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J Reed

Publications and source records attributed to J Reed.

At least 91 records · Page 5Linked to original sources
PubMed1995

Investigation of the structural components governing the polarity-dependent refolding of a CD4-binding peptide from gp120.

The conformational switch at the principle CD4-binding domain of gp120 from HIV1 exhibits a highly cooperative folding transition from beta-sheet to helix triggered within a very narrow range of solvent polarity. The physical basis of this folding behaviour is of interest because it is unusual and because it is closely connected with biological function, i.e. binding to the CD4 receptor. Previous work revealed two primary structural elements, an N-terminal LPCR tetrad and a tryptophan residue eight residues C-terminal to this, that were essential for the helical and for the beta-sheet conformation, respectively. Attempts to construct synthetic "switch" domains using the characteristics so far identified produce peptides undergoing the transition at much higher polarity and involving fewer residues than the natural domain, in essence a lower stability of the beta-fold to apolar conditions. Introduction of a tryptophan residue reduced at the C(2)-C(3) linkage demonstrates clearly that the aromatic system of the tryptophan residue is central to beta-sheet stabilization. Residues with side-chains that might participate in electrostatic or aromatic interactions with the pi-electron system of Trp were sequentially altered to alanine. The results indicate that the "switch" properties of the CD4-binding domain arise from a poised tension between multiple interactions with the Trp aromatic ring stabilizing the beta-structure and the tendency of the LPCR tetrad to act as a template for a helical fold. Under polar conditions the former dominate. Lowering the polarity alters this both by weakening the aromatic interactions and by simultaneously increasing the helical propensities of the isoleucine and valine side-chains. Tryptophan seems uniquely suited to act as a polarity-sensitive conformational sensor.

Amino Acid Sequence↗
PubMed1995

Characterization of putative polyphosphoinositide binding motifs from phospholipase C beta 2.

Several phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-regulated actin-binding proteins and most phosphoinositide-specific phospholipases C (PI-PLCs) comprise a basic amino acid motif (KxxxKxKK, where x denotes any amino acid), which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have shown earlier that a peptide corresponding to amino acids 448-464 of human PLC beta 2 (LPSPEDLRGKILIKNKK, peptide P1) markedly and specifically stimulated the activity of this enzyme [Simões et al. (1993) FEBS Lett. 331, 248]. Here, we present a detailed analysis of the effects of various peptides related to peptide P1 aimed at understanding the mechanisms of peptide-mediated PLC beta 2 stimulation. Peptide KILIKNKK (P2), which comprises only the basic amino acid consensus motif, also stimulated PLC beta 2, although higher concentrations were required to observe this stimulatory effect. The effects of P1 and P2 were not additive, indicating that the two peptides affect PLC beta 2 activity via the same mechanism. Peptide LPSPEDLRG (P3), composed of the amino-terminal half of P1, did not affect the activity of PLC beta 2. Peptide KILIKNKKQFSGPTSS (P4), which includes the nine amino acids flanking the carboxy-terminus of the KILIKNKK motif within the sequence of PLC beta 2, stimulated the enzyme but was indistinguishable in potency from P2. Circular dichroism analysis revealed that peptide P1 changes its conformation in the presence of PtdInsP2 but not in the presence of other phospholipids including phosphatidylinositol 4-phosphate. The results suggest that the basic amino acid sequence physically interacts with PtdInsP2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Solvent polarity-dependent structural refolding: a CD and NMR study of a 15 residue peptide.

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a beta-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular beta-sheet to a helical secondary structure is known as "switch" behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the "switch." The tetrad has a strong beta-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3(10)-helix suggesting that the tetrad adopts a type III beta-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3(10)-helix at a critical polarity favoring intrachain hydrogen bonds.

Amino Acid Sequence↗
PubMed1995

Using a group project to teach research methods.

Teaching research methods is a problematic area in nurse education primarily because of the range of research methodologies that nursing research utilises, and also because there is some ambiguity about the purpose of the teaching-whether it is to enable students to read research or to do research. The strategy described in this paper, which is to use a group project, goes some way towards resolving these problems, as it focuses on the types of decisions made in research, rather than the results. By taking students through these decisions in a practicum-type environment, they gain insight into the 'hidden' research process which both informs their reading and any future studies they may conduct.

Education, Nursing, Baccalaureate↗
PubMed1995

[Specification of glial lineage in invertebrates].

Glial cells in invertebrates have been known for a long time, however, a systematic analysis of the development and the role of these cells has been missing. In the last few years, the development of new markers and the identification of genes specifically expressed in glial cells have shed some light on the mechanisms leading to gliogenesis in Drosophila melanogaster. The organisation of glial cells in the central and in the peripheral nervous system has been described and the origin of some of these cells has also been assessed. Lineage studies have shown that glial cells display different mechanisms of differentiation depending on their position in the nervous system. In some cases, glial differentiation seems intimately associated with neurogenesis, as has already been shown in the vertebrate peripheral nervous system. This suggests that similar developmental mechanisms have been conserved during evolution and that Drosophila can be used as a model system to study glial differentiation in vertebrates.

Animals↗
PubMed1995

Offline assessment of atherosclerotic coronary calcium from electron beam tomograms.

Coronary calcium screening using electron beam computed tomography (EBCT) is being applied clinically and for research purposes. We compared the accuracy of a specialized image analysis system with the standard proprietary software in the scanner's host computer. Sixty-seven symptomatic patients underwent coronary angiography and EBCT. Tomograms were analyzed using the proprietary software included in the scanner and with a specialized coronary calcium scoring work station. Sensitivities, specificities, and receiver operating characteristic curve areas were calculated for the proprietary software and the specialized system using the angiographic definition of disease of at least one stenosis causing greater than 50% luminal narrowing. There were no significant differences between the proprietary and the specialized software's accuracy. Receiver operating characteristic curve areas were 0.84 and 0.82 for proprietary software, respectively. During a 50 minute analysis session, the average number of studies analyzed were 12.6 +/- 1.7 using the proprietary software and 23.2 +/- 5.7 using the specialized software (P = .02).Image analysis was thus found to be more rapid using the specialized software. The specialized coronary calcium analysis system is as accurate as the proprietary software for scoring EBCT for coronary calcium. The reduction in analysis time makes the specialized system the preferable method.

Adult↗
PubMed1995

Evidence against a direct role for the induction of c-jun expression in the mediation of drug-induced apoptosis in human acute leukemia cells.

Previous reports have demonstrated that a variety of anticancer drugs, e.g., 1-beta-D-arabinofuranosylcytosine (ara-C), mitoxantrone, etoposide, camptothecin, and cisplatin, induce the expression of c-jun oncogene in leukemic cells prior to producing internucleosomal DNA fragmentation and the morphological features of apoptosis. This has led to the impression that the induction of c-jun expression may be directly involved in the molecular signaling of the final common pathway of programmed cell death or apoptosis. In the present study, we examined the role of c-jun expression in three different settings of anticancer drug-induced apoptosis in human leukemic cells. First, exposure of human myeloid leukemia HL-60 cells to high-dose ara-C for 4 h produced internucleosomal DNA fragmentation preceded by c-jun induction. However, pretreatment of HL-60 cells with staurosporine, a protein kinase C inhibitor, repressed c-jun yet enhanced DNA fragmentation and apoptosis due to ara-C. Second, in human pre-B leukemia 697/BCL-2 cells which are transfected with the cDNA of the bcl-2 oncogene and overexpress p26BCL-2, although ara-C or mitoxantrone treatment caused greater c-jun induction than in the 697/neo cells, significantly reduced endonucleolytic DNA fragmentation and apoptosis was observed in 697/BCL-2 cells. Finally, taxol-induced internucleosomal DNA fragmentation and morphological features of apoptosis in HL-60 cells were not associated with the induction of c-jun expression. These lines of evidence indicate that the induction of c-jun expression may not have a direct role in the molecular signaling of anticancer drug-induced apoptosis, and that the anticancer drug-induced apoptosis can occur by a mechanism that does not involve the induction of c-jun expression.

Antineoplastic Agents↗
PubMed1994

Inhibitors of the conformational switch involved in CD4 binding by the env glycoprotein gp120 from human immunodeficiency virus type 1 (HIV1).

A 15-residue fragment within the major continuous domain of gp120 from HIV1 that can bind independently to the CD4 receptor conserves the property of behaving as a polarity-triggered conformational switch despite displaying over 50% variability between strains. As this switch behavior (the ability to flip abruptly from beta-sheet to alpha-helix as the medium polarity is lowered past a critical point) is closely linked to CD4-binding ability, it presents a potential strain-independent target for intervention. A number of compounds have been tested for their ability to function as switch inhibitors. All those that displayed switch inhibitory activity also have been shown to act to prevent CD4 binding and/or viral infectivity. In addition, all compounds testing positive as switch inhibitors have certain chemical characteristics in common. The groundwork has thus been established for the design of strain-independent blockers of CD4 binding based on the strategy of switch inhibition.

Amino Acid Sequence↗
PubMed1994

Identification of serine-143 as the most likely precursor of dehydroalanine in the active site of histidine ammonia-lyase. A study of the overexpressed enzyme by site-directed mutagenesis.

The gene coding for histidase (histidine ammonia-lyase, HAL, EC 4.3.1.3) was isolated from a lambda-EMBL3 genomic library from Pseudomonas putida nicII and subcloned into the expression vector pT7-7. Transformation of Escherichia coli BL21 (DE3) cells with the recombinant vector led to the expression of catalytically active histidase amounting to 20-30% of the total soluble protein in the crude cell extract. A new rapid and highly efficient isolation procedure is described leading to electrophoretically homogeneous histidase within 1.5 days. Six grams of E. coli BL21 (DE3) cells (wet weight) gives approximately 100 mg of homogeneous histidase with a specific activity of 27 IU/mg. To investigate the possible role of serine as a precursor of dehydroalanine in the active site of histidase, each of the four serines, conserved in all known histidases and phenylalanine ammonia-lyases, was consecutively changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7-7 and were assayed for histidase activity. The catalytic activities of the four mutants and of wild-type histidase were compared. The Km and Vmax values of the overexpressed mutants S112A, S393A, and S418A and wild-type histidase did not show any significant differences. Mutant S143A, however, was devoid of catalytic activity (< 0.01%), pointing to the outstanding importance of this serine for the formation of an active enzyme. We conclude that serine-143 is the most probable precursor of the active-site dehydroalanine. The role of serine-143 in the biosynthesis of active histidase is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1994

The impact of the medical model on nursing practice and assessment.

In its extreme form, the medical model with its concerns of diagnosis, treatment and cure, has been criticized for the narrow and unsatisfactory view it takes of health care. Proponents of nursing theory, in contrast, attempt to develop a conceptual structure which offers a more humanistic approach to patient care, where nurses attempt to move beyond the influences of medical values in the way that they work. This study indicates, however, that the medical model is occasionally compatible with nurses' values, and in certain settings can enhance and support nursing care. In other settings, however, the medical model, although in accord with nursing values, has little to offer practice, and indeed may have a negative effect on the development of alternative approaches to care.

Activities of Daily Living↗
PubMed1994

Phenomenology without phenomena: a discussion of the use of phenomenology to examine expertise in long-term care of elderly patients.

Phenomenological approaches to research have gained popularity in nursing research over past years, in particular the use of critical incident technique. Phenomenology can be traced back to existentialist philosophy where it is expounded in the work of Husserl and Heidegger. One of the most notable examples of phenomenological research in nursing has been the work of Benner who has used this approach to examine expertise in nursing. This paper is an account of a study which attempted to adapt phenomenological methods to the investigation of expertise in nurses working in long-term care settings, which was curtailed by the apparent inability of nurses in the study to identify any significant incidents. The paper examines this problem in the light of existentialist philosophy and suggests that the apparent lack of expertise identified in the nurses might be due more to a tendency of phenomenological studies to focus more on articulation than on attunement or potential, the other elements of dasein. The paper concludes that attention to these elements is required when phenomenology is used.

Aged↗