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1.
Mol Endocrinol ; 11(7): 851-8, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9178745

ABSTRACT

Two different activating PTH/PTH-related peptide (PTHrP) receptor mutations, H223R and T410P, were recently identified as the most likely cause of Jansen's metaphyseal chondrodysplasia. To assess the functional importance of either amino acid position in the human PTH/PTHrP receptor, H223 and T410 were individually replaced by all other amino acids. At position 223, only arginine and lysine led to agonist-independent cAMP accumulation; all other amino acid substitutions resulted in receptor mutants that lacked constitutive activity or were uninformative due to poor cell surface expression. In contrast, most amino acid substitutions at position 410 conferred constitutive cAMP accumulation and affected PTH/PTHrP receptor expression not at all or only mildly. Mutations corresponding to the H223R or T410P exchange in the human PTH/PTHrP receptor also led to constitutive activity when introduced into the opossum receptor homolog, but showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor. The PTH/PTHrP receptor residues mutated in Jansen's disease are conserved in all mammalian members of this family of G protein-coupled receptors. However, when the equivalent of either the H223R or the T410P mutation was introduced into several other related receptors, including the PTH2 receptor and the receptors for calcitonin, secretin, GH-releasing hormone, glucagon-like peptide I, and CRH, the resulting mutants failed to induce constitutive activity. These studies suggest that two residues in the human PTH/PTHrP receptor, 223 and 410, have critical roles in signal transduction, but with different sequence constrains.


Subject(s)
Cyclic AMP/metabolism , Gene Expression Regulation/genetics , Osteochondrodysplasias/genetics , Point Mutation/genetics , Receptors, Parathyroid Hormone/genetics , Amino Acid Sequence , Animals , COS Cells , DNA/genetics , Dose-Response Relationship, Drug , Humans , Immune Sera/immunology , Molecular Sequence Data , Rabbits , Rats , Receptors, Parathyroid Hormone/biosynthesis , Receptors, Parathyroid Hormone/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Signal Transduction/physiology
2.
Endocrinology ; 137(9): 3936-41, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8756569

ABSTRACT

Inverse agonists, ligands that suppress spontaneous receptor signaling activity, have been described for a growing number of G protein-coupled receptors; however, none have been reported for the PTH/calcitonin/secretin receptor family. We took advantage of the constitutive signaling activity of two mutant forms of the PTH/PTH-related peptide (PTHrP) receptor, recently identified in patients with Jansen's metaphyseal chondrodysplasia, to screen for PTH and PTHrP analogs with inverse agonist activity. Two antagonist peptides, [Leu11, D-Trp12]hPTHrP(7-34)NH2 and [D-Trp12, Tyr34]bPTH-(7-34)NH2, displayed inverse agonist activity and reduced cAMP in COS-7 cells expressing either mutant receptor by 30-50% (EC50 approximately 50 nM). These data demonstrate that the concept of inverse agonism can be extended to this distinct family of G protein-coupled receptors and their cognate antagonist peptide ligands. Such ligands shall be useful probes of the multi-state conformational equilibria proposed for these receptors and could lead to new approaches for treating human diseases caused by receptor activating mutations.


Subject(s)
Mutation , Receptors, Parathyroid Hormone/agonists , Receptors, Parathyroid Hormone/genetics , Cell Line , Cyclic AMP/biosynthesis , Humans , Ligands , Receptor, Parathyroid Hormone, Type 1 , Receptors, Parathyroid Hormone/metabolism , Time Factors
3.
Hematol Oncol Clin North Am ; 6(2): 297-322, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1533857

ABSTRACT

The origin of the malignant stem cell in multiple myeloma, despite years of investigation by many laboratories, remains elusive. We have described a population of monoclonal circulating B-lineage lymphocytes that has been detected in all myeloma patients analyzed, both at diagnosis and after chemotherapy, and that has many properties consistent with its definition as either a stem cell compartment or an intermediate between the stem cell and the bone marrow localized plasma cells. On average, 40% to 50% of peripheral blood mononuclear cells are abnormal B cells that express CD10 and PCA-1 in conjunction with B-lineage markers CD19, CD20, and CD24 and variable expression of CD5. The B cells are monoclonal by Southern blot analysis and represent a highly pleiomorphic population. The migratory patterns of these cells are unknown, and their presence in blood may reflect cells in transit from a parent organ such as spleen to bone marrow for terminal differentiation, or they may originate in the bone marrow prior to circulation and seeding of other skeletal or extraskeletal sites. The working hypothesis underlying this work postulates that these abnormal B cells originate outside the marrow, giving rise to plasma cells only after migration to the bone marrow, which provides a microenvironment conducive to terminal plasma cell differentiation. Bone marrow plasma cells do not include an actively proliferating component and are terminally differentiated end stage cells. In contrast, the circulating abnormal B cells include proliferating cells and appear to be heterogeneous in differentiation stage. Analysis of CD45 isoform expression indicates a population continuously differentiating from a late B-cell stage through the early plasma cell stages to an end stage plasma cell. Quantitative and qualitative expression of CD45 has been shown to characterize B-cell development, with a high density of the CD45RA isoform on mature resting B cells, a transition to CD45R0 on activated B cells, and a gradual loss of total CD45, predominantly of the CD45R0 isoform, during plasma cell development until, on end stage plasma cells, all CD45 expression is lost. In myeloma patients, all of these B-cell stages are represented, with the least differentiated B cells occurring in blood, intermediate stages in both blood and marrow, the most differentiated B and/or plasma cells in the bone marrow.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Antigens, CD/analysis , B-Lymphocytes/pathology , Histocompatibility Antigens/analysis , Multiple Myeloma/blood , Antibodies, Monoclonal , B-Lymphocytes/immunology , Cell Adhesion , Cell Differentiation , Humans , Immunophenotyping , Integrins/metabolism , Leukocyte Common Antigens
4.
Scand J Plast Reconstr Surg Hand Surg ; 32(4): 365-71, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9862103

ABSTRACT

The inflammatory recruitment of leucocytes is a main cause of tissue damage in ischaemia/reperfusion (I/R) injury. Under appropriate flow conditions, E-selectin and L-selectin participate in the initial deceleration of neutrophils (PMNs) on inflamed endothelial cells before transmigration of PMNs into the surrounding tissue. Previous work from our lab showed increased survival of I/R injured myocutaneous flaps after treatment with anti-E/L-selectin. In this study, we have evaluated a combined antibody to E-selectin and L-selectin (EL-246) in porcine pure skin flaps exposed to I/R injury. Buttock skin flaps were exposed to eight hours of ischaemia and 20 hours of reperfusion. EL-246 or saline was given intra-arterially into the flaps. Estimated surviving area was not improved in the treated group. The lack of effect of EL-246 supports our suspicion that different mechanisms are involved in I/R injury in myocutaneous flaps compared with pure skin flaps. As a certain shear stress must be present for the selectins to exert their effect, a possible explanation for the diverse results in muscle and skin might be different reflow patterns.


Subject(s)
E-Selectin/physiology , L-Selectin/physiology , Reperfusion Injury/physiopathology , Skin Transplantation/physiology , Surgical Flaps/physiology , Animals , Antibodies, Monoclonal/pharmacology , E-Selectin/immunology , Graft Survival/drug effects , Graft Survival/physiology , L-Selectin/immunology , Leukocytes, Mononuclear/physiology , Muscle, Skeletal/physiology , Peroxidase/metabolism , Reperfusion Injury/immunology , Swine
5.
Ugeskr Laeger ; 153(44): 3088-90, 1991 Oct 28.
Article in Danish | MEDLINE | ID: mdl-1949340

ABSTRACT

Two different techniques of small intestinal examination were assessed in 29 patients. Comparison between small intestinal enema and small intestinal follow-through examination ("fluoroscopic" small intestinal meal) did not reveal any difference in the diagnostic values.


Subject(s)
Contrast Media/administration & dosage , Enema/methods , Intestine, Small/diagnostic imaging , Administration, Oral , Cold Temperature , Crohn Disease/diagnostic imaging , Evaluation Studies as Topic , Humans , Intestinal Obstruction/diagnostic imaging , Radiography
9.
Comp Immunol Microbiol Infect Dis ; 31(6): 487-500, 2008 Nov.
Article in English | MEDLINE | ID: mdl-17915321

ABSTRACT

Yeast culture is widely used in animal feed and has been linked to beneficial effects on animal health and production. This study examined the anti-oxidant and immunomodulating effects of a consumable yeast culture, XP, in vitro. An aqueous extract of XP contained anti-oxidants able to enter living cells and quench free radicals. The XP extract induced an increased expression of CD69 and CD25 on NK and NKT cells, and an increased cytotoxic response to K562 tumor cells. The XP extract amplified ProteinA-induced B cell activation in vitro, as measured by induction of the CD86 antigen on B lymphoblasts in 7-day cultures. The data show an anti-inflammatory effect of the XP extract in conjunction with activation of NK cells and B lymphocytes in vitro. Further in vivo studies are needed to examine the impact of XP in animals with bacterial and viral infections, as well as around the time of vaccination.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Killer Cells, Natural/drug effects , Lymphocyte Activation , Natural Killer T-Cells/drug effects , Yeasts/immunology , Adult , Anti-Inflammatory Agents/immunology , Anti-Inflammatory Agents/metabolism , Antigens, CD/drug effects , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/drug effects , Antigens, Differentiation, T-Lymphocyte/immunology , Antigens, Differentiation, T-Lymphocyte/metabolism , Antioxidants/metabolism , Antioxidants/pharmacology , B7-2 Antigen/drug effects , B7-2 Antigen/immunology , B7-2 Antigen/metabolism , Cell Line, Tumor , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/drug effects , Interferon-gamma/immunology , Interleukin-2 Receptor alpha Subunit/drug effects , Interleukin-2 Receptor alpha Subunit/immunology , Interleukin-2 Receptor alpha Subunit/metabolism , K562 Cells , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lectins, C-Type , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Middle Aged , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Reactive Oxygen Species/analysis , Reactive Oxygen Species/immunology , Yeasts/chemistry , Yeasts/metabolism
10.
Am J Hematol ; 41(3): 199-208, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1384318

ABSTRACT

In order to fully understand the complexity of the monoclonal B lineage cells in multiple myeloma, it is necessary to evaluate the extent to which these cells are resident in solid lymphoid tissues and the phenotypic differences and similarities as compared to the circulating or bone marrow derived B lineage cells. Peripheral blood mononuclear cells from a patient with multiple myeloma were obtained 8 and 3 days prior to death, and mononuclear cells from lymph nodes, spleen, and bone marrow were obtained at autopsy. Rapid changes in the stage of differentiation of blood late-stage B lineage cells towards mature end-stage plasma cells were observed during the last week prior to death. Lymphoid cells within the blood comprised very few T cells, sub-normal numbers of monocytes, and 80% of B lineage cells which were at a late stage of differentiation. Shortly before death, plasma cells were found in the peripheral blood, indicating progression to plasma cell leukemia. At autopsy, the monoclonal B lineage cells in lymph node, spleen, and bone marrow represented different stages of terminal B cell differentiation. In each tissue, the B lineage cells were at an earlier differentiation stage, as defined phenotypically, than the circulating B lineage cells found in blood 3 days prior to death. Analysis of B cell markers and CD45 was used to define the differentiation stage of the relevant B cell populations, revealing a series of differentiation stages. The least mature B lineage cells (CD45hi) were found in lymph node. However, the CD45 isoform expressed was CD45R0, unlike most normal lymph node B cells. More differentiated B lineage cells (CD45med) were found in the bone marrow, and three sequential stages of pre-plasma cells were found in the spleen (CD45bright, CD45moderate, and CD45low-neg), all of which were CD45R0+. The B cells in normal spleen and bone marrow are CD45RA+. The presence of monoclonal B lineage cells in spleen was confirmed by Southern blotting. The B lineage cells from peripheral blood 3 days prior to death were approaching an end-stage plasma cell stage (CD45low/-). On B lineage cells from the various myeloma tissues, a concomitant loss of CD11b and increasing density of CD29 were observed as a function of progression to terminally differentiated stages.


Subject(s)
B-Lymphocytes/pathology , Blood Cells/pathology , Bone Marrow/pathology , Cell Transformation, Neoplastic/pathology , Lymph Nodes/pathology , Multiple Myeloma/pathology , Spleen/pathology , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antigens, CD/analysis , Antigens, CD19 , Antigens, Differentiation, B-Lymphocyte/analysis , Autopsy , B-Lymphocytes/immunology , Blotting, Southern , CD11 Antigens , CD18 Antigens , Cell Separation , Female , Fluorescent Antibody Technique , Humans , Integrin beta1 , Isomerism , Leukocyte Common Antigens/analysis , Middle Aged , Multiple Myeloma/mortality , Phenotype , Tumor Cells, Cultured/pathology
11.
Biochem Biophys Res Commun ; 217(3): 1145-50, 1995 Dec 26.
Article in English | MEDLINE | ID: mdl-8554569

ABSTRACT

We report the induction of intracellular calcium mobilization [Ca2+]i in normal peripheral blood mononuclear cells (PBMC) and Daudi cells following binding with the L-selectin monoclonal antibody FMC46. The [Ca2+]i signal was mediated directly by binding of FMC46 without cross-linking antibodies. Increased [Ca2+]i was not induced by other L-selectin antibodies tested (TQ1, Leu8, Lam1.3). The increase in [Ca2+]i was rapid and was blocked completely by BAPTA, an agent which chelates intracellular calcium. The increase in [Ca2+]i was observed in calcium-containing as well as calcium free medium, suggesting that FMC46 caused release of Ca2+ from intracellular stores. In both PBMC and Daudi cells, previous signaling via L-selectin still allowed signaling through cross-linking of surface antigen receptor. These data provide evidence for direct alteration of the state of lymphocytes after ligation of a specific L-selectin epitope. L-selectin-mediated signaling does not desensitize signaling through the antigen-receptor and could therefore play a role in preactivating lymphocytes during endothelial transmigration into lymphoid tissues.


Subject(s)
Calcium/metabolism , L-Selectin/physiology , Leukocytes, Mononuclear/metabolism , T-Lymphocytes/metabolism , Antibodies, Monoclonal , Cell Compartmentation , Cytoplasm/metabolism , Humans , Immunologic Techniques , Receptors, Antigen, T-Cell/physiology , Receptors, Lymphocyte Homing/physiology , Signal Transduction , Tumor Cells, Cultured
12.
Neuroradiology ; 35(4): 319-21, 1993.
Article in English | MEDLINE | ID: mdl-8492905

ABSTRACT

Whether a history of headache or "early" versus "late" ambulation (no bed rest or bed rest for 24 h) influence the occurrence of headache after lumbar iohexol myelography was studied by blinded interviews in 158 consecutive patients referred for elective lumbar myelography (LM) because of suspected lumbar disc prolapse or spinal stenosis. Headache after LM occurred more often in patients with a history of headache (57%) than in patients without such a history (29%), P < 0.001. Patients with normal myelographic findings complained of headache after LM more often (55%) than patients with abnormal myelograms (31%), P < 0.008. No difference in the incidence of headache after LM was demonstrated in early versus late ambulation.


Subject(s)
Early Ambulation , Headache/chemically induced , Iohexol/adverse effects , Lumbar Vertebrae/diagnostic imaging , Myelography , Adolescent , Adult , Female , Headache/prevention & control , Humans , Intervertebral Disc Displacement/diagnostic imaging , Male , Middle Aged , Risk Factors , Spinal Stenosis/diagnostic imaging
13.
J Immunol ; 147(3): 830-7, 1991 Aug 01.
Article in English | MEDLINE | ID: mdl-1830599

ABSTRACT

The integrin beta 1 (CD29) is a marker for total very late activation Ag integrins on cells, and exhibits considerable fluctuation in cell surface density at various stages of T cell development. We have analyzed beta 1 integrin expression on subsets of human thymus, and on T cells from healthy babies and children, in comparison to healthy adults aged 26 to 75. T cells from adult peripheral blood include a CD29-, a CD29lo, and a CD29hi set. Compared with adults, PBMC T cells from children have reduced numbers of both CD29lo and CD29hi subsets but equivalent numbers of CD29- T cells. The number of CD29hi T cells increases gradually with age, achieving adult levels only at about 26 yr of age; in aged adults (69 to 75 yr), nearly all T cells have a CD29hi phenotype. Most thymocytes and cord blood T cells, in contrast, have a single peak of CD29 staining that is intermediate to the two peaks seen in adults. Multi-negative progenitor and CD45RO- thymocytes (presumptive thymic generative line-age) are 98% CD29hi. Progenitor thymocytes and adult PBMC T cells express equivalent amounts of beta 1 and alpha 4, but progenitors are alpha 5hi, whereas PBMC T cells are alpha 5lo. T cells from children have reduced beta 1hi and alpha 5lo, but nearly comparable numbers of alpha 4hi. This suggests that the major very late activation Ag integrins during childhood may be alpha 5 beta 1 and alpha 4 complexed with an alternate beta chain. In children, the majority of CD29hi cells are also CD45RAhi, in contrast to the pattern in adults, in whom the majority of CD29hi T cells are CD45RA-. This suggests that in children, the main defense against infection may reside in the CD29hi45RAhi T cells, which have not yet made the transition to CD45RO and to bona fide memory status. The proliferative response to tetanus toxoid of 4- to 6-mo-old babies correlates with the number of CD29hi45RAhi T cells, suggesting that it derives at least in part from cells that do not express a "memory" phenotype. These observations show a pattern of alternating high and low density CD29 during T cell development, which is consistent with the idea that CD29 is a marker for functionally defined T cell sets. Analysis of the CD29 expression of CD29hi thymocytes developing in vitro supports this view. We suggest that the intensity of CD29 expression on a T cell varies, dependent upon the microenvironmental interactions required by a differentiating T cell.


Subject(s)
Antigens, CD/biosynthesis , Histocompatibility Antigens/biosynthesis , Integrins/biosynthesis , T-Lymphocyte Subsets/immunology , Adolescent , Adult , Aged , Aging/immunology , Cell Differentiation , Child , Child, Preschool , Fetal Blood/immunology , Humans , Infant , Infant, Newborn , Integrins/analysis , Isoantigens , Leukocyte Common Antigens , Middle Aged , T-Lymphocytes/immunology , Thymus Gland/immunology
14.
Int Immunol ; 1(3): 229-36, 1989.
Article in English | MEDLINE | ID: mdl-2535062

ABSTRACT

We have demonstrated a transition in the expression of CD45 isoforms, from the expression of the high molecular weight CD45R to the low molecular weight CD45 p180 isoform on normal and on monoclonal (possibly malignant) B cells undergoing differentiation into plasma cells. The differentiation into plasma cells was shown by the loss of CD20 and CD21 surface antigens, a reduced expression, but in our system not a complete loss, of CD19, and the expression of the plasma cell marker PCA-1. We used three-color immunofluorescence to demonstrate the shift from CD45R to CD45 p180 on CD19+CD20-CD21- cells in cultures of normal cells stimulated by pokeweed mitogen (PWM). Furthermore, tissue sections of extramedullary plasmacytomas, where plasma cells were defined by morphology and expression of cytoplasmic Ig, showed a complete loss of CD45R and CD45 p180 antigens, although the cells retained expression of CD45 common determinants. Finally, we have analysed PBMC from patients with Waldenström's macroglobulinemia (WM) at various stages of disease, and demonstrated that the monoclonal B cell subset present in their peripheral blood is heterogeneous in the expression of CD45 isoforms, including cells bearing only CD45R, those with a transitional CD45R+CD45 p180+ phenotype, and those expressing only CD45 p180. Upon stimulation in vitro these cells show greatly increased expression of CD45 p180. The differential expression of CD45 isoforms within a clonal B cell subset in the blood of these patients suggests that the monoclonal B cells in WM represent a continuously differentiating lineage of CD45R+ mature B cells giving rise to CD45 p180+ pre-plasma cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antigens, Differentiation , B-Lymphocytes/immunology , Histocompatibility Antigens , Antigens, Differentiation/chemistry , B-Lymphocytes/cytology , Cell Differentiation , Histocompatibility Antigens/chemistry , Humans , In Vitro Techniques , Interleukin-6/pharmacology , Leukocyte Common Antigens , Molecular Weight , Plasma Cells/cytology , Plasma Cells/immunology , Plasmacytoma/immunology , Plasmacytoma/pathology , Waldenstrom Macroglobulinemia/immunology , Waldenstrom Macroglobulinemia/pathology
15.
Abdom Imaging ; 20(5): 436-9, 1995.
Article in English | MEDLINE | ID: mdl-7580778

ABSTRACT

BACKGROUND: Patient discomfort 0-24 h after double-contrast barium enema (DCBE) was investigated in two ways. METHODS: In part 1, 139 patients, not previously informed, were contacted by telephone to assess symptom rates without bias. In part 2, designed as a prospective randomized double-blind trial, the effect of carbon dioxide (CO2) as an insufflating gas was compared with conventional atmospheric air (AA). RESULTS: Part 1: 10% experienced severe abdominal pain, and 18% severe abdominal distention. Part 2: Low discomfort rates were found for both severe pain (7% for AA vs. 2% for CO2) and severe distention (13% for AA vs. 8% for CO2); the differences were not significant. In both parts of the study, female patients with a history of abdominal discomfort of "colon irritable" type were significantly overrepresented in the severely symptomatic groups. Equal numbers of patients experiencing severe abdominal distention for the first time were found in both the AA and CO2 groups, ruling out AA as the sole cause of these symptoms. CONCLUSION: Abdominal post-DCBE discomfort seems to be less frequent than previously reported and is not effectively eliminated by CO2. We still find the use of AA in DCBEs justified.


Subject(s)
Abdominal Pain/epidemiology , Barium Sulfate , Carbon Dioxide , Enema/adverse effects , Pneumoradiography/adverse effects , Abdominal Pain/etiology , Aged , Double-Blind Method , Female , Humans , Male , Middle Aged , Prospective Studies , Sex Distribution
16.
J Biol Chem ; 271(33): 19888-93, 1996 Aug 16.
Article in English | MEDLINE | ID: mdl-8702701

ABSTRACT

Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.


Subject(s)
Parathyroid Hormone/chemistry , Proteins/chemistry , Receptors, Parathyroid Hormone/agonists , Amino Acid Sequence , Animals , Cell Line , Chlorocebus aethiops , Cyclic AMP/metabolism , Humans , Ligands , Molecular Sequence Data , Parathyroid Hormone-Related Protein , Protein Binding , Receptor, Parathyroid Hormone, Type 2 , Recombinant Fusion Proteins , Recombinant Proteins , Signal Transduction , Structure-Activity Relationship
17.
Mol Pharmacol ; 47(2): 330-9, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7870041

ABSTRACT

To help define essential interactions of cGMP with the catalytic site, we tested a series of cGMP analogs as competitive inhibitors of each cyclic nucleotide phosphodiesterase (PDE) family known to hydrolyze cGMP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to binding by the catalytic sites of each isozyme. The results indicate that the N1-nitrogen of cGMP contributes to binding at the catalytic site of all PDEs, probably as a hydrogen donor. All PDEs tested, with the exception of PDE2, also use the 6-oxo group, probably as a hydrogen acceptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-hydroxyl groups of cGMP are not major requirements for binding to any PDE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggesting that these PDEs are not sterically hindered with bulky 8-position substitutions and that they do not preferentially bind the anti-conformation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these substitutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding to the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to the catalytic, compared with noncatalytic, binding domains of PDEs. Even larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase and the cGMP-gated cation channel.


Subject(s)
Cyclic GMP/metabolism , Phosphoric Diester Hydrolases/metabolism , Animals , Baculoviridae/genetics , Catalysis , Cattle , Cells, Cultured , Cloning, Molecular , Cyclic GMP/analogs & derivatives , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Protein Conformation , Spodoptera
18.
Mol Pharmacol ; 47(2): 340-7, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7870042

ABSTRACT

To define essential interactions of cAMP with the catalytic sites of cyclic nucleotide phosphodiesterases (PDEs) and to begin to map the topology of the sites, we have tested a series of cAMP analogs as competitive inhibitors of the PDEs that hydrolyze cAMP with high efficiency (PDE1, PDE2, PDE3, and PDE4). Comparisons of IC50 values, relative to cAMP, were used to predict which functional groups on cAMP interact with each isozyme. Common to all PDEs tested, except for the calcium/calmodulin-dependent PDE (CaM-PDE, PDE1), is an interaction at the N1-position of cAMP and a distinct lack of binding to the 2'-hydroxyl group of the ribose moiety. Only the cGMP-stimulated (PDE2) and cAMP-specific (PDE4) PDEs appear to interact strongly at the N7-position. The cGMP-inhibited PDE (cGI-PDE, PDE3) may interact less strongly with this nitrogen. The PDE4 and PDE3 both interact with cAMP through the 6-amino group, which most likely serves as a hydrogen bond donor. PDE4 and PDE3 appear to be able to bind to the anti-conformer of cAMP, whereas the PDE1 and PDE2 bind the syn-conformer. The CaM-PDE exhibits no appreciable specificity for any of the analogs tested, showing little or no interaction with the 6-amino group or with any of the ring nitrogens. Large differences exist in the nucleotide-binding requirements for the PDE catalytic sites, compared with the regulatory sites of cAMP-dependent protein kinase and the catabolite activator protein.


Subject(s)
Cyclic AMP/metabolism , Phosphoric Diester Hydrolases/metabolism , Proteins/metabolism , Animals , Binding Sites , Catalysis , Cell Line , Cyclic AMP/analogs & derivatives , Protein Binding , Protein Conformation
19.
Am J Hematol ; 43(1): 29-36, 1993 May.
Article in English | MEDLINE | ID: mdl-7686332

ABSTRACT

We have previously reported the presence of monoclonal, tumor-related B lineage cells in the blood of myeloma patients. The cells are continuously differentiating, and the majority are at a very late stage of B cell differentiation into plasma cells, consistent with the hypothesis that they comprise a precursor cell subset responsible for disseminating and possibly for relapse of the disease. The pattern of beta 1 integrin expression on monoclonal B lineage cells from blood and bone marrow of myeloma patients was evaluated using multiparameter flow cytometry in comparison to normal blood or tissue B cells and malignant B cells from B-CLL, B lymphoma, or plasma cell leukemia. The alpha 4 and beta 1 chains were found on the majority of normal B cells, usually with a higher expression of alpha 4 compared to beta 1. alpha 5 was detectable at low density on B cells from lymph node, bone marrow, and lamina propria. the alpha 2 and alpha 6 chains are absent on B cells localized in normal lymphoid tissues as well as on normal blood B cells and in vitro activated B cells. In myeloma, the blood B cells express alpha 2, alpha 5, and alpha 6, suggesting important functional differences between these tumor-related B cells and their normal counterparts. The plasma cells located in myeloma bone marrow express no alpha 2, and almost no alpha 6, although they have variable expression of alpha 4, alpha 5, and beta 1. Thus the end-stage plasma cells appear to lack receptors that would support a propensity for invasion of basement membranes and exit to extravascular spaces. In contrast, the circulating plasmablasts in a patient with plasma cell leukemia make up a large subset of early plasma cells expressing all integrin receptors analyzed, including alpha 2 and alpha 6. Malignant cells from B-CLL and B lymphoma express only the alpha 4 and beta 1 integrins, and some B-CLL have very low levels of alpha 3, but no alpha 2, alpha 5, or alpha 6, suggesting that they may be limited to the vascular spaces and do not extravasate, at least for the stages of disease analyzed here.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
B-Lymphocytes/immunology , Integrins/analysis , Multiple Myeloma/blood , Multiple Myeloma/immunology , Antigens, CD/blood , Antigens, CD19 , Antigens, Differentiation, B-Lymphocyte/blood , B-Lymphocyte Subsets/immunology , Bone Marrow/immunology , Bone Marrow/pathology , Flow Cytometry , Humans , Integrins/biosynthesis , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Plasma Cell/blood , Leukemia, Plasma Cell/immunology , Lymphocyte Activation , Lymphoma, B-Cell/blood , Lymphoma, B-Cell/immunology , Multiple Myeloma/pathology
20.
Blood ; 78(3): 711-9, 1991 Aug 01.
Article in English | MEDLINE | ID: mdl-1830500

ABSTRACT

The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B-lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.


Subject(s)
Antigens, CD/analysis , Antigens, Differentiation/analysis , Antigens, Neoplasm/analysis , B-Lymphocytes/immunology , Histocompatibility Antigens/analysis , Multiple Myeloma/immunology , Paraproteinemias/immunology , Antibodies, Monoclonal , Antigens, CD/genetics , Fluorescent Antibody Technique , Histocompatibility Antigens/genetics , Humans , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Leukocyte Common Antigens , Multiple Myeloma/blood , Neprilysin , Paraproteinemias/blood , Restriction Mapping
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