Glucocorticoids improve erythroid progenitor maintenance and dampen Trp53 response in a mouse model of Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.

p16 immunohistochemistry can be used to detect human papillomavirus in oral cavity squamous cell carcinoma.

PURPOSE: Human papillomavirus (HPV) is of etiologic significance in the development of oral squamous carcinoma and is noted to result in p16 overexpression. Identification of HPV is clinically important because the presence of HPV has prognostic and epidemiologic associations. Detection of HPV by polymerase chain reaction (PCR) is expensive and not widely accessible. The authors examined p16 immunohistochemistry (IHC) as a surrogate marker for high-risk HPV and its use as an alternative test to PCR. PATIENTS AND METHODS: A retrospective cohort of patients with oral squamous cell carcinoma underwent surgery and then analysis with p16 IHC and HPV PCR. The p16 IHC staining intensity was graded from 0 to 3+, and these results were compared with PCR. Descriptive and frequency statistics were performed by comparing HPV PCR results with p16 IHC, patient age, gender, and outcome. RESULTS: Eighty-one cases were included in the study. Forty-four study patients were men and 37 were women (mean age, 63.9 yr). Forty-five cases (55.6%) had 0 staining, 22 cases (27.2%) had 1+ staining, and 7 cases (8.6%) had 2+ staining. Seven cases (8.6%) had 3+ staining, all of which were positive for HPV serotype 16 by PCR. Three of 7 HPV PCR-positive cases had keratinization typical of an oral cavity location and not the basaloid growth of HPV oropharyngeal tumors. There was a statistical correlation (P < .001) among HPV PCR positivity, 3+ staining, and younger age. CONCLUSION: p16 3+ staining correlates with HPV PCR positivity. p16 IHC is a technically simple and widely available test, and this study establishes the use of p16 IHC as an alternative test to HPV PCR. Given the clinical significance of HPV in oral squamous carcinoma, p16 IHC should be performed in all cases and included in the pathology report.

A Case of Chronic Thrombocytopenia in a 17-Year-Old Female.

Storage pool deficiency (SPD) is a group of rare platelet disorders that result from deficiencies in α-granules, δ-granules, or both. One type of α-SPD is gray platelet syndrome (GPS), caused by mutations in the neurobeachin-like 2 (NBEAL2) gene that results in a bleeding diathesis, thrombocytopenia, splenomegaly, and progressive myelofibrosis. Due to the lack of α-granules, platelets have a gray and degranulated appearance by light microscopy. However, definitive diagnosis of GPS requires confirmation of α-granule deficiency by electron microscopy. Treatment is nonspecific, with the conservative utilization of platelet transfusions being the most important form of therapy. We present a case of a 17-year-old female with a past medical history of thrombocytopenia, first identified at the age of five. Her clinical symptomatology included chronic fatigue, gingival bleeding, bruising, menorrhagia, and leg pain. This report will discuss both the clinical and the pathophysiologic aspects of this rare platelet disorder.

Multi-Parameter Laser Imaging Reveals Complex Microscale Biofilm Matrix in a Thick (4,000 µm) Aerobic Methanol Oxidizing Community.

Although methanol has frequently been used as an inexpensive supplementary carbon source to support treatment processes, knowledge of the resultant microbial biofilms, their 3D architecture, microenvironments, exopolymer chemistry and populations remains limited. We supplied methanol as a supplementary carbon source to biofilms developing in rotating annular reactors. Analysis of circulation waters (1.0 l d-1) indicated that dissolved organic carbon was reduced by 25%, NO3-nitrogen by 95%, and total phosphorus by 70%. Analyses of populations using culture based techniques and fluorescence in situ hybridization indicated enrichment of nitrifiers, denitrifiers, and methylotrophic bacteria relative to reference biofilms not receiving methanol. The biofilms that developed were up to 4,000 µm thick. Staining with fluor conjugated lectins in combination with nucleic acid stains, revealed the presence of discrete bacterial cells inside complex globular polymeric structures. These structures were in turn surrounded by an interstitial polymer containing a variety of bacterial cell types. The globular structures bound FITC-conjugated lectins, from Canavalia ensiformis and Ulex europeaus. The FITC-lectin of Phaseolus vulgaris bound the surface of the globular structures and more generally within the matrix. Chemical analyses of the polymer paralleled the results of lectin analyses indicating that the dominant neutral sugars were glucose, galactose, mannose, rhamnose, with fucose and ribose as minor constituents. Amino sugars were not detected. Dual channel imaging with pH sensitive probes indicated that pH gradients from pH 4 to 7 occurred across the globular microcolonies. Critically for the maintenance of aerobic conditions throughout the thick biofilm it was extensively penetrated by a fine fissure network revealed by the location of fluorescent latex microbeads as detected by confocal laser scanning microscopy. Microelectrode studies confirmed the absence of any detectable Eh gradients within the biofilm. However, mobility of various size-fractionated fluorescent probes indicated that the basal region was only penetrated by the lowest molecular weight probes with a hydrated radius of 2.2 nm or less. These observations indicate the selection of a unique, thick (>4,000 µm) microbial community in which a self-organized architecture promotes the maintenance of optimal conditions and metabolism throughout the biofilm community.

The putative tumor suppressor gene EphA7 is a novel BMI-1 target.

Bmi1 was originally identified as a gene that contributes to the development of mouse lymphoma by inhibiting MYC-induced apoptosis through repression of Ink4a and Arf. It codes for the Polycomb group protein BMI-1 and acts primarily as a transcriptional repressor via chromatin modifications. Although it binds to a large number of genomic regions, the direct BMI-1 target genes described so far do not explain the full spectrum of BMI-1-mediated effects. Here we identify the putative tumor suppressor gene EphA7 as a novel direct BMI-1 target in neural cells and lymphocytes. EphA7 silencing has been reported in several different human tumor types including lymphomas, and our data suggest BMI1 overexpression as a novel mechanism leading to EphA7 inactivation via H3K27 trimethylation and DNA methylation.

Evaluation of liquid chromatography-negative ion electrospray mass spectrometry for the determination of selected resin acids in river water.

A liquid chromatography-negative ion electrospray mass spectrometric (LC-ESI-MS) method was evaluated for detection of four prevalent softwood-derived resin acids in natural water. Method detection limits based on a signal-to-noise ratio of 3:1 in river water samples of 0.40, 0.40, 0.30 and 0.25 microg l(-1) for abietic, dehydroabietic, isopimaric and pimaric acids, respectively, are comparable or lower than reported GC methods. Unlike the majority of GC methods, however, the three structural resin acid isomers (abietic, isopimaric and pimaric acids) do not separate sufficiently under the various LC conditions evaluated in this work. Therefore, LC-ESI-MS may not be suitable for instances where measurement of individual isomeric resin acids is required. However, the method is suitable for trace analysis of resin acids in natural waters where isomeric speciation is not required.

Determination of dissolved naphthenic acids in natural waters by using negative-ion electrospray mass spectrometry.

Naphthenic acids (NAs) have been implicated as some of the most toxic substances in oil sands leachates and identified as priority substances impacting on aquatic environments. As a group of compounds, NAs are not well characterized and comprise a large group of saturated aliphatic and alicyclic carboxylic acids found in hydrocarbon deposits (petroleum, oil sands bitumen, and crude oils). Described is an analytical method using negative-ion electrospray ionization mass spectrometry (ES/MS) of extracts. Preconcentration was achieved by using a solid-phase extraction procedure utilizing a crosslinked polystyrene-based polymer with acetonitrile elution. Recovery of the Fluka Chemicals NA mixture was highly pH-dependent, with 100% recovery at pH 3.0, but only 66 and 51% recoveries at pHs 7 and 9, respectively. The dissolved phase of the NA was very dependent on sample pH. It is thus critical to measure the pH and determine the appropriate mass profiles to identify NAs in natural waters. The ES/MS analytical procedure proved to be a fast and sensitive method for the recovery and detection of NAs in natural waters, with a detection limit of 0.01 mg/L.

High grade primitive neuroectodermal tumor of the uterus: A case report.

•Primitive neuroectodermal tumor of the uterus is extremely rare.•Diagnosis requires timely evaluation with molecular analysis.•Different combinations of adjuvant chemotherapy have been reported.

Microphthalmia transcription factor regulates pancreatic ß-cell function.

Precise regulation of ß-cell function is crucial for maintaining blood glucose homeostasis. Pax6 is an essential regulator of ß-cell-specific factors like insulin and Glut2. Studies in the developing eye suggest that Pax6 interacts with Mitf to regulate pigment cell differentiation. Here, we show that Mitf, like Pax6, is expressed in all pancreatic endocrine cells during mouse postnatal development and in the adult islet. A Mitf loss-of-function mutation results in improved glucose tolerance and enhanced insulin secretion but no increase in ß-cell mass in adult mice. Mutant ß-cells secrete more insulin in response to glucose than wild-type cells, suggesting that Mitf is involved in regulating ß-cell function. In fact, the transcription of genes critical for maintaining glucose homeostasis (insulin and Glut2) and ß-cell formation and function (Pax4 and Pax6) is significantly upregulated in Mitf mutant islets. The increased Pax6 expression may cause the improved ß-cell function observed in Mitf mutant animals, as it activates insulin and Glut2 transcription. Chromatin immunoprecipitation analysis shows that Mitf binds to Pax4 and Pax6 regulatory regions, suggesting that Mitf represses their transcription in wild-type ß-cells. We demonstrate that Mitf directly regulates Pax6 transcription and controls ß-cell function.

Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to K(IR)6.2, ligand binding, and oligomerization.

ATP-sensitive K(+) (K(ATP)) channels consist of two types of subunits, K(IR)6.x that form the pore, and sulfonylurea receptors (SURs) that serve as regulatory subunits. SURs are ATP-binding cassette (ABC) proteins and contain, in addition to two nucleotide binding folds, the binding sites for channel openers such as diazoxide and P1075 and channel inhibitors such as glibenclamide (GBC) and repaglinide. Structurally, SURs differ from most eukaryotic ABC proteins by an additional amino-terminal transmembrane domain (TMD0); in case of SUR1, the subunit of the pancreatic K(ATP) channel, TMD0 serves as a major domain for association with K(IR). In this study we sought to elucidate the roles of TMD0 in SUR2B, the smooth muscle gating subunit, in the coupling between SUR2B and K(IR)6.2, in the self-association of SUR2B and in channel modulator binding to SUR2B. SUR2B has a weaker affinity for sulfonylureas thus SUR2B(Y1206S), with a higher affinity for GBC, but an equivalent opener binding was used. Association of SUR2B(YS)Δ, lacking TMD0, with K(IR)6.2 was shown by immunoprecipitation; however, no evidence for formation of functional channels was obtained. SUR2B(YS)Δ self-associates like SUR2B(YS) and binds GBC, repaglinide, and P1075 with slightly reduced affinities. The binding profile of the SUR2B(YS)Δ/K(IR)6.2 complex differs slightly but significantly from that of SUR2B(YS)Δ alone showing impaired allosteric coupling of binding sites. We conclude that TMD0 is not required for oligomerization of SUR2B, is of only minor importance in ligand binding, but is essential for both functional and allosteric coupling of SUR2B to K(IR)6.2.

Importance of the Kir6.2 N-terminus for the interaction of glibenclamide and repaglinide with the pancreatic K(ATP) channel.

The pancreatic K(ATP) channel, SUR1/Kir6.2, couples insulin secretion to the plasma glucose level. The channel is an octamer with four Kir6.2 subunits forming the pore and four sulphonylurea receptors (SUR1) regulating channel activity. SUR1 is an ABC protein with adenosine triphosphate (ATP)ase activity which activates the channel. It also contains the binding site for antidiabetic drugs like glibenclamide and repaglinide which close the channel by disrupting the stimulatory effect SUR-ATPase (MgATP-dependent) and by stabilising a long-lived closed channel state (MgATP-independent). In this study, we examined the effects of progressive truncation of the Kir6.2 N-terminus up to 20 amino acids on equilibrium binding and channel closure by glibenclamide and repaglinide, on the channel activating effect of the opener, 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4thiadiazine 1,1-dioxide (NNC 55-0462), and on the binding kinetics of [(3)H]glibenclamide. Kir and SUR were transiently coexpressed in HEK cells and [(3)H]glibenclamide binding and patch-clamp experiments were performed in whole cells at 37°C and in isolated inside/out patches at 22°C. Truncation of the first 5 N-terminal amino acids abolished most of the affinity increase for glibenclamide and repaglinide that is produced by the association of Kir6.2 with SUR1. Progressive truncation continuously reduced the potency and efficacy of these drugs in closing the channel and impaired the ability to stabilise the closed state more than the ability to disrupt channel stimulation by SUR-ATPase. The effects of NNC 55-0462 were unchanged. Progressive truncation also speeded up dissociation of [(3)H]glibenclamide from the channel when dissociation was induced by an excess of (unlabelled) glibenclamide. This suggests the existence of a putative low affinity glibenclamide site on the channel whose affinity increases upon truncation. The data show that progressive truncation of the Kir6.2 N-terminus impairs the transduction of drug binding into channel closure more strongly than drug binding but leaves the effect of the opener NNC 55-0462 unchanged.

Synthesis of benzofuran, benzothiophene, and benzothiazole-based thioamides and their evaluation as K(ATP) channel openers.

Several series of benzofurans, benzothiophenes, and benzothiazoles, all featuring the thioamide group, were synthesized and tested as novel K(ATP) channel openers in artificial cell systems: CHO cells transfected with SUR1/Kir6.2, and HEK 293 cells transfected with SUR2B/Kir6.1; these served as model systems for insulin-secreting pancreatic ß cells and smooth muscle cells, respectively. All compounds were investigated with respect to their binding affinity for the SUR2B-type K(ATP) channels using [(3)H]P1075 as radioligand. Selected compounds were also tested as agonists in intact cells using DiBAC(4)(3) and DyeB (R7260) as membrane potential dyes. Remarkable affinity for SUR2B/Kir6.1 channels in the single-digit micromolar range was observed. In addition, benzothiazole-derived thioamides with sterically demanding, lipophilic substituents showed >100-fold selectivity in favor of SUR2B/Kir6.1. A one-carbon spacer between the heterocyclic skeleton and the thioamide moiety was observed to be crucial for affinity and selectivity. Two of the most potent and selective compounds were studied by crystal structure analyses.

Substitution of the Walker A lysine by arginine in the nucleotide-binding domains of sulphonylurea receptor SUR2B: effects on ligand binding and channel activity.

Sulphonylurea receptors (SURs) serve as regulatory subunits of ATP-sensitive K(+) channels. SURs are members of the ATP-binding cassette (ABC) protein superfamily and contain two conserved nucleotide-binding domains (NBDs) which bind and hydrolyse MgATP; in addition, they carry the binding sites for the sulphonylureas like glibenclamide (GBC) which close the channel and for the K(ATP) channel openers such as P1075. Here we have exchanged the conserved Lys in the Walker A motif by Arg in both NBDs of SUR2B, the regulatory subunit of the vascular K(ATP) channel. Then the effect of the mutation on the ATPase-dependent binding of GBC and P1075 to SUR2B and on the activity of the recombinant vascular (Kir6.1/SUR2B) channel was assessed. Surprisingly, in the absence of MgATP, the mutation weakened binding of P1075 and the extent of allosteric inhibition of GBC binding by P1075. The mutation abolished most, but not all, of the MgATP effects on the binding of GBC and P1075 and prevented nucleotide-induced activation of the channel which relies on SUR reaching the posthydrolytic (MgADP-bound) state; the mutant channel was, however, opened by P1075 at higher concentrations. The data provide evidence that mutant SUR2B binds MgATP but that the posthydrolytic state is insufficiently populated. This suggests that the mutation locks SUR2B in an MgATP-binding prehydrolytic-like state; binding of P1075 may induce a posthydrolytic-like conformation to open the channel.

Analysis of two KCNJ11 neonatal diabetes mutations, V59G and V59A, and the analogous KCNJ8 I60G substitution: differences between the channel subtypes formed with SUR1.

beta-Cell-type K(ATP) channels are octamers assembled from Kir6.2/KCNJ11 and SUR1/ABCC8. Adenine nucleotides play a major role in their regulation. Nucleotide binding to Kir6.2 inhibits channel activity, whereas ATP binding/hydrolysis on sulfonylurea receptor 1 (SUR1) opposes inhibition. Segments of the Kir6.2 N terminus are important for open-to-closed transitions, form part of the Kir ATP, sulfonylurea, and phosphoinositide binding sites, and interact with L0, an SUR cytoplasmic loop. Inputs from these elements link to the pore via the interfacial helix, which forms an elbow with the outer pore helix. Mutations that destabilize the interfacial helix increase channel activity, reduce sensitivity to inhibitory ATP and channel inhibitors, glibenclamide and repaglinide, and cause neonatal diabetes. We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle. The substituted channels have increased P(O) values, decreased sensitivity to inhibitors, and impaired stimulation by phosphoinositides but retain sensitivity to Ba(2+)-block. The V59G and V59A channels are either not, or poorly, stimulated by phosphoinositides, respectively. Inhibition by sequestrating phosphatidylinositol 4,5-bisphosphate with neomycin and polylysine is reduced in V59A, and abolished in V59G channels. Stimulation by SUR1 is intact, and increasing the concentration of inhibitory ATP restores the sensitivity of Val-59-substituted channels to glibenclamide. The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas. The results suggest the interfacial helix dynamically links inhibitory inputs from the Kir N terminus to the gate and that sulfonylureas stabilize an inhibitory configuration.

Synthesis of modified 4H-1,2,4-benzothiadiazine-1,1-dioxides and determination of their affinity and selectivity for different types of K(ATP) channels.

4H-1,2,4-Benzothiadiazine-1,1-dioxides with various substituents in positions 3, 5, and 7 were synthesized and tested as K(ATP) channel agonists in artificial cell systems (CHO cells transfected with SUR1/Kir6.2, and HEK 293 transfected with SUR2B/Kir6.1) as model systems for insulin-secreting pancreatic beta-cells and for smooth muscle cells, respectively. The effects of agonists were tested in intact cells using DiBAC4(3) [bis-(1,3-dibarbituric acid)trimethine oxanol] as a membrane potential dye, and the results compared with their binding affinity for the SUR2B-type K(ATP) channels using the radioligand [(3)H]P1075. Compounds with cycloalkyl and (cycloalkyl)methyl side chains in position 3 had higher affinities towards the SUR2B/Kir6.1 receptor compared with the parent compound diazoxide (1 a). Compounds with bulky, nonpolar residues in position 3 exhibited remarkable selectivity for SUR2B-type K(ATP) channels. The compound substituted with a bulky (1-adamantyl)methyl residue exhibited micromolar affinity and activity on SUR2B-type K(ATP) channels without being able to activate the SUR1-type K(ATP) channels.

Testing the bipartite model of the sulfonylurea receptor binding site: binding of A-, B-, and A + B-site ligands.

ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulfonylurea receptors (SURx). Subtypes of K(ATP) channels are expressed in different organs. The sulfonylureas and glinides (insulinotropes) close the K(ATP) channel in pancreatic beta-cells and stimulate insulin secretion. The insulinotrope binding site of the pancreatic channel (Kir6.2/SUR1) consists of two overlapping (sub)-sites, site A, located on SUR1 and containing Ser1237 (which in SUR2 is replaced by Tyr1206), and site B, formed by SUR1 and Kir6.2. Insulinotropes bind to the A-, B-, or A + B-site(s) and are grouped accordingly. A-ligands are highly selective in closing the pancreatic channel, whereas B-ligands are nonselective and insensitive to the mutation S1237Y. We have examined the binding of insulinotropes representative of the three groups in [(3)H]glibenclamide competition experiments to determine the contribution of Kir6.x to binding affinity, the effect of the mutation Y1206S in site A of SUR2, and the subtype selectivity of the compounds. The results show that the bipartite nature of the SUR1 binding site applies also to SUR2. Kir6.2 as part of the B-site may interact directly or allosterically with structural elements common to all insulinotropes, i.e., the negative charge and/or the adjacent phenyl ring. The B-site confers a moderate subtype selectivity on B-ligands. The affinity of B-ligands is altered by the mutation SUR2(Y1206S), suggesting that the mutation affects the binding chamber of SUR2 as a whole or subsite A, including the region where the subsites overlap.

Determination of molecular formulas and structural regularities of low molecular weight fulvic acids by size-exclusion chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry.

Size-exclusion chromatography was coupled to a quadrupole time-of-flight mass spectrometer using electrospray ionization in the negative ion mode to analyze Suwannee River fulvic acid. About 220 exact masses of low molecular weight fulvic acids (FAs) were measured in the range from m/z 190 to 350, and molecular formulas could be deduced for all these molecular masses. All molecular formulas can be described by the superimposition of three homologous series in the FA mixture: (a) a series of hydrogen homologs constituted of five to six members for a given number of carbons and oxygens; (b) a series of alkyl chain homologs with stepwise addition of one methylene group; and (c) isobaric compounds that differ by the formal replacement of an oxygen atom by a methane group. Product ion spectra of the FA species reflect the repeated parallel losses of carbon dioxide and water. The minimum number of carboxylate groups and the maximum number of hydroxy moieties could be determined from the product ion spectra. Furthermore, it was obvious that the structural differences between homologs in the three series are located in the carbon backbone of the FA rather than in its extremities that are expelled as neutral fragments. These structural regularities reduce the complexity of FA mixtures to a certain set of yet unknown basic structures.