Biomedical subjects

K A Walsh

Publications and source records attributed to K A Walsh.

At least 19 recordsLinked to original sources

PubMed2001

Pollen on grass clippings: putting the suspect at the scene of the crime.

In a case of alleged sexual assault, the pollen content of samples of grass clippings and soil from the suspect's clothing and shoes was compared to that of a sample of grass clippings from the alleged crime scene (a grassy area) to determine whether or not the suspect had been at the scene. The clothing and shoe samples showed a very strong correlation with each other and with the sample from the alleged crime scene in the combination of the different types of pollen present, very strongly supporting the contention that the suspect had been at the scene.

Adult↗

PubMed2000

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior.

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Animals↗

PubMed1999

Quality of life assessment in patients undergoing head and neck surgery as evaluated by lay caregivers.

OBJECTIVES/HYPOTHESIS: Rehabilitation following head and neck cancer surgery has steadily moved into the outpatient realm and become dependent on caregivers with no formal medical background (lay caregivers.) Satisfactory rehabilitation and quality of life (QOL) depend on successful relationships between patients and the lay caregivers. This study evaluates the QOL assessments of patients by themselves and their primary lay caregivers before head and neck surgery. STUDY DESIGN: Institutional Review Board-approved study using validated QOL assessment instrument. MATERIALS AND METHODS: The preoperative QOL status in 50 patients undergoing extensive head and neck surgery was evaluated using the self-administered Medical Outcomes Survey Short Form 36 (SF-36). The patient's primary lay caregiver (spouse, child, or friend) completed a similar questionnaire evaluating the patient's status. RESULTS: Thirty-three (66%) questionnaires were returned. Twenty-five (50%) questionnaire sets were successfully completed by both parties and employable for comparison. Sixty percent of the caregivers were within the 90% confidence interval of the patient's assessment for six or more of the eight parameters evaluated by the SF-36. Likewise, caregiver assessments for specific parameters were consistently congruent with patient evaluation, except for the parameters of bodily pain and general health, for which caregivers demonstrated a trend for overrating pain and underestimating general health. Caregivers of the same generation as the patient demonstrated significantly higher congruence (P = .007). Similarly, a trend for higher congruence was noted in patients with recurrent disease. CONCLUSIONS: The importance of the lay caregiver has increased in the era of greater outpatient rehabilitation. This pilot study indicates that QOL assessment by lay caregivers may be examined with existing instruments and highlights QOL parameters critical to both the head and neck surgery patient and his or her primary lay caregiver.

Adolescent↗

PubMed1999

Locating and identifying posttranslational modifications by in-source decay during MALDI-TOF mass spectrometry.

A technique is described for identifying and locating posttranslational modifications (PTMs) in peptides and proteins of known sequence by interpretation of c(n) ion signals generated by in-source decay during delayed ion extraction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sites of phosphorylation in seven synthetic peptides were determined, as was the location of both the heme group and N,N,N-trimethyllysine in yeast cytochrome c. A semi-automated data analysis process facilitates the identification of segments of the sequence on each side of the PTM, permitting its placement at the junction of the segments and definition of the added mass. A graphical display facilitates illustration of both the location and mass of the PTM.

Amino Acid Sequence↗

PubMed1997

Stability enhancement for peptide analysis by electrospray using the triple quadrupole mass spectrometer.

Electrospray ionization sources, used with triple quadrupole mass spectrometers from PE/Sciex (API III+), Micromass (Quattro II), and Finnigan (TSQ 7000), were modified with a 35-gauge stainless steel needle. The dimensions of the needle were 63 microm i.d. by 145 microm o.d. with variable length, depending on the specific instrument. This modification led to enhanced signal stability, improved signal/noise ratios, and lowered sample consumption for a wide range of peptides. Stable baselines were observed with flow rates in the range of 50 nL/min to 5 microL/min. An alternative design, based on a metal wire housed within a fused silica capillary, led to the most stable signals of all during infusion, but caused excessive peak broadening with capillary chromatography. The Finnigan interface was further modified with an external postcolumn addition tee, used in conjunction with capillary liquid chromatography columns of 30 and 50 microm internal diameter. The best results with the modified Finnigan interface were acquired using the 50-microm column at a flow rate of 150 to 200 nL/min.

Factor VII↗

PubMed1997

Monitoring calcium-induced conformational changes in recoverin by electrospray mass spectrometry.

Recoverin is a calcium-binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N-acylated by myristoyl and related fatty acyl residues that are thought to act as "calcium-myristoyl switches," whereby, in the presence of Ca2+, the N-terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely associated with the protein. Here we use electrospray ionization mass spectrometry (ESI/MS) to examine hydrogen isotopic exchange rates for specific regions of both acylated and nonacylated recoverin in the presence and absence of calcium. The deuterium exchange rates of three regions in the hydrophobic myristoyl binding pocket of acylated recoverin decreased in the absence of calcium. This effect is most likely due to the closer association of the acyl group with the protein under these conditions. In contrast, rates of deuterium incorporation increased in the absence of calcium for other regions, including the two functional calcium-binding sites. In addition to supporting the calcium-myristoyl switch hypothesis, a comparison of the behavior of acylated and unacylated recoverin revealed that the N-acyl group (N-lauroyl or N-myristoyl) exerts a significant stabilizing influence on the dynamics of recoverin. We demonstrate that the new technique of monitoring hydrogen isotopic exchange by ESI/MS can be used to obtain useful information concerning protein structures in solution using smaller amounts of protein and under more physiologically relevant conditions than is typically possible with NMR or X-ray crystallography.

Amino Acid Sequence↗

PubMed1997

Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction.

Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.

Amino Acid Sequence↗

PubMed1997

Electrocardiographic markers of late sudden death risk in postoperative tetralogy of Fallot children.

Following surgery for tetralogy of Fallot (TOF), children may develop late onset ventricular arrhythmias. Many patients have both depolarization and repolarization abnormalities, including right bundle branch block (RBBB) and QT prolongation. The goal of this study was to improve prospective risk-assessment screening for late onset sudden death. Resting ECG markers including QRS duration, QTc, JTc, and interlead QT and JT dispersion were statistically analyzed to identify those patients at risk for ventricular arrhythmias and sudden cardiac death. To determine predictive markers for future development of arrhythmia, we examined 101 resting ECGs in patients (age 12 +/- 6 years) with postoperative TOF and RBBB, 14 of whom developed late ventricular tachycardia (VT) or sudden death. These ECGs were also compared with an additional control group of 1000 age- and gender-matched normal ECGs. The mean QRS (+/- SD) in the VT group was 0.18 +/- 0.02 seconds versus 0.14 +/- 0.02 seconds in the non-VT group (P < 0.01). QTc and JTc in the VT group was 0.53 +/- 0.05 seconds and 0.33 +/- 0.03 seconds compared with 0.50 +/- 0.03 seconds and 0.32 +/- 0.03 seconds in the non-VT group (P = NS). There was no increase in QT dispersion among TOF patients with VT or sudden death compared with control patients or TOF patients without VT, although JT dispersion was more common in the TOF groups. A prolonged QRS duration in postoperative TOF with RBBB is more predictive than QTc, JTc, or dispersion indexes for identifying vulnerability to ventricular arrhythmias in this population, while retaining high specificity. The combination of both QRS prolongation and increased JT dispersion had very good positive and negative predictive values. These results suggest that arrhythmogenesis in children following TOF surgery might involve depolarization in addition to repolarization abnormalities. Prospective identification of high-risk children may be accomplished using these ECG criteria.

Child↗

PubMed1997

Interpreting DNA mixtures.

The interpretation of mixed DNA stains is explained in the context of likelihood ratios. The probabilities for the mixed-stain profile are evaluated under alternative explanations that specify the numbers of contributors and the profiles of any known contributors. Interpretations based simply on the frequencies with which random members of a population would not be excluded from a mixed-stain profile do not make use of all the information, and may overstate the strength of the evidence against included people. The effects of the numbers of contributors depends on whether all the alleles at a locus are present in the mixed stain. A general equation is given to allow likelihood ratios to be calculated, and includes the "2p" modification suggested by the 1996 NRC report. This modification is not always conservative. A computer program to perform calculations is available.

Alleles↗

PubMed1996

Autophosphorylation of type Ibeta cGMP-dependent protein kinase increases basal catalytic activity and enhances allosteric activation by cGMP or cAMP.

Autophosphorylation of purified bovine Ibeta isozyme of cGMP-dependent protein kinase (Ibeta cGK) in the presence of cGMP or cAMP increased basal kinase activity (-cGMP) as much as 4-fold and reduced the Ka for both cGMP and cAMP; maximum catalytic activity (+cGMP) was not altered. Autophosphorylation proceeded with at least two rate components. The faster rate correlated with phosphorylation of Ser-63. The slower rate, as well as the increase in basal kinase activity and decrease in Ka for cyclic nucleotides, correlated with phosphorylation of Ser-79. Autophosphorylation of either residue was an intramolecular reaction. Autophosphorylation of a proteolytically generated Ibeta cGK monomer lacking amino-terminal residues 1-64 increased basal activity (3-fold) and decreased Ka for cAMP (15-fold). This indicated that autophosphorylation of Ser-79 did not require dimeric cGK and that the phosphorylation of Ser-79 in the monomer was sufficient to alter enzymatic characteristics of Ibeta cGK. These studies suggested that increases in intracellular cGMP or cAMP could result in autophosphorylation of Ibeta cGK, which would increase basal kinase activity as well as the sensitivity of cGK to activation by cGMP or to cross-activation by cAMP. Autophosphorylation could also prolong the increased kinase activity after decline of the second messenger.

Allosteric Regulation↗

PubMed1996

Arginine 75 in the pseudosubstrate sequence of type Ibeta cGMP-dependent protein kinase is critical for autoinhibition, although autophosphorylated serine 63 is outside this sequence.

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Ibeta cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (approximately 0.8 phosphate/cGK monomer) approximately 3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (approximately 67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Ibeta cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.

Amino Acid Sequence↗

PubMed1996

Structural and enzymatic aspects of rhodopsin phosphorylation.

Photoactivated rhodopsin (Rho*) is phosphorylated near the C terminus at multiple sites, predominantly at Ser334, Ser338, and Ser343. We systematically examined the sites of phosphorylation upon flash activation of Rho in rod outer segment (ROS) homogenates. Addition of an inhibitory antibody against rhodopsin kinase (RK) lowered phosphorylation at Ser334, Ser338, and Ser343, without changing the ratio between phosphorylation sites. In contrast, no effect of protein kinase C was detected after stimulation (by a phorbol ester), inhibition (with H7), or reconstitution of protein kinase C with purified ROS membranes. The stoichiometry and the ratio between different phosphorylation sites in purified Rho were also reproduced using RK, purified to apparent homogeneity from ROS or from an insect cell expression system. Thus, we conclude that light-dependent phosphorylation of Rho is mediated primarily by RK. Depalmitoylation of Rho at Cys322 and Cys323 altered the conformation of the C terminus of Rho, as observed by phosphorylation by casein kinase I, but did not affect phosphorylation by RK. The sites of phosphorylation were influenced, however, by the presence of four conserved amino acids at the C terminus of Rho. The accumulation of phosphorylated Ser334 observed in vivo could result from slower dephosphorylation of this site as compared with dephosphorylation of Ser338 and Ser343. These data provide a molecular mechanism for the site-specific phosphorylation of Rho observed in vivo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Sherpa: a Macintosh-based expert system for the interpretation of electrospray ionization LC/MS and MS/MS data from protein digests.

A computer program, Sherpa, has been implemented to aid in the elucidation of electrospray ionization liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry data from protein digests. The program differs from existing analysis software in its ability to search concurrently against multiple protein sequences, to search and compare two LC/MS files concurrently, to search for glycosylated and phosphorylated peptides, and to give a simple evaluation of the quality of a match between the data and a prediction.

Amino Acid Sequence↗

PubMed1996

Microheterogeneity of human filaggrin: analysis of a complex peptide mixture using mass spectrometry.

Filaggrin is the product of posttranslational processing of the large, epidermal protein profilaggrin, which consists of 10 or more tandem filaggrin domains plus an amino and a carboxyl domain. According to fragmentary cDNA sequences, the filaggrin domains in the human protein vary at 40% of the amino acid positions; hence, mature filaggrin is a population of homologous but heterogeneous proteins, even within one individual. Available gene sequences give only a limited picture of the heterogeneity of human filaggrin protein because no complete human profilaggrin gene has been sequenced. Questions about the extent of heterogeneity of filaggrin within and between individuals have not been answered, nor have questions concerning the limited proteolytic cleavage of human profilaggrin that generates filaggrin in vivo. In order to address these questions and to provide an analysis of the primary structure of human filaggrins, we employed various methods of mass spectrometry. The intact protein and a tryptic digest of the mixture of human filaggrins were examined by matrix-assisted laser desorption time-of-flight mass spectrometry. Tryptic digests of human filaggrin from single individuals were also separated and analyzed by liquid chromatography/mass spectrometry (LC/MS) (using electrospray mass spectrometry), and specific peptides were identified by tandem mass spectrometry (MS/MS). A robust data analysis program, Sherpa, was developed to facilitate the interpretation of both LC/MS and MS/MS. These experiments show that human filaggrin includes heterogeneity not yet seen in cDNA sequences, but that much structure is highly conserved. Interestingly, we found that the heterogeneity is conserved among individuals. An approximation of the regions linking filaggrins in human profilaggrin is developed. These investigations provide a unique test of the limits of tryptic mapping of complex mixtures using mass spectrometry.

Amino Acid Sequence↗

PubMed1996

Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli.

Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, beta-methylthio-aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome.

Aspartic Acid↗

PubMed1995

Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.

Human furin catalyzes the proteolytic maturation of many proproteins in the exocytic and endocytic secretory pathways by cleavage at the C-terminal side of the consensus sequence-ArgXaaLys/ArgArg decreases -. Both the trans-Golgi network (TGN) concentration and intracellular routing of furin require sequences in its 56 amino acid cytoplasmic tail. Here, we show that the furin cytoplasmic tail contains multiple trafficking signals. Localization to the TGN requires a cluster of acidic amino acids that, together with a pair of serine residues, forms a casein kinase II (CK II) phosphorylation site. We show that CK II efficiently phosphorylates these serines in vitro, and using a permeabilized cell system we provide evidence that CK II is the in vivo furin kinase. Analysis by mass spectrometry shows that, in vivo, furin exists as di-, mono- and non-phosphorylated forms. Finally, employing (i) furin constructs that mimic either non-phosphorylated or phosphorylated furin and (ii) the phosphatase inhibitor tautomycin, we show that the phosphorylation state of the furin cytoplasmic tail modulates retrieval of the endoprotease to the TGN. Thus, routing of furin is a two-tiered process combining a set of trafficking signals comprised of the primary amino acid sequence of the tail with its phosphorylation state.

Amino Acid Sequence↗

PubMed1995

Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein.

Profilaggrin, a highly phosphorylated protein synthesized in mammalian cornified epithelia, is the precursor of filaggrin, a protein that is involved in aggregation of keratin during terminal differentiation. Possible functions for the phosphorylation include preventing premature aggregation of keratin, packing profilaggrin into a storage granule, association of other proteins with the granule, and/or regulating proteolytic processing of profilaggrin. As a first step in characterizing the phosphorylation of rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC and analyzed by ionspray mass spectrometry. Nine putative phosphopeptides were identified as those with masses 80 Da (or multiples of 80 Da) greater than the predicted unphosphorylated masses. The six that were phosphorylated to a high stoichiometry were analyzed further. Several multiply phosphorylated peptides underwent neutral loss of H3PO4 during collisional activation, complicating interpretation of the MS/MS spectra. In order to circumvent this problem, an alternative strategy was applied in which peptide mixtures were treated with Ba(OH)2, resulting in beta-elimination of H3PO4 and generation of dehydrated serine or threonine at the site of phosphorylation. Peptides containing dehydrated serine or threonine fragmented well, providing unequivocal identification of multiple phosphorylation sites in peptides as long as 39 amino acids. The phosphopeptides (with phosphorylated residues underlined) were GQQHSGHPQVYYYGVEETEDESDAQQGHHQQQQQQR, GGQAGSHSESEASGGQAGR, HTSRPEQSPDTAGR, GESPAGQQSPDR, EASASQSSDSEGHSGAHAGIGQGQTSTTHR, and GSSESQASDSEGHSDYSEAHTQGAHGGIQTSSQR.

Amino Acid Sequence↗

PubMed1995

Identification of the amino terminus of human filaggrin using differential LC/MS techniques: implications for profilaggrin processing.

Filaggrin, the intermediate filament aggregating protein of epidermis, is the product of proteolytic processing of the precursor profilaggrin, which consists of 10-20 tandem filaggrin domains. The proteolytic processing sites in mouse and rat profilaggrin have been previously reported. Mouse filaggrin is N-terminally blocked. Rat filaggrin is N-terminally ragged, making it heterogeneous. Human filaggrin, in addition to being N-terminally blocked and potentially ragged at the amino terminus, is heterogeneous due to sequence variation between one filaggrin domain and another along the profilaggrin gene. This complexity has made more difficult the analysis of processing sites in human profilaggrin. We have identified the amino terminus of human filaggrin by applying a general method we have developed for the recognition of amino-terminal peptides in digests of N-terminally blocked proteins. This method compares the peptides in an acetylated and an unacetylated tryptic digest of the protein during their separation by liquid chromatography on-line with electrospray mass spectrometry. In this comparison only the original blocked amino-terminal peptides appear unchanged between the two profiles. Human filaggrin was found to have a heterogeneous N-terminus, as a result both of sequence heterogeneity and of ragged processing; it is blocked by a pyrrolidonecarboxyl group derived from glutamine. By comparison to the termini of rat and mouse filaggrins, implications for the processing of human profilaggrin are discussed.

Amino Acid Sequence↗