Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability.

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability.

N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum.

Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system.

Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors.

Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.

Acute promyelocytic leukemia, centre, experience, Turkey.

Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia (AML) and has distinct hematopathologic, cytogenetic, clinical and molecular features. This study was a retrospective review of 18 adult patients (10 male, 8 female; mean age of 32.17 ± 5.66 (15-61 years) with APL at our department from January 2006 to December 2011. Following induction therapy, 17 patients achieved CR, 1 of 18 patients died of result bleeding within thirty-sixth hours of admission. In two of 18 patients developed RAS. The relapse rate was 27% (5/18). Fourteen of 18 patients (77%) have been followed in remission. APL is a malignancy requiring quick diagnosis, efficient treatment and supportive care system. ATO, one of the important therapy option in the treatment of APL, cannot be obtained easily in developing countries. This may lead to an increase in the mortality rates. The studies should be made with more number of patients and a longer period of time for accurate results.

Gender differences in severity of sickle cell diseases in non-smokers.

OBJECTIVE: To find out gender differences in severity of sickle cell diseases (SCDs) in non-smokers. METHODS: Three groups of SCDs patients on the basis of red blood cell (RBC) transfusions were included. Less than 10 units in their lives were kept in Group-1, Ten units of higher in Group-2 and 50 units or higher as the Third Group. Patients with a history of using one pack of cigarettes -year or above were excluded. RESULTS: The study included 269 patients. Mean ages of the groups were similar (28.4, 28.5, and 28.9 years, respectively). Prevalences of cases without any RBC transfusion in their lives were 7.2% and 3.7% in females and males, respectively (p<0.05). Prevalences of cases without any painful crisis were 13.8% and 6.0% in females and males, respectively (p<0.001). There was progressive increase according to mean painful crises, clubbing, chronic obstructive pulmonary disease (COPD), leg ulcers, stroke, chronic renal disease (CRD), pulmonary hypertension, and male ratio from the first towards the third groups (p<0.05, nearly for all). Mean ages of mortal cases were 29.1 and 26.2 years in females and males, respectively (p>0.05). CONCLUSION: The higher painful crises per year, digital clubbing, COPD, leg ulcers, stroke, CRD, pulmonary hypertension, and male ratio of the third group, lower male ratio of patients without any RBC transfusion, lower male ratio of patients without any painful crisis, lower mean ages of male SCDs patients with mortality, and longer overall survival of females in the world could not be explained by well known strong atherosclerotic effects of smoking alone, instead it may be explained by the dominant role of male sex in life.

White coat hypertension may be an initial sign of the metabolic syndrome.

AIM: to understand the role and significance of WCH in definition of the metabolic syndrome. METHODS: the study was performed in the Internal Medicine Polyclinic of the Dumlupinar University between August 2005 and March 2007. We took consecutive patients at and above the age of 20 years. Their medical histories including smoking habit, DM, dyslipidemia, and already used medications were learnt, and a routine check up procedure including fasting plasma glucose (FPG), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and an electrocardiography was performed. Comparison of proportions was used as the method of statistical analysis. RESULTS: the study included 1,068 cases. Prevalences of excess weight increased from the third (28.7%) up to the seventh decades (87.0%), gradually (p<0.05 nearly in all steps), and then decreased in the eighth (78.5%, p<0.05) decade of life. The most significant increase was seen during the passage from the third to the fourth decades (28.7% versus 63.6%, p<0.001) with a similar fashion to smoking. Hyperbetalipoproteinemia, hypertriglyceridemia, dyslipidemia, impaired glucose tolerance (IGT), and WCH had a similar fashion with excess weight by increasing until the seventh decade and decreasing afterwards (p<0.05 nearly in all steps). Whereas hypertension (HT), type 2 diabetes mellitus (DM), and coronary heart disease (CHD) always increased without any decrease by decades (p<0.05 nearly in all steps), indicating their irreversible natures. CONCLUSION: WCH may be an initial sign of the systemic atherosclerotic process that can be detected easily and prevented by a trend towards weight loss.

Mercury toxicity: a family case report.

BACKGROUND: Environmental pollution exposes humans to toxic substances. Herein we present 5 family members aged20-54 years that were poisoned by liquid mercury. CASE REPORTS: Case 1 presented to our clinic with cough, fever, and night sweats. The patient had neutropenia, anemia,and pneumonia, rapidly developed acute respiratory distress syndrome (ARDS), and died on day 4 of hospitalization.Her WBC count was 0.4 × 10³ mm-3 (normal range: 4.3-10.3 × 103 mm-3) and Hb was 10.8 g dL-1 (normal range: 11.5-16.0 g dL-1). Case 2 presented with bicytopenia; the leukocyte count was 1.3 × 103 mm-3 (normal range: 4.3-10.3 × 103mm-3) and the PLT count was 88 × 103 mm-3 (normal range: 150-400 × 103 mm-3). Cases 2 and 3 had toxic peripheralneuropathy. The PLT count in case 3 was 123 × 103 mm-3 (normal range: 150-400 × 103 mm-3). Cases 4 and 5 presentedwith fatigue and headache; these 2 patients did not have positive findings, apart from high levels of mercury in theblood. We have written informed consent. CONCLUSION: We think that heavy metal exposure-although rare-should be considered in patients that present withnumerous symptoms involving multiple systems, including the cardiovascular, respiratory, and neurological systems.The present report is unique in that in describes mercury poisoning in 5 members of the same family.

A mechanistic review of Parkin activation.

Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phospho-ubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation. This review focusses on recent, structural findings on the regulation of Parkin activity. What follows is a mechanistic introduction to the family of E3 ligases that includes Parkin, followed by a brief description of structural elements unique to Parkin that lock the enzyme in an autoinhibited state, contrasted with emerging models that have shed light on possible mechanisms of Parkin activation.

Thrombotic thrombocytopenic purpura: A single - Centre experience.

We aimed to describe the characteristics, treatment regime, and 6-month all-cause mortality of thrombotic thrombocytopenic purpura (TTP) patients treated with total plasma exchange in the our clinic. Thirteen patients were included in the study. Mortality rates of TTP have improved over the last three decades but they are still too high according to modern therapy expectations. Etiology directed treatment should be added to total plasma exchange in secondary TTP cases. Based on TTPs' immunologic etiology, immune modulator and immune suppressor agents have been applied together with total plasma exchange, but mostly in anecdotal case reports or with questionable responses.

A missense mutation in the catalytic domain of O-GlcNAc transferase links perturbations in protein O-GlcNAcylation to X-linked intellectual disability.

X-linked intellectual disabilities (XLID) are common developmental disorders. The enzyme O-GlcNAc transferase encoded by OGT, a recently discovered XLID gene, attaches O-GlcNAc to nuclear and cytoplasmic proteins. As few missense mutations have been described, it is unclear what the aetiology of the patient phenotypes is. Here, we report the discovery of a missense mutation in the catalytic domain of OGT in an XLID patient. X-ray crystallography reveals that this variant leads to structural rearrangements in the catalytic domain. The mutation reduces in vitro OGT activity on substrate peptides/protein. Mouse embryonic stem cells carrying the mutation reveal reduced O-GlcNAcase (OGA) and global O-GlcNAc levels. These data suggest a direct link between changes in the O-GlcNAcome and intellectual disability observed in patients carrying OGT mutations.

An intellectual disability syndrome with single-nucleotide variants in O-GlcNAc transferase.

Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families. OGT encodes O-GlcNAc transferase (OGT), an essential enzyme that catalyses O-linked glycosylation with ß-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins. In this review, we compile the work from the last few years that clearly delineates a new syndromic form of ID, which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG). We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions.

Structural basis of generic versus specific E2-RING E3 interactions in protein ubiquitination.

Protein ubiquitination is a fundamental regulatory component in eukaryotic cell biology, where a cascade of ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes assemble distinct ubiquitin signals on target proteins. E2s specify the type of ubiquitin signal generated, while E3s associate with the E2~Ub conjugate and select the substrate for ubiquitination. Thus, producing the right ubiquitin signal on the right target requires the right E2-E3 pair. The question of how over 600 E3s evolved to discriminate between 38 structurally related E2s has therefore been an area of intensive research, and with over 50 E2-E3 complex structures generated to date, the answer is beginning to emerge. The following review discusses the structural basis of generic E2-RING E3 interactions, contrasted with emerging themes that reveal how specificity can be achieved.

Thrombotic thrombocytopenic purpura associated with Brucella infection.

Thrombotic thrombocytopenic purpura (TTP) is characterized by disseminated thrombotic occlusions located in the microcirculation and a syndrome of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, fever, and renal and neurologic abnormalities. Although several factors such as viral and bacterial pathogens, pancreatitis, drugs, collagen-vascular diseases, cancers, and pregnancy have been reported to be associated with TTP, brucellosis is an exceptional cause of this disorder. We represent a 19-year-old woman applying to our outpatient department with the complaints of headache, fever, sweat, malaise, and jaundice. Clinical signs and laboratory findings were consistent with TTP. Brucella agglutination was found to be 1/320 positive. After the administration of therapeutic plasma exchange, all symptoms and laboratory abnormalities improved dramatically. Antibiotic therapy directed to Brucella infection was initiated and no recurrence of TTP was seen.

The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

O-linked ß-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis.

Spuriously high phosphate level which is promptly resolved after plasmapheresis in a patient with multiple myeloma.

We present a 57 year old female patient with IgG multiple myeloma and marked hyperphosphatemia. The patient had no clinical symptoms related to hyperphosphatemia. Serum inorganic phosphate measurements were repeated on sulfosalicylic acid deproteinized serum samples, yielding normal phosphate levels. We realized that this hyperphosphatemia was spurious because of high paraprotein levels. Afterwards, therapeutic plasma exchange (TPE) was administered due to hyperviscosity and bleeding tendency. After the administration of TPE, serum phosphate was reduced to normal level. Therapeutic plasma exchange resulted in steady-state serum phosphate levels during the three months follow up period. We concluded that high phosphate levels must be confirmed by measuring in deproteinized serum samples in multiple myeloma patients in order to distinguish pseudohyperphosphatemia from the true ones. In addition, TPE effectively reduces the spuriously elevated phosphate levels possibly by removing paraproteins.

Trace elements and some extracellular antioxidant proteins levels in serum of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic progressive autoimmune disorder with a wide spectrum of clinical and immunological abnormalities. In this study, we aimed to investigate the levels of serum zinc (Zn), copper (Cu), magnesium (Mg), manganese (Mn), iron (Fe), ceruloplasmin (Cp), transferrin (Trf), and albumin (Alb) in SLE and whether it is related to the severity of the clinical condition of this chronic disease. Cp and Cu levels were higher, while Trf, Alb, Zn, Mg, Mn, and Fe levels were lower in serum of patients with SLE (n = 27) compared with healthy controls (n = 20). The mechanisms by which these alterations occur in certain inflammatory conditions need to be elucidated. It is also obscure whether these alterations are a cause or a consequence of the inflammation. As a conclusion, alterations in the levels of the parameters in SLE may not be a reason for, but in fact a consequence of the disease itself.

Levels of soluble E-selectin in patients with active Behçet's disease.

Behçet's disease is a systemic vasculitis of unknown aetiology. Endothelial cell injury plays an important role in the pathogenesis and immunopathology of Behçet's disease. E-selectin is expressed by activated endothelial cells. Because the selectin adhesion molecules are shed from activated cells, soluble forms of these proteins can be used as activation markers of endothelium (E-selectin). The pathogenesis of Behçet's disease (BD) is closely related to endothelial cells, leucocyte functions and immunity. The aim of this study was to investigate circulating E-selectin adhesion molecules, which are known to play a significant part in the immune response especially by regulating interaction of the leucocytes with endothelium in BD. Plasma E-selectin concentrations were evaluated in 23 patients with BD and 20 healthy control subjects. The disease activity was evaluated by clinical manifestations (oral aphthous ulcer, genital ulceration, positive pathergy test, skin lesions, eye involvement, thrombophlebitis and arthritis) and by laboratory investigations [erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)]. The patients were newly or previously diagnosed cases not taking any drug for BD. Levels of E-selectin were measured with commercially available sandwich enzyme- linked immunosorbent assay (ELISA) kits using human sE-selectin (cat. no: BMS 205). Plasma E-selectin concentrations of patients and controls were compared with the Mann-Whitney U test. Statistical significance was assigned to p values lower than 0.05. Serum levels (mean±SD) of soluble E-selectin (sE-selectin) were significantly higher in 23 patients with BD than in 20 healthy controls (53.2 ± 18.2 ng/ml vs 33.8 ± 7.5 ng/ml, p < 0.0001). A statistically significant positive correlation was observed between sE-selectin levels and CRP and ESR in patients with BD (r = 0. 78, p < 0.001 and r = 0.56, p < 0.01, respectively). Increases in the E-selectin in BD may be a direct consequence of the leucocyte and endothelium activations observed during the disease process. The noninvasive investigations can be used as biochemical markers for inflammation. This may provide additional information regarding disease activity along with the traditional indices such as ESR and CRP.

Levels of soluble E-selectin in patients with active Behcet's disease.

Behcet's disease is a systemic vasculitis of unknown aetiology. Endothelial cell injury plays an important role in the pathogenesis and immunopathology of Behcet's disease. E-selectin is expressed by activated endothelial cells. Because the selectin adhesion molecules are shed from activated cells, soluble forms of these proteins can be used as activation markers of endothelium (E-selectin). The pathogenesis of Behcet's disease (BD) is closely related to endothelial cells, leucocyte functions and immunity. The aim of this study was to investigate circulating E-selectin adhesion molecules, which are known to play a significant part in the immune response especially by regulating interaction of the leucocytes with endothelium in BD. Plasma E-selectin concentrations were evaluated in 23 patients with BD and 20 healthy control subjects. The disease activity was evaluated by clinical manifestations (oral aphthous ulcer, genital ulceration, positive pathergy test, skin lesions, eye involvement, thrombophlebitis and arthritis) and by laboratory investigations [erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)]. The patients were newly or previously diagnosed cases not taking any drug for BD. Levels of E-selectin were measured with commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits using human sE-selectin (cat. no: BMS 205). Plasma E-selectin concentrations of patients and controls were compared with the Mann-Whitney U test. Statistical significance was assigned to p values lower than 0.05. Serum levels (mean+/-SD) of soluble E-selectin (sE-selectin) were significantly higher in 23 patients with BD than in 20 healthy controls (53.2+/-18.2 ng/ml vs 33.8+/-7.5 ng/ml, p<0.0001). A statistically significant positive correlation was observed between sE-selectin levels and CRP and ESR in patients with BD (r=0.78, p<0.001 and r=0.56, p<0.01, respectively). Increases in the E-selectin in BD may be a direct consequence of the leucocyte and endothelium activations observed during the disease process. The noninvasive investigations can be used as biochemical markers for inflammation. This may provide additional information regarding disease activity along with the traditional indices such as ESR and CRP.

Glutathione peroxidase, glutathione reductase, Cu-Zn superoxide dismutase activities, glutathione, nitric oxide, and malondialdehyde concentrations in serum of patients with chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a rare neoplasm that comprises a substantial proportion of all leukemias in middle-aged persons and is the most common type among elderly persons. The major causes are not known nor is there a detailed understanding about how the elusive origin(s) may relate to clinical expression, basic biological mechanisms, or pathogenesis. METHODS: Glutathione peroxidase (GSH-Px), glutathione reductase (GRD), Cu-Zn superoxide dismutase (Cu-Zn SOD) activities, glutathione (GSH), nitric oxide (NO(*), and malondialdehyde (MDA) concentrations were measured in serum of patients with CLL and a healthy control group. RESULTS: Serum GSH-Px, Cu-Zn SOD activities, GSH concentration were lower in patients with CLL while serum NO(*) and MDA concentrations were higher in these patients compared with the control group. Serum GRD activity was not statistically significant in patients with CLL compared with the control. However, there was no statistically significant difference in the parameters on the basis of stages in these patients. Serum GSH concentration negatively correlated with serum MDA (r=30.63, p<0.05) and NO(*) concentrations (r=0.72, p<0.05) in patients with advanced stage (III+IV). However, no other correlation could be found among the parameters in healthy controls and patients with CLL CONCLUSIONS: There is significant changes in antioxidant defense system in CLL cases, which may lead to enhanced action of oxygen radical, resulting in lipid peroxidation.