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J Connor

Publications and source records attributed to J Connor.

At least 73 records · Page 4Linked to original sources
PubMed1990

Involvement of Rh blood group polypeptides in the maintenance of aminophospholipid asymmetry.

The human erythrocyte (RBC) Rh blood group system consists of a complex of distinct integral membrane polypeptides with physical properties common to the aminophospholipid transporter responsible for the transbilayer movement of phosphatidylserine (PS) in RBC. To assess the involvement of Rh polypeptides in PS translocation, the aminophospholipid translocase was labeled with a photoactivatable PS analogue, 125I-azido-PS, and with an inhibitor of PS transport, 125I-labeled 2-(2-pyridyldithio)ethylamine. The ability of monoclonal Rh antibodies to immunoprecipitate the labeled transporter was determined. Immunoprecipitated Rh polypeptides were found to be labeled with the aminophospholipid translocase markers, suggesting that Rh proteins are involved in the transbilayer movement of PS.

Biological Transport↗
PubMed1990

Maintenance of lipid asymmetry in red blood cells and ghosts: effect of divalent cations and serum albumin on the transbilayer distribution of phosphatidylserine.

The maintenance of lipid asymmetry in the plasma membrane of human red blood cells (RBC) was investigated by assessing the equilibrium distribution of exogenously inserted NBD-labeled phosphatidylserine (PS) and endogenous PS in RBC and hypotonically lysed ghosts. PS distribution was determined by the ability to 'back-exchange' NBD-lipids into acceptor membranes and bovine serum albumin, and by prothrombinase complex assay for endogenous PS. To maintain the normal asymmetric distribution of PS in RBC, ghosts required Mg2+ in the lysis buffer. The inclusion of Ca2+, even in the presence of Mg2+ resulted in complete randomization of endogenous and exogenously inserted PS. These results indicate that NBD-labeled PS analogs faithfully monitor the distribution of endogenous PS during ghost preparation. In contrast, treatment of RBC with bovine serum albumin had no effect on the distribution of endogenous PS, although it resulted in a time-dependent movement of NBD-labeled PS from the inner to the outer leaflet (flop). This phenomenon was dependent on continuous incubation in the presence of albumin and could not be duplicated when pure acceptor membranes were used.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Aminophospholipid translocation in erythrocytes: evidence for the involvement of a specific transporter and an endofacial protein.

The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown [Connor & Schroit (1988) Biochemistry 27, 848-851] to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. Our results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with 125I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Similarly, treatment of RBC with 5,5'-dithiobis(2-nitrobenzoic acid), which depletes endogenous glutathione and induces oxidation of endofacial proteins [Reglinski et al. (1988) J. Biol. Chem. 263, 12360-12366], inhibited NBD-PS transport in a manner analogous to diamide. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids resides on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Proton and divalent cations induce synergistic but mechanistically different destabilizations of pH-sensitive liposomes composed of dioleoyl phosphatidylethanolamine and oleic acid.

Protons and divalent cations show synergistic effects on the destabilization of liposomes composed of unsaturated phosphatidylethanolamine and oleic acid (Düzgünes et al., Biochemistry (1985) 24, 3091). We have extended these observations and investigated the effects of Ca2+ and Mg2+ on the proton-induced destabilization of dioleoyl phosphatidylethanolamine/oleic acid (DOPE/OA) (4:1 molar ratio) liposomes. Temperature-induced aggregation was measured by 90 degrees light scattering. Lipid mixing was used to monitor vesicle destabilization and freeze-fracture electron microscopy was used to examine the structures formed from DOPE/OA vesicles in the presence of Ca2+ and/or protons. Both Mg2+ and Ca2+ shift the pH required for 50% lipid mixing to higher values. Temperature-induced vesicle aggregation occurs at lower temperatures in the presence of divalent cations and/or protons, indicating that intervesicular repulsions are decreased. Freeze-fracture electron micrographs show that the structures formed from DOPE/OA in the presence of Ca2+ differ significantly from those found in the presence of protons. In general, protons induce the formation of hexagonal phase, while the presence of Ca2+ leads to the formation of extensive regions of lamellar sheets with numerous lipidic particles. The synergistic effect of divalent cations and proton may be important for the maximal biological activity of DOPE/OA liposomes.

Calcium↗
PubMed1989

Transbilayer movement of phosphatidylserine in nonhuman erythrocytes: evidence that the aminophospholipid transporter is a ubiquitous membrane protein.

A 31-32-kDa integral membrane protein has been previously identified in erythrocytes as the protein most likely to be responsible for the transbilayer movement of phosphatidylserine (PS) [Connor & Schroit (1988) Biochemistry 27, 848-851]. Using similar techniques, we have identified analogous proteins of identical molecular weights in bovine, equine, ovine, porcine, canine, caprine, and rhesus red blood cells. Similar to human red blood cells, all of the mammalian cells were able to specifically transport an exogenously supplied fluorescent PS analogue from their outer-to-inner membrane leaflet. In addition, transport could be reversibly inhibited with the sulfhydryl-specific inhibitor pyridyldithioethylamine (PDA). PDA-sensitive PS transport was also observed in nucleated human and murine cell lines. Analysis of isolated plasma membranes from 125I-PDA-labeled cells revealed marked labeling of a 32,000-Da component. Attempts to inhibit PS transport by treating the cells with proteases, lectins, or antibody suggested that the 32-kDa polypeptide is an integral membrane protein that does not contain sites critical to its function at the cell surface.

Animals↗
PubMed1989

Gene expression in response to acute unilateral ureteral obstruction.

Acute unilateral ureteral obstruction results in differential growth characteristics of both the ipsilateral and contralateral kidney. The obstructed kidney undergoes cellular atrophy following an initial phase of interstitial proliferation while the contralateral kidney hypertrophies. To evaluate the molecular events occurring in both kidneys after obstruction, we examined the expression of growth related (c-fos, c-myc, cH-ras, HSP 70), cell maintenance (beta-actin), and cellular damage (TRPM-2) genes at the mRNA level. In the contralateral kidney an early and transitory induction of c-fos and c-myc expression occurred while a bimodal induction was noted in the obstructed kidney. The patterns of cH-ras, HSP 70 and actin expression also differed in both kidneys. Induction of TRPM-2 was noted only in the obstructed kidney. Rapid gene activation is evident in both the contralateral and obstructed kidney following unilateral ureteral obstruction. The patterns of expression are distinct and may reflect the cellular response to stress (cell death and stromal proliferation) in the obstructed kidney versus a response to a systemic stimulus resulting in cellular hypertrophy in the contralateral kidney.

Acute Disease↗
PubMed1989

Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.

Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS--e.g., PS exposure in the outer leaflet of the cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells--serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival of pathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7- to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages.

4-Chloro-7-nitrobenzofurazan↗
PubMed1988

Transbilayer movement of phosphatidylserine in erythrocytes: inhibition of transport and preferential labeling of a 31,000-dalton protein by sulfhydryl reactive reagents.

A series of labeled thiolation reagents were synthesized on the basis of the parent structure pyridyldithioethylamine (PDA). These compounds specifically and reversibly inhibit the active intrabilayer transport of phosphatidylserine (PS) in human red blood cells. The binding of PDA to cells can be quantified since the thiol-disulfide exchange reaction yields a chromophore. In addition, the presence of a primary amine makes it amenable to derivatization with a variety of compounds. An iodinated derivative of PDA preferentially labeled a 31,000-dalton red blood cell peptide. The labeled component, which may represent the PS transporter, comigrated with integral membrane protein band 7.

Carrier Proteins↗
PubMed1988

Calcium channel antagonists delay regression of androgen-dependent tissues and suppress gene activity associated with cell death.

Androgen deprivation subsequent to castration of an adult male rat results in the regression of sexual accessory tissues. Regression of these tissues involves the massive death of androgen-dependent cells. Using the rat ventral prostate gland as a model to study androgen-programed cell death, we have characterized a series of molecular events that accompany its regression. This analysis has shown that there was a sequential induction of specific gene transcripts in the ventral prostate gland following castration. The first event in this cascade was an abrupt induction of transcripts encoding c-fos. Since c-fos expression has been linked to perturbations in intracellular Ca2+ levels, we investigated whether membrane-mediated Ca2+ flux might be an early physiological step involved in the death of prostatic cells. To test this, rats were treated simultaneously upon castration with either verapamil or nifedipine, two different calcium channel antagonist drugs. Compared to the ventral prostate glands of untreated castrated rats, the glands of the calcium channel antagonist-treated rats showed a significant delay in all parameters associated with regression (loss of wet weight and DNA content and delay in histological changes associated with prostatic regression). The seminal vesicle glands of treated rats also showed signs of delayed regression. Furthermore, calcium channel antagonists suppressed the induction of transcripts encoding both c-fos and testosterone-repressed prostate message-2 (TRPM-2), a gene expressed exclusively by dying cells, during the first 62 hr following castration. These findings further support a role for calcium ion influx in the pathway leading to hormonally programed prostate cell death, and suggest the intriguing possibility that modulation of this activity can alter the process by which cells die.

Androgens↗
PubMed1987

Determination of lipid asymmetry in human red cells by resonance energy transfer.

This report describes the application of a resonance energy transfer assay to determine the transbilayer distribution of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipid analogues. The validity of this technique was established by determining the relationship between the distance of separation of lissamine rhodamine B labeled phosphatidylethanolamine (N-Rho-PE) acceptor lipid and NBD-labeled donor lipid and energy transfer efficiency. By determination of the distance between probes at 50% transfer efficiency (R0), the distance between fluorophores distributed symmetrically (outer leaflet label) and asymmetrically in artificially generated vesicles was determined. Calculation of the average distance between probes revealed a 14-A difference between NBD-lipid and N-Rho-PE localized in the same leaflet and in opposing leaflets, respectively. Application of this technique to the study of the transbilayer distribution of NBD-lipid in human red blood cells (RBC) showed that exogenously supplied NBD-phosphatidylserine (NBD-PS) was selectively transported to the inner leaflet, whereas NBD-phosphatidylcholine remained in the outer leaflet. In contrast, pretreatment of the RBC with diamide (a SH cross-linking reagent) blocked the transport of NBD-PS. The absence or presence of NBD-PS in the outer leaflet was independently verified by employing "back-exchange", trinitrobenzenesulfonic acid derivatization, and decarboxylation with PS decarboxylase experiments. These control experiments yielded results which confirmed the lipid distributions determined by the resonance energy transfer assay.

4-Chloro-7-nitrobenzofurazan↗
PubMed1987

Hematuria induced by urethral catheterization.

The amount of traumatic hematuria induced by urethral catheterization was studied by comparing the pre- and post-catheterization urinalyses of 93 patients presenting to the emergency department with abdominal complaints of an acute nature. Thirty-nine male patients ranging in age from 4 to 91 years and 51 female patients ranging in age from 1 to 70 years were studied. Catheterizations were performed by nurses (54%), resident house-staff (38%), and nurse practitioners (8%). Overall, hematuria was induced in 16 of 93 patients (17.2%) and, when present, never exceeded 3 RBC/hpf.

Adolescent↗
PubMed1986

Biodistribution of pH-sensitive immunoliposomes.

Liposomes composed of either dioleoylphosphatidylethanolamine and oleic acid (pH-sensitive) or dioleoylphosphatidylcholine and oleic acid (pH-insensitive) were injected into C3H and Balb/c mice in order to determine the tissue distribution of both the lipid and the aqueous content. The lipid component was monitored by use of [3H]cholestanyl ether and the aqueous content was monitored by use of encapsulated 125I-tyraminyl-inulin. The pH-insensitive liposomes injected into both types of mice were rapidly cleared from the blood stream followed by accumulation primarily in the liver, followed by the spleen. The presence of a monoclonal antibody on the liposome surface caused a slight acceleration in liver accumulation, though generally gave the same profile as the antibody-free liposomes. pH-sensitive liposomes were leaky upon exposure to the mouse plasma following injection. The lipid component, though, displayed a large amount (e.g., 50-70% in C3H mice) of accumulation in the lung for up to 6 h, followed by a subsequent appearance in the liver and spleen. The presence of monoclonal antibody had no effect on the tissue distribution profile. These results indicate that the pH-sensitive liposomes, although ineffective as an aqueous drug delivery agent, may be effective as a means of delivering lipophilic drugs to the lung.

Animals↗