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J A Wise

Publications and source records attributed to J A Wise.

At least 19 recordsLinked to original sources
PubMed2006

Influence of beta-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity.

Muscle carnosine synthesis is limited by the availability of beta-alanine. Thirteen male subjects were supplemented with beta-alanine (CarnoSyn) for 4 wks, 8 of these for 10 wks. A biopsy of the vastus lateralis was obtained from 6 of the 8 at 0, 4 and 10 wks. Subjects undertook a cycle capacity test to determine total work done (TWD) at 110% (CCT(110%)) of their maximum power (Wmax). Twelve matched subjects received a placebo. Eleven of these completed the CCT(110%) at 0 and 4 wks, and 8, 10 wks. Muscle biopsies were obtained from 5 of the 8 and one additional subject. Muscle carnosine was significantly increased by +58.8% and +80.1% after 4 and 10 wks beta-alanine supplementation. Carnosine, initially 1.71 times higher in type IIa fibres, increased equally in both type I and IIa fibres. No increase was seen in control subjects. Taurine was unchanged by 10 wks of supplementation. 4 wks beta-alanine supplementation resulted in a significant increase in TWD (+13.0%); with a further +3.2% increase at 10 wks. TWD was unchanged at 4 and 10 wks in the control subjects. The increase in TWD with supplementation followed the increase in muscle carnosine.

Adult↗
PubMed2006

The absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis.

Beta-alanine in blood-plasma when administered as A) histidine dipeptides (equivalent to 40 mg . kg(-1) bwt of beta-alanine) in chicken broth, or B) 10, C) 20 and D) 40 mg . kg(-1) bwt beta-alanine (CarnoSyn, NAI, USA), peaked at 428 +/- SE 66, 47 +/- 13, 374 +/- 68 and 833 +/- 43 microM. Concentrations regained baseline at 2 h. Carnosine was not detected in plasma with A) although traces of this and anserine were found in urine. Loss of beta-alanine in urine with B) to D) was <5%. Plasma taurine was increased by beta-alanine ingestion but this did not result in any increased loss via urine. Pharmacodynamics were further investigated with 3 x B) per day given for 15 d. Dietary supplementation with I) 3.2 and II) 6.4 g . d(-1) beta-alanine (as multiple doses of 400 or 800 mg) or III) L-carnosine (isomolar to II) for 4 w resulted in significant increases in muscle carnosine estimated at 42.1, 64.2 and 65.8%.

Adult↗
PubMed2005

Perceptual and physiological responses to the visual complexity of fractal patterns.

Fractals have experienced considerable success in quantifying the complex structure exhibited by many natural patterns and have captured the imagination of scientists and artists alike. With ever widening appeal, they have been referred to both as "fingerprints of nature" and "the new aesthetics." Our research has shown that the drip patterns of the American abstract painter Jackson Pollock are fractal. In this paper, we consider the implications of this discovery. We first present an overview of our research from the past five years to establish a context for our current investigations of human response to fractals. We discuss results showing that fractal images generated by mathematical, natural and human processes possess a shared aesthetic quality based on visual complexity. In particular, participants in visual perception tests display a preference for fractals with mid-range fractal dimensions. We also present recent preliminary work based on skin conductance measurements that indicate that these mid-range fractals also affect the observer's physiological condition and discuss future directions based on these results.

Arousal↗
PubMed2002

Effects of an 8-week weight-loss program on cardiovascular disease risk factors and regional body composition.

OBJECTIVE: To determine the influence of weight loss on multiple cardiovascular disease (CVD) risk factors. DESIGN: Overweight women (n = 12; mean 44.2% fat) and men (n = 10; mean 30.7% fat) participated in an 8 week weight-loss program that included dietary, exercise, multi-vitamin/mineral supplementation, and behavior modification components. Measurement of total and regional body composition assessed using dual energy X-ray absorptiometry (DEXA), circumferences and blood sampling for total cholesterol, LDL cholesterol, HDL cholesterol, triacylglycerols, homocysteine, insulin and leptin were performed before and after the weight loss intervention. RESULTS: Subjects increased their physical activity and decreased their energy intake, resulting in a mean decrease in body mass of - 4.3 +/- 3.4 kg in women and -4.7 +/- 3.1 kg in men. Fat accounted for 88 and 58% of the decrease in body mass in men and women, respectively. Proportionally, men lost significantly more fat mass from the trunk region compared to women. Serum total and LDL cholesterol were significantly decreased in men (-11 and -14%, respectively) but not women (-3 and -3%, respectively) and there were no changes in HDL cholesterol and triacylglycerols. Serum leptin was significantly decreased (-36%) and highly correlated to fat mass (r= 0.839). There were no changes in serum insulin and plasma homocysteine. CONCLUSIONS: These data indicate that short-term weight loss resulting from reducing percentage energy from fat, increasing physical activity and vitamin/mineral supplements including folic acid has a favorable effect on regional body composition and total and LDL cholesterol with minimal effects on HDL cholesterol, triacylglycerols, homocysteine and insulin and the effects are greater in men compared to women. Supplementation with folic acid or emphasis on folic acid-rich foods may be an important component of a weight loss program to prevent increases in homocysteine.

Absorptiometry, Photon↗
PubMed2001

Activation of a cryptic 5' splice site by U1 snRNA.

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.

Base Pairing↗
PubMed2000

Evidence for splice site pairing via intron definition in Schizosaccharomyces pombe.

Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5' junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5' junctions can compete effectively with the standard 5' splice site if they are closer to the 3' splicing signals. Taken together, our data suggest that pairing of splice sites in S. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Systematic mutagenesis of the fission yeast Srp54 protein.

The signal recognition particle (SRP) is a ribonucleoprotein required for targeting a subset of nascent pre-secretory proteins to the endoplasmic reticulum membrane. Of the six SRP polypeptides, the most highly conserved is Srp54p, a modular protein consisting of an amino-terminal (N) domain of unknown function, a central GTPase (G) domain, and a carboxyl-terminal (M) domain implicated in the recognition of both signal sequences and SRP RNA. To identify regions of Srp54p that interact with other SRP subunits or regulatory proteins, we carried out systematic mutagenesis of the fission yeast homolog, principally using a "clustered charged-to-alanine" strategy. Of the 35 alleles examined, 13 are unable to support growth, two confer cold-sensitivity, five confer heat-sensitivity, and 15 produce no discernible phenotype. The lethal and conditional mutations map throughout the protein to several conserved regions, confirming that these motifs play critical roles in Srp54p function. The effects of the amino-acid substitutions are analyzed with reference to the recently determined tertiary structures of the N/G domain and the intact protein from a thermophilic bacterium.

Amino Acid Sequence↗
PubMed1999

Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: differential sensitivity of introns to mutational inactivation.

The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro. To expand our understanding of how this protein functions in vivo, we have created a null allele of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and employed it in a variety of genetic complementation assays. First, analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations. Second, despite extensive structural conservation, we find that metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59. Third, because the activity of U2AF65 in vitro involves binding to the 3' polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575). Our data indicate that all four introns tested, including two that lack extensive runs of pyrimidines between the branchpoint and 3' splice site, show splicing defects upon shifting to the nonpermissive condition. In all cases, splicing is blocked prior to the first transesterification reaction in the mutants, consistent with the role inferred for human U2AF65 based on in vitro experiments.

Amino Acid Sequence↗
PubMed1997

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.

Alleles↗
PubMed1996

Molecular analysis of a novel schizosaccharomyces pombe gene containing two RNP consensus-sequence RNA-binding domains.

Proteins containing RNP consensus-sequence RNA-binding domains (CS-RBDs) play diverse roles in many aspects of RNA metabolism. Using a PCR strategy, we cloned portions of six new Schizosaccharomyces pombe genes encoding RBD proteins, including a putative homolog of the mammalian splicing factor SAP49. The genomic locus corresponding to a second PCR product, designated rnp24a, was cloned and characterized in detail. Sequence analysis revealed that the Rnp24 protein is highly charged and contains a second RBD with an unusually long Loop-3 sequence. Strains containing a disrupted copy of the rnp24 gene display neither loss of viability nor any discernible growth defects under a variety of conditions, suggesting that the function of Rnp24p overlaps with that of another fission yeast protein. Although database searches did not identify proteins that share extensive amino-acid identity with Rnp24p, phylogenetic analysis suggests that its closest relatives are metazoan hnRNP proteins. The lack of an observable phenotype in S. pombe cells lacking Rnp24p is consistent with this classification, since hnRNP proteins in higher cells include several distinct subfamilies with similar sequences and RNA-binding specificities.

Amino Acid Sequence↗
PubMed1996

Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo.

The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated. To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe. Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast.

Base Sequence↗
PubMed1994

Genetic and biochemical analysis of the fission yeast ribonucleoprotein particle containing a homolog of Srp54p.

Mammalian signal recognition particle (SRP), a complex of six polypeptides and one 7SL RNA molecule, is required for targeting nascent presecretory proteins to the endoplasmic reticulum (ER). Earlier work identified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical properties to mammalian SRP. The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further characterization of the subunit structure, subcellular distribution, and assembly of fission yeast SRP. S. pombe SRP RNA and Srp54p co-sediment on a sucrose velocity gradient and coimmunoprecipitate, indicating that they reside in the same complex. In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E. coli SRP RNA, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs. Moreover, the association of S. pombe Srp54p with SRP RNA in vivo is disrupted by conditional mutations not only in domain IV, which contains its binding site, but in domains I and III, suggesting that the particle may assemble cooperatively. The growth defects conferred by mutations throughout SRP RNA can be suppressed by overexpression of Srp54p, and the degree to which growth is restored correlates inversely with the severity of the reduction in protein binding. Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cell fractionation. Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from [35S]-labeled fission yeast cells suggests that five additional polypeptides are complexed with Srp54p; each of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.

Base Sequence↗
PubMed1994

Molecular evolution of SRP cycle components: functional implications.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane. We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP. Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway. The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution. The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria. Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein. Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif. Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E. coli EF-Tu. The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha. Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases. The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin.

Amino Acid Sequence↗
PubMed1994

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast.

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-alpha-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A-Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-alpha-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.

Acid Phosphatase↗
PubMed1994

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.

Amino Acid Sequence↗
PubMed1993

Functional interchangeability of the structurally similar tetranucleotide loops GAAA and UUCG in fission yeast signal recognition particle RNA.

Signal recognition particle (SRP) RNA exhibits significant primary sequence conservation only in domain IV, a bulged hairpin capped by a GNRA (N, any nucleotide; R, purine) tetranucleotide loop except in plant homologs. Tetraloops conforming to this sequence or to the consensus UNCG enhance the stability of synthetic RNA hairpins and have strikingly similar three-dimensional structures. To determine the biological relevance of this similarity, as well as to assess the relative contributions of sequence and structure to the function of the domain IV tetraloop, we replaced the GAAA sequence in fission yeast SRP RNA with UUCG. Haploid strains harboring this substitution are viable, providing experimental evidence for the functional equivalence of the two tetraloops. We next tested the two sequences found in plant SRP RNAs at this location for function in the context of the Schizosaccharomyces pombe RNA. While substitution of CUUC does not allow growth, a viable strain results from replacing GAAA with UUUC. Although the viable tetraloop substitution mutants exhibit wild-type growth under normal conditions, all three express conditional defects. To determine whether this might be a consequence of structural perturbations, we performed enzymatic probing. The results indicate that RNAs containing tetraloop substitutions exhibit subtle differences from the wild type not only in the tetraloop itself, but also in the 3-base pair adjoining stem. To directly assess the importance of the latter structure, we disrupted it partially or completely and made the compensatory mutations to restore the helix. Surprisingly, mutant RNAs with as little as one Watson-Crick base pair can support growth.

Alleles↗