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Biomedical subjects

R L Sloan

Publications and source records attributed to R L Sloan.

At least 19 recordsLinked to original sources
PubMed2002

Percutaneous endoscopic gastrostomy feeding in a district rehabilitation service.

OBJECTIVES: To examine gender, diagnosis, age, reasons for feeding, nutritional status, complications, outcome and duration of feeding in patients who have required a percutaneous endoscopic gastrostomy (PEG) for nutritional support at a district rehabilitation unit in the six years since the service was established. To identify potential for improvements in the management of future patients. DESIGN: Retrospective case note review of cases from 1992 to 1998. SETTING: District rehabilitation service for ages 16-64 serving the population of Fife, Scotland (population circa 350 000). SUBJECTS: All patients (n = 42) who had been fed via a PEG feeding tube in the previous six years. RESULTS: Forty-four PEG tube insertions had been conducted for 43 episodes of feeding in 42 patients. Five episodes of feeding were because of persistent vegetative state or low awareness state and 38 because of neurological swallowing impairment. Twenty-six (60%) patients were nutritionally depleted when PEG feeding was commenced. Twenty-seven (64%) patients experienced minor complications and 15 (34%) had no complications. At three months post procedure four (9.5%) patients had died and 21 (50%) had been discharged home. The mean duration of feeding on 31 October 1998 of the 20 patients (48%) who continued feeding at that date was 3.19+/-1.89 (mean +/- SD) years. CONCLUSIONS: Patients requiring PEG feeding in a district rehabilitation service have a range of diagnoses and the main indication for intervention is neurological swallowing impairment. The majority of patients were nutritionally depleted when feeding commenced and the reasons for this require further investigation.

Enteral Nutrition↗
PubMed2001

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton.

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.

Autoantibodies↗
PubMed2000

Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 degrees C.

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts at 37 degrees C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4, 4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport V(max) by using the same resealed ghosts. Incubation of membranes at 37 degrees C caused the dissociation of band 3 dimers to monomers but only after a lag period lasting approx. 50 h. The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 degrees C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 degrees C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 degrees C.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Characterization of the pH dependence of hemoglobin binding to band 3. Evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex.

The pH dependence of hemoglobin binding to inside-out red cell membrane vesicles was studied using 90 degrees light scattering (Salhany, J.M. et al., Biochemistry 19 (1980) 1447-1454). Hyperbolic binding curves were observed for high-affinity hemoglobin binding to the cytoplasmic domain of band 3 (CDB3) within the intact transporter. Analysis of these saturation curves yielded the apparent Kd and the maximum light scattering signal change (DeltaLSmax ). The apparent Kd for hemoglobin binding did not change substantially between pH 5.5 and 7.0, while at pH 8, there is no binding. In contrast, DeltaLSmax decreased by about 20-fold between pH 5.5 and 7.0, with an apparent pK of 6.5. These results suggest that hemoglobin binds to CDB3 with high affinity at both neutral and acid pH, a suggestion that was confirmed using a centrifugation method. Thus, the pK for the light scattering signal change is significantly lower than the pK for the actual binding process. We show that the change in DeltaLSmax is not related to a change in band 3 binding capacity, which remains constant as a function of pH. We also show that hemoglobin binding to non-band 3 sites contributes less than 10% to DeltaLSmax under our specific experimental conditions. On the basis of these and previous fluorescence quenching results in the literature, we propose a new model for hemoglobin binding to band 3, where raising the pH from 6 and 7 causes the CDB3-hemoglobin complex to undergo a conformational change leading to the movement of the bound hemoglobin away from the surface of the bilayer. The possible implication of this new mechanistic interpretation is discussed briefly.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

Fluoxetine as a treatment for post-stroke emotionalism.

The effectiveness of fluoxetine compared to placebo in the treatment of post-stroke emotionalism was investigated in 20 patients using a double-blind protocol. A total of 19 patients completed the study, as one subject in the treatment group had to be withdrawn. The subjects receiving fluoxetine showed a statistically and clinically significant improvement in their emotionalism compared to the placebo group, which was apparent in the majority of subjects from the third day of treatment.

Aged↗
PubMed1998

Multiple sclerosis in Fife.

Patients in Fife with multiple sclerosis were identified from three sources: a postal questionnaire to all general practices in Fife, hospital discharge data, rehabilitation service database. A total of 508 patients were identified in a population of 354,273 giving a crude prevalence rate of 143/100,000. The Standardised Prevalence Rate was 178/100,000. The sex ratio was 2.43 females to males. The prevalence was higher than identified in southern parts of the United Kingdom and similar to northern Scotland. A total of 200 (40%) cases were identified as having relapsing-remitting disease and of these 151 were ambulant, making them eligible for treatment with interferon beta-lb under current licensing criteria. Only 90 (60%) of the 151 currently eligible for the drug were under the care of a specialist and almost half the practices felt that they would change their referring habits. These factors may have implications for the workload of specialist clinics.

Adolescent↗
PubMed1997

Mild traumatic brain injury--the Fife perspective.

Results are reported from a study to identify patients residing in Fife with mild traumatic brain injury in the 16-65 year old age group, who attended an accident and emergency department following their brain injury. Over a two month period 161 such patients attended with minor head trauma, of which 33 entered our study. The major cause of mild traumatic brain injury was assault. We found that over two-thirds of patients in the study had persisting post-concussive symptoms six months post injury. Neuropsychological testing showed problems of concentration and memory, but not at a level that was significantly different from that expected in an average population. Other studies have shown that symptom rates are higher when patients get no explanation of their symptoms and we feel that better co-ordination of services for brain injured patients in Fife is required, to provide the necessary information, education and support.

Adolescent↗
PubMed1996

Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854-->Leu).

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56-->Glu within the cytoplasmic domain, and Pro-854-->Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4'-di-isothiocyanatodihydro-2, 2'-stilbenedisulphonate) to 'lysine A' (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'off' rate constant, but no difference was found between mutant and control when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854-->Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Kinetic evidence for ternary complex formation and allosteric interactions in chloride and stilbenedisulfonate binding to band 3.

The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4'-dibenzamidostilbene-2,2'-dibenzamidostilbene-2,2'-disu lfonate) and H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) by more than 10-fold at constant ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Evidence for the development of an intermonomeric asymmetry in the covalent binding of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to human erythrocyte band 3.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies have identified two oligomeric forms of band 3 whose proportions on gel profiles were modulated by the particular ligand occupying the intramonomeric stilbenedisulfonate site during intermonomeric cross-linking by BS3 [bis-(sulfosuccinimidyl) suberate] [Salhany et al. (1990) J. Biol. Chem. 265, 17688-17693]. When DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) was irreversibly attached to all monomers, BS3 covalent dimers predominated, while with DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) present to protect the intramonomeric stilbenedisulfonate site from attack by BS3, a partially cross-linked band 3 tetramer was observed. In the present study, we investigate the structure of the protected stilbenedisulfonate site within the tetrameric complex by measuring the ability of patent monomers to react irreversibly with DIDS. Our results show two main populations of band 3 monomers present after reaction with DNDS/BS3: (a) inactive monomers resulting from the displacement of reversibly bound DNDS molecules and subsequent irreversible attachment of BS3 to the intramonomeric stilbenedisulfonate site and (b) residual, active monomers. All of the residual activity was fully inhibitable by DIDS under conditions of reversible binding, confirming expectations that all of the monomers responsible for the residual activity have patent stilbenedisulfonate sites. However, within this active population, two subpopulations could be identified: (1) monomers which were irreversibly reactive toward DIDS and (2) monomers which were refractory toward irreversible binding of DIDS at pH 6.9, despite being capable of binding DIDS reversibly. Increasing the pH to 9.5 during treatment of DNDS/BS3-modified cells with 300 microM DIDS did not cause increased irreversible transport inhibition relative to that seen for cells treated at pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

In situ cross-linking of human erythrocyte band 3 by bis(sulfosuccinimidyl)suberate. Evidence for ligand modulation of two alternate quaternary forms: covalent band 3 dimers and noncovalent tetramers formed by the association of two covalent dimers.

Treatment of intact human erythrocytes with bis(sulfosuccinimidyl)suberate converted band 3 to two species with lower electrophoretic mobility in sodium dodecyl sulfate (SDS). The presence of the noncovalent anion transport inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonate, promoted the lowest mobility form, while a closely related analogue, 4,4'-diisothiocyano-2,2'-stilbenedisulfonate, did not. Ferguson analysis of the electrophoretic behavior of the two slowly migrating bands strongly suggested that they represented dimers and tetramers of band 3. Increasing the temperature of the SDS solution to greater than 60 degrees C quantitatively converted the tetrameric species to the dimeric form. We conclude that band 3 can be intermonomerically cross-linked by bis(sulfosuccinimidyl)suberate as covalent dimers within two alternate quaternary forms in a manner modulated by the ligand occupying the intramonomeric stilbenedisulfonate site. In one form, band 3 covalent dimers are noncovalently associated as a SDS-resistant tetramer, while in the other form, covalent dimers are not so associated. There is no obvious relationship between ligand stereochemistry and the resulting quaternary form, suggesting that the two forms reflect alternate allosterically modulated porter quaternary structures. The significance of these two quaternary states to the transport or the ankyrin binding functions of band 3 is unknown.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Direct evidence for modulation of porter quaternary structure by transport site ligands.

The transport inhibitor DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) changes the bis(sulfosuccinimidyl)suberate (BS3) crosslinking pattern of band 3 protein from a mixture of dimer-crosslinkable (DC) and tetramer-crosslinkable (TC) states to the TC-state as the exclusive crosslinked product for reactions occurring in membranes of intact human erythrocytes. Pretreatment of cells with DNDS followed by extensive washing restores the original DC to TC proportionality indicating that the two states are reversibly interconvertible. We suggest a model wherein band 3 transport site ligands allosterically modulate the global conformation of a tetrameric porter between two reversibly interconvertible quaternary structures. These transitions in quaternary structure may be important to transmembrane signaling of events between the exofacial ligand binding site and the sites on the porter extension which bind ankyrin and hemoglobin.

Allosteric Regulation↗
PubMed1988

Partial covalent labeling with pyridoxal 5'-phosphate induces bis(sulfosuccinimidyl)suberate crosslinking of band 3 protein tetramers in intact human red blood cells.

Partial covalent labeling of band 3 protein lysines with pyridoxal 5'-phosphate (a substrate and affinity probe) changes the bis(sulfosuccinimidyl)suberate crosslinking pattern of band 3 in intact red cells from a mixture of dimers and tetramers to all tetramers as the exclusive crosslinked product. This is the first demonstration of band 3 crosslinkage to the tetrameric level within membranes of intact red cells. The possible implications of the ligand-induced change in the band 3 crosslinking pattern are discussed.

Affinity Labels↗
PubMed1987

Characterization of pyridoxal 5'-phosphate affinity labeling of band 3 protein. Evidence for allosterically interacting transport inhibitory subdomains.

Pyridoxal 5'-phosphate (PLP) is a substrate of band 3, the erythrocyte anion transport protein. It competitively inhibits anion transport and labels two exofacial chymotryptic domains (the 17-kDa (CH17) and the 35-kDa (CH35) integral fragments). Two mol of PLP are bound/mol of each fragment at saturation. PLP labeling of both domains is competitive with chloride at constant ionic strength. Addition of DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), protects PLP labeling of CH35 but exposes new, nonoverlapping sites on CH17.4,4'-Diisothiocyanostilbene-2,2'-disulfonate reduces PLP labeling to both domains with time, while NAP-taurine (N(-4-azido-2-nitrophenyl)2-aminosulfonate) has no effect on either domain. At low chloride (balance citrate) and high DNDS, we can strongly suppress CH35 labeling and selectively titrate CH17 with PLP. Correlation of fractional transport inhibition with fractional PLP covalent coverage of CH17, quantitatively follows the 1:2 correlation line indicating that full coverage of CH17 sites (which constitute half of the total PLP-labeling sites on band 3) exactly inhibits one-half of transport. PLP labeling of CH35 sites accounts for the other half of inhibition. The inhibition-labeling correlation plots are nonlinear in the absence of DNDS, indicating the presence of allosteric interactions between the domains. We conclude that CH17 and CH35 compose nonoverlapping, functionally equivalent, allosterically linked transport inhibitory subdomains on band 3.

Affinity Labels↗
PubMed1987

Alterations in pyridoxal 5'-phosphate inhibition of human erythrocyte anion transport associated with osmotic hemolysis and resealing.

When pyridoxal 5'-phosphate (PLP) is covalently bound to band 3 protein in intact red blood cells and those cells are subjected to the osmotic hemolysis and resealing process, a significant reduction in the original PLP anion transport inhibitory potency occurs. We show that partial deinhibition is not due to the development of a second anion transport pathway in resealed ghosts. Rather, partial deinhibition arises from a hemolysis-induced conformational change in CH17 (17-kDa integral chymotryptic domain of band 3). This change causes the extracellular exposure of new transport inhibitory sites. Exposure of the new sites leads to a 2-fold increase in PLP labeling of CH17 in resealed ghosts compared with CH17 in intact red cells. The hemolysis and resealing process has no effect on the labeling of CH35 (35-kDa integral chymotryptic fragment of band 3). Double-labeling studies show restoration of transport inhibitory potency to near red cell levels when the newly exposed CH17 sites are labeled with PLP in resealed ghosts. The results support the view that CH17 contains PLP transport inhibitory sites. They show that a major conformational change occurs in band 3 with hemolysis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Evidence for multisite allosteric interactions on the band 3 monomer.

Pyridoxal-5'-phosphate is known to label the two integral, chymotryptic domains (CH17 and CH35) of the erythrocyte anion exchange protein known as band 3. The CH35 sites are mutually exclusive with stilbene disulfonate binding, while the CH17 sites are not. Selective, irreversible pyridoxal-5'-phosphate labeling of CH17, reduces the transport inhibitory potency due to reversible stilbene disulfonate binding to vacant, nonoverlapping CH35 sites. We conclude that multisite allosteric interactions can occur on one band 3 monomer.

Allosteric Site↗
PubMed1978

Primary structure of the major beta-chain of rat haemoglobins.

The amino acid sequence of the major beta-chain, (II)beta, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the epsilon-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.

Amino Acid Sequence↗