Biomedical subjects

R D Nelson

Publications and source records attributed to R D Nelson.

At least 19 recordsLinked to original sources

PubMed2000

The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site.

Vacuolar H(+)-ATPase (V-ATPase) binds actin filaments with high affinity (K(d) = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.

Actin Cytoskeleton↗

PubMed2000

Kinetic and genetic bases for the heteroclitic recognition of mouse cytochrome c by mouse anti-pigeon cytochrome c monoclonal antibodies.

The B lymphocyte response to pigeon cytochrome c (CYT) in BALB/c mice was previously shown to initiate as a heteroclitic response specific for the self antigen mouse CYT. As the immune response progressed, the mAb that were produced became less heteroclitic and often bound pigeon CYT with higher affinity than they bound mouse CYT [Minnerath, J. et al., 1995. Proc. Natl. Acad. Sci. USA 92, 12379-12383]. To study the basis for heterocliticity and its loss in this system, the H and L chain amino acid sequences of anti-pigeon CYT mAb obtained from the primary and secondary Ab responses were first compared. The most frequent somatic mutations and Ig gene joints were then introduced into an engineered single-chain Fv (scFv) that expressed the germline-encoded V(H) and V(L) amino acid sequences. The effects of those changes on the on- rate, off-rate, and affinity constants in binding both mouse and pigeon CYT were determined by surface plasmon resonance. In this system, the heterocliticity of primary mAb was largely due to a decreased on-rate constant for binding pigeon CYT relative to mouse CYT. Various combinations of the three frequently occurring H chain somatic mutations (H31, H56, and H58) increased the on-rate constant to maximal levels. Only one of the mutations (H58) decreased the off-rate constant when in combination with the other mutations and the effect of H58 was greater for scFv binding mouse CYT than pigeon CYT. Consequently, the mutated scFv and many secondary mAb remained heteroclitic, although their affinities for pigeon CYT increased. Secondary mAb that were no longer heteroclitic expressed non-canonical amino acid sequences in the V(H)-D-J(H) joint in the context of the canonical V genes or expressed different V genes. In addition to providing insight into the molecular basis for heterocliticity, our findings confirm that both faster on-rate and slower off-rate constants are favored during affinity maturation of the Ab response.

Amino Acid Sequence↗

PubMed2000

Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens.

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [(3)H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.

Animals↗

PubMed1999

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles.

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.

Animals↗

PubMed1999

Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness.

H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause distal renal tubular acidosis, a condition characterized by impaired renal acid secretion resulting in metabolic acidosis. Patients with ATP6B1 mutations also have sensorineural hearing loss; consistent with this finding, we demonstrate expression of ATP6B1 in cochlea and endolymphatic sac. Our data, together with the known requirement for active proton secretion to maintain proper endolymph pH, implicate ATP6B1 in endolymph pH homeostasis and in normal auditory function. ATP6B1 is the first member of the H+-ATPase gene family in which mutations are shown to cause human disease.

Acidosis, Renal Tubular↗

PubMed1999

The Cre/loxP system and gene targeting in the kidney.

The Cre/loxP and Flp/FRT systems mediate site-specific DNA recombination and are being increasingly utilized to study gene function in vivo. These systems allow targeted gene disruption in a single cell type in vivo, thereby permitting study of the physiological and pathophysiological impact of a given gene product derived from a particular cell type. In the kidney, the Cre/loxP system has been employed to achieve gene deletion selectively within principal cells of the collecting duct. Disruption of target genes in the collecting duct, such as endothelin-1 or polycystic kidney disease-1 (PKD1), could lead to important insights into the biological roles of these gene products. With selection of the appropriate renal cell-specific promoters, these recombination systems could be used to target gene disruption to virtually any renal cell type. Although transgenic studies utilizing these recombination systems are promising, they are in their relative infancy and can be time consuming and expensive and yield unanticipated results. It is anticipated that continued experience with these systems will produce an important tool for analyzing gene function in renal health and disease.

Animals↗

PubMed1999

Modular assembly of voltage-gated channel proteins: a sequence analysis and phylogenetic study.

Voltage-sensitive cation-selective ion channels of the voltage-gated ion channel (VGC) superfamily were examined by a combination of sequence alignment and phylogenetic tree construction procedures. Segments of the alpha-subunits of K+-selective channels homologous to the structurally elucidated KcsA channel of Streptomyces lividans were multiply aligned, and this alignment provided the database for computer-assisted structural analyses and phylogenetic tree construction. Similar analyses were conducted with the four homologous repeats of the alpha-subunits from representative Ca2+- and Na+-selective channels, as well as with the ensemble of K+, Ca2+ and Na+ channels. In both the single subunit of the K+ channels and the individual repeats of the Ca2+ and Na+ channels, the analyses suggest the occurrence of at least two tandemly arranged modules corresponding to the predicted voltage-sensor domain and the pore domain. The phylogenetic analyses reveal strict clustering of segments according to cation-selectivity and repeat unit. We surmise that the pore module of the prokaryotic K+ channel was the primordial polypeptide upon which other modules were superimposed during evolution in order to generate phenotypic diversity. These observations may prove applicable to all members of the VGC family yet to be discovered throughout the prokaryotic and eukaryotic kingdoms.

Amino Acid Sequence↗

PubMed1998

Site-specific recombination using an epitope tagged bacteriophage P1 Cre recombinase.

Since the original description of Cre mediated site-specific recombination in bacteriophage P1 (Sternberg, N., Hamilton, D., 1981 J. Mol. Biol., 150, 467-487), the Cre-lox system of recombination has been widely used to manipulate prokaryotic and eukaryotic genomes. Unfortunately, there are few means available to measure Cre protein expression in vivo. We have constructed an expression vector wherein the Cre protein is tagged at the carboxy terminus with an 11-amino-acid epitope to the herpes simplex virus (HSV) glycoprotein D coat protein (Isola, V.J., Eisenberg, R.J., Siebert, G.R., Heilman, C.J., Wilcox, W.C., Cohan, G.H., 1989. J. Virol. 63, 2325-2334). The epitope tag facilitates detection of Cre expression in vitro and in vivo using immunofluorescent labeling with a commercially available antibody. The epitope tag does not interfere with Cre recombinase activity or alter recombination efficiency between loxP sites. We have shown in mice that a transgene expressing our tagged Cre is capable of excising a loxP flanked sequence contributed by another transgenic mouse. In summary, we have developed an epitope-tagged Cre recombinase that is fully active and readily detectable.

3T3 Cells↗

PubMed1998

Dimerization of P-selectin glycoprotein ligand-1 (PSGL-1) required for optimal recognition of P-selectin.

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with alpha1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.

Alanine↗

PubMed1998

Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice.

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5'-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5'-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.

Animals↗

PubMed1997

NMR study of the galactomannans of Trichophyton mentagrophytes and Trichophyton rubrum.

Around 90% of chronic dermatophyte infections are caused by the fungi Trichophyton mentagrophytes and Trichophyton rubrum. One of the causes of the chronic infection resides in the immunosuppressive effects of the cell-wall components of these organisms. Therefore we have attempted to identify the chemical structure of galactomannan, one of the major cell-wall components. The cell-wall polysaccharides secreted by T. mentagrophytes and T. rubrum were isolated from the culture medium and fractionated into three subfractions by DEAE-Sephadex chromatography. Analysis of each subfraction by NMR indicated that there are two kinds of polysaccharides present, i.e. mannan and galactomannan. The mannan has a linear backbone consisting of alpha1,6-linked mannose units, with alpha1,2-linked mannose units as side chains. The core mannan moiety of the galactomannan was analysed by a sequential NMR assignment method after removing the galactofuranose units by acid treatment. The result indicates that the mannan moiety has a linear repeating structure of alpha1,2-linked mannotetraose units connected by an alpha1,6 linkage. The H-1 signals of the two intermediary alpha1, 2-linked mannoses of the tetraose unit showed a significant upfield shift (Deltadelta=0.05-0.08 p.p.m.), due to the steric effect of an alpha1,6-linked mannose unit. The attachment point of the galactofuranose units was determined at C-3 of the core mannan by the assignment of the downfield-shifted 13C signals of the galactomannan compared with those of the acid-modified product. In these galactomannans there were no polygalactofuranosyl chains which have been found in Penicillium charlesii and Aspergillus fumigatus.

Carbohydrate Conformation↗

PubMed1997

Anti-inflammatory action of dapsone: inhibition of neutrophil adherence is associated with inhibition of chemoattractant-induced signal transduction.

Dapsone has clinical utility as an anti-inflammatory agent but the mechanism of this action remains unknown. We have previously reported that dapsone inhibits beta2 integrin (CD11b/CD18)-mediated adherence of human neutrophils in vitro and now describe studies designed to discover how dapsone-mediated inhibition of this neutrophil function occurs. Results indicate that dapsone interferes with the activation or function of the G-protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli. They also show that dapsone-mediated suppression of this pathway inhibits the generation of second messengers essential to the activation of beta2 integrin molecules, as well as respiratory and secretory functions of neutrophils exposed to chemoattractants. We propose that the inhibition of chemoattractant-induced signal transduction by dapsone suppresses neutrophil recruitment and local production of toxic respiratory and secretory products in the affected skin of dermatitis herpetiformis and other neutrophilic dermatoses.

Anti-Infective Agents↗

PubMed1997

High-resolution backscatter electron detection of cell surface molecules on human platelets using the double-layer coating method and cryo field emission scanning electron microscopy.

A model system utilizing cryo scanning electron microscopy (SEM) for the detection of putative cell adhesion molecule(s) on the surface of human platelets is described. Plunge freezing was used for cryoimmobilization of unactivated and activated platelets after prefixation. Extracellular ice was removed by sublimation to expose the surface of the platelet membrane. Cryosamples were coated by the double-layer method, in which undirectional shadowing is performed at an angle of 45 degrees with 2 nm of platinum by thermal evaporation, followed by evaporation of 5 nm of carbon at an angle of 90 degrees for stabilization of the platinum film. The topography of the extracellular surface of the unstimulated platelet membrane was dominated by small spherical protrusions, while that of the activated platelet had not only similar spherical projections, but also possessed numerous rod-like protrusions, presumably representing the upregulation of the cell adhesion molecule, P-selectin, from intracellular a granules. These results clearly demonstrate that cryo field-emission SEM can detect molecular topography on the extracellular surface of cells consistent with the dimensions and shape of membrane cell adhesion molecules.

Blood Platelets↗

PubMed1997

Receptors for the chemoattractants C5a and IL-8 are clustered on the surface of human neutrophils.

We have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N-formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.

Animals↗

PubMed1997

Preservation of intercalated cell H(+)-ATPase in two patients with lupus nephritis and hyperkalemic distal renal tubular acidosis.

In patients with Sjögren's syndrome and a secretory-defect distal renal tubular acidosis (dRTA), absence of vacuolar H(+)-ATPase from collecting duct intercalated cells has been reported. The H(+)-ATPase was examined in two patients with lupus nephritis and hyperkalemic (presumed voltage defect) dRTA. Both patients had a positive urine anion gap, alkaline urine despite acidemia, no rise in urine PCO2 with alkaluria, a urine pH > 5.5, and urine potassium excretion rate not significantly increased after 80 mg of intravenous furosemide. In both patients, immunocytochemistry of renal biopsy frozen sections with an anti-H(+)-ATPase monoclonal antibody showed bright staining of the proximal tubule brush border and collecting duct intercalated cells. In one patient, routine immunofluorescence analysis of a frozen section of her kidney biopsy with antihuman IgG showed staining of the collecting duct, indicative of autoantibodies to this segment. Moreover, rat kidney sections incubated with her serum showed labeling of the intercalated cells. On immunoblots of human kidney microsomal membranes performed with serum from both patients, an immunoreactive polypeptide was observed at M(r) approximately 56 kD that was not seen with control serum. Neither patient's sera reacted with affinity-purified bovine H(+)-ATPase or with lysates from 293 cell fibroblasts in which either of both isoforms of the human H(+)-ATPase B subunit (56 kD) were expressed. These findings demonstrate that the spectrum of dRTA includes the preservation of H(+)-ATPase in intercalated cells, in patients with presumed voltage defect dRTA. Moreover, some patients may have autoantibodies to the intercalated cells that are not directed to subunits of the H(+)-ATPase.

Acid-Base Equilibrium↗

PubMed1996

Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.

We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.

Animals↗

PubMed1995

A central role for microvillous receptor presentation in leukocyte adhesion under flow.

Leukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation ("tethering"), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking.

Animals↗

PubMed1995

Cytokine-stimulated chemotaxis of human neutrophils in a 3-D conjoined fibrin gel assay.

The ability of neutrophils to migrate through three-dimensional (3-D) tissues in response to chemical stimuli is critical to their host defense function. However, studies characterizing stimulated migration in vitro have been largely limited to two-dimensional (2-D) surfaces. In this study, we have employed direct observation methods to quantify human neutrophil migration in 3-D fibrin gel using time-lapse video microscopy and automated cell tracking methods. A novel 3-D conjoined gel assay was developed to establish experimentally quantifiable and theoretically predictable diffusion gradients of chemotactic factors. This assay was used to measure objective migration parameters, namely the random motility and chemotaxis coefficients, in response to the cytokine, interleukin-8 (IL-8). The random motility coefficient, mu, showed a biphasic dependence on IL-8 concentration with a maximum of 1.1 x 10(-8) cm2/s at 5 x 10(-8) M IL-8; no significant motility was observed in the absence of IL-8. We further established the dependence of cell orientation bias, phi, on the concentration and gradient steepness (i.e., specific gradient, SG) of IL-8. Results indicate that phi increases with increasing SG, provided the concentration is maintained sufficiently low, which we conjecture to result from minimizing IL-8 receptor down-regulation. The chemotaxis coefficient, chi, was maximum at an intermediate SG for both IL-8 concentrations studied. We also examined the applicability of this assay to estimate mu and chi from indirect measurements of chemotaxis, namely the simpler measurement of cell redistribution after a prescribed incubation time, as opposed to direct cell tracking measurements. By virtue of measuring chi, this is the first quantitatively objective study of mammalian cell chemotaxis in a physiologically relevant 3-D gel and, in particular, of neutrophil chemotaxis on any substratum in response to the physiologically relevant chemotactic factor, IL-8.

Chemotactic Factors↗