Biomedical subjects

J P MacManus

Publications and source records attributed to J P MacManus.

At least 19 recordsLinked to original sources

PubMed1995

DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat.

DNA extracted from regional brain samples of hypoxic/ischemic neonatal rats showed internucleosomal cleavage indicative of apoptosis. Cells containing cleaved DNA were identified by in situ labelling in the cortex, hippocampus, striatum and thalamus of the ipsilateral hemisphere. When the effects of increasing the length of the hypoxia were examined, increases were seen in the amount of internucleosomally cleaved DNA and in the number of labelled cells.

Animals↗

PubMed1995

An adherent cell model to study different stages of apoptosis.

Apoptosis in the classical thymocyte model occurs very rapidly making it difficult to study the intermediate steps in the process. An alternative adherent cell model is characterized and proposed in this paper. HT29 cells treated with a teniposide were collected at various times for morphological and biochemical assessments. Large DNA breaks (450-500, 350-400, 100-200 kb) were observed in these cells between 6 and 24 h. The larger DNA breaks appeared initially and in progression such that the smaller DNA break of 100-200 kb became apparent by 24 h. These changes in DNA corresponded with an increase in cell diameter and a gradual rounding and detaching of cells from each other but not from the tissue culture plates. The smallest DNA break of 23-50 kb appeared at 48 h and persisted throughout the 96 h of incubation. DNA ladders of 180- to 200-bp oligomers were also observed between 48 and 96 h and these coincided with the presence of small floating cells. Changes in cell adherence after teniposide treatment have permitted the consistent isolation of cells in four distinct morphological and biochemical stages of apoptosis: (1) "preapoptotic," (2) "swelling," (3) "rounding," and (4) "floating." The main advantages of this adherent cell model are: (1) apoptosis occurs very slowly (minimum of 48 h) permitting the observation of progressive changes; (2) cells from four stages of apoptosis can be used to study the sequence of events of other biochemical and genetic factors involved in the process; and (3) extracellular matrix proteins are present in this model so their participation in apoptosis, if any, can be determined.

Adenocarcinoma↗

PubMed1995

A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate.

OBJECTIVES: The objective of this work was to demonstrate the utility of luminescence from lanthanides bound to a mutant of the Ca2+ binding protein, oncomodulin, to monitor protease activity. DESIGN AND METHODS: A mutant of oncomodulin with a cysteine residue at position 57 located in the CD binding loop was conjugated to a salicylic acid group. The luminescence of Tb3+ resulting from electronic energy transfer from the salicylic acid group was monitored using time resolved lanthanide luminescence in the presence of proteolytic enzymes. RESULTS: Low detection limits for subtilisin (150 pg), chymotrypsin (2.5 ng), cathepsin B (3.5 ng), and HIV-1 protease (25 ng) were found. CONCLUSION: The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.

Animals↗

PubMed1995

Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.

The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.

Animals↗

PubMed1994

Detection of calcium binding proteins on polyacrylamide gels using time-resolved lanthanide luminescence photography.

Methods were developed for using the luminescent lanthanides Tb3+ and Eu3+ for the specific staining of calcium-binding proteins, as well as the nonspecific staining of proteins, on polyacrylamide gels. These methods involve equilibration of the gel after electrophoresis in solutions containing the appropriate lanthanide and a weak competitive chelating agent, such as N-(2-hydroxyethyl)iminodiacetic acid or nitrilotriacetic acid. This staining has the potential for complete reversibility using stronger chelating agents such as EDTA or diethylenetriaminepentaacetic acid, to allow for recovery of the protein. Specific staining produces an intense luminescent signal from those metal-binding proteins which have been modified either chemically or via site-directed mutagenesis. Gels were photographed using a time-resolved fluorescence camera system.

Acrylic Resins↗

PubMed1994

DNA damage consistent with apoptosis in transient focal ischaemic neocortex.

Transient focal ischaemia was produced in rat right neocortex by temporary middle cerebral artery occlusion. DNA damage was visualized in situ in cells of this right hemisphere but not in the contralateral hemisphere. The extracted damaged DNA exhibited laddered fragmentation which is indicative of apoptotic degradation. The amount of DNA damage was quantified by an end-labelling technique and shown to increase with the duration of the ischaemic insult. We conclude that the neurodegeneration resulting from focal ischaemia has an apoptotic component.

Animals↗

PubMed1993

Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain.

Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.

Animals↗

PubMed1993

A novel peptide designed for sensitization of terbium (III) luminescence.

Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.

Amino Acid Sequence↗

PubMed1993

Self-association of Ca(2+)-binding peptides induced by lanthanide ions: a fluorescence study.

In this study a Ca(2+)-binding 14mer peptide was synthesized with the sequence GDKNADGCIEFEEL, allowing covalent attachment of sulfhydryl-reactive fluorescent molecules at position 7 of the 12-residue, metal-binding loop (underlined). This provided the opportunity to select donor molecules with suitable spectral characteristics for sensitized excitation of chelated terbium (Tb3+) or europium (Eu3+) ions. N-(1-Pyrene)-iodoacetamide and 7-diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin were attached to the peptide and titrations carried out with terbium or europium stock solutions. It was possible to observe lanthanide ion binding to the loop in stoichiometric quantities, but maximal lanthanide luminescence was achieved with a large excess of lanthanide present, due to metal-induced peptide association. Obtaining maximal lanthanide luminescence is important in the development of systems for use in sensitive clinical diagnostic and time-resolved luminescence-based immunoassay applications.

Amino Acid Sequence↗

PubMed1992

A new intrinsic fluorescent probe for proteins. Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin.

The tryptophan analog, 5-hydroxytryptophan (5HW), has a significant absorbance between 310-320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca2+ binding protein, oncomodulin. Fluorescence anisotropy and time-resolved fluorescence decay measurements of the interaction between anti-oncomodulin antibodies and the 5HW-incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use of 5HW as an intrinsic probe in the study of structure and dynamics of protein-protein interactions.

5-Hydroxytryptophan↗

PubMed1992

Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.

The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the Förster equation in approximating distances in these systems.

Amino Acid Sequence↗

PubMed1992

Differentiation of HT-29 human colonic adenocarcinoma cells correlates with increased expression of mitochondrial RNA: effects of trehalose on cell growth and maturation.

The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.

Adenocarcinoma↗

PubMed1991

Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.

Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗

PubMed1990

Structure of oncomodulin refined at 1.85 A resolution. An example of extensive molecular aggregation via Ca2+.

The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.

Amino Acid Sequence↗

PubMed1990

A calcium-specific conformational response of parvalbumin.

The single tryptophan containing isotype III parvalbumin from codfish (Gadus callarius) was purified by a modified procedure and was shown to be homogeneous by a number of biochemical techniques. Sequence analysis established the location of the single tryptophan in position 102 of the 108 amino acid primary sequence. Atomic absorption spectroscopy showed that trichloroacetic acid (TCA) precipitation was more effective in parvalbumin decalcification compared to the more commonly used method of EGTA treatment. Magnesium induced steady-state fluorescence spectral changes of the EGTA-treated, but not the TCA-treated, parvalbumin. Steady-state fluorescence and circular dichroism spectra showed that calcium, but not magnesium, induced a conformational response in the TCA-treated protein. The fluorescence decay of the calcium-loaded native (holo) cod III parvalbumin was best described by two decay time components. By contrast, three lifetime components were necessary to describe the fluorescence decay of the metal-free (apo) protein. The decay-associated spectra of each temporal component were obtained. Collectively, these results demonstrate that it is possible for a parvalbumin to display a calcium-specific response.

Amino Acid Sequence↗

PubMed1990

Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102.

The calcium-induced conformational changes of the 108-amino acid residue proteins, cod III parvalbumin and oncomodulin, were compared using tryptophan as a sensitive spectroscopic probe. As native oncomodulin is devoid of tryptophan, site-specific mutagenesis was performed to create a mutant protein in which tryptophan was placed in the identical position (residue 102) as the single tryptophan residue in cod III parvalbumin. The results showed that in the region probed by tryptophan-102, cod III parvalbumin experienced significantly greater changes in conformation upon decalcification compared to the oncomodulin mutant, F102W. Addition of 1 eq of Ca2+ produced greater than 90% of the total fluorescence response in F102W, while in cod III parvalbumin, only 74% of the total was observed. Cod III parvalbumin displayed a negligible response upon Mg2+ addition. In contrast, F102W did respond to Mg2+, but the response was considerably less when compared to Ca2+ addition. Time-resolved fluorescence showed that the tryptophan in both proteins existed in at least two conformational states in the presence of Ca2+ and at least three conformational states in its absence. Comparison with quantum yield measurements indicated that the local electronic environment of the tryptophan was significantly different in the two proteins. Collectively, these results demonstrate that both cod III parvalbumin and oncomodulin undergo Ca2(+)-specific conformational changes. However, oncomodulin is distinct from cod III parvalbumin in terms of the electronic environment of the hydrophobic core, the magnitude of the Ca2(+)-induced conformational changes, and the number of calcium ions required to modulate the major conformational changes.

Amino Acid Sequence↗

PubMed1990

Terbium luminescence in synthetic peptide loops from calcium-binding proteins with different energy donors.

Fourteen 14-mer peptides corresponding to a consensus sequence of metal-binding loops from proteins of the calmodulin family were synthesized. The effect of varying both the position in the binding loop, and the type of aromatic side chains as energy donors for enhancement of terbium luminescence, was studied. It was concluded that tryptophan in loop position 7 gave optimal luminescence enhancement, and that the additional inclusion of a tyrosine in the loop at positions 2 or 4 could further boost emission from the bound terbium. In all further cases energy transfer from aromatic residues at positions other than 7 was markedly less efficient. These results suggest that the peptides assume a configuration which allows a hexadentate ligand structure around the bound terbium ion. This is consistent with a Dexter-type electron exchange model of energy transfer.

Amino Acid Sequence↗

PubMed1990

Calcium- and magnesium-binding properties of oncomodulin. Direct binding studies and microcalorimetry.

Ca2+ binding to the wild type recombinant oncomodulin was studied by equilibrium flow dialysis in the absence and presence of 1, 2, and 10 mM Mg2+. Direct Mg2(+)-binding experiments were carried out by the Hummel-Dryer gel filtration technique. These studies revealed that in the absence of Mg2+ oncomodulin binds two Ca2+ with KCa = 2.2 x 10(7) and 1.7 x 10(6) M-1, respectively. In the absence of Ca2+ the protein binds only one Mg2+ with KMg = 4.0 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding at the high affinity site according to the rule of direct competition. Ca2+ binding to the low affinity site is only slightly affected by Mg2+, so that in the presence of 2-3 mM Mg2+ the two sites have apparently an equal affinity for Ca2+. Microcalorimetry showed that, in spite of the different affinities of the two Ca2(+)-binding sites, delta H0 for the binding of each Ca2+ is identical and exothermic for -18.9 kJ/site. It follows that the entropy gain upon binding of Ca2+ is +77.1 J K-1 site-1 for the high affinity Ca2(+)-Mg2+ site and +56.0 J K-1 site-1 for the low affinity Ca2(+)-specific site. Mg2+ binding is endothermic for +13 kJ/site with an entropy change of +111 J K-1 site-1. The thermodynamic characteristics of the Ca2(+)-Mg2+ site resemble most those of site II (the so-called EF domain) of toad alpha-parvalbumin. The characteristics of Ca2+ binding to the specific site (likely the CD domain) are different from those of the Ca2+ specific sites in troponin C and in calmodulin and suggest that in oncomodulin hydrophobic forces do not play a predominant role in the binding process at the specific site.

Animals↗