Biomedical subjects

P J Jansen

Publications and source records attributed to P J Jansen.

11 recordsLinked to original sources

PubMed2002

Determination of relative UV response factors for HPLC by use of a chemiluminescent nitrogen-specific detector.

Ultraviolet (UV) absorbance is the most widely used detection method for high-performance liquid chromatography (HPLC) separations. In pharmaceutical analysis, purity determinations often include quantitation of related impurities based on relative HPLC peak areas obtained at a specific wavelength. In order for this quantitation to accurately reflect weight percentages of impurities, the relative UV response factors (absorptivities) at the given wavelength must be known. In this work, we present a convenient method for determining relative UV response factors on-line, without isolation or purification of impurities, without standards, and without requiring known analyte concentrations. The procedure described makes use of a chemiluminescent nitrogen-specific HPLC detector (CLND) in conjunction with a UV detector. The CLND response is directly proportional to the number of moles of nitrogen in each eluting peak, and can, therefore, be used to determine relative amounts of each nitrogen-containing impurity present in the sample, provided the molecular formulas are known (e.g. from exact mass LC-MS). It is a simple matter, then, to determine the relative UV response factors from the UV area ratios obtained for the same sample. The feasibility and accuracy of this method is demonstrated for gradient HPLC separations of commercially available compounds of widely varying structures. Finally, the method's utility in obtaining accurate mass balance is demonstrated by application to photodegradation of nifedipine.

Animals↗

PubMed2000

CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux.

CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5'-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22.Grb2.Shc.SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The degradation of the antitumor agent gemcitabine hydrochloride in an acidic aqueous solution at pH 3.2 and identification of degradation products.

A study of the degradation kinetics of gemcitabine hydrochloride (2'-deoxy-2',2'-difluorocytidine) in aqueous solution at pH 3.2 was conducted. The degradation of gemcitabine followed pseudo first-order kinetics, and rate constants were determined at four different temperatures. These rates were used to construct an Arrhenius plot from which degradation rates at lower temperatures were extrapolated and activation energy calculated. Four major degradation products were identified. Only one of these degradation products, the uridine analogue of gemcitabine, was a known degradation product of gemcitabine and was identified by comparison with synthesized material. The other three degradation products were isolated and characterized by spectroscopic techniques. Two of these products were determined to be the diastereomeric 6-hydroxy-5, 6-dihydro-2'-deoxy-2',2'-difluorouridines, and the other product was determined to be O(6),5'-cyclo-5,6-dihydro-2'-deoxy-2', 2'-difluorouridine. The mechanisms of formation of these degradation products are discussed.

Chromatography, High Pressure Liquid↗

PubMed2000

CD19 regulates Src family protein tyrosine kinase activation in B lymphocytes through processive amplification.

CD19 regulates constitutive and antigen receptor-induced signaling thresholds in B lymphocytes through its unique cytoplasmic domain. Herein, we demonstrate a novel molecular mechanism where interactions between CD19 and Lyn amplify basal and antigen receptor-induced Src family kinase activation. Lyn expression was required for CD19 tyrosine phosphorylation in primary B cells. Experiments with purified proteins demonstrated that CD19-Y513 was Lyn's initial phosphorylation and binding site. This led to processive phosphorylation of CD19-Y482, which recruited a second Lyn molecule, allowing for transphosphorylation and amplification of Lyn activation. In vivo, CD19 deficiency impaired, and CD19 overexpression enhanced, Lyn kinase activity. Thus, CD19 functions as a specialized adapter protein for the amplification of Src family kinases that is crucial for intrinsic and antigen receptor-induced signal transduction.

Amino Acid Sequence↗

PubMed1999

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.

Animals↗

PubMed1998

Characterization of impurities formed by interaction of duloxetine HCl with enteric polymers hydroxypropyl methylcellulose acetate succinate and hydroxypropyl methylcellulose phthalate.

Duloxetine hydrochloride ((S)-N-methyl-3-(1-naphthalenyloxy)-2-thiophenepropanamine hydrochloride) has been found to react with polymer degradation products or residual free acids present in the enteric polymers hydroxypropyl methylcellulose acetate succinate (HPMCAS) and hydroxypropyl methylcellulose phthalate (HPMCP) in dosage formulations to form succinamide and phthalamide impurities, respectively. The rate of formation of the impurities is accelerated by heat and humidity. The structures were deduced using molecular weights obtained from LC-MS experiments and confirmed by comparison of UV spectra, HPLC retention times, and electrospray mass spectra to independently synthesized material. It is proposed that polymer-bound succinic and phthalic substituents can be cleaved from the polymer, resulting in the formation of either the free acids or the anhydrides. It is postulated that the reaction is enabled by migration of either (1) the free acid or anhydride or (2) the parent drug through the formulation. The formation of these impurities was minimized by increasing the thickness of the physical barrier separating the enteric coating from the drug.

Adrenergic Uptake Inhibitors↗

PubMed1997

CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte signaling.

B cell development and humoral immune responses are controlled by signaling thresholds established through the B lymphocyte antigen receptor (BCR) complex. BCR signaling thresholds are differentially regulated by the CD22 and CD19 cell surface receptors in vivo. B cells from CD22-deficient mice exhibit characteristics of chronic stimulation and are hyper-responsive to BCR crosslinking with augmented intracellular Ca2+ responses. By contrast, B cells from CD19-deficient mice are hypo-responsive to transmembrane signals. To identify signaling molecules involved in the positive and negative regulation of signaling thresholds, the signal transduction pathways activated after BCR crosslinking were examined in CD22- and CD19-deficient B cells. These comparisons revealed that tyrosine phosphorylation of Vav protein was uniquely augmented after BCR or CD19 crosslinking in CD22-deficient B cells, yet was modest and transient after BCR crosslinking in CD19-deficient B cells. Ligation of CD19 and CD22 in vivo is likely to positively and negatively regulate BCR signaling, respectively, because CD19 crosslinking was more efficient than BCR crosslinking at inducing Vav phosphorylation. However, simultaneous crosslinking of CD19 with the BCR resulted in a substantial decrease in Vav phosphorylation when CD22 was expressed. Thus, the differential regulation of Vav tyrosine phosphorylation by CD19 and CD22 may provide a molecular mechanism for adjusting BCR signaling thresholds.

Animals↗

PubMed1997

CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19.

Establishing signal transduction thresholds that regulate B lymphocyte responses to foreign Ags and tolerance to self Ags is critical for humoral immune responses. The effects of altered signaling thresholds in B lymphocytes were examined in CD19-deficient mice and transgenic mice that expressed human CD19 at varying densities. Human CD19 restored normal B cell function and development to CD19-deficient mice when expressed at levels comparable to those of circulating human B cells. While CD19 expression levels were found to be developmentally regulated and tightly controlled in normal mice, two- or threefold changes in cell surface CD19 expression in transgenic mice dramatically affected B cell development, mitogen responses, serum Ig levels, humoral immune responses, and germinal center formation. B cells from mice that overexpressed CD19 also had decreased levels of surface IgM and a cell surface phenotype consistent with increased signaling in these cells. These results suggest that CD19 may serve similar functions in humans and mice and that CD19 defines signaling thresholds in vivo for the Ag receptor as well as other cell surface receptors that regulate B lymphocyte selection, activation, and differentiation.

Animals↗

PubMed1996

P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is a muclin-like glycoportein ligand for P- and E-selectin on myeloid cells and a subset of lymphocytes. We used flow cytometry and immunohistochemistry to examine expression of PSGL-1 on minor leukocyte populations, differentiating hematopoletic cells, and nonhematopoietic tissues using two monoclonal antibodies to distinct protein epitopes on PSGL-1. In the bone marrow, PSGL-1 was expressed on myeloid cells from the myeloblast stage to the segmented neutrophil, but was not detected on erythroblasts or megakaryocytes. All types of circulating myeloid cells expressed PSGL-1, and PSGL-1 was retained after extravasation of myetoid cells into tissues. PSGL-1 was also expressed on circulating dendritic cells, monocyte-derived dendritic cells, dendritic cells in lymphoid tissues and epidermis, and follicular dendritic cells. All types of lymphoid cells examined expressed PSGK-1, including immature and mature thymocytes, naive and memory T cells, gamma/delta T cells, netural killer cells, B cells and CD34+ progenitor cells. However, PSGL-1 levels were substantially lower on tonsillar lymphocytes than on circulating lymphocytes, suggesting that PSGL-1 expression is down regulated during or after entry of lymphocytes into secondary lymphoid tissue. Although PSGL-1 antigen was detected primarily on hamatopoietic cells, it was also present on time epithelium of the fallopian tube. Furthermore, PSGL-1 antigen gen was detected sporadically on microvascular endothelium in some pathologic tissues. This suggests that PSGL-1 may have functions other than mediating leukocyte adhersion.

Bone Marrow↗

PubMed1996

CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice.

B cell activation following antigen receptor cross-linking can be augmented in vitro by ligation of cell surface CD22, which associates with the SHP1 protein tyrosine phosphatase. The targeted deletion of CD22 in mice demonstrated that CD22 differentially regulates antigen receptor signaling in resting and antigen-stimulated B lymphocytes. B cells from CD22-deficient mice exhibited the cell surface phenotype and augmented intracellular calcium responses characteristic of chronically stimulated B cells, as occurs in SHP1-defective mice. Thus, CD22 negatively regulates antigen receptor signaling in the absence of antigen. However, activation of CD22-deficient B lymphocytes by prolonged IgM cross-linking resulted in modest B cell proliferation, demonstrating that CD22 positively regulates antigen receptor signaling in the presence of antigen.

Animals↗

PubMed1994

Two different dihydroorotate dehydrogenases in Lactococcus lactis.

The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic.

Amino Acid Sequence↗