Molecular cloning and characterization of a novel regulator of G-protein signaling from mouse hematopoietic stem cells.

A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH(2) and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.

The L-shaped osteotomy for vertical callus distraction in the molar region of the mandible: a technical note.

The following report refers to a well described surgical procedure using a prototype vertical distractor developed in Cologne in cooperation with Martin Medizintechnik GmbH. The surgical technique has been refined by an L-shaped osteotomy stabilized posteriorly by a miniplate to limit the amount of callus regeneration in the molar region where no increase in vertical height is required. The surgical technique with its advantages and one complication is described as used in four patients undergoing six distraction treatments.

Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny.

Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter.

Endoscopy of the middle ear through the eustachian tube: anatomic possibilities and limitations.

Hypothesis: This anatomic study was designed to provide otosurgeons with a detailed description of the technique of transtubal middle ear endoscopy and anatomic landmarks facilitating orientation. To establish the role of transtubal endoscopy, its anatomical potentials and limitations were studied.

Background: Transtubal endoscopy has recently been described as an atraumatic tool for exploring the tympanic cavity.

Two-port endoscopy of the middle ear: endoscopic anatomy.

Objectives: To shed light on the endoscopic anatomy of the middle ear seen on 2-port endoscopy and to describe potential clinical applications.

Design: Anatomical structures were visualized by transmeatal or transtympanic rigid scopes of different angles and by a flexible scope in the eustachian tube. This arrangement ensured reciprocal guidance of the scopes and provided access to regions not seen otherwise.

Guanidine hydrochloride unfolding of a transmembrane beta-strand in FepA using site-directed spin labeling.

We have used the electron spin resonance (ESR) site-directed spin-labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn-HCl) induced denaturation of several sites along a transmembrane beta-strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the beta-strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027-13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a beta-barrel motif comprised of a series of antiparallel beta-strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria.

Ligand-induced conformational change in the ferric enterobactin receptor FepA as studied by site-directed spin labeling and time-domain ESR.

A mutant of the ferric enterobactin receptor, FepA, containing a valine to cysteine (V338C) substitution was made and the purified protein selectively modified with a sulfhydryl-specific nitroxide spin label. In reconstituted liposomes, interaction of the attached spin label with a combination of water-soluble and lipid-soluble relaxation agents indicated that the V338C site was located in the polar headgroup region of the membrane, approximately 1.5-4.

Helios, a T cell-restricted Ikaros family member that quantitatively associates with Ikaros at centromeric heterochromatin.

The Ikaros gene encodes multiple protein isoforms that contribute critical functions during the development of lymphocytes and other hematopoietic cell types. The intracellular functions of Ikaros are not known, although recent studies have shown that Ikaros proteins colocalize with inactive genes and centromeric heterochromatin. In this study, Ikaros proteins were found to be components of highly stable complexes.

[Combined effect of temperature a(W) and pH on proteases from Pseudomonas and Bacillus spp].

The factors affecting protease activity were investigated on certain media. Five strains of Bacillus (B.) cereus, four strains of B.

A framework for improving quality. Using project study teams, Providence Health System tackles problem areas.

To identify issues for quality improvement and monitoring, an organization first articulates its service mission and objectives. The organization uses internal measurements and self-examination with external accountability and benchmarks to select a manageable number of projects for study. Supported by collected evidence, published data, and broad-based approval, projects focus on the process or outcomes of care, frequency of services, or patient populations.

Slowed enzymatic turnover allows characterization of intermediates by solid-state NMR.

EPSP (5-enolpyruvylshikimate-3-phosphate) synthase catalyzes condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor to the aromatic amino acids. S3P and [2-13C]POP were bound to mutant or wild type E. coli forms of the enzyme prior to lyophilization.

Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes.

The generation of lymphoid cells in mice depends on the function of the Ikaros protein. Ikaros has been characterized as a lymphoid-restricted, zinc-finger transcription factor that is derived from an alternatively spliced message. Ikaros knockout mice have defects in multiple cell lineages, raising the question of whether the protein regulates multiple committed progenitors and/or multipotent stem cells.

Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling.

Electron paramagnetic resonance (EPR) site-directed spin-labeling (SDSL) has been used to characterize a proposed transmembrane beta-strand of the Escherichia coli ferric enterobactin receptor, FepA. Each of nine consecutive residues was mutated to cysteine and subsequently labeled with the sulfhydryl-specific spin-label methanethiosulfonate (MTSL) and the purified protein reconstituted into liposomes. Continuous wave (CW) power saturation methods were used to determine exposure of the nitroxide side chains to a series of paramagnetic relaxation agents, including nickel acetylacetonate (NiAA), nickel ethylenediaminediacetate (NiEDDA), chromium oxalate (CROX), and molecular oxygen.

Closed form of liganded glutamine-binding protein by rotational-echo double-resonance NMR.

Rotational-echo double-resonance NMR has been used to determine internuclear distances in the complex of glutamine-binding protein and its ligand, l-glutamine. The distances between the ligand and Tyr185 are consistent with the results of molecular dynamics simulations constrained by three REDOR-determined distances to His156. This model is also consistent with six other REDOR-determined internuclear distances, most of which agree with values from the first report of an X-ray structure of the complex of glutamine-binding protein and l-glutamine.

A minimally invasive technique for endoscopic middle ear surgery.

Middle ear endoscopy should be considered a useful adjunctive or alternative method to microscopic surgical exploration for middle ear pathology. This minimally invasive technique provides excellent visualization for viewing the surgical micromorphology and pathological findings of the middle ear. Selected patients underwent middle ear endoscopy using a transtympanic approach.

Distance between phosphine-sulfide sidechains of a disubstituted peptide by DRAMA 31P NMR.

Dipolar restoration at the magic angle (DRAMA) has been used to measure the 31P-31P internuclear distance between phosphine-sulfide substituted sidechanins on the fourth and eighth residues of a 12-residue helical peptide. The 7.4 A distance is the same for the peptide lyophilized in bulk or isolated in a cryo- and lyoprotected matrix of poly(ethylene glycol) and sucrose.

In situ analysis of peptidyl DOPA in mussel byssus using rotational-echo double-resonance NMR.

Rotational-echo double-resonance (REDOR) 13C NMR spectra with 2H dephasing have been obtained from plaques and threads from the byssus of the marine mussel Mytilus edulis labeled by sea-water exposure to L-[ring-4-(13)C]tyrosine and L-[ring-d4]tyrosine for 2 days. Specific isotopic enrichment of tyrosine in protein reached 25% in both 13C and 2H. Fifteen percent of the total 13C label was incorporated as diphenolic carbon.

Ligand geometry of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR.

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances.

Clinical assessment of periodontal conditions in patients treated with nifedipine.

Calcium antagonists are widely used in treating acute and chronic coronary insufficiency disorders. A major side effect of long-term treatment is gingival hyperplasia. In the present study, 70 patients taking nifedipine for at least six months and 70 controls similar in age, gender, approximal hygiene and systemic disease with at least 6 anterior teeth in upper and lower arches were examined.

Denaturant unfolding of the ferric enterobactin receptor and ligand-induced stabilization studied by site-directed spin labeling.

FepA is an integral outer membrane protein that is the specific receptor for the siderophore, ferric enterobactin, and is thus primarily responsible for iron uptake in many Gram-negative bacteria. A site-specific mutant of FepA, containing a single introduced cysteine in the ligand-binding domain, was spin labeled and used to examine the denaturant-induced unfolding of this receptor with guanidine hydrochloride (Gdn-HCl) and urea. Electron spin resonance (ESR) spectra showed conversion of the spin label from a motionally-restricted, immobilized environment to a freely-accessible, rotationally-mobile state upon denaturation.

Regional induction of tumor necrosis factor alpha expression in the mouse brain after systemic lipopolysaccharide administration.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine that is responsible, in part, for several aspects of the acute-phase response to inflammation, including the generation of fever. TNF-alpha has direct effects on central nervous system neurons deep within the hypothalamus that are involved in producing the febrile response, but the blood-brain barrier prevents circulating TNF-alpha from having access to these sites. We therefore have hypothesized that TNF-alpha may be produced in the brain and used as a mediator in the cerebral components of the acute-phase response.

The v-abl tyrosine kinase negatively regulates NF-kappa B/Rel factors and blocks kappa gene transcription in pre-B lymphocytes.

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. We demonstrate that nontransformed pre-B cells expanded from the mouse bone marrow efficiently transcribe unrearranged kappa alleles.

Cytomegalovirus reactivation and tumour necrosis factor.

Tumour necrosis factor-alpha (TNF) stimulates cytomegalovirus (CMV) activity in a transfected human monocytic cell line. We assessed whether this finding is relevant in vivo by evaluating the frequency of active CMV infection in patients with diseases that enhance plasma TNF. In septic disease, peripheral blood mononuclear cells of almost all patients studied were positive for CMV.

Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation.

The Oct-2 protein is a tissue-specific POU-homeodomain transcription factor. It has been considered to represent a developmental regulator of immunoglobulin gene expression by virtue of its interaction with a functionally essential octamer element found in immunoglobulin gene promoters. This proposal has been most strongly challenged by several in vitro transcription analyses which have shown that the related ubiquitous factor Oct-1 can activate transcription from immunoglobulin gene promoters as efficiently as Oct-2.

Baking stability of acesulfame K.

The stability of acesulfame K during baking was investigated at different baking temperatures and baking times. The contents of acesulfame K in baked and unbaked doughs were determined by HPLC. The recovery rate of acesulfame K was independent of the baking conditions chosen and correlated with the recovery rate of acesulfame K in the unbaked doughs.