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S E Clark

Publications and source records attributed to S E Clark.

At least 19 recordsLinked to original sources
PubMed2004

Effect of adding and removing N-glycosylation recognition sites on the thermostability of barley alpha-glucosidase.

The thermostability of alpha-glucosidase is important because the conversion of starch to fermentable sugars during the industrial production of beer and fuel ethanol typically occurs at relatively high temperatures (60-75 degrees C). Barley (Hordeum vulgare) alpha-glucosidase is unstable at these elevated temperatures; however, the alpha-glucosidase from sugar beet (Beta vulgaris) is stable at these temperatures. An alignment of the deduced amino acid sequences of barley and sugar beet alpha-glucosidases revealed considerable differences in the number and position of N-glycosylation recognition sites (NGRS). Other researchers have shown that additions or removals of NGRS resulted in either the stabilization or destabilization of the enzymes at elevated temperatures. NGRS present in the barley sequence and absent in the sugar beet sequence were removed via site-directed mutagenesis from the barley protein. Recognition sites absent in the barley sequence and present in the sugar beet sequence were added via mutagenesis into the barley alpha-glucosidase. Two mutations significantly increased thermostability, one mutation significantly decreased thermostability and five mutations had little effect on alpha-glucosidase thermostability.

Alanine↗
PubMed2002

The effect of proline insertions on the thermostability of a barley alpha-glucosidase.

The thermal stability of alpha-glucosidase is important because the conversion of starch to fermentable sugars during industrial production of ethanol (e.g. brewing, fuel ethanol production) typically takes place at temperatures of 65-73 degrees C. In this study we investigate the thermostability of alpha-glucosidases from four plant species, compare their deduced amino acid sequences, and test the effect of substituting a proline for the residue present in the wild-type enzyme on the thermostability of alpha-glucosidase. The alpha-glucosidase from barley (Hordeum vulgare) was significantly less thermostable than the other three alpha-glucosidases. A comparison of the published deduced amino acid sequences of these four alpha-glucosidases revealed conserved proline residues in the three most thermostable alpha-glucosidases that were not found in the barley enzyme. Site-directed mutagenesis was done on recombinant barley alpha-glucosidase to create proteins with prolines at these conserved positions. The thermostability (T(50)) of one of these mutant enzymes, T340P, was 10 degrees C higher than the non-mutated enzyme.

Amino Acid Sequence↗
PubMed2001

Postnatal alterations of GABA receptor profiles in the rat superior colliculus.

Midbrain sections taken from Sprague-Dawley rats of varying ages within the first four postnatal weeks were used to determine, immunocytochemically, putative changes of GABA(A) receptor beta2/3 subunits, GABA(B) receptor (R1a and R1b splice variants), and GABA(C) receptor rho1 subunit expression and distribution in the superficial, visual layers of the superior colliculus. Immunoreactivity for the GABA(A) receptor beta2/3 subunits was found in the superficial grey layer from birth. The labelling changed with age, with an overall continuous reduction in the number of cells labelled and a significant increase in the labelling intensity distribution (neuropil vs soma). Further analysis revealed an initial increase in the labelling intensity between postnatal days 0 and 7 in parallel with an overall reduction of labelled neurones. This was followed by a significant decrease in labelling intensity distribution between postnatal days 7 and 16, and a subsequent increase in intensity between postnatal days 16 and 28. The labelling profiles for GABA(B) receptors (R1a and R1b splice variants) and GABA(C) receptors (rho1 subunit) showed similar patterns. Both receptors could be found in the superficial layers of the superior colliculus from birth, and the intensity and distribution of labelling remained constant during the first postnatal month. However, the cell body count showed a significant decrease between postnatal days 7 and 16. These changes may be related to the time-point of eye opening, which occurred approximately two weeks after birth. For all three receptor types, the cell body count remained constant after postnatal day 16. By four weeks of age, there was no significant difference between the cell numbers obtained for the different receptors. Both GABA itself and neurofilament labelling were also obtained in the superficial superior colliculus at birth. Neurofilament, although found at birth, showed very little ordered arrangement until 16days after birth. When slices were double labelled for GABA(C) receptors and neurofilament, some overlap was observed. Double labelling for the presynaptic protein synaptophysin and GABA(C) receptors showed proximity in some places, indicative of a partly synaptic location of GABA(C) receptors. When GABA(C) and GABA(A) receptors were labelled simultaneously, some but not all neurones showed immunoreactivity for both receptor types. In conclusion, all three GABA receptor types were found to be present in the superior colliculus from birth, and all show some form of postnatal modification, with GABA(A) receptors demonstrating the most dramatic changes. However, GABA(B) and GABA(C) receptors are modified significantly around the onset of input-specific activity. Together, this points towards a contribution of the GABAergic system to processes of postnatal maturation in the superficial superior colliculus.

Aging↗
PubMed2001

Meristems: start your signaling.

All of the cells of the shoot apex are derived from a small number of stem cells in the center of the shoot meristem. Hence, cell lineage plays no role in establishing pattern formation or cell fate at the shoot apex. Growing evidence has implicated a number of cell signaling pathways in regulating shoot meristem development and organ formation. These signaling pathways include receptor-mediated signaling, protein movement via plasmodesmata, and potential feedback loops.

Arabidopsis↗
PubMed2001

Selecting lineup foils in eyewitness identification experiments: experimental control and real-world simulation.

Experimental research on eyewitness identification follows a standard principle of experimental design. Perpetrator-present and perpetrator-absent lineups are constructed with the same foils, so that the two conditions are identical except for the presence or absence ofthe trueperpetrator ofthe crime. However, this aspect of the design simulates conditions that do not correspond to those of real criminal investigations. Specifically, these conditions can create perp-absent lineups in which the foils are selected based on their similarity to an unknown person--the real perpetrator. Analysis of the similarity relations predicts that when foils for perp-absent lineups are selected based on their match to the perpetrator the false identification rate will be lower than if the foils are selected based on their match to the innocent suspect. This prediction was confirmed in an experiment that compared these two perp-absent lineup conditions. These results suggest that false identification rates in previous experiments would have been higher if the foils had been selected based on their match to the innocent suspect, rather than the absent perpetrator.

Adult↗
PubMed2001

Cell signalling at the shoot meristem.

The regulation of cell differentiation at meristems is crucial to developmental patterning in plants. Rapid progress has been made in identifying the genes that regulate differentiation and the receptor-mediated signalling events that have a key role in this process. In particular, we are now learning how the CLAVATA receptor kinase signalling pathway promotes stem cell differentiation in balance with the initiation of stem cells by the transcription factor WUSCHEL.

Arabidopsis↗
PubMed2001

REVOLUTA regulates meristem initiation at lateral positions.

While the shoot apical meristem (SAM) is indirectly responsible for the initiation of all above-ground postembryonic organs, in most plants the vast majority of these organs are directly initiated by lateral meristems. In Arabidopsis thaliana, the lateral meristems include flower meristems (FMs), which form on the flanks of the SAM, and lateral shoot meristems (LSMs), which develop in leaf axils. While significant progress has been made on the molecular genetic basis of SAM initiation during embryo development, relatively little is known about the initiation of meristems at lateral positions. Here we have characterized the phenotypic consequences and genetic interactions of mutations in the REVOLUTA (REV) gene, with an emphasis on the role of REV in lateral meristem initiation. Our observations indicate that REV is required for initiation of both LSMs and FMs, and likely acts in the same pathway as, and upstream of, known meristem regulators. We identified the REV gene and found it encodes a predicted homeodomain/leucine zipper transcription factor that also contains a START sterol-lipid binding domain. REV is the same as the IFL gene. REV was expressed at the earliest stages of LSM and FM formation. Within the inflorescence shoot meristem, REV expression appeared to predict 3--5 incipient flower primordia on the flanks of the SAM, and REV expression at stage 1 and stage 2 matched that of WUS and STM, respectively. We propose that REV acts at lateral positions to activate the expression of known meristem regulators.

Arabidopsis↗
PubMed2000

CLAVATA3, a multimeric ligand for the CLAVATA1 receptor-kinase.

The CLAVATA1 (CLV1) and CLAVATA3 (CLV3) proteins form a potential receptor and ligand pair that regulates the balance between cell proliferation and differentiation at the shoot meristem of Arabidopsis. CLV1 encodes a receptor-kinase, and CLV3 encodes a predicted small, secreted polypeptide. We demonstrate that the CLV3 and CLV1 proteins coimmunoprecipitate in vivo, that yeast cells expressing CLV1 and CLV2 bind to CLV3 from plant extracts, and that binding requires CLV1 kinase activity. CLV3 only associates with the presumed active CLV1 protein complex in vivo. More than 75% of CLV3 in cauliflower extracts is bound with CLV1, consistent with hypotheses of ligand sequestration. Soluble CLV3 was found in an approximately 25-kilodalton multimeric complex.

Alleles↗
PubMed2000

Overexpression, purification, and characterization of a barley alpha-glucosidase secreted by Pichia pastoris.

alpha-Glucosidases (EC 3.2.1.20) are recognized as important in starch degradation during cereal seed germination. A barley (Hordeum vulgare) alpha-glucosidase expressed in Pichia pastoris was cultured in flasks; however, the yield was low necessitating the use of multiple batches. Problems arose because of significant variation between batches. We solved these problems by switching to a fermentation system producing a sufficient quantity of a uniform sample. Here we present the expression and purification of a recombinant alpha-glucosidase grown under fermentation conditions. We also present the results of experiments to characterize the thermostability, pH optimum, and substrate specificity of the recombinant enzyme. The optimal pH for the hydrolysis of maltose by recombinant alpha-glucosidase is between 3.5 and 4.5. The thermostability of recombinant alpha-glucosidase was determined at pH 4, where activity is optimal, and at pH 5 and 6, which better mimic the conditions used to convert barley starch to fermentable sugars during industrial processing. The results indicate the enzyme is most thermolabile at pH 4. However, the enzyme is protected from heat inactivation at pH 4 by high concentrations of sucrose. The purified enzyme hydrolyzed maltose three times more rapidly than nigerose and 20 times more rapidly than trehalose and isomaltose. Concentrations of maltose greater than 20 mM inhibited maltose hydrolysis. This is the first report of substrate inhibition for any alpha-glucosidase. The results indicate that the only significant difference between the recombinant enzyme and the previously characterized barley isoforms was the V(max) for maltose hydrolysis.

Disaccharides↗
PubMed2000

Does the Conners' Continuous Performance Test aid in ADHD diagnosis?

The performance of clinic-referred children aged 6-11 (N = 100) was examined using the Conners' Continuous Performance Test (CPT) and measures of auditory attention (Auditory Continuous Performance Test; ACPT), phonological awareness, visual processing speed, and visual-motor competence. The Conners' CPT overall index was unrelated to measures of visual processing speed or visual-motor competence. Although the Conners' CPT converged with the ACPT, the latter demonstrated age and order effects. Significant variance in Conners' CPT parameters was predicted by phonological awareness measures, suggesting that Reading Disordered (RD) children could be "false positives" on the Conners' CPT. The Conners' CPT overall index, phonological awareness, and visual-motor measures were submitted to a 2 x 2 MANCOVA (ADHD vs. RD, covarying for age and socioeconomic status): a main effect for RD status was found. Children with ADHD did not have higher Conners' CPT scores than did clinical controls; however, children with Reading Disorders did. Phonological measures distinguished RD children from ADHD children and other clinical controls. ADHD children who failed the Conners' CPT were rated by teachers as more hyperactive. Despite the strengths of the Conners' CPT, its utility for differential diagnosis of ADHD is questioned.

Attention↗
PubMed2000

Selecting foils for identification lineups: matching suspects or descriptions?

Two experiments directly compare two methods of selecting foils for identification lineups. The suspect-matched method selects foils based on their match to the suspect, whereas the description-matched method selects foils based on their match to the witness's description of the perpetrator. Theoretical analyses and previous results predict an advantage for description-matched lineups both in terms of correctly identifying the perpetrator and minimizing false identification of innocent suspects. The advantage for description-matched lineups should be particularly pronounced if the foils selected in suspect-matched lineups are too similar to the suspect. In Experiment 1, the lineups were created by trained police officers, and in Experiment 2, the lineups were constructed by undergraduate college students. The results of both experiments showed higher suspect-to-foil similarity for suspect-matched lineups than for description-matched lineups. However, neither experiment showed a difference in correct or false identification rates. Both experiments did, however, show that there may be an advantage for suspect-matched lineups in terms of no-pick and rejection responses. From these results, the endorsement of one method over the other seems premature.

Adult↗
PubMed2000

Group collaboration in recognition memory.

Group collaboration was examined in item and associative recognition. The present study distinguishes between group effects versus collaborative processes and defines the latter as interactive information exchange among group members. By that definition, many group effects do not involve collaboration. For example, group performance can exceed individual performance by pooling the increased resources of the group. Specifically, a group advantage can be obtained by deferring to a majority vote or to the group's best member. For both item and associative recognition, a group advantage was obtained that could not be accounted for by resource pooling. Collaborative facilitation was shown reliably in recognizing targets but not for rejecting distractors.

Adolescent↗
PubMed2000

POLTERGEIST functions to regulate meristem development downstream of the CLAVATA loci.

Mutations at the CLAVATA loci (CLV1, CLV2 and CLV3) result in the accumulation of undifferentiated cells at the shoot and floral meristems. We have isolated three mutant alleles of a novel locus, POLTERGEIST (POL), as suppressors of clv1, clv2 and clv3 phenotypes. All pol mutants were nearly indistinguishable from wild-type plants; however, pol mutations provided recessive, partial suppression of meristem defects in strong clv1 and clv3 mutants, and nearly complete suppression of weak clv1 mutants. pol mutations partially suppressed clv2 floral and pedicel defects in a dominant fashion, and almost completely suppressed clv2 phenotypes in a recessive manner. These observations, along with dominant interactions observed between the pol and wuschel (wus) mutations, indicate that POL functions as a critical regulator of meristem development downstream of the CLV loci and redundantly with WUS. Consistent with this, pol mutations do not suppress clv3 phenotypes by altering CLV1 receptor activation.

Arabidopsis↗
PubMed1999

Genetic and physical characterization of a region of Arabidopsis chromosome 1 containing the CLAVATA1 gene.

With the advance of Arabidopsis as a model system for understanding plant genetics, development and biochemistry, a detailed description of the genome is necessary. As such, focused projects are underway to map and sequence the Arabidopsis nuclear genome. We have characterized a region of chromosome 1, surrounding the CLAVATA1 (CLV1) locus. Three (RFLP) clones were mapped relative to clv1-1, and were used to construct an ca. 700 kb yeast artificial chromosome (YAC) contig. Three cosmids spanning the CLV1 locus were analyzed and ca. 24 kb of genomic DNA was sequenced, including a continuous stretch of 18 kb. In addition to generating clones in this region of chromosome 1, we have analyzed the size, spacing and organization of several contiguous genes.

Arabidopsis↗
PubMed1999

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.

Amino Acid Sequence↗
PubMed1999

The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein.

The CLAVATA1 (CLV1) and CLAVATA3 (CLV3) genes are required to maintain the balance between cell proliferation and organ formation at the Arabidopsis shoot and flower meristems. CLV1 encodes a receptor-like protein kinase. We have found that CLV1 is present in two protein complexes in vivo. One is approximately 185 kD, and the other is approximately 450 kD. In each complex, CLV1 is part of a disulfide-linked multimer of approximately 185 kD. The 450-kD complex contains the protein phosphatase KAPP, which is a negative regulator of CLV1 signaling, and a Rho GTPase-related protein. In clv1 and clv3 mutants, CLV1 is found primarily in the 185-kD complex. We propose that CLV1 is present as an inactive disulfide-linked heterodimer and that CLV3 functions to promote the assembly of the active 450-kD complex, which then relays signal transduction through a Rho GTPase.

Arabidopsis Proteins↗
PubMed1998

CLAVATA2, a regulator of meristem and organ development in Arabidopsis.

Mutations at the CLAVATA2 (CLV2) locus of Arabidopsis result in enlarged shoot and flower meristems, as well as alterations in the development of the gynoecia, flower pedicels, and stamens. The shoot and flower meristem phenotypes of clv2 mutants are similar to weak clv1 and clv3 mutants. We present genetic analysis that CLV2 may function in the same pathway as CLV1 and CLV3 in the regulation of meristem development, but function separately in the regulation of organ development. We also present evidence that clv2 phenotypes are altered when the mutants are grown under short-day light conditions. These alterations include flower-to-shoot transformations, as well as a nearly complete suppression of the flower phenotypes, indicating that the requirement for CLV2 changes in response to different physiological conditions. The stm-1 mutation dominantly suppresses clv2, and clv2 mutations suppress the strong stm-1 allele, but not the weak stm-2 allele.

Alleles↗