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Biomedical subjects

A M Smith

Publications and source records attributed to A M Smith.

At least 145 records · Page 8Linked to original sources
PubMed1998

A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and phytoglycogen.

In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

Amylopectin↗
PubMed1998

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos.

Mutations at the rug5 (rugosus5) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B2 and B3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B2 and B3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed.

Amylopectin↗
PubMed1998

Alterations in PBP 1A essential-for high-level penicillin resistance in Streptococcus pneumoniae.

High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.

Bacterial Proteins↗
PubMed1998

Rapid detection of penicillin-resistant Streptococcus pneumoniae in cerebrospinal fluid by a seminested-PCR strategy.

A seminested-PCR assay, based on the amplification of the pneumococcal penicillin-binding protein 2B gene (pbp2B), was developed for the detection of penicillin-resistant and -susceptible pneumococci in cerebrospinal fluid (CSF) specimens. Species-specific primers (P5 and P6) which amplified a 682-bp conserved region of the transpeptidase-encoding region of the pbp2B gene were used. Four "resistance" primers were designed to bind to altered areas of the pbp2B gene identified in penicillin-resistant South African wild-type strains. Together with the downstream primer P6, the upstream resistance primers amplified fragments which were used to detect the presence of penicillin resistance. This system identified all 35 of the S. pneumoniae isolates evaluated, including strains of 11 different serotypes and a range of penicillin-resistant and -susceptible strains. The specificity of the assay was demonstrated by its inability to amplify DNA from other bacterial species which commonly cause meningitis. It was possible to detect pneumococcal DNA from culture-negative CSF inoculated with 2.5 pg of purified DNA or 18 CFU. Analysis of 285 CSF specimens showed that PCR detected the pneumococcus in 18 samples positive by culture, including the identification of four penicillin-resistant isolates. The positive predictive value and the negative predictive value of the assay were each 100%.

Aminoacyltransferases↗
PubMed1998

Sir proteins, Rif proteins, and Cdc13p bind Saccharomyces telomeres in vivo.

Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.

Binding Sites↗
PubMed1998

Does additional lubrication affect condom slippage and breakage?

The risk of condom slippage (1.94%) and breakage (0.89%) among 3607 condoms was analysed with respect to the use of additional lubricant. Whether or not lubricant was used, the site at which it was applied and the type of lubricant used were all found to vary significantly with the type of sexual act(s) for which the condoms were used. Little evidence was found for differing effects of type of additional lubricant (water-based, saliva or other) or site of lubricant use (on penis/inside condom, on condom, in vagina/anus). The use of lubricant more than doubles the risk of slippage for vaginal sex. While anal sex is associated with much higher risks of slippage the use of lubricant for this practice actually reduces the risk of slippage to that similar for vaginal sex where lubricant is used. No significant effect of additional lubricant on condom breakage was observed. It is recommended that education messages concerning the use of additional lubricant may need to change to take into account the varied nature of lubricant use practices and the differential effects of lubricant with respect to sexual practices. Specifically, if the use of additional lubricant has little or no impact on condom breakage but increases condom slippage then encouraging its use may be counterproductive if condom users consider slippage to be a reason not to use condoms.

Adult↗
PubMed1998

Does penis size influence condom slippage and breakage?

This study examined the effect of penis dimensions on the probability of complete condom slippage and condom breakage in actual use. Men were recruited through advertising, used the condoms supplied and completed a diary sheet for each condom used. A total of 3658 condoms were used by 184 men of which 1.34% broke and 2.05% slipped off. No significant effect was demonstrated for penile dimensions on the probability of complete condom slippage. However, condom breakage was strongly associated with penile circumference. These findings suggest that condom manufacturers may need to increase the range of condom sizes available, or some aspects of their performance, in order to ensure that condoms meet the needs of all men without unduly exposing them to risk.

Condoms↗
PubMed1998

AIDS is ... reflections on the Australian research response to the HIV and AIDS epidemics.

Drawing on institutional history, biography, and interviews with key informants from the range of academic disciplines that have contributed to the Australian response to the HIV and AIDS epidemics, this article provides critical insights into the factors that have shaped the Australian research response. The author demonstrates conflicts between disciplines that are rooted in epistemological differences. While the conflicts and tensions are often expressed in terms of access to resources, the disputes are grounded in the relative power of academic disciplines, most particularly the preeminence of biomedicine, and are related to the weight attributed to the various knowledge claims of those disciplines.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Revisiting sexual styles: patterns of adolescents' self-perceptions.

Recently, Buzwell and Rosenthal proposed that sexual styles were a useful way of understanding young people's constructions of a sexual identity. In this work we replicated with a sample of 522 15- to 16-yr.-olds in broad terms, the five styles identified by Buzwell and Rosenthal.

Adolescent↗
PubMed1998

Psychologic, situational, and physiologic variables and on-ice performance of youth hockey goalkeepers.

OBJECTIVE: To determine the relationship between psychologic, situational, and physiologic variables and on-ice performance of youth hockey goalkeepers. DESIGN: This study was structured to identify relationships and predictors of goalie performance. MATERIAL AND METHODS: Because athletes playing solo positions in team sports have not been analyzed in depth in terms of precompetition anxiety and because goalkeeper performance is critical to game outcome, we undertook a study of 43 goalies at a hockey camp. These goalies completed psychometric inventories to assess trait and state anxiety, confidence, life stress, and social support. Holter monitors measured heart rate while the goalies rotated through on-ice stations. Goalies were videotaped at the puck-shooting machine station, and performance (percent saves) was calculated. RESULTS: Trait (somatic) anxiety and positive mood state (ability to share) had different but significant relationships with on-ice performance. Heart rates ranged from 88 to 208 beats/min at the on-ice stations. Mean heart rate for older goalies (14 to 18 years of age) was 164 beats/min at the puck-shooting machine and 176 beats/min at other stations such as the slap-shot station. CONCLUSION: Older goalies performed well at a high level of arousal. Better performing goalies were more experienced, had faster heart rates "in the net," and had lower scores on all measures of anxiety.

Adolescent↗
PubMed1998

Functional magnetic resonance imaging: the basics of blood-oxygen-level dependent (BOLD) imaging.

There are 2 principal techniques of functional MRI (fMRI): the blood-oxygen-level dependent (BOLD) technique, which is the favoured method because no intravenous contrast medium is required, and the dynamic or exogenous technique. The BOLD technique takes advantage of the fact that the change from diamagnetic oxyhemoglobin to paramagnetic deoxyhemoglobin that takes place with brain activation results in decreased signal intensity on MRI. Commercially available scanners can be used to conduct single-slice BOLD fMRI experiments, but echo-planar hardware is needed for multislice wholebrain experiments. Sequence choices in BOLD fMRI include spin-echo and gradient-echo sequences, to which rapid acquisition with relaxation enhancement and echoplanar techniques may be applied. Optimal imaging parameters (echo time, slice thickness, field of view and flip angle) are important in maximizing signal-to-noise ratios. Various statistical techniques and software programs have been developed to interpret the large amounts of data gathered from BOLD fMRI experiments, which presents one of the biggest challenges in performing this technique with clinical MR units. Controversy exists regarding the effects of draining veins on cortical mapping, of inflow of blood into the imaging slice or volume, and of motion artifact. BOLD fMRI has demonstrated good correlation with positron emission tomography, magneto-encephalography and electrocorticographic recordings of motor responses. It has been used to study cortical activity of visual, motor, auditory and speech tasks as well as brain centres for smell, motor imagery, complex motion and memory. As such, it holds promise for the study of brain function, but must be subjected to larger studies comparing it with the gold standard of electrocorticographic mapping.

Arousal↗
PubMed1997

Parallin, a cerebellar granule cell protein the expression of which is developmentally regulated by Purkinje cells: evidence from mutant mice.

In this paper we report on monoclonal antibody 3H6 with unique specificities for development of the cerebellum. Immunohistochemical studies on normal and mutant mice suggest that it is primarily located in or on granule cell parallel fibers in the cerebellum. The only other region showing immunoreactivity is a small region of the hippocampus. The antigen is detected immunohistochemically as early as postnatal day 11 in the molecular layer of the cerebellum. In adult wild-type mice parallin expression is seen in the molecular layer and to a lesser degree in the internal granular layer. In the cerebella of two neurological granule cell-deficient mutants, weaver (wv) and staggerer (sg), parallin is not detected. However, in two Purkinje cell-deficient mutants, Purkinje cell degeneration (pcd) and nervous (nr), a more complex and interesting pattern is observed. These two mutants do have granule cells and parallel fibers and 3H6 immunoreactivity is observed. However, in both of these Purkinje cell-deficient mutants the 3H6 immunoreactivity is drastically reduced in regions where Purkinje cells have degenerated. Furthermore, in nr mutants, the antigen appears to be concentrated in regions of the parallel fiber that are in close proximity to Purkinje cells, suggesting its possible association with synapses. Taken together these results suggest that parallin is a marker of granule cells and their parallel fibers, its onset correlates with the formation of granule cell synapses on developing Purkinje cells, and it requires Purkinje cells for the maintenance of expression.

Animals↗
PubMed1997

Identification of serine/threonine protein kinases secreted by Trichinella spiralis infective larvae.

Serine/threonine protein kinase activity was identified in excretory/secretory (ES) products of Trichinella spiralis infective larvae, via phosphorylation of exogenous and endogenous substrates. Protein kinase activity was identified as an authentic secretory product via blockade of release into culture medium by brefeldin A. Enzyme activity was reductant-dependent, and the relative resistance to a panel of inhibitors suggested that it could not be readily assigned to any of the major documented subfamilies of serine/threonine protein kinases. There was no evidence for protein tyrosine kinase activity in ES products. The major phosphorylated proteins in this compartment resolved at 50 and 55 kDa by SDS-PAGE, and are therefore designated pp50/55. These proteins contained mainly phosphoserine, and appear to represent differentially glycosylated variants of a 35 kDa polypeptide, modified via the addition of three and four N-linked oligosaccharides, respectively. An autophosphorylation assay following separation by SDS-PAGE identified two protein kinases of 70 and 135 kDa in ES products.

Animals↗
PubMed1997

Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2.

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of beta-sheets and random coil. Based on long range distance constraints, a number of beta-strands were aligned into two three-stranded, anti-parallel beta-sheets.

Allergens↗
PubMed1997

Quantitation of regional cerebral blood flow increases during motor activation: a steady-state arterial spin tagging study.

Steady-state arterial spin tagging MRI approaches were used to quantitate regional cerebral blood flow increases during finger tapping tasks in seven normal subjects. Statistically significant increases in cerebral blood flow were observed in the contralateral primary sensorimotor cortex in all seven subjects and in the supplementary motor area in five subjects. The intrinsic spatial resolution of the cerebral blood flow images was approximately 4 mm. If no spatial filtering was applied, the average increase in cerebral blood flow in the activated primary sensorimotor cortex was 60 +/- 10 cc/100 g/min (91 +/- 32%). If the images were filtered to a spatial resolution of 15 mm, the average increase in cerebral blood flow in the activated primary sensorimotor cortex was 23 +/- 7 cc/100 g/min (42 +/- 15%), in agreement with previously reported 133Xe and PET results.

Adult↗