to delineate the role of various Vif regions, determining if they are important for binding CBFß or A3G/F. Furthermore, our studies reveal an important potential mechanism of CBFß that has not been shown before. Our results suggest that CBFß may serve as a molecular chaperone to enable Vif to adopt an appropriate conformation for interaction with the Cul5-based E3 ligase. This study advances our understanding of how CBFß facilitates the Vif-mediated degradation of APOBEC3 proteins.

α-Synuclein can inhibit SNARE-mediated vesicle fusion through direct interactions with lipid bilayers.

The native function of α-synuclein is thought to involve regulation of synaptic vesicle trafficking. Recent work has also implicated a role in neurotransmission, possibly through interactions with the proteins involved in synaptic vesicle fusion. Here, we demonstrate that α-synuclein inhibits SNARE-mediated vesicle fusion through binding the membrane, without a direct interaction between α-synuclein and any of the SNARE proteins. This work supports a model in which α-synuclein plays a role in the regulation of vesicle fusion by modulating properties of the lipid bilayer.

The conformational ensembles of α-synuclein and tau: combining single-molecule FRET and simulations.

Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson's and Alzheimer's diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.

Health-Beneficial Phenolic Aldehyde in Antigonon leptopus Tea.

Tea prepared from the aerial parts of Antigonon leptopus is used as a remedy for cold and pain relief in many countries. In this study, A. leptopus tea, prepared from the dried aerial parts, was evaluated for lipid peroxidation (LPO) and cyclooxygenase (COX-1 and COX-2) enzyme inhibitory activities. The tea as a dried extract inhibited LPO, COX-1 and COX-2 enzymes by 78%, 38% and 89%, respectively, at 100 µg/mL. Bioassay-guided fractionation of the extract yielded a selective COX-2 enzyme inhibitory phenolic aldehyde, 2,3,4-trihydroxy benzaldehyde. Also, it showed LPO inhibitory activity by 68.3% at 6.25 µg/mL. Therefore, we have studied other hydroxy benzaldehydes and their methoxy analogs for LPO, COX-1 and COX-2 enzymes inhibitory activities and found that compound 1 gave the highest COX-2 enzyme inhibitory activity as indicated by a 50% inhibitory concentration (IC(50)) at 9.7 µg/mL. The analogs showed only marginal LPO activity at 6.25 µg/mL. The hydroxy analogs 6, 7 and 9 showed 55%, 61% and 43% of COX-2 inhibition at 100 µg/mL. However, hydroxy benzaldehydes 3 and 12 showed selective COX-1 inhibition while compounds 4 and 10 gave little or no COX-2 enzyme inhibition at 100 µg/mL. At the same concentration, compounds 14, 21 and 22 inhibited COX-1 by 83, 85 and 70%, respectively. Similarly, compounds 18, 19 and 23 inhibited COX-2 by 68%, 72% and 70%, at 100 µg/mL. This is the first report on the isolation of compound 1 from A. leptopus tea with selective COX-2 enzyme and LPO inhibitory activities.

Liposomal reconstitution of monotopic integral membrane proteins.

In spite of considerable progress in the methodology for reconstitution of membrane proteins into the liposomes, a successful reconstitution still appears to be more an art than a science. Reconstitution of membrane proteins into bilayers is required for establishing several aspects of the functions of membrane proteins and lipids and for elaborating models of naturally occurring membranes.Cyclooxygenase (COX)-1 and -2 (also prostaglandin endoperoxide H(2) synthase, PGHS-1 and -2) belong to the class of monotopic membrane proteins. Membrane-binding domains of both COX-1 and -2 contain four short, consecutive, amphipathic alpha-helices (A, B, C, and D). Crystal structures of the COXs indicate that basic, hydrophobic, and aromatic residues in the membrane-binding domain are oriented away from the protein core and form a surface on the enzyme, which has been proposed to interact with the lipid bilayer (1).In this chapter, we describe a fast and efficient method for direct incorporation of COX-1 and -2 isozymes - as models for monotopic integral membrane proteins - into preformed liposomes containing fatty acids without loss of activity.

Anthocyanin content, lipid peroxidation and cyclooxygenase enzyme inhibitory activities of sweet and sour cherries.

Cherries contain bioactive anthocyanins that are reported to possess antioxidant, anti-inflammatory, anticancer, antidiabetic and antiobese properties. The present study revealed that red sweet cherries contained cyanidin-3-O-rutinoside as major anthocyanin (>95%). The sweet cherry cultivar "Kordia" (aka "Attika") showed the highest cyanidin-3-O-rutinoside content, 185 mg/100 g fresh weight. The red sweet cherries "Regina" and "Skeena" were similar to "Kordia", yielding cyanidin-3-O-rutinoside at 159 and 134 mg/100 g fresh weight, respectively. The yields of cyanidin-3-O-glucosylrutinoside and cyanidin-3-O-rutinoside were 57 and 19 mg/100 g fresh weight in "Balaton" and 21 and 6.2 mg/100 g fresh weight in "Montmorency", respectively, in addition to minor quantities of cyanidin-3-O-glucoside. The water extracts of "Kordia", "Regina", "Glacier" and "Skeena" sweet cherries gave 89, 80, 80 and 70% of lipid peroxidation (LPO) inhibition, whereas extracts of "Balaton" and "Montmorency" were in the range of 38 to 58% at 250 microg/mL. Methanol and ethyl acetate extracts of the yellow sweet cherry "Rainier" containing beta-carotene, ursolic, coumaric, ferulic and cafeic acids inhibited LPO by 78 and 79%, respectively, at 250 microg/mL. In the cyclooxygenase (COX) enzyme inhibitory assay, the red sweet cherry water extracts inhibited the enzymes by 80 to 95% at 250 microg/mL. However, the methanol and ethyl acetate extracts of "Rainier" and "Gold" were the most active against COX-1 and -2 enzymes. Water extracts of "Balaton" and "Montmorency" inhibited COX-1 and -2 enzymes by 84, and 91 and 77, and 87%, respectively, at 250 microg/mL.

Withanolide sulfoxide from Aswagandha roots inhibits nuclear transcription factor-kappa-B, cyclooxygenase and tumor cell proliferation.

Investigation of the methanol extract of Aswagandha (Withania somnifera) roots for bioactive constituents yielded a novel withanolide sulfoxide compound (1) along with a known withanolide dimer ashwagandhanolide (2) with an S-linkage. The structure of compound 1 was established by extensive NMR and MS experiments. Compound 1 was highly selective in inhibiting cyclooxygenase-2 (COX-2) enzyme by 60% at 100 microm with no activity against COX-1 enzyme. The IC(50) values of compound 1 against human gastric (AGS), breast (MCF-7), central nervous system (SF-268) and colon (HCT-116) cancer cell lines were in the range 0.74-3.63 microm. Both S-containing dimeric withanolides, 1 and 2, completely suppressed TNF-induced NF-kappaB activation when tested at 100 microm. The isolation of a withanolide sulfoxide from W. somnifera roots and its ability to inhibit COX-2 enzyme and to suppress human tumor cell proliferation are reported here for the first time. In addition, this is the first report on the abrogation of TNF-induced NF-kappaB activation for compounds 1 and 2.

Force-velocity curves of motor proteins cooperating in vivo.

Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule "force-velocity curves" have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force-velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37 degrees C, three parameters were measured--velocity (v), radius (a), and effective cytoplasmic viscosity (eta')--as they applied to moving vesicles. These parameters were combined in Stokes' equation, F = 6piaeta'v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force-velocity curve for a single motor agrees with previously reported in vitro single motor force-velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities.

Lipid peroxidation and cyclooxygenase enzyme inhibitory activities of acidic aqueous extracts of some dietary supplements.

The botanical supplement market is growing at a fast pace with more and more people resorting to them for maintaining good health. Echinacea, garlic, ginkgo, ginseng, Siberian ginseng, grape seed extract, kava kava, saw palmetto and St John's wort are some of the popular supplements used for a variety of health benefits. These supplements are associated with various product claims, which suggest that they possess cyclooxygenase (COX) enzyme and lipid s inhibitory activities. COX enzymes are found to be at elevated levels in inflamed and cancerous cells. To test some of the product claims, selected supplements were analysed for their ability to inhibit COX-1 and -2 enzymes and lipid peroxidation in vitro. The supplements were extracted with acidified water (pH 2) at 37 degrees C to simulate the gastric environment. The supplements tested demonstrated varying degrees of COX enzyme inhibition (5-85% for COX-1 and 13-28% for COX-2). Interestingly, extracts of garlic (Meijer), ginkgo (Solaray), ginseng (Nature's Way), Siberian ginseng (GNC, Nutrilite, Solaray, Natrol), kava kava (GNC, Sundown, Solaray) and St John's wort (Nutrilite) selectively inhibited COX-2 enzyme. These supplements also inhibited lipid peroxidation in vitro (5-99%). The results indicated that the consumption of these botanical supplements studied possess health benefits.