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Biomedical subjects

J M Bradshaw

Publications and source records attributed to J M Bradshaw.

At least 19 recordsLinked to original sources
PubMed2006

Higher cortical deficits influence attentional processing in dementia with Lewy bodies, relative to patients with dementia of the Alzheimer's type and controls.

BACKGROUND: Attentional dysfunction is believed to be a prominent and distinguishing neuropsychological feature of dementia with Lewy bodies (DLB); yet, the specific nature of the attentional deficit and factors that can potentially influence attentional processing in DLB have not been fully defined. AIMS: To clarify the nature of the attentional deficit in early-stage DLB relative to patients with early-stage dementia of the Alzheimer's type (DAT) and elderly controls, and examine the effect of task complexity and type of cognitive load on attentional processing in DLB. METHODS: Attentional impairment and fluctuating attention were investigated in three groups of subjects--patients with clinical features of early probable DLB (n = 20), a group with early probable DAT (n = 19) and healthy elderly controls (n = 20)--using an experimental computerised reaction time paradigm. RESULTS: Patients with DLB showed greater attentional impairment and fluctuations in attention relative to patients with DAT and elderly controls. The attentional deficit was generalised in nature but increased in magnitude as greater demands were placed on attentional selectivity. Attentional deficits in DLB were most pronounced under task conditions that required more active recruitment of executive control and visuospatial cognitive processes. CONCLUSIONS: Attentional deficits in DLB are widespread and encompass all aspects of attentional function. Deficits in higher cortical function influence the degree of attentional impairment and fluctuating attention, suggesting that attentional processing in DLB is mediated by interacting cortical and subcortical mechanisms. These findings serve to clarify the nature of the attentional deficit in DLB and have potentially important ramifications for our understanding of the neurocognitive underpinnings of fluctuations.

Aged↗
PubMed2004

Feline necrotising sialometaplasia: a report of two cases.

Unilateral swelling of submandibular salivary gland in two cats was diagnosed as necrotising sialometaplasia. Histological features that differentiate the disease from other salivary gland lesions, particularly neoplasia are: lobular necrosis of salivary tissue; squamous metaplasia conforming to duct and/or acinar outlines; preservation of salivary lobular morphology; and variable inflammation and granulation tissue.

Animals↗
PubMed2001

Inconsistent detection of PrP in extraneural tissues of cats with feline spongiform encephalopathy.

Feline spongiform encephalopathy (FSE), a transmissible spongiform encephalopathy or prion disease of cats, first reported in Great Britain in 1990, is believed to result from the consumption of food contaminated by the agent of bovine spongiform encephalopathy (BSE). The accumulation of PrP in non-neural tissues of cats diagnosed as suffering from FSE was investigated by immunohistochemistry. In the majority of the cats no disease-specific PrP was detected in lymphoid tissues. Small amounts of PrP were detected in the spleen of only two of 13 samples examined, in Peyer's patches of one of the two cases for which suitable material was available, but in the myenteric plexus of all four cats in which sections of intestine were examined. In addition PrP immunostaining was found in the kidney of all the cats with FSE whose kidneys were examined.

Animals↗
PubMed2000

Role of electrostatic interactions in SH2 domain recognition: salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase.

SH2 domains are small protein domains that bind specifically to tyrosyl-phosphorylated sequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulated that electrostatic interactions may play an important role in SH2 domain recognition. To test this hypothesis, we have examined the salt dependence of the interaction between tyrosyl-phosphorylated peptides and SH2 domains. The dependence of the binding constant, K(obs), on [NaCl] was shown to be strong for binding of the tandem SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptides derived from immune-receptor tyrosine activation motifs (dpITAMs): the slopes of plots of log(K(obs)) versus log [NaCl], designated SK(obs), ranged from -2.6 +/- 0.1 to -3.1 +/- 0.2. Binding of the single SH2 domain of the Src kinase to its consensus singly phosphorylated peptide (sequence pYEEI where pY indicates a phosphotyrosine) was also highly dependent on [NaCl] with a SK(obs) value of -2.4 +/- 0.1. The ability of salt to disrupt the interactions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependent with the inhibitory effect following the order: phosphate > Cl(-) > F(-). For the Syk-tSH2 system, interactions in the pY-binding pockets were shown to be responsible for a large portion of the total salt dependence: removal of either phosphate from the dpITAM peptide reduced the magnitude of SK(obs) by 40-60% and weakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single amino acid Ac-pY-NH(2) was characterized by a large salt dependence of binding and was also dependent on the identity of the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicated in determining the specificity of the phosphopeptide-SH2 domain interaction, was next probed by comparing the binding of the Src SH2 domain to a peptide containing the pYEEI sequence with that of a lower affinity variant pYAAI peptide: the magnitude of SK(obs) for the variant peptide was reduced to -1.3 +/- 0.1 as compared to -2.4 +/- 0.1 for the pYEEI peptide, indicating that in addition to pY, residues conferring peptide binding specificity contribute significantly to the salt dependence of SH2 domain binding. This study shows that electrostatic interactions play important roles not only in mediating pY recognition and binding but also in contributing to the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.

Amino Acid Sequence↗
PubMed2000

Mutational investigation of the specificity determining region of the Src SH2 domain.

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.

Amino Acid Substitution↗
PubMed1999

Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (DeltaGo (dephosphorylated)=-3.6 kcal/mol, DeltaGo (phosphoserine) >-3.7 kcal/mol); however, the DeltaGo value of pTyr binding is more favorable (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg betaB5 resulted in a large increase in DeltaGo (DeltaDeltaGo=3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (DeltaDeltaGo<1.4 kcal/mol) reduction in affinity, indicating that Arg betaB5 is the single most important determinant of pTyr recognition. However, mutation of Cys betaC3, a residue unique to the Src SH2 domain, surprisingly increased affinity by eightfold (DeltaDeltaGo=-1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue's DeltaDeltaGo value upon mutation with that residue's sequence conservation among SH2 domains revealed only a modest correlation between a residue's energetic contribution to pTyr recognition and its conservation throughout evolution. The results of this investigation highlight the importance of a single critical interaction, the buried ionic bond between the phosphate of the pTyr and Arg betaB5 of the SH2 domain, driving the binding of SH2 domains to tyrosine phosphorylated targets.

Amino Acid Substitution↗
PubMed1999

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics.

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees </= 0.6 kcal/mol) to conservative substitutions at all three peptide positions. However, mutation to Ala resulted in moderate reductions in DeltaG degrees, with the substitution at the +3 position showing the largest loss in affinity (DeltaDeltaG degrees = 1.4 kcal/mol), followed by the +2 (DeltaDeltaG degrees = 1.0 kcal/mol) and +1 (DeltaDeltaG degrees = 0.5 kcal/mol) positions. This hierarchy of binding was not reflected in the values of the heat capacity change, since only the peptide substituted to Ala at the +3 position showed a DeltaCp degrees that was reduced in magnitude compared to wild-type. To assess the degree of cooperation upon binding (or coupling) between the amino acids of the EEI sequence, the binding of a series of singly, doubly, and triply Ala substituted phosphopeptides was examined and analyzed using double mutant cycles. It was revealed that the effects of the Ala substitutions on DeltaG degrees were additive. However, nonadditive binding enthalpies were observed between the +1 Glu and +3 Ile, as well as the +2 Glu and +3 Ile, suggesting that communication occurs between residues of the EEI motif upon binding.

Alanine↗
PubMed1999

Physician participation in research surveys. A randomized study of inducements to return mailed research questionnaires.

The authors randomly selected 400 physicians from a population of 1,545 practicing physicians providing follow-up care to patients who received bone marrow or blood stem cell transplants at the Fred Hutchinson Cancer Research Center to determine interest in receiving Internet-based transplant information. In a two-factor completely randomized factorial design, the 400 physicians were assigned to receive mailed surveys with either no compensation or a $5 check and either no follow-up call or a follow-up call 3 weeks after mailing. Overall, 51.5% of the physicians returned the mailed surveys. Comparison of logit models showed that inclusion of a $5 check in the mailer significantly (p = .016) increased the probability of returning the surveys (57.5% vs. 45.5%). In contrast, the telephone follow-up had no overall effect. The authors concluded a modest financial reward can significantly improve physician response rates to research surveys but a telephone follow-up may be inefficient and even ineffective.

Adult↗
PubMed1999

A neuropsychological-genetic profile of atypical cri du chat syndrome: implications for prognosis.

Cri du chat syndrome is associated with a deletion on the short arm of chromosome 5. The main diagnostic feature is a high pitched, cat-like cry which has recently been localised to 5p15.3 and is separate from the remaining clinical features of the syndrome, which have been localised to 5p15.2. The present study describes a family of four who have a deletion slightly distal (5p15.3) to the critical region. Detailed neuropsychological evaluations indicated a similar pattern of cognitive performance to that reported for subjects with typical CDCS but with only minimal intellectual impairment. In addition, in this family the 5p deletion is transmitted in an autosomal dominant fashion, contrasting with most cases of CDCS, which are either de novo or occur as an unbalanced product of a balanced translocation in a normal parent. This study confirms the importance of differentiating between 5p deletions that coincide with the typical cri du chat phenotype which includes severe to profound learning disability and deletions that only delete the distal critical region that coincides with a milder degree of cognitive impairment and a much improved prognosis.

Adolescent↗
PubMed1998

Calorimetric investigation of proton linkage by monitoring both the enthalpy and association constant of binding: application to the interaction of the Src SH2 domain with a high-affinity tyrosyl phosphopeptide.

The binding of Src homology 2 (SH2) domains to tyrosyl phosphopeptides depends on electrostatic interactions between the phosphotyrosine and its binding site. To probe the role of these interactions, we have used isothermal titration calorimetry to study the pH dependence of the binding of the SH2 domain of the Src kinase to a high-affinity tyrosyl phosphopeptide. Two independent approaches were employed. In a first series of experiments that focused on determining the peptide's association constant between pH 5.0 and 9.0, two ionizable groups were characterized. One group, with free and bound pKas of 6.2 and 4.4, respectively, could be identified as the phosphate in the phosphotyrosine while the other group, with free and bound pKas of 8.2 and 8.5, respectively, could be only tentatively assigned to a cysteine in the phosphotyrosine binding pocket. Further information on the linkage between peptide binding and protonation of the phosphotyrosine was obtained from a second series of experiments, which focused on determining the peptide binding enthalpy at low values of pH in several buffers with different ionization enthalpies. These data provided free and bound pKa values for the phosphotyrosine identical to those derived from the first series of experiments, and hence demonstrated for the first time that the two approaches provide identical information regarding proton linkage. In addition, the second series of experiments also determined the intrinsic enthalpy of binding of both the protonated and deprotonated phosphate forms of the peptide. These two sets of experiments provided a complete energetic profile of the linkage between phosphate ionization and peptide binding. From this profile, it was determined that the PO32- form of the peptide binds 2.3 kcal mol-1 more favorably than the PO3H1- form due entirely to a more favorable entropy of binding.

Animals↗
PubMed1998

Mass spectrometric and thermodynamic studies reveal the role of water molecules in complexes formed between SH2 domains and tyrosyl phosphopeptides.

BACKGROUND: SH2 domains have a fundamental role in signal transduction. These domains interact with proteins containing phosphorylated tyrosine residues and, in doing so, mediate the interactions of proteins involved in tyrosine kinase signalling. The issue of specificity in SH2 domain interactions is therefore of great interest in terms of understanding tyrosine kinase signal-transduction pathways and in the discovery of drugs to inhibit them. Water molecules are found at the interfaces of many complexes, however, to date little attention has been paid to their role in dictating specificity. RESULTS: Here we use a combination of nanoflow electrospray ionization mass spectrometry (ESI-MS), isothermal titration calorimetry and structural data to investigate the effect of water molecules in complexes formed between the SH2 domain of tyrosine kinase Src and tyrosyl phosphopeptides. Binding studies have been performed using a series of different peptides that were selected to allow changes in the water content at the complex interface and demonstrate changes in specificity. ESI-MS enables quantification of the number of water molecules that interact with a higher affinity than those generally found solvating the biomolecular complex. CONCLUSIONS: Comparing the interactions of different peptides, we show that an intricate network of water molecules have a key role in dictating specificity. The use of mass spectrometry to quantify tightly bound water molecules may prove of general use in structural biology, where an independent determination of the water molecules associated with a structure would be advantageous. Furthermore, the ability to assess whether given water molecules are important in high-affinity binding could make this method a precious tool in drug design.

Crystallography, X-Ray↗
PubMed1998

Probing the "two-pronged plug two-holed socket" model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.

Src homology 2 (SH2) domains are protein modules that specifically bind to tyrosyl phosphorylated peptides on signaling proteins. X-ray crystallographic studies of the SH2 domain of the Src kinase have probed the mechanism of binding, leading to the "two-pronged plug two-holed socket" mechanism whereby binding is hypothesized to resemble a two-pronged plug (the peptide) inserting into a two-holed socket (the SH2 domain). This binding model predicts (1) a hydrophobic basis for high-affinity binding largely determined by the level of insertion of the third residue C-terminal to the phosphotyrosine in the peptide into a primarily hydrophobic pocket (the +3 binding pocket) of the SH2 domain, and (2) a binding mechanism involving no significant conformational changes in the SH2 domain. In this study, we have probed these predictions by using isothermal titration calorimetry to extract complete thermodynamic profiles (Delta G degrees, Delta H degrees, Delta S degrees, Delta Cp degrees) for the binding of the Src SH2 domain to two series of tyrosyl phosphopeptides. One series consisted of peptides that have been determined by X-ray crystallography to have different levels of insertion of the peptide's +3 position into the +3 binding pocket. The other series consisted of peptides with progressively smaller hydrophobic side chains (I, L, V, and A) at the +3 position. Consistent with a binding mechanism that does not involve substantial conformational changes, the Delta Cp degrees values for all peptides were small and, at least for the high-affinity interactions, similar to the Delta Cp degrees values predicted from surface area calculations. However, unexpectedly, this study reveals that high-affinity binding was only partially determined by the interactions between the +3 residue in the peptide and the +3 binding pocket. Furthermore, the Delta Cp degrees values for all peptides studied were similar, implying similar degrees of desolvation of the +3 binding pocket upon binding. These results indicate that the "two-pronged plug two-holed socket" model is an oversimplification of the Src SH2 domain binding mechanism.

Animals↗
PubMed1998

CARE-PARTNER: a computerized knowledge-support system for stem-cell post-transplant long-term follow-up on the World-Wide-Web.

Evidence-based practice in medicine promotes the performance of medicine based upon proven and validated practice. The CARE-PARTNER system presented here is a computerized knowledge-support system for stem-cell post-transplant long-term follow-up (LTFU) care on the WWW, which means that it monitors the quality of the knowledge both of its own knowledge-base and of its users. Its aim is to support the evidence-based practice of the LTFU clinicians and of the home-town physicians who actually care for the transplanted patients. Currently, three fundamental characteristics of CARE-PARTNER are accountable for its knowledge-support function: the quality of its knowledge-base, its availability on the WWW, and its learning from experience capability. As a matter of fact, the integration of a case-based reasoner in the reasoning framework enables the system to introspectively study its results, and to learn from its successes and failures, thus confronting the quality of the guidelines and pathways it reuses to the reality and complexity of the clinical cases.

Artificial Intelligence↗
PubMed1996

Dysuria associated with urethral caruncle in the dog.

Three cases of urethral caruncle were recognized in bitches with a history of chronic dysuria. Clinical and radiological examinations revealed the presence of inoperable lesions involving much of the urethra in all three cases. At post-mortem examination of Case 1, an oval swelling, 1.5 x 1.0 cm, was detected within the wall of the urethra close to the vagino-urethral orifice. In Case 2, firm, mottled yellow, white and red tissue formed a thickening between the urethra and vagina. In Case 3, a cylindrical cream mass, 8 cm long and 3 cm in diameter, surrounded the urethra and impinged on the wall of the vagina. Histologically, glandular structures lined by a single layer of epithelial cells and a mixed granulomatous inflammatory reaction were present in the wall of the urethra of all three cases.

Animals↗
PubMed1982

The timing and possible mechanisms of cervical opening in the oestrous rat.

The time that the retention of luminal fluid begins and its release from the uterus in pro-oestrous rats was determined precisely using a dye injection technique. Oestradiol was found to cause dye retention in ovariectomized rats when provided continuously from an implant of 5 mg. Injections of 0.5 microgram/d X 4 were ineffective. Administration or progesterone to rats bearing implants of oestrogen interfered with the ability of the uterus to retain dye. Treatment with relaxin for 28 h prevented retention of eye in pro-oestrous rats whereas treatment at the time of dye injection was without effect. Mechanical stimulation of the cervix did not cause loss of injected dye. It is suggested that the mechanisms of opening of the cervix at coitus and at the end of oestrous may differ.

Animals↗
PubMed1982

Plasma concentrations of prolactin, progesterone, relaxin and oestradiol-17 beta in sows treated with progesterone, bromocriptine or indomethacin during late pregnancy.

Pregnant gilts (3/group) were given no treatment, 10 mg bromocriptine twice daily by mouth, from Day 111 of pregnancy to 1 day post partum, 25 mg progesterone s.c. at 6-h intervals from Days 111 to 116 inclusive or 400 mg indomethacin by mouth at 6-h intervals from Day 111 to 116 inclusive. Before spontaneous delivery maternal plasma prolactin and relaxin concentrations started to rise almost simultaneously between 58 and 47 h before the first piglet and both hormones reached peak values when the plasma progesterone concentration had started to decline rapidly (approximately 21-23 h). Suppression of prolactin levels by bromocriptine prevented the onset of lactation completely but had no obvious influence on changes of the other hormone concentrations and the course of parturition. Progesterone treatment delayed the onset of expulsion of the piglets but did not delay the simultaneous increase in prolactin and relaxin concentrations. These changes in hormone levels were prevented by indomethacin treatment but occurred essentially unchanged when the treatment was ended. The results support the concept that parturition in the pig is preceded by a biphasic increase of plasma prostaglandin levels.

Animals↗
PubMed1981

Relaxin inhibits myometrial activity in the ovariectomized non-pregnant ewe.

Myometrial activity was abolished abruptly but reversibly in 4 out of 5 ewes by the intravenous injection of 1 mg (500 GPU) porcine relaxin. Recovery began only after about 90 min and was not complete until 3-4 h after the injection. During the relaxin-induced inhibition the myometrium responded to oxytocin administered intravenously in doses of 250 mU. One ewe received intrauterine infusions of 2.5 and 5.0 microgram PGF-2 alpha per min during the period of relaxin inhibition: the former dose evoked a slight, and the latter a marked, response from the myometrium. The rate of rise of intrauterine pressure and the mean amplitude of pressures cycles were significantly depressed at 1, 1.5 and 2 h after the relaxin injection.

Animals↗