Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains.

Distinct interaction of human and guinea pig histamine H2-receptor with guanidine-type agonists.

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H2-receptor (H2R) agonists are lower at the H2R of human neutrophils than at the H2R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H2R (hH2R) and guinea pig H2R (gpH2R), respectively, and the short splice variant of Gsalpha (GsalphaS) expressed in Sf9 cells. The potencies and efficacies of small H2R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH2R-GsalphaS and gpH2R-GsalphaS were similar.

Radiographic diagnosis of dental caries.

The purpose of this report was to respond to aspects of the RTI/UNC systematic review relating to the radiographic diagnosis of dental caries. The systematic review was commissioned as part of the NIH Consensus Development Conference on Diagnosis and Management of Dental Caries Throughout Life. The systematic review evaluated the dental literature from 1966 to 1999.

Characterization of MTMR3. an inositol lipid 3-phosphatase with novel substrate specificity.

Inositol lipids play key roles in many fundamental cellular processes that include growth, cell survival, motility, and membrane trafficking. Recent studies on the PTEN and Myotubularin proteins have underscored the importance of inositol lipid 3-phosphatases in cell function. Inactivating mutations in the genes encoding PTEN and Myotubularin are key steps in the progression of some cancers and in the onset of X-linked myotubular myopathy, respectively.

Homoarcyriaflavin: Synthesis of Ring-Expanded Arcyriaflavin Analogues.

The construction of the ring-expanded carbazole system, forming arcyriaflavin homologues, is efficiently accomplished by the reaction of 2,2'-bridged bis-indoles with 3,4-dibromo-2,5-dihydro-1H-2,5-pyrroledione derivatives under Grignard conditions. A ring size of up to nine members in the central ring is achievable. Substitutions either at the indole system or at the imide-N are also possible.

AtPIP5K1, an Arabidopsis thaliana phosphatidylinositol phosphate kinase, synthesizes PtdIns(3,4)P(2) and PtdIns(4,5)P(2) in vitro and is inhibited by phosphorylation.

PtdIns phosphate kinases (PIPkins), which generate PtdInsP(2) isomers, have been classified into three subfamilies that differ in their substrate specificities. We demonstrate here that the previously identified AtPIP5K1 gene from Arabidopsis thaliana encodes a PIPkin with dual substrate specificity in vitro, capable of phosphorylating PtdIns3P and PtdIns4P to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) respectively. We also show that recombinant AtPIP5K1 is phosphorylated by protein kinase A and a soluble protein kinase from A.

Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa.

A number of transcriptional regulators mediate their effects through direct contact with the sigma(70) subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of sigma(70) that harbors conserved region 4. This region of sigma contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma(70)-dependent promoters directly.

Comparison of five methods for the derivation of spectra for a constant potential dental X-ray unit.

Objectives: To compare the diagnostic X-ray spectra derived by different methods for a constant potential dental X-ray unit.

Materials And Methods: Five methods of deriving X-ray spectra for a constant potential dental X-ray unit were compared: measurement by spectrometer using cadmium-zinc-telluride (CZT) detector, calculation by Monte Carlo simulation, calculation by two different, semi-empirical methods and estimation from transmission data. The dental X-ray set was a Heliodent MD unit (Sirona, Charlotte, NC, USA) operable at 60 or 70 kV.

Antineutrophil cytoplasm autoantibodies from patients with systemic vasculitis activate neutrophils through distinct signaling cascades: comparison with conventional Fcgamma receptor ligation.

In systemic vasculitis, interactions between antineutrophil cytoplasm autoantibodies (ANCAs) and neutrophils initiate endothelial and vascular injury. ANCAs directed against either myeloperoxidase (MPO) or proteinase 3 (PR3) can activate cytokine-primed neutrophils by binding cell surface-expressed MPO or PR3, with the concurrent engagement of Fcgamma receptors (FcgammaR). Because roles for phospholipase D (PLD) and phosphatidylinositol 3 kinase (PI3K) have been demonstrated in FcgammaR activation of neutrophils, this study investigated the hypothesis that ANCA stimulation of neutrophils involved a similar engagement of FcgammaR and activation of PLD and PI3K.

Modification and benchmarking of MCNP for low-energy tungsten spectra.

The MCNP Monte Carlo radiation transport code was modified for diagnostic medical physics applications. In particular, the modified code was thoroughly benchmarked for the production of polychromatic tungsten x-ray spectra in the 30-150 kV range. Validating the modified code for coupled electron-photon transport with benchmark spectra was supplemented with independent electron-only and photon-only transport benchmarks.

Magnitude of the CREB-dependent transcriptional response is determined by the strength of the interaction between the kinase-inducible domain of CREB and the KIX domain of CREB-binding protein.

The activity of the transcription factor CREB is regulated by extracellular stimuli that result in its phosphorylation at a critical serine residue, Ser133. Phosphorylation of Ser133 is believed to promote CREB-dependent transcription by allowing CREB to interact with the transcriptional coactivator CREB-binding protein (CBP). Previous studies have established that the domain encompassing Ser133 on CREB, known as the kinase-inducible domain (KID), interacts specifically with a short domain in CBP termed the KIX domain and that this interaction depends on the phosphorylation of Ser133.

Mechanism for a transcriptional activator that works at the isomerization step.

Transcriptional activators in prokaryotes have been shown to stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a postbinding step known as the isomerization step. Evidence suggests that activators that affect initial binding can work by a cooperative binding mechanism by making energetically favorable contacts with RNAP, but the mechanism by which activators affect the isomerization step is unclear. A well-studied example of an activator that normally exerts its effect exclusively on the isomerization step is the bacteriophage lambda cI protein (lambdacI), which has been shown genetically to interact with the C-terminal region of the final sigma(70) subunit of RNAP.

Measurement and validation of benchmark-quality thick-target tungsten X-ray spectra below 150 kVp.

Pulse-height distributions of two constant potential X-ray tubes with fixed anode tungsten targets were measured and unfolded. The measurements employed quantitative alignment of the beam, the use of two different semiconductor detectors (high-purity germanium and cadmium-zinc-telluride), two different ion chamber systems with beam-specific calibration factors, and various filter and tube potential combinations. Monte Carlo response matrices were generated for each detector for unfolding the pulse-height distributions into spectra incident on the detectors.

Using measured 30-150 kVp polychromatic tungsten x-ray spectra to determine ion chamber calibration factors, Nx (Gy C(-1)).

Two methods for determining ion chamber calibration factors (Nx) are presented for polychromatic tungsten x-ray beams whose spectra differ from beams with known Nx. Both methods take advantage of known x-ray fluence and kerma spectral distributions. In the first method, the x-ray tube potential is unchanged and spectra of differing filtration are measured.

The outer membrane protein, antigen 43, mediates cell-to-cell interactions within Escherichia coli biofilms.

Transcription of the agn43 locus, which specifies an outer membrane protein of Escherichia coli, is regulated in a phase-variable fashion by the OxyR-DNA binding protein and Dam methylase. Despite its well-characterized regulation, the function of Ag43 has remained elusive until now. Previous studies indicated that Ag43 mediates autoaggregation of certain strains of E.

Mechanical loading stimulates differentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes.

The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal.

Structure-activity relationships of neuropeptide Y Y1 receptor antagonists related to BIBP 3226.

Analogues of BIBP 3226, (R)-N(alpha)-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated for Y1 antagonism (Ca2+-assay, HEL cells) and binding on Y1, Y2 and Y5 receptors. Replacing the benzylamino by a tetrahydrobenzazepinyl group preserves most of the Y1 activity. Combination with a N(G)-phenylpropyl arginine and a N(alpha)-p-biphenylylacetyl moiety shifted the NPY receptor selectivity towards Y5.

Analysis of sensitivity and specificity of a new digital subtraction system: an in vitro study.

Objective: The purpose of this study was to compare a new digital subtraction system with conventional radiograph images for the detection of periapical and periodontal bone lesions.

Study Design: Periapical and periodontal bone lesions were simulated with cortical bone chips of varying sizes placed on a human dry mandible. Radiographic film images were acquired from varying projections and were subsequently digitized, registered, and subtracted.

Clinical validation of a new subtraction radiography technique for periodontal bone loss detection.

Background: Diagnostic subtraction radiography (DSR) is a new digital radiographic image subtraction method designed to enhance detection of crestal or periapical bone density changes and to help evaluate caries progression in teeth. In this clinical study, the performance of the DSR method was evaluated for its ability to detect periodontal bone loss and was compared with that of conventional evaluation of radiographs and the standardized cephalostat-guided image acquisition and subtraction technique (LRA) which served as the "gold standard."

Methods: In each of 25 subjects with alveolar crestal bone loss created by periodontal surgery, one set of DSR radiographs and one set of LRA radiographs were obtained before and after the surgery.

Pharmacology and quantitative structure-activity relationships of imidazolylpropylguanidines with mepyramine-like substructures as non-peptide neuropeptide Y Y1 receptor antagonists.

The design of non-peptide, Y1-selective antagonists of neuropeptide Y (NPY) as pharmacological tools is in progress and is increasingly important as therapeutic applications are expected. Starting from the potent histamine H2 agonist and weak NPY Y1 antagonist arpromidine, 16 imidazolylpropylguanidine derivatives were synthesized and tested for Y1 antagonistic activity (inhibition of NPY-stimulated Ca2+ increase in human erythroleukemic cells), where the pheniramine-like moiety of arpromidine was replaced with 2-pyridylaminoalkyl, benzyl-(2-pyridyl)aminoalkyl, and phenyl-(2-pyridyl)alkylaminoalkyl partial structures derived from mepyramine. The pA2 values of the most active compounds are in the range of 6.

Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion.

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion.

Isolation of peptide aptamers that inhibit intracellular processes.

We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible P(BAD) promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo.

SAC1 encodes a regulated lipid phosphoinositide phosphatase, defects in which can be suppressed by the homologous Inp52p and Inp53p phosphatases.

The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity. The activity of Sac1p and its relationship to these phenotypes are unresolved. We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate.

Salinity and hyperosmotic stress induce rapid increases in phosphatidylinositol 4,5-bisphosphate, diacylglycerol pyrophosphate, and phosphatidylcholine in Arabidopsis thaliana cells.

In animal cells, phosphoinositides are key components of the inositol 1,4,5-trisphosphate/diacylglycerol-based signaling pathway, but also have many other cellular functions. These lipids are also believed to fulfill similar functions in plant cells, although many details concerning the components of a plant phosphoinositide system, and their regulation are still missing. Only recently have the different phosphoinositide isomers been unambiguously identified in plant cells.