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D Witt

Publications and source records attributed to D Witt.

At least 19 recordsLinked to original sources
PubMed2001

Self-association of facially amphiphilic methylene bridged glycoluril dimers.

[structure: see text] Facially amphiphilic derivatives of methylene bridged glycoluril dimers are a versatile model system for systematic studies of self-assembly in water. Thorough physical organic characterization, including analytical ultracentrifugation, a technique rarely used in synthetic self-assembly studies, allows us to conclude that this class of molecules undergoes hydrophobically driven self-association to yield tightly associated discrete dimeric assemblies.

Alkynes↗
PubMed2000

Diastereoselective Formation of Methylene-Bridged Glycoluril Dimers.

The acid-catalyzed formation of methylene-bridged glycoluril dimers yields the C(2)(v)()-diastereomer selectively. Product resubmission experiments establish that the selectivity is the result of thermodynamic control. A modified synthetic route is presented that allows for the preparation of unsymmetrically substituted dimers. We present the X-ray crystal structures of both diastereomers. This class of compounds is useful for studies of self-assembly in aqueous solution.

Journal Article↗
PubMed1999

Tumor necrosis factor alpha regulates CXC chemokine receptor expression and function.

The alpha chemokine family is central to the participation of neutrophils in the acute inflammatory response. These substances interact with neutrophils through two cell surface receptors, CXCR-1 and CXCR-2 (formally known as IL-8R-1 and IL-8R-2). We investigated the possible regulatory effects of tumor necrosis factor alpha (TNFalpha) pretreatment on CXCR-1 and CXCR-2. To this end, we examined these receptors with flow cytometry, radioligand binding, Northern blot analyzes, calcium mobilization, and chemotaxis experiments on human neutrophils. In flow cytometry experiments, TNFalpha pretreatment substantially decreased cell surface CXCR-2 receptor levels but showed partial recovery at 120 min. On the other hand, CXCR-1 receptor levels had a sharp decline at 15 min and maintained that level to 120 min. Northern blot analyzes showed that mRNA levels of both IL-8 receptors were essentially unchanged after 45 min of TNFalpha pretreatment, but declined markedly following 2 h of pretreatment. Chemotaxis experiments on cells treated with TNFalpha for 5-120 min showed a substantial down-regulation of chemotaxis to IL-8 and GROalpha. This was noted to be much greater than the decline in cell surface receptors. Calcium mobilization experiments revealed minimal inhibition of the IL-8-induced increase in calcium after pretreatment with TNFalpha, but the response to NAP-2 was substantially inhibited. The data demonstrate differential regulation of the IL-8 receptor.

Blotting, Northern↗
PubMed1998

Defining the interleukin-8-binding domain of heparan sulfate.

Interleukin-8, a member of the CXC chemokine family, has been shown to bind to glycosaminoglycans. It has been suggested that heparan sulfate on cell surfaces could provide specific ligand sites on endothelial cells to retain the highly diffusible inflammatory chemokine for presentation to leukocytes. By using selectively modified heparin and heparan sulfate fragments in a nitrocellulose filter trapping system, we have analyzed sequence requirements for interleukin-8 binding to heparin/heparan sulfate. We demonstrate that the affinity of a monomeric interleukin-8 molecule for heparin/heparan sulfate is too weak to allow binding at physiological ionic strength, whereas the dimeric form of the protein mediates binding to two sulfated domains of heparan sulfate. These domains, each an N-sulfated block of approximately 6 monosaccharide units, are contained within an approximately 22-24-mer sequence and are separated by a region of </=14 monosaccharide residues that may be fully N-acetylated. Binding to interleukin-8 correlates with the occurrence of the di-O-sulfated disaccharide unit -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. We suggest that the heparan sulfate sequence binds in horseshoe fashion over two antiparallel-oriented helical regions on the dimeric protein.

Binding Sites↗
PubMed1998

The effects of dilution on the outcome of pooled plasma testing with HIV type 1 (HIV-1) RNA genome amplification as compared to the outcome of individual-unit testing with other HIV-1 markers.

BACKGROUND: Proposed testing of large plasma pools with genome amplification technology (GAT) for detection of transfusion-transmissible viruses may have unanticipated complications not associated with individual unit testing. One such potential complication, the effect of dilution resulting from pool formation, was the subject of the present study. STUDY DESIGN AND METHODS: Specimens from three plasma donor HIV type 1 (HIV-1) seroconversion panels were tested with a quantitative HIV-1 RNA GAT assay (lower detection limit, 400 copies). GAT results were compared to HIV-1 p24 antigen and anti-HIV-1/2 enzyme immunoassay results. Effects of dilution on the detection of GAT-positive panel specimens were assessed by terminal dilution with pooled volunteer-donor EDTA plasma samples. RESULTS: Low HIV-1 RNA copy numbers (755 and 890 copies/0.1-mL input) that were detectable in two individual plasma specimens before HIV-1 p24 antigen were subsequently undetectable by GAT upon dilution with an equal volume of nonreactive plasma from a single donor. HIV-1 RNA at higher copy numbers (15,500 copies/0.1-mL input) in an HIV-1 p24 antigen-reactive and anti-HIV-1/2-nonreactive specimen was undetectable when diluted to 1-in-50 (1-in-50). Terminal dilution of seven HIV-1 RNA-containing plasma panel specimens indicated a proportional loss of HIV-1 RNA detectability with increasing dilution. CONCLUSION: GAT for detection of HIV-1 RNA in individual specimens was more sensitive than other HIV markers. For pooled plasma testing, GAT-independent constraints, such as effects of dilution, may preclude the use of GAT detection as a replacement for individual unit testing with HIV serologic assays.

Gene Amplification↗
PubMed1997

beta-phosphorylated nitroxides in the pyrrolidine series: reduction by ascorbate.

Reduction-resistant nitroxides are particularly interesting for biomedical applications. beta-Phosphorylated pyrrolidinyl nitroxides, a new series of stable pyrrolidinoxyl radicals prepared in our laboratory, were tested toward ascorbate reduction in phosphate buffer at pH 7.4. The kinetics of decay were monitored by ESR and compared to those of two reference nitroxides, PCA and Proxyl. The introduction of a beta-phosphoryl group on a pyrrolidinoxyl structure resulted in a moderate increase of the reduction rate constant. However, inside the phosphorylated series, slight structural modifications can induce significant changes in the rate constants.

Ascorbic Acid↗
PubMed1997

High-affinity binding of corticosterone to mammalian neuronal membranes: possible role of corticosteroid binding globulin.

The signal transduction mechanisms mediating rapid steroid actions are poorly understood. To characterize corticosteroid interaction with neuronal membranes in a species with rapid behavioral responses to corticosterone, we examined [3H]corticosterone binding to membranes prepared from prairie vole brains. At 22 degrees C, the rates of association and dissociation of [3H]corticosterone with well-washed synaptosomal membranes were very rapid. Specific binding was characterized by high affinity (Kd = 6.01 nM) and low density (Bmax = 63.1 fmol/mg protein). The binding sites were highly specific for naturally occurring glucocorticoids and the density of binding sites appeared to vary by neuroanatomical region. Unlike most G-protein-coupled receptors, the high-affinity binding of [3H]corticosterone to vole brain membranes was unaffected by the addition of Mg2+ or guanyl nucleotides. Surprisingly, saline perfusion of vole brains before tissue homogenization greatly reduced high-affinity binding. In addition, the affinity and specificity of corticosteroid binding sites were similar in vole neuronal membranes and vole plasma. These data suggest that corticosteroid binding globulins may facilitate [3H]corticosterone binding to neuronal membranes. However, the addition of blood to perfused brains before homogenization did not restore high-affinity binding, so the role of plasma binding globulins is unclear. Whether these binding phenomena represent a technical artifact or a regulatory mechanism for corticosteroid action has yet to be determined.

Animals↗
PubMed1997

Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma.

To define the optimal blood collection parameters for plasma human immunodeficiency virus type 1 (HIV-1) viral load testing, plasma HIV-1 RNA levels were quantitated with the NASBA HIV-1 RNA QT System from blood specimens that were collected, processed, and stored under a variety of conditions that might have affected HIV-1 RNA stability. We determined that when whole blood was processed within 2 h of specimen collection the levels of HIV-1 RNA detected in EDTA-, heparin-, and acid citrate dextrose (ACD)-anticoagulated plasma samples were comparable. The levels of HIV-1 RNA in serum specimens (mean = 4.126 log units) were significantly lower (P < 0.01) than the levels in corresponding plasma samples (mean = 4.501 log units). One cycle of freeze-thaw (-70 degrees C) did not significantly reduce the level of HIV-1 RNA detected in EDTA-, heparin-, or ACD-anticoagulated plasmas. The EDTA-anticoagulated plasmas showed the smallest decrease in HIV-1 RNA copies (0.050 log units). HIV-1 RNA levels decreased over a 6-month time period in serum as well as in EDTA-, ACD-, and heparin-anticoagulated plasmas stored at -70 degrees C. However, the only significant decreases were for serum (mean decrease = 0.317 log units) and heparin-anticoagulated samples (mean decrease = 0.384 log units). A comparison of the levels of HIV-1 RNA in cell-free plasma collected in VACUTAINER EDTA Plasma Preparation Tubes and in standard VACUTAINER EDTA tubes determined that HIV-1 RNA levels were stable for up to 30 h after collection when stored at either room temperature (mean standard deviation [SD] = +/- 0.101 log units) or at 4 degrees C (mean SD = +/- 0.102 log units) as cell-free plasma or as EDTA-anticoagulated whole blood (mean SD = +/- 0.109 log units). These data indicate that EDTA-anticoagulated plasma is the most suitable and stable matrix for HIV-1 RNA quantitation.

Anticoagulants↗
PubMed1996

Clinical phenotypes of different MPZ (P0) mutations may include Charcot-Marie-Tooth type 1B, Dejerine-Sottas, and congenital hypomyelination.

Hereditary demyelinating peripheral neuropathies consist of a heterogeneous group of genetic disorders that includes hereditary neuropathy with liability to pressure palsies (HNPP), Charcot-Marie-Tooth disease (CMT), Dejerine-Sottas syndrome (DSS), and congenital hypomyelination (CH). The clinical classification of these neuropathies into discrete categories can sometimes be difficult because there can be both clinical and pathologic variation and overlap between these disorders. We have identified five novel mutations in the myelin protein zero (MPZ) gene, encoding the major structural protein (P0) of peripheral nerve myelin, in patients with either CMT1B, DSS, or CH. This finding suggests that these disorders may not be distinct pathophysiologic entities, but rather represent a spectrum of related "myelinopathies" due to an underlying defect in myelination. Furthermore, we hypothesize the differences in clinical severity seen with mutations in MPZ are related to the type of mutation and its subsequent effect on protein function (i.e., loss of function versus dominant negative).

Adult↗
PubMed1993

Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs.

Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

Base Sequence↗
PubMed1992

Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity.

Interleukin-8 (IL-8) is a key mediator in the migration of neutrophils from the circulation to the site of inflammation in the tissue. IL-8 is secreted by many cell types in response to proinflammatory stimuli such as interleukin 1, tumor necrosis factor, and lipopolysaccharide and is a potent chemoattractant and activator of neutrophils. Neutrophil activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA/GRO) are structurally and functionally related to IL-8 and, like IL-8, bind to specific G protein-coupled receptors on neutrophils. In the present study two closely related cloned IL-8 receptor subtypes are characterized by expression of the cDNA clones in monkey kidney cells (COS-7) or chinese hamster ovary cells and analysis of their ligand binding profiles. Both receptor subtypes bind 125I-labeled IL-8 with similar high affinity, however, the F3R receptor binds IL-8 exclusively, while the 4Ab receptor binds both IL-8 and MGSA/GRO with high affinity and NAP-2 with lesser affinity. Furthermore, we demonstrate with the use of intersubtype chimeric receptors that the specificity of ligand binding to both IL-8 receptor subtypes is dictated by the heterogeneous NH2-terminal domain. The F3R receptor is representative of a restricted IL-8 receptor subtype, and 4Ab represents a nonrestricted receptor subtype. It is proposed that these subtypes be named IL-8 receptors alpha and beta, respectively.

Amino Acid Sequence↗
PubMed1992

Ribosomal RNA and rDNA sequence analyses.

The potential of ribosomal (r) RNA and the encoding genes (rDNA) to elucidate natural relationships has been dramatically extended by improved sequencing approaches and the application of polymerase chain reaction. Sequence information on 16S and 23S rRNA/DNA from 69 strains of 53 Streptomyces species allows determination of regions that can be used as target sites for diagnostic probes, and for amplification and sequencing primers. To generate phylogenetic trees, sequence similarities are converted into distance values. The topologies of the trees based on different parts of the molecule are compared among each other and to the numerical phenotypic clustering of the strains.

Base Sequence↗
PubMed1991

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.

Base Sequence↗
PubMed1990

Ammonia and lactate: differential information on monitoring training load in sprint events.

Two hypotheses were tested concerning training relevance of ammonia in capillary blood compared with lactate after extensive and intensive high-paced runs. 1. There is a load threshold at which ammonia concentration deviates statistically significantly from the pre-load level, while lactate indicates already anaerobic metabolic processes at extensive training loads. Increased ammonia concentration is understood as the activation of type IIb fibers. 2. The increase in ammonia is primarily a consequence of increasing training load intensity. The hypotheses were tested in a field experiment with male sprinters n = 13. The experimental plan followed a design of repeated measures in a 4 x 4 Latin-square. The subjects completed four high-pace runs (300 m) at 82.5%, 85%, 87.5%, and 90% of the maximum running intensity (identified in a contest). The results confirmed our first hypothesis. Significant increase in ammonia concentration was not found until after four runs at an intensity of 87.5% or after one 300-m run at maximum running speed. In contrast to this, a glycolytic reaction became evident in one run at 82.5% of the maximum intensity. Lactate concentration exceeded the anaerobic threshold at this point. Definite instructions for training practice cannot be made at the present state of research.

Adenosine Monophosphate↗
PubMed1990

Amycolatopsis methanolica sp. nov., a facultatively methylotrophic actinomycete.

The generic position of a gram-positive, facultatively methylotrophic actinomycete known as Nocardia sp. strain 239 was determined by comparing reverse transcriptase sequences of 16S rRNA. The assignment of the organism to the genus Amycolatopsis was strongly supported by chemotaxonomic and morphological data. A comparison with the type strains of validly described Amycolatopsis species showed that the organism formed the nucleus of a new species. The name proposed for this new species is Amycolatopsis methanolica. The organism has been deposited in the National Collection of Industrial Bacteria as NCIB 11946.

Actinomycetales↗
PubMed1989

Nucleotide sequence of 16S rRNA and phylogenetic position of the green sulfur bacterium Clathrochloris sulfurica.

The almost complete primary structure of the 16S rRNA from the green sulfur bacterium "Clathrochloris sulfurica" was determined by reverse transcriptase sequencing. Comparison of defined invariable parts of the molecule from representatives of 9 major lines of descent from the eubacterial kingdom shows C. sulfurica to be highly related to Chlorobium vibrioforme. The relationship between "Clathrochloris" and Chlorobium is in accord with the present allocation of these two genera into the family Chlorobiaceae.

Base Sequence↗
PubMed1988

Clinical use of the total dose intravenous infusion of iron dextran.

Eighty-seven patients with anemia and absent bone marrow hemosiderin were given treatment with total dose intravenous infusions of iron dextran. The effect of rate of infusion and premedication with diphenhydramine, aspirin, and methylprednisolone on acute and delayed reactions was assessed. All patients were monitored for 72 hours after infusion. Two patients reacted to the test dose. One responded with generalized body pain that lasted approximately 5 minutes. In one an anaphylactoid reaction developed, which was promptly terminated by intravenous methylprednisolone, subcutaneous epinephrine, and intravenous diphenhydramine. Transient delayed adverse reactions easily controlled by nonsteroidal anti-inflammatory drugs occurred in 37 patients. The most common delayed reaction was a syndrome characterized by arthralgia, myalgia, and fever. Seven subjects had a chronic disease in addition to anemia with absent bone marrow iron. In all seven normal hemoglobin and hematocrit values were attained after treatment. The results of our experience with total dose intravenous iron dextran therapy suggest that it be the preferred method of replenishment in clinical situations where parenteral administration of iron is indicated. An unexpected benefit was the efficaciousness of this method of administration in patients with iron deficiency and coexisting chronic disease. A protocol for its administration is proposed.

Anaphylaxis↗