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S E Clark

Publications and source records attributed to S E Clark.

53 records · Page 3Linked to original sources
PubMed1991

Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins.

We demonstrate that the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP of Photosystem II), encoded by a Type I gene, contains distinct determinants for processing at two sites during in vitro import into the chloroplast. Using precursors from both pea and wheat, it is shown that primary site processing, and release of a approximately 26-kD peptide, depends on an amino-proximal basic residue. Substitution of an arginine at position -4 resulted in an 80% reduction in processing, with the concomitant accumulation of a high molecular weight intermediate. Cleavage occurred normally when arginine was changed to lysine. The hypothesis that a basic residue is a general requirement for transit peptide removal was tested. We find that the precursors for the small subunit of Rubisco and Rubisco activase do not require a basic residue within seven amino acids of the cleavage site for maturation. In the wheat LHCP precursor, determinants for efficient cleavage at a secondary site were identified carboxy to the primary site, beyond what is traditionally called the transit peptide, within the sequence ala-lys-ala-lys (residues 38-41). Introduction of this sequence into the pea precursor, which has the residues thr-thr-lys-lys in the corresponding position, converted it to a substrate with an efficiently recognized secondary site. Our results indicate that two different forms of LHCP can be produced with distinct NH2-termini by selective cleavage of a single precursor polypeptide.

Amino Acid Sequence↗
PubMed1990

Scintigraphic evaluation of glomus tumours.

The current investigations of choice for a suspected glomus tumour are either direct or indirect angiography to include digital subtraction followed by computerized tomography (CT) or magnetic resonance imaging (MRI) or, if available, CT and MRI with gadolinium alone. Although these modalities confirm the diagnosis and give anatomical information to facilitate accurate staging, they do not provide functional data. The use of radionuclide scintigraphy can add an extra physiological dimension to glomus tumour imaging. Iodine-131/123 metaiodobenzylguanidine (MIBG) is a tumour imaging agent which has been used to diagnose head and neck neuroendocrine tumours to include paragangliomata and medullary carcinoma of the thyroid (MCT). However, it is expensive and the new head and neck tumour imaging agent technetium-99 m (Tc99m) (v) dimercaptosuccinic acid (DMSA) has superceded it as the imaging agent of choice to evaluate MCT. We report a patient with a glomus jugulare tumour which was evaluated with I131/I123-MIBG and Tc99m (v) DMSA. The tumour was functional and is the first reported case exhibiting positive accumulation of both I131-MIBG and Tc99m (v) DMSA. The patient was subsequently treated with a therapeutic dose of I131-MIBG. The significance of these results is discussed.

3-Iodobenzylguanidine↗
PubMed1990

List-strength effect: II. Theoretical mechanisms.

Ratcliff, Clark, and Shiffrin (1990) examined the list-strength effect: the effect of strengthening (or weakening) some list items upon memory for other list items. The list-strength effect was missing or negative in recognition, missing or positive in cued recall, and large and positive in free recall. We show that a large number of current models fail to predict these findings. A variant of the SAM model of Gillund and Shiffrin (1984), involving a differentiation hypothesis, can handle the data. A variant of MINERVA 2 (Hintzman, 1986, 1988) comes close but has some problems. Successful variants of a variety of composite and network models were not found (e.g., Ackley, Hinton, & Sejnowski, 1985; Anderson, 1972, 1973; Metcalfe Eich, 1982; Murdock, 1982; Pike, 1984). The results suggest constraints on the future development of such models.

Cues↗
PubMed1990

Loss of efficient import and thylakoid insertion due to N- and C-terminal deletions in the light-harvesting chlorophyll a/b binding protein.

C-terminally truncated precursors of wheat light-harvesting chlorophyll a/b binding protein (LHCP) were synthesized to investigate the origin of the two forms (about 25 kD and 26 kD) of the mature protein observed upon in vitro import into the chloroplast. Precursors p delta 13 and p delta 27, lacking 13 and 27 amino acids, respectively, were successfully imported, and both gave rise to two smaller forms proportional to the size of their deletions. These results demonstrate that there are two N-terminal sites that are cleaved during import of the LHCP precursor, undoubtedly contributing to the heterogeneity of LHCP found in vivo. Significantly, p delta 27 yielded only 50% of mature LHCP when compared with wild type. Although the products of p delta 27 import were localized to the thylakoids, in contrast to p delta 13 they were not correctly inserted into the membranes, indicating that residues essential for this step are missing. p delta 27 is distinguished from p delta 13 by lacking the carboxy end of a domain highly conserved between LHCP of photosystems II and I. Other specific precursor mutants with larger C-terminal deletions were not efficiently transported into the organelle in time course experiments, nor did they insert directly into the thylakoids using chloroplast lysates, in an assay independent of translocation across the envelope. In addition, the mutant p delta 18n, lacking the first 18 amino acids of mature LHCP, was only found bound to the chloroplast envelope. However, both p delta 18n and the mature protein, i.e., LHCP, synthesized in vitro without its 34-amino acid transit peptide inserted into the thylakoids in chloroplast lysates. The overall conformation of the mutant precursor polypeptides was probed using the chloroplast soluble processing enzyme in an organelle-free reaction optimized for the LHCP precursor and the more general protease trypsin. A tightly folded, protease-resistant conformation was not apparent to explain the loss of efficient import.

Amino Acid Sequence↗
PubMed1990

List-strength effect: I. Data and discussion.

Extra items added to a list cause memory for the other items to decrease (the list-length effect). In one of the present studies we show that strengthening (or weakening) some items on a list harms (helps) free recall of the remaining list items. This is termed the list-strength effect. However, in seven recognition studies the list-strength effect was either absent or negative. This held whether strengthening was accomplished by extra study time or extra repetitions. The seven studies used various means to control rehearsal strategies, thereby providing evidence against the possibility that the findings were due to redistribution of rehearsal or effort from stronger to weaker items within a list. Current models appear unable to predict these results. We suggest that different retrieval operations underlie recall and recognition, as in the SAM model of Gillund and Shiffrin (1984), which can be made to fit the results with certain relatively minor modifications.

Cues↗
PubMed1989

Mutations at the transit peptide-mature protein junction separate two cleavage events during chloroplast import of the chlorophyll a/b-binding protein.

We have shown previously that during in vitro import into chloroplasts, the precursor of the major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and approximately 26 kDa) which are inserted into the thylakoids. However, during incubation of the LHCP precursor with a chloroplast-soluble extract in an organelle-free processing reaction, the NH2 terminus is cleaved, yielding only a 25-kDa peptide. In the present study, mutations at the transit peptide-mature protein junction were introduced in the LHCP precursor to investigate the relationship between the two peptides and the determinants of proteolytic processing. Mutant p delta 3 lacks 3 amino acids including Met34 at the primary cleavage site thought to give rise to the 26-kDa peptide. It is still processed during import and in the organelle-free reaction yielding in both assays only a 25-kDa peptide. Mutant p + 4 has 4 amino acids inserted immediately after Met34 and a proline that disrupts the alpha-helix predicted by the Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120) to extend through this region. Although p + 4 is imported, it is inefficiently processed; both a 25- and 26-kDa peptide are found, but at least 60% of the imported precursor remains uncleaved. Less than 5% is processed in the organelle-free assay. Replacement of the predicted alpha-helix in the mutant p + 4 alpha restores processing upon import into the chloroplast, but this mutant, which also has a 4-amino acid insert, yields only a 26-kDa peptide. p + 4 alpha is not processed in the organelle-free reaction. These results provide evidence that the two forms of LHCP obtained during import are the result of independent processing at two cleavage sites: the first site at Met34, and a second approximately 10 amino acids downstream within what has been designated the NH2 terminus of the mature protein. Whereas p delta 3 has the first site removed but retains a functional second site, in p + 4 alpha only the first site, or one very near it, is accessible to the processing enzyme during import. The conditions of the organelle-free reaction are specific for processing at only the secondary site. We discuss the implications of these findings in terms of the heterogeneity of LHCP in vivo.

Amino Acid Sequence↗
PubMed1989

Rules and exemplars in categorization, identification, and recognition.

Subjects learned to classify perceptual stimuli varying along continuous, separable dimensions into rule-described categories. The categories were designed to contrast the predictions of a selective-attention exemplar model and a simple rule-based model formalizing an economy-of-description view. Converging evidence about categorization strategies was obtained by also collecting identification and recognition data and by manipulating strategies via instructions. In free-strategy conditions, the exemplar model generally provided an accurate quantitative account of identification, categorization, and recognition performance, and it allowed for the interrelationship of these paradigms within a unified framework. Analyses of individual subject data also provided some evidence for the use of rules, but in general, the rules seemed to have a great deal in common with exemplar storage processes. Classification and recognition performance for subjects given explicit instructions to use specific rules contrasted dramatically with performance in the free-strategy conditions and could not be predicted by the exemplar model.

Adult↗
PubMed1989

Properties of a Chloroplast Enzyme that Cleaves the Chlorophyll a/b Binding Protein Precursor : Optimization of an Organelle-Free Reaction.

The major light-harvesting chlorophyll a/b binding protein (LHCP) of higher plant chloroplasts is nuclear-encoded, synthesized as a precursor, and processed upon import. We have previously (GK Lamppa, M Abad [1987] J Cell Biol 105: 2641-2648) identified a soluble enzyme that cleaves the LHCP precursor (pLHCP). In this study, we describe the conditions for optimal recovery of the processing activity and provide evidence that the N terminus of pLHCP is indeed cleaved, removing the transit peptide. Two pLHCP deletions were made from a cloned pLHCP gene removing 13 and 21 amino acids, respectively, from the carboxy terminus of the protein. After organelle-free processing, the cleavage products showed a shift in mobility during SDS-PAGE proportional to the size of the precursor truncations, as predicted for N-terminal processing. Unexpectedly, a third truncated precursor lacking 91 residues of the C-terminus was not cleaved although the transit peptide domain was intact, suggesting that this deletion disrupted conformational features of the precursor necessary for processing. The pLHCP processing enzyme is inhibited by 2 millimolar EDTA and the metal chelator 1, 10 phenanthroline at 0.4 millimolar, while being inhibited by EGTA only at high concentrations and insensitive to iodoacetate. Optimal processing occurs at pH 8 to 9, and 26 degrees C. Gel filtration chromatography shows that the pLHCP processing enzyme has an apparent molecular weight of about 240,000. The identical column fractions that process pLHCP also convert the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase to its mature form.

Journal Article↗
PubMed1989

Clinical chemistry outside laboratories: report of a second survey.

The growth in the use of small portable analysers outside hospital laboratories has been a cause of concern to laboratory practitioners. This has been partly because of a perceived threat to laboratory careers and partly because of fears that the quality of testing would be poor, with a consequent danger of patients being placed at risk. The evidence in this paper indicates that the use of equipment outside laboratories is still increasing in many areas. Despite the fears which have been expressed, little attention is being given to operator training, service costs, safety and many other aspects; only rarely has responsibility for the quality of results been accepted.

Chemistry, Clinical↗
PubMed1988

Tibial nerve branching in the tarsal tunnel.

Tarsal tunnel syndrome is an infrequent and probably underdiagnosed clinical condition. Diagnosis and treatment depend on understanding the tibial branching pattern within the tarsal tunnel. A total of 68 foot dissections were performed. Bifurcation into the medial and lateral plantar nerves occurred within the tunnel in 93% and proximal in 7%. Proximal bifurcation may predispose to tarsal tunnel syndrome, and its infrequent occurrence correlates with the infrequent clinical diagnosis. Nine different calcaneal branching patterns were noted; they provide an anatomical explanation for heel sparing. An understanding of anatomic variations should aid in providing complete surgical release and in avoiding accidental heel denervations.

Ankle↗
PubMed1983

Actin polymerization and synthesis in cultured neurones.

The protein content of sympathetic neurones explanted from 10-11-day old chick embryos into culture medium containing nerve growth factor (NGF) increases steadily from about 100 to about 400 pg/cell in 7 days. Actin remains at close to 5% of the total protein during this period, but the proportion of unpolymerized actin falls. As measured by the inhibition of DNase I activity, rounded neurones without neurites contain 70 +/- 7% of their total actin in monomeric form, whereas cells in mature, neurite-bearing cultures contain 39 +/- 7%. When allowance is made for the increase in size of the neuronal cell bodies, the actin present in the neurites ('axons') alone is found to be almost entirely in filamentous form. Cultures exposed to radioactive leucine rapidly incorporate radioactivity into both sedimentable and non-sedimentable forms of actin. Actin-specific activities in the two fractions--estimated after isolation of the actin on small DNase I--Sepharose affinity columns--are similar after labelling for less than 1 h. Direct incorporation of newly-synthesized actin into filaments is suggested from these results. Pulse-chase experiments show that non-sedimentable protein in cultured sympathetic neurones turns over more rapidly than sedimentable protein. However, this is not true for actin, which shows a similar specific activity in sedimentable and non-sedimentable forms--even after 6 days of cold chase. This anomalous behaviour is simply explained by an exchange of actin molecules between filamentous and non-filamentous forms. Control experiments indicate that exchange does not occur to this degree during preparation of subcellular fractions. It is consequently attributed to exchange processes in the living cell.

Actins↗
PubMed1981

The construction of the red cell cytoskeleton.

The state of actin in the erythrocyte membrane cytoskeleton has been examined. The presence of ADP, rather than ATP, as the predominant nucleotide species reinforces the view that actin occurs in the polymerized form. Redeterminations of the amounts of the three cytoskeletal proteins, spectrin, actin and 4.1, present in the cell allow evaluation of some of the stoichiometric constraints on the construction of the cytoskeleton. The available evidence is compatible with a network consisting of spectrin tetramers as the structural members, attached at both ends to junctions consisting of 4.1 and short filaments (or 'protofilaments') of actin. Electron micrographs of isolated cytoskeletons support such a picture. The dynamic state of the actin has been studied, using the DNAase assay method. The results indicate that whereas native monomeric actin is present in the cell, only a small proportion of the subunits of the protofilament can enter into an equilibrium with this pool. A considerable proportion of the cytoskeletal actin is not liberated under conditions that dissociate F-actin and is evidently tightly associated with spectrin and 4.1. Attention is drawn to the possible consequences of ATP depletion on the state of the actin and thus of the cytoskeleton.

Actins↗
PubMed1979

A modified non-radioisotope method for measurement of platelet production time.

Platelet production time (PPT), based on the measurement of malondialdehyde (MDA) production prior to and after the intake of acetylsalicylic acid (ASA), was determined in 20 healthy subjects. High MDA levels were produced by stimulating platelet lipid peroxidation with arachidonic acid, resulting in a reliable, sensitive and accurate technique. PPT correlated well with platelet survival time measured by 51Cr autologous labelled platelets when both methods were used simultaneously in the same patients. This modified non-radioisotope method might serve as a useful aid in the diagnosis of platelet disorders, thromboembolic diseases associated with increased platelet consumption and for the evaluation of drugs affecting platelet function.

Aspirin↗