Structure and function of N-acetylglucosaminyltransferase V (GnT-V).

BACKGROUND: The ß1,6-GlcNAc branch in N-glycans, produced by a glycosyltransferase N-acetylglucosaminyltransferase V (GnT-V or MGAT5), is associated with cancer and autoimmune diseases. SCOPE: Here, we summarize the structure and activity regulation of GnT-V. We also describe the roles of the ß1,6-GlcNAc branch on glycoproteins in cells and the phenotypes of Mgat5-deficient mice, focusing on cancer and the immune system. MAJOR CONCLUSIONS: GnT-V has a unique structure for substrate recognition, and its activity and function are regulated by shedding. The glycans produced by GnT-V play pivotal roles in the differentiation of neural cells, cancer malignancy and immunotherapy, and the development of autoimmune diseases by regulating the functions and cell surface residency of glycoproteins. GENERAL SIGNIFICANCE: Controlling the expression or activity of GnT-V could be a therapeutic option against cancer and autoimmune diseases. Future work should clarify how GnT-V selectively modifies the specific glycoproteins or N-glycosylation sites in vivo.

The cancer-associated glycosyltransferase GnT-V (MGAT5) recognizes the N-glycan core via residues outside its catalytic pocket.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) is a glycosyltransferase involved in cancer metastasis that creates the ß1,6-branch on N-glycans of target proteins such as cell adhesion molecules and cell surface receptors. The 3D structure of GnT-V and its catalytic site, which are critical for the interaction with the N-glycan terminal, have already been revealed. However, it remains unclear how GnT-V recognizes the core part of N-glycan or the polypeptide part of the acceptor. Using molecular dynamics simulations and biochemical experiments, we found that several residues outside the catalytic pocket are likely involved in the recognition of the core part of N-glycan. Furthermore, our simulation suggested that UDP binding affects the orientation of the acceptor due to the conformational change at the Manα1,6-Man linkage. These findings provide new insights into how GnT-V recognizes its glycoprotein substrates.

Structure-based design of UDP-GlcNAc analogs as candidate GnT-V inhibitors.

BACKGROUND: N-Glycan branching regulates various functions of glycoproteins. N-Acetylglucosaminyltransferase V (GnT-V) is a GlcNAc transferase that acts on N-glycans and the GnT-V-producing branch is highly related to cancer progression. This indicates that specific GnT-V inhibitors may be drug candidates for cancer treatment. To design novel GnT-V inhibitors, we focused on the unique and weak recognition of the donor substrate UDP-GlcNAc by GnT-V. On the basis of the catalytic pocket structure, we hypothesized that UDP-GlcNAc analogs with increasing hydrophobicity may be GnT-V inhibitors. METHODS: We chemically synthesized 10 UDP-GlcNAc analogs in which one or two phosphate groups were replaced with hydrophobic groups. To test these compounds, we set up an HPLC-based enzyme assay system for all N-glycan-branching GlcNAc transferases in which GnT-I-V activity was measured using purified truncated enzymes. Using this system, we assessed the inhibitory effects of the synthesized compounds on GnT-V and their specificity. RESULTS: Several UDP-GlcNAc analogs inhibited GnT-V activity, although the inhibition potency was modest. Compared with other GnTs, these compounds showed a preference for GnT-V, which suggested that GnT-V was relatively tolerant of hydrophobicity in the donor substrate. Docking models of the inhibitory compounds with GnT-V suggested the mechanisms of how these compounds interacted with GnT-V and inhibited its action. CONCLUSIONS: Chemical modification of the donor substrate may be a promising strategy to develop selective inhibitors of GnT-V. GENERAL SIGNIFICANCE: Our findings provide new insights into the design of GnT inhibitors and how GnTs recognize the donor substrate.

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan ß1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.

Recognition of glycan and protein substrates by N-acetylglucosaminyltransferase-V.

BACKGROUND: N-Glycosylation is crucial for protein folding, trafficking, and functions. N-Glycans have a different number of N-acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the ß1,6-linked GlcNAc branch on specific proteins produced by N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins. METHODS: Based on our structural model, we hypothesized that GnT-V interacts with the N-glycan core or polypeptide moiety as well as the accepter site of N-glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants. RESULTS: Our in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N-glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells. CONCLUSIONS: We identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids. GENERAL SIGNIFICANCE: Our data provide new insights into how GnT-V recognizes glycoproteins.

The accuracy of an injection technique for Flexor hallucis longus tendon sheath: A cadaveric study.

BACKGROUND: Corticosteroid injections are used in the conservative treatment of Flexor hallucis longus (FHL) tendinopathy. Studies for imaging guided injection are done, however, the accuracy of blind injection has not yet been studied. PURPOSE: The aim of this study was to determine the accuracy of a blind injection technique into the FHL tendon sheath. HYPOTHESIS: We hypothesize that a blind injections technique into the FHL tendon sheath based on clinical examination has a high accuracy. STUDY DESIGN: Descriptive cadaveric study. METHODS: Ten ankles of human cadavers were blindly injected with radiologic contrast mixed with methylene blue into the FHL tendon sheath. After injection, a CT scan of each ankle was performed to evaluate the location of contrast material. CT scans were reviewed by an experienced musculoskeletal radiologist blinded to the procedure. Anatomic dissection was undertaken to assess the location of the injection fluid. RESULTS: In nine ankles the radiological contrast was injected in the FHL tendon sheath. In one cadaver there was a technical problem and was therefore excluded. CONCLUSION: This study shows that the FHL tendon sheath can be blindly injected based on only clinical examination with high accuracy. LEVEL OF EVIDENCE: Descriptive cadaveric study.

On the origin of the highest ozone episodes in Spain.

The 2000-2015 occurrences of the highest ozone (O3) pollution episodes in Spain were evaluated to investigate their origin. To this end, data series available for urban and regional background (UB and RB), traffic (TR) and industrial (IN) sites were analysed separately and intercompared. Results evidenced that during these 16years mean O3 levels in the RB sites did not change significantly, and remained constantly high. However, there is a clear increase at the TR and UB sites. Although sensitivity analysis is needed to interpret the cause of this increasing trend, this might be caused probably by the lower O3 titration intensity due to the preferential abatement of NO vs NO2, as supported from the neutral trend of OX (NO2+O3) at these sites. We found that the exceedances of the hourly information threshold for O3 (>180µg/m3) are recorded mostly at UB and IN sites located in seven areas of Spain (specific hotspots or at the tail end of large urban plumes), and that these increased during summer heatwaves (i.e. 2003 and 2015). Although the external contribution of regional-to-subcontinental transported O3 might be relevant during the highest O3 episodes in the Western Mediterranean, our results evidenced that in the above specific areas, regional-local O3 production decisively contributes to the exceedances of the information threshold. Also that the human protection threshold and the AOT40 are more frequently exceeded in the Central, Southern and Mediterranean sides of the Iberian Peninsula. The design of effective episode abatement measures is quite complex in those conditions, due to both the nonlinearity of the chemical processes of O3 formation and destruction, and to the interplay with the complex meteorological setting, causing frequent recirculation and in situ aging of air masses. However, the combination of meteorological forecasting of the main recirculation processes and sensitivity analysis of NOX/VOC emission abatement measures might be powerful tools to evaluate the effectiveness of potential O3 mitigation strategies. Finally we would like to highlight that the current UB, RB, IN and TR classification (somewhat subjective) is not adequate to interpret the origin of O3 exceedances in complex areas of Southern Europe. Thus, a UB station recording exceedances, and located in a small city in the tail end of an urban plume of a large city, receives not only the contribution from its own UB, but mainly from the specific high O3 RB caused by the urban plume transport.

Fluid Structural Analysis of Urine Flow in a Stented Ureter.

Many urologists are currently studying new designs of ureteral stents to improve the quality of their operations and the subsequent recovery of the patient. In order to help during this design process, many computational models have been developed to simulate the behaviour of different biological tissues and provide a realistic computational environment to evaluate the stents. However, due to the high complexity of the involved tissues, they usually introduce simplifications to make these models less computationally demanding. In this study, the interaction between urine flow and a double-J stented ureter with a simplified geometry has been analysed. The Fluid-Structure Interaction (FSI) of urine and the ureteral wall was studied using three models for the solid domain: Mooney-Rivlin, Yeoh, and Ogden. The ureter was assumed to be quasi-incompressible and isotropic. Data obtained in previous studies from ex vivo and in vivo mechanical characterization of different ureters were used to fit the mentioned models. The results show that the interaction between the stented ureter and urine is negligible. Therefore, we can conclude that this type of models does not need to include the FSI and could be solved quite accurately assuming that the ureter is a rigid body and, thus, using the more simple Computational Fluid Dynamics (CFD) approach.

Epigallocatechin-3-gallate treatment reduces thermal hyperalgesia after spinal cord injury by down-regulating RhoA expression in mice.

BACKGROUND: (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent found in green tea. It has been reported that may be a natural agent for reducing thermal and mechanical pain after nervous system injuries. However, the molecular pathways implicated in these beneficial effects have not been completely elucidated. This study aimed to assess the EGCG treatment effects on thermal hyperalgesia, spinal cord gliosis and modulation of Ras homologue gene family member A (RhoA), fatty acid synthase (FASN) and tumour necrosis factor alpha (TNF-α) expression after spinal cord contusion in mice. METHODS: Animals were subjected to a spinal cord contusion. Thirty minutes after contusion and daily during the first week post-surgery, animals were treated with EGCG or dimethyl sulfoxide-saline (DMSO-saline). At 7 and 14 days post-operation, motor recovery was evaluated using the Basso Mouse Scale, and nociceptive response was evaluated using the Hargreaves test. Furthermore, at 14 days, the expression of RhoA, FASN and TNF-α proteins was quantified in the lesion site of spinal cord by Western blot technique. Finally, spinal cord samples were processed by immunohistochemical techniques for observing astrocytes, microglia and afferent nerve fibres. RESULTS: At short time, EGCG treatment reduced significantly thermal hyperalgesia but had no effect on locomotor recovery in spinal cord injured mice. Furthermore, EGCG treatment down-regulated the RhoA, FASN and TNF-α proteins expression, and decreased astro- and microglia reactivity in spinal cord. CONCLUSION: These findings suggest that at short time EGCG treatment reduces thermal hyperalgesia and gliosis via FASN and RhoA pathway, causing a decrease in cytokines in spinal cord.

2001-2012 trends on air quality in Spain.

This study aims at interpreting the 2001-2012 trends of major air pollutants in Spain, with a major focus on evaluating their relationship with those of the national emission inventories (NEI) and policy actions. Marked downward concentration trends were evidenced for PM10, PM2.5 and CO. Concentrations of NO2 and NOx also declined but in a lesser proportion at rural and traffic sites. At rural sites O3 has been kept constant, whereas it clearly increased at urban and industrial sites. Comparison of the air quality trends and major inflection points with those from NEIs, the National Energy Consumption and the calendar of the implementation of major policy actions allowed us to clearly identify major benefits of European directives on power generation and industrial sources (such as the Large Combustion Plants and the Integrated Pollution Prevention and Control Directives). This, together with a sharp 2007-2008 decrease of coal consumption has probably caused the marked parallel decline of SO2, NOx and for PM2.5 concentrations. Also the effect of the EURO 4 and 5 vehicle emission standards on decreasing emissions of PM and CO from vehicles is noticeable. The smooth decline in NO2-NOx levels is mostly attributed to the low efficiency of EURO 4 and 5 standards in reducing real life urban driving NO2 emissions. The low NOx decrease together with the complexity of the reactions of O3 formation is responsible for the constant O3 concentrations, or even the urban increase. The financial crisis has also contributed to the decrease of the ambient concentration of pollutants; however this caused a major reduction of the primary energy consumption from 2008 to 2009, and not from 2007 to 2008 when ambient air PM and SO2 sharply decreased. The meteorological influence was characterized by a 2008-2012 period favorable to the dispersion of pollutants when compared to the 2001-2007.

[The objective structured clinical evaluation of teaching in anaesthesiology and resuscitation]. / La evaluación clínica objetiva estructurada en la docencia en Anestesiología y Reanimación.

OBJECTIVES: To find out the acquirement of professional competencies of Anaesthesiology and Resuscitation medical residents at the end of their training period using the Objective Structured Clinical Evaluation (OCSE) tool. MATERIAL AND METHODS: Six competency components to evalúate were defined as follows: clinical interview (communication), technical ability and relationship abilities (leadership, decision making, work in a team), diagnostic assessment, therapeutic management, and medical records. Different methodologies were determined depending on the knowledge and skills to evaluate. Twelve clinical cases were developed that were performed in 12 stations. A total of 107 Ítems, specified within the stations, evaluated the competency components. A total of 43 residents were invited to participate in the last 4 months of their training in hospitals in Andalusia and Extremadura. RESULTS: A total of 33 residents participated. The overall mean of the classifications obtained in the 12 stations was 64.2 out of a maximum of 100. The medical residents demonstrated higher competency in obstetrics, paediatric anaesthesia, and that associated with difficult airway. The main competency gaps were detected in the area of one-day surgery, chronic pain, and literature management, in which approximately half passed the test. CONCLUSIONS: We believe that training evaluations, such as the OCSE, help in determining the skill levels of the medical resident, making it easier to continually improve the training of the future anaesthesiologist.

[Prostatic enucleation with holmium laser]. / Enucleación prostática con láser holmium.

OBJECTIVES: Benign prostatic hyperplasia (BPH) affects a growing percentage of males over the age of 40 years, increasing with age. Currently, we have a new therapeutic tool available: the holmium laser. METHODS: We retrospectively collected data of 300 patients undergoing holmium laser enucleation of the prostate (HoLEP) in our center. RESULTS: The results are: mean hospital stay 1.8 days (range 1-15 days, median 1.8); mean bladder catheter time 30.6 hours (range 12-312, median 30.3), total operative room time 75 minutes (range 38-150, median 71), maximal flow rate at six months 24.7 ml/sec. and 23.9 ml/sec. at 12 months. Surgical performance, number of grams resected per minute, is 0.48 for the whole group. We observed a variation in data from the first 20 cases, with worse results in this group. CONCLUSIONS: In our opinion holmium laser enucleation is an adequate method that the guarantees optimal results, comparable to those obtained with classic endoscopic and open surgical techniques, with a low rate of complications, which benefits the patient by diminishing the need for transfusions, catheterization time, and hospital stay; conversely, it has a learning curve of around 20 procedures, which may be associated with complications that may discourage the surgeon and stop the project of technique implementation in a center, having easy, accessible, established alternative procedures.

Carcinoma basocelular: el valor de los factores pronosticos / Carcinoma to basocelular: the value of the pronosticos factors

El carcinoma basocelular (CBC) es el tumor de presentación más común de la clínica dermatológica, siendo los individuos con mas riesgo a padecerlos aquellos de sexo masculino, mayores de 50 años y de raza caucásica. En este trabajo se estudiaron 60 biopsias de piel 30 pertenecientes a la variante sólida y 30 a la variante agresiva o ulcus rodens utilizando técnicas histológicas de rutina y especial de histoquímica (PAS, Alcian Blue PH: 0,5, ATO y lectinas ABL y EML) e inmunohistoquíomica para la detención de la oncoproteína bcl-2 y ULex europaeus. La observación con microscopio óptico permitió confirmar los cambios histopatológicos en las diferentes variantes clínico-patológicas coincidiendo en general con las descripciones dada por los autores consultados, aportando con la metodología aplicada, detalles y elementos de interés para el conocimiento histopatológico de esta enfermedad

[Giant stone in a Bricker urinary diversion. Obstruction uropathy as the presenting form]. / Litiasis gigante en derivación urinaria tipo Bricker. Uropatía obstructiva como forma de presentación.

Bowel is used in urological surgery to replace the bladder, either as a conduit to drain urine to the abdominal wall as a urinary stoma or refashioned to form a substitute bladder. Many factors contribute to stone formation, being urinary stasis, mucus production and bacteriuria the most important. Metabolic changes induced by exposure of segments of the alimentary tract to urine promote struvite, calcium oxalate and calcium phosphate stone formation. Generally, the majority of patients with stones in a urinary diversion can be treated with minimally invasive techniques. Open surgical removal is considered when other modality of treatments cannot be accomplished safely and expeditiously.

Clinical anatomy of the arterial supply of the human patellar ligament.

The arterial supply of the human patellar ligament has been systematized on 20 knee joints. After intravascular injection of colored natural latex, the blood supply to the extensor apparatus of the knee was studied by anatomical dissection and tissue transparentation techniques. Three arterial pedicles (superior, middle and inferior) were observed placed on each side of the patellar ligament. Medial pedicles had their origin from the descending and the inferior medial genicular arteries. The lateral pedicles took their origin from the lateral genicular arteries and the recurrent tibial anterior artery. Two main vascular arches anastomosed with these pedicles: the retropatellar and the supratubercular. Both arterial pedicles and anastomotic arches gave rise to a peritendinous network, characterized by a high vascular density next to poles of the patellar ligament. Only the anastomotic arches gave rise to collateral vessels that pierced the tendon, which revealed two vascular segments in the arterial supply of the patellar ligament (bipolar pattern). The upper segment was supplied by deep vessels from the retropatellar arch, whereas the inferior segment received superficial vessels from collaterals of the supratubercular arch. These intratendinous vessels anastomosed in the middle third of the patellar ligament.

[Retroperitoneal cystic lymphangioma. A silent disease in adults]. / Linfangioma quístico retroperitoneal. Una patología silente en adultos.

Primary retroperitoneal tumours may arise from different structures as neural, mesodermal, urogenital ridge, or embryonic remnant tissues. Lymphangioma is a rare benign tumour of the lymphatic tissue. They result from a developmental failure of the lymphatic system. Although benign, they can compress and infiltrative vital structures. The size of the lesion is more important than its location to the symptomatology development. Intraabdominal and retroperitoneal lymphangioma are the rarest tumour, specially when occurring in adults. The tumour can occur at any age and most are asymptomatic. Preoperative diagnosis is facilitated by ultrasonography and computed tomography. In order to correctly diagnose of these neoplasms it is essential to carry on ultrasound and CT examination. Treatment of choice is always surgical and a complete extirpation should be performed, unless vital structures were are involved.

Influence of a stationary magnetic field on water relations in lettuce seeds. Part I: theoretical considerations.

The theoretical calculation about the dependence of the ionic current density across the cellular membrane on the intensity of the magnetic field applied to cellular tissue is presented. This interaction induces changes in the magnitude of the ionic current density across the cellular membrane and in the ionic concentration, and it also causes alterations in the osmotic pressure and in the capacity of the cellular tissues to absorb water. The magnetic field dependence of the ionic current densities J(p) (B) (positive ions) and J(n) (B) (negative ions), the membrane conductivity sigma (B), the ionic concentration in both membrane sides c(B), the osmotic pressure pi (B), and the water uptake rate by seeds k(w) (B) are presented. The increase in water uptake rate due to the applied magnetic field may be the explanation of the recently reported increase in the germination speed of the seeds treated with stationary magnetic fields.