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Biomedical subjects

J E Bell

Publications and source records attributed to J E Bell.

At least 109 records · Page 6Linked to original sources
PubMed1989

Interaction of terbium and calcium with chicken cystatin.

The emission intensity of the fluorescent lanthanide, terbium, is shown to be enhanced upon binding to chicken cystatin. Fluorescence titrations indicate the presence of a single high affinity binding site per molecule. Binding of the terbium results in a 29% quenching of the fluorescence of the single tryptophan residue in the molecule. Calcium displaces the terbium from cystatin as judged by the decrease of terbium fluorescence in competition titrations. Similar titrations with magnesium or strontium demonstrate that the metal binding site of cystatin exhibits specificity for calcium or terbium. Analysis of the N-terminal sequence of chicken cystatin suggests the presence of a putative consensus sequence for a metal binding site between residues 13 and 24. Calcium causes a 17% decrease in the tryptophan fluorescence of cystatin, indicating that an induced conformational change accompanies metal binding. The increased quenching observed with terbium appears to be the result of resonance energy transfer from tryptophan to terbium. From the critical distance for energy transfer from tryptophan to terbium, it is estimated that the terbium binding site lies approximately 12 A from the single tryptophan residue in the molecule.

Animals↗
PubMed1989

Development of the cerebellum with particular reference to cellular differentiation in the external granular layer.

Immunocytochemical evidence of differentiation in developing human cerebellum is presented in this study. Antibodies to neuron specific enolase, neurofilament protein, glial fibrillary acidic protein, vimentin, cytokeratin, epithelial membrane antigen and lymphoid markers, DLC and Leu 7 were used. The external granular layer showed positivity with neuronal markers between 27 weeks gestation and 4 months postnatal, but was negative for all other markers including glial fibrillary acidic protein. Characteristic staining reactions were noted in the other cerebellar layers. Monoclonal antibodies, UJ13A (pan-neuroectodermal marker) and G10 (localising microtubule-associated protein MAP1x) were also used in a limited number of cryostat sections and were positive and negative, respectively, in the external granular layer. The results of this study are discussed in relation to the theory that the external granular layer may be one source of medulloblastomas.

Biomarkers↗
PubMed1989

Differentiation in medulloblastomas and other primitive neuroectodermal tumours.

Fifty-four cases of primitive neuroectodermal tumour (PNET) including 47 medulloblastomas, were examined for evidence of neuronal and glial differentiation, using antibodies to neuron-specific enolase (NSE), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP). In 30 of the cases, antibodies to vimentin, alphafetoprotein, cytokeratin, epithelial membrane antigen and lymphoid markers were also used. Most of the 47 medulloblastomas in the group were NSE positive but NF negative; about half were GFAP positive and three of them were positive for both neuronal markers and for GFAP. Vimentin was demonstrated in four cases and was not always co-expressed with GFAP. Medulloblastomas were negative for all the other markers. Supratentorial PNETs were sometimes positive with neuronal markers but were GFAP negative. The cell specificity of these markers and the interpretation of immunocytochemical findings are discussed in relation to differentiation potential in primitive neuroectodermal tumours.

Adolescent↗
PubMed1988

Kinetic properties and regulation of biliverdin reductase.

In kinetic studies of the dual nucleotide enzyme biliverdin reductase, product inhibition patterns obtained with bilirubin as the inhibitor, using either the NADPH-linked reaction at pH 8.7 or the NADH-linked reaction at pH 7.0, are consistent with a random order of substrate addition and product release at either pH. With NAD(P) as the product inhibitor, the inhibition patterns suggest the existence of an enzyme-NAD(P)-biliverdin abortive complex. In the absence of added products, substrate inhibition observed at high biliverdin concentrations is also consistent with such a complex. Direct evidence for the existence of enzyme-NADP-biliverdin abortive complexes has been obtained at both pH 7.0 and 8.7 using the fluorescent NADP analog nicotinamide-1-N-6-ethenoadenine dinucleotide phosphate. Etheno-NADP fluorescence is enhanced in enzyme:etheno-NADP complexes but quenched in enzyme:biliverdin:etheno-NADP complexes. The effects of iron hematoporphyrin and cobalt protoporphyrin on the activity of biliverdin reductase have been examined. Iron-hematoporphyrin is a competitive inhibitor with respect to coenzyme at either pH. Comparison of Km values for biliverdin and Ki values for bilirubin or iron-hematoporphyrin indicate that while the binding of biliverdin is relatively pH independent, bilirubin binds more tightly at pH 7.0 than pH 8.7. Conversely, Fe-hematoporphyrin binds considerably tighter at pH 8.7 than pH 7.0. With cobalt-protoporphyrin, however, activation is observed, suggesting the existence of a regulatory site on biliverdin reductase for porphyrin derivatives. Resonance energy transfer measurements, from the fluorescent coenzyme analog etheno-NADP to biliverdin, iron-hematoporphyrin, or cobalt-protoporphyrin indicate that while the biliverdin site is located approximately 15 A from the etheno moiety of the coenzyme, both porphyrin derivatives are located 23.5 A away. This suggest the existence of a separate porphyrin binding site on biliverdin reductase which, depending upon the nature of the porphyrin binding, can produce activation or inhibition of the enzyme.

Algorithms↗
PubMed1988

"Bullet"-shaped head in fetuses with spina bifida: a pointer to the spinal lesion.

A "bullet"-shaped head, characterized by flattening in the parietal regions with pointing of the frontal bones, was noted in ultrasound examinations of fetuses with neural tube defects. In a series of 12 fetuses with spinal defects, this finding was present in nine. This abnormal head shape is a useful pointer to the spinal lesion.

Female↗
PubMed1988

3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers.

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.

Enzyme Activation↗
PubMed1988

Use of immunohistochemical methods in the differential diagnosis between primary cerebellar haemangioblastoma and metastatic renal carcinoma.

The potential role of immunohistochemistry in making the distinction between primary cerebellar haemangioblastoma and metastatic renal carcinoma was investigated by examining the reaction pattern of 10 cerebellar haemangioblastomas (seven women, three men, aged 20-40 years) and 10 primary renal carcinomas (six men, four women, aged 49-82 years) to a panel of epithelial, glial, and neural/neuroendocrine antisera. The tumour cell membranes of the renal carcinomas stained strongly with epithelial membrane antigen (EMA); membrane staining was totally absent in the haemangioblastomas. Strong neurone specific enolase (NSE) and S100 staining were also seen in haemangioblastomas but were more variable than EMA staining in renal carcinomas. It is concluded that a panel of antisera is required to distinguish between histologically similar areas in primary haemangioblastomas and metastatic renal carcinomas, and that while complementing conventional histological techniques, new problems of interpretation result which must be taken into account.

Adult↗
PubMed1988

Mammalian blastocyst: transport functions in a developing epithelium.

This article reviews the current state of knowledge concerning morphological and physiological mechanisms important to growth and differentiation of the mammalian blastocyst between compaction and implantation. Morphological processes occur in conjunction with major changes in transport systems that control the movement of substances into and out of the embryo. Compaction is a morphological development that is associated with the formation of an outer squamous epithelium, the trophectoderm, which regulates the composition of the medium bathing the presumptive embryo (the inner cell mass). Implantation involves the interaction of two epithelia, the adhesion between the trophectoderm and the maternal endometrium. Before adhesion, the blastocyst lies free in the uterine fluid and exchanges occur between this fluid and the embryo. Apposition of these epithelia is brought about in part by expansion of the blastocyst and removal of the uterine fluid. Blastocyst physiology is an inherently important field because vectorial transport system development and the genes that regulate it can be studied.

Animals↗
PubMed1987

A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase.

The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.

Animals↗
PubMed1987

Interaction of Zn2+ and Eu3+ with bovine liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase is potently inhibited by Zn2+ ions. At pH 7.0 a kinetic dissociation constant for Zn2+ of 18 microM is obtained. The fluorescent lanthanide Eu3+ competes for the Zn2+-binding site and relieves the Zn2+-induced inhibition, but does not cause inhibition. Studies on the effects of Zn2+ or Eu3+ on the tertiary and quaternary structure of the enzyme by the use of protein fluorescence, heat-stability and re-activation after guanidinium chloride denaturation indicate that, whereas Zn2+ affects both tertiary and quaternary structure, Eu3+ does not affect either, consistent with its lack of effect on enzymic properties. Eu3+ fluorescence had a strong excitation peak at 395 nm with emission at 456 nm. In the presence of glutamate dehydrogenase the fluorescence emission is shifted to 501 nm. Eu3+, with high-affinity binding site and distinctive fluorescence properties after binding, would appear to be an ideal fluorophore for use in conformational studies or resonance-energy-transfer studies.

Animals↗
PubMed1986

An exponential gradient marker for use with minigel polyacrylamide electrophoresis systems.

Minigel electrophoresis has reduced the required sample size and cost of running polyacrylamide gels without loss of resolution. The many problems incurred in the minigel system when pouring multiple gradient gels are eliminated by pouring individual gels with the described economical, exponential gradient marker, which can accurately deal with the small gel volumes. The advantages of exponential gradients are discussed.

Buffers↗
PubMed1986

Inositol-1,4,5-trisphosphate injection mimics fertilization potentials in sea urchin eggs.

The effects of inositol-1,4,5-trisphosphate (IP3) and of diacylglycerol (DAG) and its analogues on the membrane potential of eggs from the sea urchin Strongylocentrotus purpuratus were examined. Injection of IP3 into eggs resulted in a change in membrane potential that was similar in magnitude and time course to the fertilization potential elicited by sperm attachment. In low-calcium seawater, IP3 injection elicited a partial response. DAG and its analogues phorbol myristyl acetate and 1-oleoyl-2-acetylglycerol did not affect membrane potential either when applied by perfusion or when injected. The results indicate that IP3, but not DAG or its analogues, may be involved in the generation of the fertilization potential triggered by the interaction of sperm with sea urchin eggs.

Animals↗
PubMed1986

Neutral amino acid influx in developing rabbit blastocysts.

The influx of the neutral amino acids glycine, aminoisobutyric acid (AIB), and leucine into rabbit blastocysts was measured. In day 6 postcoitus (pc) embryos, glycine influx was Na+ independent, whereas AIB and leucine influx involved both Na+-dependent and independent components. From days 5 to 7 pc, the leucine and AIB influx remained constant, although the Na+-dependent fraction decreased and the Na+-independent fraction increased with age. None of the Na+-independent influx was inhibited by methylaminoisobutyric acid (MeAIB), an amino acid analogue specific for the system A of neutral amino acid uptake. In addition, MeAIB influx was Na+ independent, implying that system A is not involved in leucine or AIB uptake. All Na+-dependent influx is thus considered to occur via system ASC. System L contributed only to the influx of leucine at days 6 and 7 pc, as measured by inhibition of Na+-independent influx by 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid.

Amino Acids↗
PubMed1985

Investigation of the effects of crosslinking glutamate dehydrogenase with dimethyl pimelimidate.

Chemical crosslinking with dimethyl pimelimidate has been used to examine the quaternary structure and conformational mobility of bovine liver glutamate dehydrogenase. Crosslinking patterns are shown to be consistent with either a stacked or staggered dimer of trimers structure of the hexamer. Crosslinking in the absence of coligands results in a small loss of activity but an almost complete loss of GTP inhibitory effects. Protection experiments show that the active site can be protected by a variety of ligand combinations, and that the loss of GTP inhibition is protected by several complexes containing either NADH or NADPH, indicating that the second coenzyme site per subunit (which preferentially binds NADH) is not involved in the protection process. A significant loss of ADP activation occurs during crosslinking which is not protected against by any combination of protecting ligands tried, including those which involve second coenzyme site binding, showing that the ADP site is functionally distinct from the GTP site and from the second coenzyme binding site. Crosslinking in the presence of protecting ligands gives similar gel patterns to those obtained in the absence of protection. Affinity chromatography experiments show that the crosslinked enzyme still binds GTP despite the loss of GTP inhibition, and hysteresis experiments show that the second coenzyme site is left functional if protected with either coenzyme. A model is presented where crosslinking affects the conformational linkage between various ligand binding sites involved in GTP inhibition rather than the sites themselves.

Adenosine Diphosphate↗
PubMed1985

Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes.

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

Binding Sites↗
PubMed1984

Catalytic activity of bovine glutamate dehydrogenase requires a hexamer structure.

Previous workers have shown that the hexamers of glutamate dehydrogenase are dissociated first into trimers and subsequently into monomers by increasing guanidinium chloride concentrations. In renaturation experiments it is shown that trimers of glutamate dehydrogenase can be reassociated to give the hexamer form of the enzyme, with full regain of activity. Monomeric subunits produced at high guanidinium chloride concentrations cannot be renatured. The trimer form of the enzyme is shown to have no catalytic activity, although the hexamer form in guanidinium chloride has full activity.

Animals↗
PubMed1984

The lactose synthase acceptor site: a structural map derived from acceptor studies.

A pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either alpha or beta-, but alpha- is somewhat preferred. In the absence of alpha-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of alpha-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (beta-configuration). However, upon alpha-lactalbumin binding the aglycone specificity allows for axial (alpha-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that alpha-lactalbumin binds to a hydrophobic site some distance from the C-2 group.

Acetylglucosamine↗