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Biomedical subjects

A R Clarke

Publications and source records attributed to A R Clarke.

At least 19 recordsLinked to original sources
PubMed1999

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine.

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N'-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine. By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.

Animals↗
PubMed1999

Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations.

Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly alpha-helical to beta-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native alpha conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in beta structure. The soluble beta form (beta-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to beta-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation.

Circular Dichroism↗
PubMed1999

A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys-Drash syndrome.

The Wilms tumor-suppressor gene, WT1, plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys-Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and/or Wilms tumor (WT). DDS patients are constitutionally heterozygous for exonic point mutations in WT1, which include mutations predicted to truncate the protein within the C-terminal zinc finger (ZF) region. We report that heterozygosity for a targeted murine Wt1 allele, Wt1(tmT396), which truncates ZF3 at codon 396, induces mesangial sclerosis characteristic of DDS in adult heterozygous and chimeric mice. Male genital defects also were evident and there was a single case of Wilms tumor in which the transcript of the nontargeted allele showed an exon 9 skipping event, implying a causal link between Wt1 dysfunction and Wilms tumorigenesis in mice. However, the mutant WT1(tmT396) protein accounted for only 5% of WT1 in both heterozygous embryonic stem cells and the WT. This has implications regarding the mechanism by which the mutant allele exerts its effect.

Animals↗
PubMed1999

P53-dependent and -independent links between DNA-damage, apoptosis and mutation frequency in ES cells.

The hypothesis that p53 deficiency enhances the survival of DNA-damage bearing cells was investigated in wild-type and p53 mutant embryonic stem (ES) cells. Following UV-C irradiation, p53 is rapidly induced in wild-type cells and p53-dependent apoptosis follows within 8 h, resulting in the death of the majority of cells within 36 h. Increasing doses of UV-irradiation resulted in enhanced clonogenic survival of null cells as compared to wild-type. Amongst surviving clones, the Hprt mutation frequency was found to be dependent upon UV dose and influenced by p53 status. Treatment with ionizing radiation led to enhanced expression of p53 but resulted in little induction of apoptosis irrespective of p53 status. However, clonogenic potential was considerably reduced, particularly in wild-type cells which showed a tenfold lower survival than null cells. In contrast to the effects of UV-irradiation, the incidence of Hprt mutation did not differ significantly between wild-type and p53 null survivors. The data confirm that p53 restricts the numbers of cells bearing mutations that survive DNA damage induced by either agent, albeit by different mechanisms.

Animals↗
PubMed1999

Engineered assembly of intertwined oligomers of an immunoglobulin chain.

Domain 1 of CD2 (CD2.D1) forms a conventional Ig fold stabilised by non-covalent antiparallel contacts between beta-strands. Removing two residues from the middle of the protein sequence, where the polypeptide chain normally folds back upon itself, stabilises an open conformation. In this modified molecule, the optimum evolved contacts between side-chains can only be satisfied through the antiparallel association of two chains to create a symmetrical pair of pseudo-domains. Here, we describe the dynamics of the switch between monomeric and dimeric states and demonstrate the extension of this novel underlying principle to trimer and tetramer formation. The ability of a protein molecule to form higher-order antiparallel structures is reminiscent of the behaviour of hairpins, duplexes, three-way and Holliday junctions in DNA.

Animals↗
PubMed1999

Effects of core mutations on the folding of a beta-sheet protein: implications for backbone organization in the I-state.

A series of core mutations were introduced into beta-strand segments of an immunoglobulin fold (the isolated first domain of CD2, CD2.d1) to examine their influence on the rapidly formed intermediate state (I-state) which transiently accumulates in the folding reaction [Parker, M. J., and Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. The residue changes were chemically conservative, each representing the removal of one or two methylene groups from aliphatic side chains. Predictably, the mutations destabilize the folded state with respect to the unfolded state by about 1.1 +/- 0.7 kcal mol-1 per methylene group removed. However, when the folding reaction is dissected by transient kinetic analysis into its component steps, six out of the nine mutations lead to a stabilization of the I-state. The direction and magnitude of these effects on the global stability of the transient intermediate are well correlated with changes in secondary structure propensity occasioned by the substitutions. The results show that, although side chain interactions are extremely weak in this early phase of folding, the beta-strand conformation of the polypeptide chain is established. In the next phase of the reaction, the rate-limiting transition state is attained by the formation of a tightly localized hydrophobic nucleus which includes residues V30, I18, and V78. Interestingly, in almost all immunoglobulin domains of extracellular proteins, the latter pair are cysteine residues which form a disulfide bridge.

Amino Acid Substitution↗
PubMed1999

Structural basis of substrate specificity in malate dehydrogenases: crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, alpha-ketomalonate and tetrahydoNAD.

The structural basis for the extreme discrimination achieved by malate dehydrogenases between a variety of closely related substrates encountered within the cell has been difficult to assess because of the lack of an appropriate catalytically competent structure of the enzyme. Here, we have determined the crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase with the alternative substrate alpha-ketomalonate and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide. Both subunits of the dimeric porcine heart, and from the prokaryotes Escherichia coli and Thermus flavus. However, large changes are noted around the active site, where a mobile loop now closes to bring key residues into contact with the substrate. This observation substantiates a postulated mechanism in which the enzyme achieves high levels of substrate discrimination through charge balancing in the active site. As the activated cofactor/substrate complex has a net negative charge, a positive counter-charge is provided by a conserved arginine in the active site loop. The enzyme must, however, also discriminate against smaller substrates, such as pyruvate. The structure shows in the closed (loop down) catalytically competent complex two arginine residues (91 and 97) are driven into close proximity. Without the complimentary, negative charge of the substrate side-chain of oxaloacetate or alpha-ketomalonate, charge repulsion would resist formation production of this catalytically productive conformation, hence minimising the effectiveness of pyruvate as a substrate. By this mechanism, malate dehydrogenase uses charge balancing to achieve fivefold orders of magnitude in discrimination between potential substrates.

Amino Acid Sequence↗
PubMed1998

EEG analysis in Attention-Deficit/Hyperactivity Disorder: a comparative study of two subtypes.

This study investigated differences in the EEG between children with Attention-Deficit/Hyperactivity Disorder of the Combined Type, Attention-Deficit/Hyperactivity Disorder of the Predominantly Inattentive Type and control subjects. All subjects were between the ages of 8 and 12 years, and groups were matched on age and gender. The EEG was recorded during an eyes-closed resting condition from 21 monopolar derivations and these were clustered into nine regions prior to analysis. One minute of trace was analysed using Fourier transformation to obtain both absolute and relative power estimates in the delta, theta, alpha and beta frequency bands. The patient groups were found to have greater levels of theta and deficiencies of alpha and beta in comparison to the control group. Children with Attention-Deficit/Hyperactivity Disorder of the Predominantly Inattentive type were found to be significantly different from those of the Combined type in the same measures, appearing to be closer to the normal profiles. The general results support a maturational lag model of the central nervous system in Attention Deficit/Hyperactivity Disorder. The differences between the subtypes suggest a difference in the severity of the disorder rather than a different neurological dysfunction.

Attention Deficit Disorder with Hyperactivity↗
PubMed1998

Asymmetry, commitment and inhibition in the GroE ATPase cycle impose alternating functions on the two GroEL rings.

The ATPase cycle of GroE chaperonins has been examined by transient kinetics to dissect partial reactions in complexes where GroEL is asymmetrically loaded with nucleotides. The occupation of one heptameric ring by ADP does not inhibit the loading of the other with ATP nor does it prevent the consequent structural rearrangement to the "open" state. However, ADP binding completely inhibits ATP hydrolysis in the asymmetric complex, i.e. ATP cannot by hydrolysed when ADP is bound to the other ring. This non-competitive inhibition of the ATPase by ADP is consistent with a ring-switching, or "two-stroke", mechanism of the type: ATP:GroEL --> ADP:GroEL --> ADP:GroEL:ATP --> GroEL:ATP --> GroEL:ADP, i.e. with respect to the GroEL rings, ATP turns over in an alternating fashion. When the ATP-stabilized, "open" state is challenged with hexokinase and glucose, to quench the free ATP, the open state relaxes slowly (0.44 s-1) back to the apo (or closed) conformation. This rate, however, is three times faster than the hydrolytic step, showing that bound ATP is not committed to hydrolysis. When GroES is bound to the GroEL:ATP complex and the system is quenched in the same way, approximately half of the bound ATP undergoes hydrolysis on the chaperonin complex showing that the co-protein increases the degree of commitment. Thus, non-competitive inhibition of ATP hydrolysis, combined with the ability of the co-protein to block ligand exchange between rings has the effect of imposing a reciprocating cycle of reactions with ATP hydrolysing, and GroES binding, on each of the GroEL rings in turn. Taken together, these data imply that the dominant, productive steady state reaction in vivo is: GroEL:ATP:GroES --> GroEL:ADP:GroES --> ATP:GroEL:ADP:GroES --> ATP:GroEL:ADP --> GroES:ATP:GroEL:ADP --> GroES:ATP:GroEL for a hemi-cycle, and that significant inhibi tion of hydrolysis may arise through the formation of a dead-end ADP:GroEL:ATP:GroES complex.

Adenosine Diphosphate↗
PubMed1998

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Amino Acid Sequence↗
PubMed1998

p53-independent DNA repair and cell cycle arrest in embryonic stem cells.

The role of p53 in DNA repair and cell cycle checkpoint after ultraviolet irradiation was investigated in an embryonic stem cell line homozygous for a targeted deletion of p53. Results indicate that loss of p53 does not alter the capacity of ES cells to respond to DNA damage. Wild-type and p53-deficient cells showed similar cessation of DNA synthesis after UV damage and similar ultimate capacity to repair a transiently transfected reporter plasmid. Interestingly, in the absence of DNA damaging treatment, the transit of p53-deficient cells through S phase was slower than wild-type cells. We suggest that this may result from the absence of a p53-dependent response to endogenous DNA damage: without p53 sensing endogenous damage leading to immediate repair, such damage may persist and thus delay DNA synthesis.

Animals↗
PubMed1998

Thermodynamic properties of transient intermediates and transition states in the folding of two contrasting protein structures.

The N-terminal domain of phosphoglycerate kinase (N-PGK) and domain 1 of the T-cell adhesion protein CD2 (CD2.d1) fold through rapidly formed and transiently populated intermediate states in reactions which have no kinetic complications arising from proline isomerization or disulfide bonding. We have evaluated the thermodynamic parameters (DeltaCp, change in heat capacity; DeltaS, entropy change; DeltaH, enthalpy change) for each experimentally accessible step in these folding reactions. Despite their different topologies and amino acid compositions, the individual steps [U-I (unfolded to intermediate state), I-t (intermediate to major transition state), and t-F (transition state to the fully folded state)] have closely similar qualitative properties in the two proteins. For both, the heat capacity changes are proportional to m-value changes (Deltam) for every step in the reaction, but the ratio DeltaCp/Deltam is lower for N-PGK, presumably owing to a much larger compliment of aromatic amino acids in the core. According to measurements of DeltaCp and Deltam, the I-states are highly condensed (65-70% for N-PGK and 40-45% dehydrated for CD2.d1), yet the changes in entropy in the U-to-I transition are small, showing that the entropy gained from desolvation must be balanced by that lost in ordering the chain. The high degree of conformational order in the I-state, implied by these measurements, is mirrored by the extensive, native secondary structure revealed by amide exchange measurements [Hosszu, L. L. P., et al. (1997) Nat. Struct. Biol. 4, 801-804; Parker, M. J., et al. (1997) Biochemistry 36, 13396-13405]. At 25 degreesC the transition state barrier has an entirely enthalpic origin, the entropic contribution being favorable. The latter observation implies that, during the consolidation of structure occurring in the I-to-F step, further dehydration (positive DeltaS) precedes side-chain locking (negative DeltaS). Only after the transition state is surmounted do we see a net entropic penalty arising from the widespread ordering of side chains.

Animals↗
PubMed1998

Short elastin-like peptides exhibit the same temperature-induced structural transitions as elastin polymers: implications for protein engineering.

Elastin is a major protein component of the vascular wall and is responsible for its unusual elastic properties. Polymers of its repeating VPGVG sequences have been synthesised and shown to exhibit an inverse temperature transition where, as temperature rises, the polymer collapses from an extended chain to a beta-spiral structure with three VPGVG units per turn, each pentamer adopting a type II beta-turn conformation. These studies, however, have not established whether the temperature-driven conformational change is an intrinsic property of the individual pentameric sequences or a global, co-operative effect of many pentamers within the beta-spiral structure. Here, we examine by circular dichroism the behaviour of elastin-like peptides (VPGVG)n, where n varies between 1 and 5. Remarkably, we find that all lengths of peptide undergo an extended left and right arrow beta-turn transition with increasing temperature, suggesting that the induction of the beta-spiral occurs at the level of single pentameric units. The origin of this effect is a positive DeltaS term for the transition. At 35 degreesC, the average transition midpoint temperature, the value of TDeltaS is about 15 kcal mol-1. With larger oligomers (n=3), there is only a modest rise in DeltaS, suggesting that the dominant entropic effect resides within the monomer and that interactions between these units make only a small contribution to the energetics of the transition. Charges at the termini, and residue replacements or additions, regulate the transitions for the short peptides in a manner similar to that observed for the longer polymers. The behaviour of the same peptides in trifluoroethanol and SDS solutions is consistent with formation of the beta-turn being driven by interactions between non-polar groups. The significance of this behaviour for the rational design of temperature-induced responses in proteins is discussed.

Amino Acid Sequence↗
PubMed1998

Efficient BLG-Cre mediated gene deletion in the mammary gland.

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70-80% of cells within the lactating mammary gland have undergone recombination. Immunohistochemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be < or = 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.

Animals↗
PubMed1998

Smad4 (DPC4)--a potent tumour suppressor?

The recently described family of Smad molecules are essential mediators of transforming growth factor beta (TGF-beta) signalling. To date, seven members of this family have been identified, each of which plays a specific and separate role in mediating TGF-beta superfamily gene transcription. At least two different Smads, Smad2 and Smad4 (DPC4), have been implicated in human cancer and appear to have tumour-suppressor functions. Loss of function of Smad4 is most strongly associated with human pancreatic and colorectal malignancy. Furthermore, work from several different groups has suggested associations between Smad4 loss and malignancy in a number of other tissues. Here, we present a review of the current state of the literature implicating the central Smad mediator, Smad4, in the development of cancer.

Animals↗
PubMed1998

The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis.

Evidence is accumulating that meiosis is subject to 'checkpoints' that monitor the quality of this complex process. In yeast, unresolved double strand breaks (DSBs) in DNA are thought to trigger a 'recombination checkpoint' that leads to pachytene arrest. In higher eukaryotes, there is evidence for a checkpoint that monitors chromosome synapsis and in mammals the most compelling evidence relates to the sex chromosomes. In normal male mice, there is synapsis between the X and Y pseudoautosomal regions; in XSxr(a)O mice, with a single asynaptic sex chromosome, there is arrest at the first meiotic metaphase, the arrested cells being eliminated by apoptosis (our unpublished data). Satisfying the requirement for pseudoautosomal synapsis by providing a pairing partner for the XSxr(a) chromosome avoids this arrest. We have considered that this 'synapsis checkpoint' may be a modification of the yeast 'recombination checkpoint' with unresolved DSBs (a corollary of asynapsis) providing the trigger for apoptosis. DSBs induced by irradiation are known to trigger apoptosis in a number of cell types via a p53-dependent pathway, and we now show that irradiation-induced spermatogonial apoptosis is also p53-dependent. In contrast, the apoptotic elimination of spermatocytes with synaptic errors proved to be p53-independent.

Animals↗