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Biomedical subjects

P A Walker

Publications and source records attributed to P A Walker.

At least 19 recordsLinked to original sources
PubMed2002

The role of trade-offs in biodiversity conservation planning: linking local management, regional planning and global conservation efforts.

Biodiversity conservation planning requires trade-offs, given the realities of limited resources and the competing demands of society. If net benefits for society are important, biodiversity assessment cannot occur without other sectoral factors "on the table". In trade-offs approaches, the biodiversity value of a given area is expressed in terms of the species or other components of biodiversity that it has that are additional to the components protected elsewhere. That "marginal gain" is called the complementarity value of the area. A recent whole-country planning study for Papua New Guinea illustrates the importance of complementarity-based trade-offs in determining priority areas for biodiversity conservation, and for designing economic instruments such as biodiversity levies and offsets. Two international biodiversity programs provide important new opportunities for biodiversity trade-offs taking complementarity into account. Both the Millennium Ecosystem Assessment and the Critical Ecosystems or "hotspots" programs can benefit from an explicit framework that incorporates trade-offs, in which a balance is achieved not only by land-use allocation among areas, but also by the crediting of partial protection of biodiversity provided by sympathetic management within areas. For both international programs, our trade-offs framework can provide a natural linkage between local, regional and global planning levels.

Animals↗
PubMed2001

The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways.

Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.

ADP-Ribosylation Factor 1↗
PubMed2000

Structure of the TPR domain of p67phox in complex with Rac.GTP.

p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.

Amino Acid Sequence↗
PubMed1998

Isotachophoretic separations on a microchip. Normal Raman spectroscopy detection.

Isotachophoretic separations of the herbicides paraquat and diquat are performed in a glass microchip etched channel and monitored on-chip by normal Raman spectroscopy. The 40-micron-wide and 75-micron-deep separation channels are chemically etched in a serpentine design to 21-cm total length. A 120-micron-thick glass cover slip is used to seal the channels. Separation field strengths up to 380 V/cm are used. The microchip is directly coupled to a Raman microprobe. No interfacing is required. Raman spectra are generated with a 2-W, 532-nm NdY-VO4 laser and collected at 8-cm-1 resolution with a holographic transmissive spectrograph and a cryogenically cooled CCD. Data acquisition is at 2-5 spectra/s. Raman isotachopherograms of the pesticides at starting concentrations as low as 2.3 x 10(-7) M (60 ppb paraquat/80 ppb diquat) are presented.

Diquat↗
PubMed1998

Capillary isotachophoresis with fiber-optic Raman spectroscopic detection. Performance and application to ribonucleotides.

A fiber-optic Raman probe fitted with a microscope objective was used to obtain on-line normal Raman spectra of adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate and uridine 5'-monophosphate separated by capillary isotachophoresis. With multimode optical fiber, the system interrograted a 40-micron length of capillary. Fiber-optic coupling facilitated use of an unmodified spectrograph and conventional capillary mounting systems. Raman spectra were excited with a 2W 532 nm NdYVO4, laser as the excitation source, with collection of 1 spectrum per second. Even at 2.10(-5) M initial concentration, Raman spectra were obtained at a good signal-to-noise ratio.

Electrophoresis↗
PubMed1997

Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue.

Small G proteins of the Rho family, which includes Rho, Rac and Cdc42Hs, regulate phosphorylation pathways that control a range of biological functions including cytoskeleton formation and cell proliferation. They operate as molecular switches, cycling between the biologically active GTP-bound form and the inactive GDP-bound state. Their rate of hydrolysis of GTP to GDP by virtue of their intrinsic GTPase activity is slow, but can be accelerated by up to 10(5)-fold through interaction with rhoGAP, a GTPase-activating protein that stimulates Rho-family proteins. As such, rhoGAP plays a crucial role in regulating Rho-mediated signalling pathways. Here we report the crystal structure of RhoA and rhoGAP complexed with the transition-state analogue GDP.AlF4- at 1.65 A resolution. There is a rotation of 20 degrees between the Rho and rhoGAP proteins in this complex when compared with the ground-state complex Cdc42Hs.GMPPNP/rhoGAP, in which Cdc42Hs is bound to the non-hydrolysable GTP analogue GMPPNP. Consequently, in the transition state complex but not in the ground state, the rhoGAP domain contributes a residue, Arg85(GAP) directly into the active site of the G protein. We propose that this residue acts to stabilize the transition state of the GTPase reaction. RhoGAP also appears to function by stabilizing several regions of RhoA that are important in signalling the hydrolysis of GTP.

Arginine↗
PubMed1997

Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP.

Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions. These proteins are clustered into distinct families but all act as molecular switches, active in their GTP-bound form but inactive when GDP-bound. The Rho family of G proteins, which includes Cdc42Hs, activate effectors involved in the regulation of cytoskeleton formation, cell proliferation and the JNK signalling pathway. G proteins generally have a low intrinsic GTPase hydrolytic activity but there are family-specific groups of GTPase-activating proteins (GAPs) that enhance the rate of GTP hydrolysis by up to 10(5) times. We report here the crystal structure of Cdc42Hs, with the non-hydrolysable GTP analogue GMPPNP, in complex with the GAP domain of p50rhoGAP at 2.7A resolution. In the complex Cdc42Hs interacts, mainly through its switch I and II regions, with a shallow pocket on rhoGAP which is lined with conserved residues. Arg 85 of rhoGAP interacts with the P-loop of Cdc42Hs, but from biochemical data and by analogy with the G-protein subunit G(i alpha1), we propose that it adopts a different conformation during the catalytic cycle which enables it to stabilize the transition state of the GTP-hydrolysis reaction.

Arginine↗
PubMed1996

Specific cleavage of the retinoblastoma protein by an ICE-like protease in apoptosis.

Interleukin 1beta-converting enzyme-like (ICE-like) proteases are important mediators of apoptosis in diverse cell types and organisms. However, the role of these proteases in apoptosis cannot be satisfactorily explained on the basis of the physiological functions of their known substrates. Here we show that the C-terminal 42 amino acid peptide of the retinoblastoma (Rb) protein, an important cell cycle regulator with a known anti-apoptotic function, is specifically cleaved off by an ICE-like protease in tumour necrosis factor (TNF)- and staurosporine-induced apoptosis. Cleavage of Rb induced by TNF was blocked in vivo and in vitro by two specific inhibitors of ICE-like proteases, and in vitro by a point mutation (Asp886 to Ala) within the ICE-like protease cleavage site of Rb, (883)DEAD(886). An antibody raised against the C-terminal 15 amino acid peptide of Rb recognized the full-length but not the cleaved form of Rb. The extent of Rb cleavage correlated directly with TNF-induced apoptosis in all tumour cell lines examined. Cleaved Rb bound cyclin D3 and inhibited the transcriptional activity of E2F-1, but failed to bind to the regulatory protein MDM2, which has been implicated in apoptosis. As Rb suppresses cell death and its C-terminus has important regulatory functions, our results suggest that Rb cleavage is an important event in apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Pulmonary disease.

This article has presented the reader with an overview of the pulmonary disorders that develop during the course of HIV disease with special emphasis on the more commonly encountered entities. This information is intended to prepare the clinician to recognize the hallmark characteristics of the various diseases as well as atypical features. Despite the advances in basic understanding of the clinicopathologic consequences of infection with HIV, a cure has not been realized. There has, however, been success in controlling some of the major pulmonary problems that adversely affect both the quality and the length of life for persons with AIDS. For most complications of HIV infection, prognosis ultimately depends not only on treatment of the specific problem, but also controlling the relentless process of progressive immunosuppression. Continued research into treatment or prevention of HIV infection itself is needed, but at present prevention, rapid diagnosis, and treatment of recognized problems remain an intermediary goal.

AIDS-Related Opportunistic Infections↗
PubMed1996

Hearing conservation in surgery for glomus jugulare tumors.

The most common ground on which surgery for glomus jugulare (GJ) tumors is criticized is the perceived risk of functional incapacity that attends possible cranial nerve (CN) loss. It is aggregate lower CN loss that is most often highlighted as particularly disabling to the quality of postsurgical survival. The documented success of both conservation surgery and operative rehabilitation of phonopharyngeal surgical deficits has, however, neutralized much of this criticism. The issue of hearing conservation (HC) in neurotologic skull base surgery, on the other hand, has not been well documented toward this end. The presence of a GJ neoplasm need not reflexly nor technically forfeit preexisting hearing. HC is, admittedly, a subordinate priority to total tumor removal, successful distal control of the internal carotid artery, and even facial nerve integrity. Yet, in appropriately selected patients, existing operative technology permits hearing preservation, a noteworthy addition to the high-grade functional outcome we have come to reasonably expect of conservation surgery. Hearing salvage further serves to define the concept of neurotologic skull base surgery. Hearing preservation in 122 GJ tumor patients is reviewed. Intuitively, as for acoustic tumor, HC appears tumor size related. Selection criteria for conservation surgery and its operative technique are detailed. Outcome is appropriately scored. The radiation therapy literature on this subject will be assiduously scrutinized for comparison.

Audiometry, Pure-Tone↗
PubMed1995

On-line Raman spectroscopy of ribonucleotides preconcentrated by capillary isotachophoresis.

Normal Raman spectroscopy is used as an on-line detector for capillary isotachophoresis (ITP) of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate in phosphate buffers. Preconcentration is from a 1 x 10(-2) M phosphate buffer (pH 7.5) into a leading electrolyte of 0.1 M KCl or Na2SO4, with a terminating electrolyte of 0.1 M 4-morpholinepropane-sulfonic acid. The ribonucleotides are concentrated to above 10(-2) M at the detection window, allowing measurement of Raman spectra with 1 s integration, from starting concentrations of 5 x 10(-6) M or higher.

Adenosine Diphosphate↗
PubMed1995

Sequence and structural determinants of the interaction between the 5'-noncoding region of picornavirus RNA and rhinovirus protease 3C.

It has previously been established that human rhinovirus 14 protease 3C binds specifically to the 5'-noncoding region of the viral RNA. A series of mutants of protease 3C and deletion or point mutants of the 5'-noncoding region of the viral RNA were analyzed to elucidate the sites of interaction between the protease and the RNA. Amino acids in protease 3C essential for RNA binding were found to be discontinuous in the amino acid sequence, and mutations which destroyed RNA binding did not affect the catalytic (proteolytic) activity of protease 3C. Based on the three-dimensional structure of rhinovirus 14 protease 3C, the RNA binding region is located in an extended area distinct from the catalytic triad. A single stem-loop structure of 27 nucleotides (stem-loop d) in the 5'-noncoding region was necessary and sufficient to bind protease 3C. Mutagenesis of either the base-paired stem or unpaired loop or bulge regions of stem-loop d suggested that the base-paired stem, but not the loop or bulge, carries important determinants of protease 3C binding. This conclusion is strengthened by the observation that rhinovirus 14 protease 3C bound specifically to the 5'-noncoding region of poliovirus RNA, and only the base-paired stem of stem-loop d is conserved between poliovirus and rhinovirus RNAs.

3C Viral Proteases↗
PubMed1994

Efficient and rapid affinity purification of proteins using recombinant fusion proteases.

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient and efficient method of affinity purification which can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant 'fusion protease' cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.

3C Viral Proteases↗
PubMed1994

The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID.

The E6 promoters of all genital human papillomaviruses have a characteristic alignment of transcription factor binding sites. Activation of the basic transcription complex at the TATA box depends upon a sequence-aberrant Sp1 site. Repression of E6 promoters is achieved by two binding sites for the viral E2 protein positioned between the Sp1 site and the TATA box. We have purified the human papillomavirus type 16 E2 protein after expression in Escherichia coli and studied its binding and repression properties with oligonucleotides representing the homologous promoter sequences. A Kd value of 3 x 10(-10) M indicated binding properties expected for a native protein. We found low cooperativity in the binding of two E2 dimers to flanking sites, both when these sites were separated by 3 nucleotides, as in the natural promoter, and when they were further apart. E2 protein, bound close to the distal Sp1 site, displaced the Sp1 factor even when the aberrant sequence was replaced by a typical Sp1 core recognition site. The high affinity of E2 protein for its binding site even led to Sp1 displacement at concentrations of E2 protein nearly 2 orders of magnitude lower than those of Sp1. Functional analyses of mutated E6 promoter sequences showed repression by this distal E2 binding site in the complete absence of binding to the proximal E2 binding site. From our findings and observations published by others, we conclude that each of the E2 binding sites in the E6 promoter of genital human papillomaviruses plays a separate role by displacing the transcription factors Sp1 and TFIID.

Amino Acid Sequence↗
PubMed1993

Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities.

Protease 3C (3Cpro) encoded by human rhinovirus type 14 was purified from recombinant Escherichia coli and shown to bind specifically to the 5'-terminal 126 nucleotides of the viral RNA (126 RNA) in addition to efficiently cleaving a synthetic peptide in trans. The binding of 3Cpro to the viral RNA may be required for the initiation of plus strand viral RNA synthesis, suggesting a second non-proteolytic function for 3Cpro. Single amino acid substitutions were generated in 3Cpro at residues that are highly conserved among picornaviruses or that lie within the putative catalytic triad. Conservative changes at Asp-85 (D85E and D85N) destroyed the ability of 3Cpro to bind specifically to the 126 RNA, whereas the D85N mutation resulted in almost wild-type levels of proteolytic activity. Conversely, substitutions at His-40, Glu-71, or Cys-146 (H40D, E71A, or C146S) gave proteolytically inactive mutants that bound to the 126 RNA. These results suggest that the highly conserved Asp-85 is essential for specific binding to the 126 RNA, but is unlikely to function in proteolysis as the acidic member of the catalytic triad. Moreover, 3Cpro appears to have different domains for the RNA binding and proteolytic activities.

3C Viral Proteases↗
PubMed1992

Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.

Amino Acid Sequence↗
PubMed1990

The Levels of Emotional Awareness Scale: a cognitive-developmental measure of emotion.

The Levels of Emotional Awareness Scale (LEAS) is based on a new cognitive-developmental model of emotional experience. The scale poses evocative interpersonal situations and elicits descriptions of the emotional responses of self and others which are scored using specific structural criteria. Forty undergraduates (20 of each sex) were tested. Interrater reliability and intratest homogeneity of the LEAS were strong. The LEAS was significantly correlated with two measures of maturity: the Washington University Sentence Completion Test (SCT) of Ego Development, and the Parental Descriptions Scale-a cognitive-developmental measure of object representation. In addition, the LEAS correlated positively with openness to experience and emotional range but not with measures of specific emotions, repression or the number of words used in the LEAS responses. These findings suggest that it is the level of emotion, not the specific quality of emotion, that is tapped by the LEAS.

Adult↗