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1.  Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein 
Background
Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.
Results
We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.
Conclusion
It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.
doi:10.1186/1472-6807-9-32
PMCID: PMC2695463  PMID: 19454024
2.  Shwachman-Bodian Diamond syndrome is a multi-functional protein implicated in cellular stress responses 
Human Molecular Genetics  2009;18(19):3684-3695.
Shwachman-Diamond syndrome (SDS; OMIM 260400) results from loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene. It is a multi-system disorder with clinical features of exocrine pancreatic dysfunction, skeletal abnormalities, bone marrow failure and predisposition to leukemic transformation. Although the cellular functions of SBDS are still unclear, its yeast ortholog has been implicated in ribosome biogenesis. Using affinity capture and mass spectrometry, we have developed an SBDS-interactome and report SBDS binding partners with diverse molecular functions, notably components of the large ribosomal subunit and proteins involved in DNA metabolism. Reciprocal co-immunoprecipitation confirmed the interaction of SBDS with the large ribosomal subunit protein RPL4 and with DNA-PK and RPA70, two proteins with critical roles in DNA repair. Function for SBDS in response to cellular stresses was implicated by demonstrating that SBDS-depleted HEK293 cells are hypersensitive to multiple types of DNA damage as well as chemically induced endoplasmic reticulum stress. Furthermore, using multiple routes to impair translation and mimic the effect of SBDS-depletion, we show that SBDS-dependent hypersensitivity of HEK293 cells to UV irradiation can be distinguished from a role of SBDS in translation. These results indicate functions of SBDS beyond ribosome biogenesis and may provide insight into the poorly understood cancer predisposition of SDS patients.
doi:10.1093/hmg/ddp316
PMCID: PMC2742402  PMID: 19602484
3.  Pluripotent stem cell models of Shwachman-Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction 
Cell stem cell  2013;12(6):727-736.
Summary
Shwachman-Diamond syndrome (SDS), a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematopoietic dysfunction, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. We created human pluripotent stem cell models of SDS by knock-down of SBDS in human embryonic stem cells (hESCs) and generation of induced pluripotent stem cell (iPSC) lines from two SDS patients. SBDS-deficient hESCs and iPSCs manifest deficits in exocrine pancreatic and hematopoietic differentiation in vitro, enhanced apoptosis and elevated protease levels in culture supernatants, which could be reversed by restoring SBDS protein expression through transgene rescue or by supplementing culture media with protease inhibitors. Protease-mediated auto-digestion provides a mechanistic link between the pancreatic and hematopoietic phenotypes in SDS, highlighting the utility of hESCs and iPSCs in obtaining novel insights into human disease.
doi:10.1016/j.stem.2013.04.002
PMCID: PMC3755012  PMID: 23602541
4.  The NIP7 protein is required for accurate pre-rRNA processing in human cells 
Nucleic Acids Research  2010;39(2):648-665.
Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman–Bodian–Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S–80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.
doi:10.1093/nar/gkq758
PMCID: PMC3025556  PMID: 20798176
5.  SBDS Expression and Localization at the Mitotic Spindle in Human Myeloid Progenitors 
PLoS ONE  2009;4(9):e7084.
Background
Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified.
Methodology/Principal Findings
We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle.
Conclusions/Significance
SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34+ hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained.
doi:10.1371/journal.pone.0007084
PMCID: PMC2738965  PMID: 19759903
6.  Altered Intracellular Localization and Mobility of SBDS Protein upon Mutation in Shwachman-Diamond Syndrome 
PLoS ONE  2011;6(6):e20727.
Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.
doi:10.1371/journal.pone.0020727
PMCID: PMC3113850  PMID: 21695142
7.  Clinical spectrum and molecular pathophysiology of Shwachman-Diamond syndrome 
Current opinion in hematology  2011;18(1):30-35.
PURPOSE OF REVIEW
Shwachman Diamond syndrome (SDS) is an inherited bone marrow failure and cancer predisposition syndrome that affects multiple organ systems. Mutations in the SBDS gene are found in the majority of patients, but the molecular function of the SBDS protein product remains unclear. Here, we review recent progress in the clinical and molecular characterization of SDS.
RECENT FINDINGS
Emerging data support a multifunctional role for the SBDS protein. Current studies indicate that SBDS functions in 60S large ribosomal subunit maturation and in mitotic spindle stabilization. Recent data suggest it may also affect actin polymerization, vacuolar pH regulation and DNA metabolism. SBDS loss results in both hematopoietic cell-intrinsic defects as well as marrow stromal abnormalities.
SUMMARY
SDS is a multisystemic disease arising from defects in a protein that participates in several essential cellular processes. Elucidating the molecular function of SBDS will provide important insights into how defects in ribosome biogenesis and mitotic spindle stabilization result in hematopoietic failure, cancer predisposition, and abnormalities.
doi:10.1097/MOH.0b013e32834114a5
PMCID: PMC3485416  PMID: 21124213
bone marrow failure; leukemia; neutropenia; ribosome; mitotic spindle
8.  Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome 
The Journal of Clinical Investigation  2008;118(4):1511-1518.
Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.
doi:10.1172/JCI33764
PMCID: PMC2263145  PMID: 18324336
9.  SBDS Protein Expression Patterns in the Bone Marrow 
Pediatric blood & cancer  2010;55(3):546-549.
Shwachman Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome caused by biallelic SBDS gene mutations. Here we examined SBDS protein levels in human bone marrow. SBDS protein expression was high in neutrophil progenitors, megakaryocytes, plasma cells and osteoblasts. In contrast, SBDS protein levels were low in all hematopoietic cell lineages from patients harboring the common SBDS mutations. We conclude that SBDS protein levels vary widely between specific marrow lineages. Uniformly low SBDS protein expression levels distinguish the majority of SDS patients from controls or other marrow failure syndromes.
doi:10.1002/pbc.22573
PMCID: PMC2913690  PMID: 20658628
Shwachman Diamond Syndrome; SBDS; bone marrow failure; neutropenia; immunohistochemistry
10.  The Shwachman–Bodian–Diamond syndrome gene mutations cause a neonatal form of spondylometaphysial dysplasia (SMD) resembling SMD Sedaghatian type 
Journal of Medical Genetics  2007;44(4):e73.
The Shwachman–Bodian–Diamond syndrome (SBDS) gene is a causative gene for Shwachman–Diamond syndrome, an autosomal recessive disorder with exocrine pancreatic insufficiency, bone marrow dysfunction and skeletal dysplasia. We report here on two patients with skeletal manifestations at the severest end of the phenotypic spectrum of SBDS mutations. An 11‐year‐old Japanese girl presented with neonatal respiratory failure necessitating lifelong ventilation support, severe short stature and severe developmental delay. She developed neutropenia in infancy, and decreased serum amylase was noted in childhood. A British boy was a stillbirth with pulmonary hypoplasia and hepatic fibrosis found on autopsy. Both cases had neonatal skeletal manifestations that included platyspondyly, lacy iliac crests and severe metaphysial dysplasia, and thus did not fall in the range of the known Shwachman–Diamond syndrome skeletal phenotype but resembled spondylometaphysial dysplasia (SMD) Sedaghatian type. The girl harboured a recurrent mutation (183TA→CT) and a novel missense mutation (79T→C), whereas the boy carried two recurrent mutations (183TA→CT and 258+2T→C). We also examined SBDS in one typical case with SMD Sedaghantian type and eight additional cases with neonatal SMD, but failed to discover SBDS mutations. Our experience expands the phenotypic spectrum of SBDS mutations, which, at its severest end, results in severe neonatal SMD.
doi:10.1136/jmg.2006.043869
PMCID: PMC2598034  PMID: 17400792
11.  Interaction between Sdo1p and Btn1p in the Saccharomyces cerevisiae model for Batten disease 
Human Molecular Genetics  2009;19(5):931-942.
Juvenile Batten disease is an autosomal recessive pediatric neurodegenerative disorder caused by mutations in the CLN3 gene. The CLN3 protein primarily resides in the lysosomal membrane, but its function is unknown. We demonstrate that CLN3 interacts with SBDS, the protein mutated in Shwachman–Bodian–Diamond syndrome patients. We demonstrate that this protein–protein interaction is conserved between Btn1p and Sdo1p, the respective yeast Saccharomyces cerevisiae orthologs of CLN3 and SBDS. It was previously shown that deletion of BTN1 results in alterations in vacuolar pH and vacuolar (H+)-ATPase (V-ATPase)-dependent H+ transport and ATP hydrolysis. Here, we report that an SDO1 deletion strain has decreased vacuolar pH and V-ATPase-dependent H+ transport and ATP hydrolysis. These alterations result from decreased V-ATPase subunit expression. Overexpression of BTN1 or the presence of ionophore carbonyl cyanide m-chlorophenil hydrazone (CCCP) causes decreased growth in yeast lacking SDO1. In fact, in normal cells, overexpression of BTN1 mirrors the effect of CCCP, with both resulting in increased vacuolar pH due to alterations in the coupling of V-ATPase-dependent H+ transport and ATP hydrolysis. Thus, we propose that Sdo1p and SBDS work to regulate Btn1p and CLN3, respectively. This report highlights a novel mechanism for controlling vacuole/lysosome homeostasis by the ribosome maturation pathway that may contribute to the cellular abnormalities associated with juvenile Batten disease and Shwachman–Bodian–Diamond syndrome.
doi:10.1093/hmg/ddp560
PMCID: PMC2816617  PMID: 20015955
12.  Bone progenitor dysfunction induces myelodysplasia and secondary leukemia 
Nature  2010;464(7290):852-857.
Mesenchymal cell populations contribute to microenvironments regulating stem cells and the growth of malignant cells. Osteolineage cells participate in the hematopoietic stem cell niche. Here, we report that deletion of the miRNA processing endonuclease Dicer1 selectively in mesenchymal osteoprogenitors induces markedly disordered hematopoiesis. Hematopoietic changes affected multiple lineages recapitulating key features of human myelodysplastic syndrome (MDS) including the development of acute myelogenous leukemia. These changes were microenvironment dependent and induced by specific cells in the osteolineage. Dicer1−/− osteoprogenitors expressed reduced levels of Sbds, the gene mutated in the human bone marrow failure and leukemia predisposition Shwachman-Bodian-Diamond Syndrome. Deletion of Sbds in osteoprogenitors largely phenocopied Dicer1 deletion. These data demonstrate that differentiation stage-specific perturbations in osteolineage cells can induce complex hematological disorders and indicate the central role individual cellular elements of ‘estroma’ can play in tissue homeostasis. They reveal that primary changes in the hematopoietic microenvironment can initiate secondary neoplastic disease.
doi:10.1038/nature08851
PMCID: PMC3422863  PMID: 20305640
13.  Loss of the Mouse Ortholog of the Shwachman-Diamond Syndrome Gene (Sbds) Results in Early Embryonic Lethality†  
Molecular and Cellular Biology  2006;26(17):6656-6663.
Mutations in SBDS are responsible for Shwachman-Diamond syndrome (SDS), a disorder with clinical features of exocrine pancreatic insufficiency, bone marrow failure, and skeletal abnormalities. SBDS is a highly conserved protein whose function remains largely unknown. We identified and investigated the expression pattern of the murine ortholog. Variation in levels was observed, but Sbds was found to be expressed in all embryonic stages and most adult tissues. Higher expression levels were associated with rapid proliferation. A targeted disruption of Sbds was generated in order to understand the consequences of its loss in an in vivo model. Consistent with recessive disease inheritance for SDS, Sbds+/− mice have normal phenotypes, indistinguishable from those of their wild-type littermates. However, the development of Sbds−/− embryos arrests prior to embryonic day 6.5, with muted epiblast formation leading to early lethality. This finding is consistent with the absence of patients who are homozygous for early truncating mutations. Sbds is an essential gene for early mammalian development, with an expression pattern consistent with a critical role in cell proliferation.
doi:10.1128/MCB.00091-06
PMCID: PMC1592835  PMID: 16914746
14.  Non-DBA Disorders of Ribosome Function: Shwachman-Diamond Syndrome and 5q- Syndrome 
Seminars in hematology  2011;48(2):136-143.
A number of human disorders, dubbed ribosomopathies, are linked to impaired ribosome biogenesis or function. These include but are not limited to: Diamond Blackfan anemia (DBA), Shwachman Diamond syndrome (SDS) and the 5q- myelodysplastic syndrome. This review focuses on the latter two non-DBA disorders of ribosome function. Both SDS and 5q- syndrome lead to impaired hematopoiesis and a predisposition to leukemia. SDS, due to bi-allelic mutations of the SBDS gene, is a multi-system disorder that also includes bony abnormalities, pancreatic and neurocognitive dysfunction. SBDS associates with the 60S subunit in human cells and has a role in subunit joining and translational activation in yeast models. In contrast, 5q- syndrome is associated with acquired haploinsufficiency of RPS14, a component of the small 40S subunit. RPS14 is critical for 40S assembly in yeast models, and depletion of RPS14 in human CD34+ cells is sufficient to recapitulate the 5q- erythroid defect. Both SDS and the 5q- syndrome represent important models of ribosome function and may inform future treatment strategies for the ribosomopathies.
doi:10.1053/j.seminhematol.2011.01.002
PMCID: PMC3072806  PMID: 21435510
15.  PHENOTYPIC AND GENETIC CHARACTERIZATION OF PATIENTS WITH FEATURES OF “NONCLASSIC” FORMS OF CYSTIC FIBROSIS 
The Journal of Pediatrics  2005;146(5):675-680.
Objective
To determine which features of incomplete or “nonclassic” forms of cystic fibrosis (CF) are associated with deleterious CF transmembrane conductance regulator gene (CFTR) mutations, and to explore other etiologies for features not associated with deleterious CFTR mutations.
Study design
Clinical features were compared between 57 patients with deleterious mutations in each CFTR and 63 with no deleterious mutations. The Shwachman Bodian Diamond syndrome gene (SBDS) was sequenced to search for mutations in patients with no deleterious CFTR mutations and steatorrhea to determine if any had unrecognized Shwachman-Diamond syndrome (SDS).
Results
The presence of a common CF-causing mutation, absence of the vas deferens, and Pseudomona aeruginosa in the sputum correlated with the presence of two deleterious CFTR mutations, whereas sweat chloride concentration, diagnostic criteria for CF, and steatorrhea did not. However, sweat chloride concentration correlated with CFTR mutation status in patients infected with P aeruginosa. One patient had disease-causing mutations in each SBDS.
Conclusions
Presence of a common CF-causing mutation, absence of the vas deferens and/or P aeruginosa infection in a patient with features of nonclassic CF are predictive of deleterious mutations in each CFTR, whereas steatorrhea in the same context is likely to have etiologies other than CF transmembrane conductance regulator (CFTR) dysfunction.
doi:10.1016/j.jpeds.2004.12.020
PMCID: PMC3380804  PMID: 15870673
16.  Disease-specific induced pluripotent stem (iPS) cells 
Cell  2008;134(5):877-886.
Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research, but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance that include: adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21 and the carrier state of Lesch-Nyhan syndrome. Such patient-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.
doi:10.1016/j.cell.2008.07.041
PMCID: PMC2633781  PMID: 18691744
17.  Shwachman-Diamond syndrome: first molecular diagnosis in a Brazilian child 
Herein the first molecular diagnosis of a Brazilian child with Shwachman-Diamond Syndrome is reported. A 6-year-old boy was diagnosed with cystic fibrosis at the age of 15 months due to recurrent respiratory infections, diarrhea and therapeutic response to pancreatic enzymes. Three sweat tests were negative. At the age of 5 years, he began to experience pain in the lower limbs, laxity of joints, lameness and frequent falls. A radiological study revealed metaphyseal chondrodysplasia. A complete blood cell count showed leukopenia (leukocytes: 3.1-3.5 x 103/µL), neutropenia (segmented neutrophils: 15-22%), but normal hemoglobin, hematocrit and platelet count. A molecular study revealed biallelic mutations in the Shwachman-Bodian-Diamond Syndrome gene (183-184TA-CT K62X in exon 2 and a 258+2T-C transition) confirming the diagnosis of Shwachman-Diamond Syndrome. A non-pathologic, silent nucleotide A to G transition at position 201 was also found in heterozygosis in the Shwachman-Bodian-Diamond Syndrome gene. This is the first report to describe a Brazilian child with molecular diagnosis of Shwachman-Diamond Syndrome, a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, intermittent or persistent neutropenia and skeletal changes. Other characteristics include immune system, hepatic and cardiac changes and predisposition to leukemia. Recurrent bacterial, viral and fungal infections are common. The possibility of Shwachman-Diamond Syndrome should be kept in mind when investigating children with a diagnosis of cystic fibrosis and normal sweat tests.
doi:10.5581/1516-8484.20130058
PMCID: PMC3789437  PMID: 24106450
Leukopenia/genetics; Exocrine pancreatic insufficiency/genetics; Cystic fibrosis; Bacterial infections; Humans; Male; Child; Case reports
18.  Alterations in the ribosomal machinery in cancer and hematologic disorders 
Ribosomes are essential components of the protein translation machinery and are composed of more than 80 unique large and small ribosomal proteins. Recent studies show that in addition to their roles in protein translation, ribosomal proteins are also involved in extra-ribosomal functions of DNA repair, apoptosis and cellular homeostasis. Consequently, alterations in the synthesis or functioning of ribosomal proteins can lead to various hematologic disorders. These include congenital anemias such as Diamond Blackfan anemia and Shwachman Diamond syndrome; both of which are associated with mutations in various ribosomal genes. Acquired uniallelic deletion of RPS14 gene has also been shown to lead to the 5q syndrome, a distinct subset of MDS associated with macrocytic anemia. Recent evidence shows that specific ribosomal proteins are overexpressed in liver, colon, prostate and other tumors. Ribosomal protein overexpression can promote tumorigenesis by interactions with the p53 tumor suppressor pathway and also by direct effects on various oncogenes. These data point to a broad role of ribosome protein alterations in hematologic and oncologic diseases.
doi:10.1186/1756-8722-5-32
PMCID: PMC3438023  PMID: 22709827
Ribosome; MDS; Anemia
19.  Crystallization of a functionally intact Hsc70 chaperone 
Success in crystallization of a functionally intact Hsp70 chaperone required genetic engineering to minimize polydispersity and modulate interdomain interactions, as well as high concentrations of the potent structure stabilizer TMAO. These approaches may be generally useful in crystallization of conformationally flexible proteins that exhibit interdomain motions.
Hsp70s are essential chaperones with roles in a variety of cellular processes and representatives in all kingdoms of life. They are comprised of a nucleotide-binding domain (NBD) and a protein substrate-binding domain (SBD). Structures of isolated NBDs and SBDs have been reported but, until recently, a functionally intact Hsp70 containing both the NBD and SBD has resisted structure determination. Here, it is reported that preparation of diffraction-quality crystals of functionally intact bovine Hsc70 required (i) deletion of part of the protein to reduce oligomerization, (ii) point mutations in the interface between the SBD and NBD and (iii) use of high concentrations of the structure-stabilizing agents glycerol and trimethylamine oxide (TMAO). The introduction of point mutations in interdomain interfaces and the use of the potent structure stabilizer TMAO may be generally useful in crystallization of multidomain proteins that exhibit interdomain motions.
doi:10.1107/S1744309105040303
PMCID: PMC2150933  PMID: 16511258
Hsp70; Hsc70; chaperones
20.  Four Amino Acids within a Tandem QxVx Repeat in a Predicted Extended α-Helix of the Smad-Binding Domain of Sip1 Are Necessary for Binding to Activated Smad Proteins 
PLoS ONE  2013;8(10):e76733.
The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFβ family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFβ/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFβ and/or BMP (de)regulated cellular, developmental and pathological processes.
doi:10.1371/journal.pone.0076733
PMCID: PMC3795639  PMID: 24146916
21.  Quality and Quantity of Information in Summary Basis of Decision Documents Issued by Health Canada 
PLoS ONE  2014;9(3):e92038.
Background
Health Canada’s Summary Basis of Decision (SBD) documents outline the clinical trial information that was considered in approving a new drug. We examined the ability of SBDs to inform clinician decision-making. We asked if SBDs answered three questions that clinicians might have prior to prescribing a new drug: 1) Do the characteristics of patients enrolled in trials match those of patients in their practice? 2) What are the details concerning the drug’s risks and benefits? 3) What are the basic characteristics of trials?
Methods
14 items of clinical trial information were identified from all SBDs published on or before April 2012. Each item received a score of 2 (present), 1 (unclear) or 0 (absent). The unit of analysis was the individual SBD, and an overall SBD score was derived based on the sum of points for each item. Scores were expressed as a percentage of the maximum possible points, and then classified into five descriptive categories based on that score. Additionally, three overall ‘component’ scores were tallied for each SBD: “patient characteristics”, “benefit/risk information” and “basic trial characteristics”.
Results
161 documents, spanning 456 trials, were analyzed. The majority (126/161) were rated as having information sometimes present (score of >33 to 66%). No SBDs had either no information on any item, or 100% of the information. Items in the patient characteristics component scored poorest (mean component score of 40.4%), while items corresponding to basic trial information were most frequently provided (mean component score of 71%).
Conclusion
The significant omissions in the level of clinical trial information in SBDs provide little to aid clinicians in their decision-making. Clinicians’ preferred source of information is scientific knowledge, but in Canada, access to such information is limited. Consequently, we believe that clinicians are being denied crucial tools for decision-making.
doi:10.1371/journal.pone.0092038
PMCID: PMC3961288  PMID: 24651766
22.  Radiographic evaluation on prevalence of Stafne bone defect: a study from two centres in Turkey 
Dentomaxillofacial Radiology  2012;41(2):152-158.
Objectives
The aim of this study was to investigate the frequency of Stafne bone defect (SBD) and to describe the clinical and radiological characteristics of detected cases.
Methods
A retrospective study was performed using panoramic radiographs from 34 221 patients undergoing dental treatment in the Department of Oral and Maxillofacial Radiology at Erciyes University and Ataturk University, Turkey. After finding an image compatible with SBD in the radiographs, multislice CT (MSCT) on seven patients and cone beam CT (CBCT) on six patients were performed to confirm the diagnosis.
Results
Of the 34 221 patients, 29 (0.08 %) had SBDs, of whom 4 were female (13.8%) and 25 were male (86.2 %). The age range of patients with SBD was 18–77 years (mean age 49.6 years). SBD was found in the lingual molar region in 28 patients and in the lingual canine–premolar region of the mandible in 1 patient. The contour of the concavities on CT images (MSCT and CBCT) was detected. The MSCT revealed glandular tissue within the defects.
Conclusions
According to our results, SBD is an uncommon anomaly. Examination of MSCT images supports the presence of aberrant submandibular glands within these mandibular defects, suggesting that pressure from submandibular gland tissue had caused the SBD, as generally thought. Both CBCT and MSCT can provide adequate support for the detection of SBDs. The CBCT could be suggested as the most suitable non-invasive diagnostic modality for this bony configuration of the mandible since it provides a lower radiation exposure dose than MSCT.
doi:10.1259/dmfr/10586700
PMCID: PMC3520381  PMID: 22074869
bone cyst; computer-assisted three-dimensional imaging; panoramic radiography; cone beam computed tomography; salivary glands
23.  PREVALENCE OF MUTATIONS IN ELANE, GFI1, HAX1, SBDS, WAS, AND G6PC3 IN PATIENTS WITH SEVERE CONGENITAL NEUTROPENIA 
British journal of haematology  2009;147(4):535-542.
SUMMARY
Severe congenital neutropenia (SCN) is a genetically heterogeneous syndrome associated with mutations of ELANE (ELA2), HAX1, GFI1, WAS, CSF3R or G6PC3. We investigated the prevalence of mutations of ELANE in a cohort of 162 SCN patients for whom blood or bone marrow samples were submitted to the North American Severe Chronic Neutropenia Tissue Repository. Mutations of ELANE were found in 90 of 162 patients (55.6%). Subsequently, we conducted an analysis of a subset of 73 of these cases utilizing a high throughput sequencing approach to determine the prevalence of other mutations associated with SCN. Among the 73 patients, mutations of ELANE were detected in 28. In the remaining 45 patients with wild type ELANE alleles, 5 patients had mutations: GFI1 (1), SBDS (1), WAS (1) and G6PC3 (2); no mutations of HAX1 were detected. In approximately 40% of our cases, the genetic basis of SCN remains unknown. These data suggest that for genetic diagnosis of SCN, ELANE genotyping should first be performed. In patients without ELANE mutations, other known SCN-associated gene mutations will be found rarely and genotyping can be guided by the clinical features of each patient.
doi:10.1111/j.1365-2141.2009.07888.x
PMCID: PMC2783282  PMID: 19775295
bone marrow failure; chronic neutropenia; DNA mutation
24.  'Diabetology & Metabolic Syndrome: providing an open access future for diabetes research' 
Diabetology & Metabolic Syndrome (D&MS), the official journal of the Brazilian Diabetes Society (SBD), is a new open access, peer reviewed journal publishing research on all aspects of the pathophysiology of diabetes and metabolic syndrome. With the many ongoing and upcoming challenges for diabetes diagnosis, treatment and care, a dedicated journal providing unrestricted access for researchers and health care professionals working in the field of diabetes is needed. Diabetology & Metabolic Syndrome aims to fulfil this need.
doi:10.1186/1758-5996-1-1
PMCID: PMC2758596  PMID: 19825197
25.  Starch-Binding Domain Affects Catalysis in Two Lactobacillus α-Amylases 
A new starch-binding domain (SBD) was recently described in α-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus α-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus α-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities.
doi:10.1128/AEM.71.1.297-302.2005
PMCID: PMC544272  PMID: 15640201

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