Biomedical subjects

R L Wright

Publications and source records attributed to R L Wright.

At least 19 recordsLinked to original sources

PubMed2000

Mutational analysis of the karmellae-inducing signal in Hmg1p, a yeast HMG-CoA reductase isozyme.

In response to elevated levels of HMG-CoA reductase, an integral endoplasmic reticulum (ER) membrane protein, cells assemble novel ER arrays. These membranes provide useful models for exploration of ER structure and function, as well as general features of membrane biogenesis and turnover. Yeast express two functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of which induces morphologically different ER arrays. Hmg1p induces stacks of paired nuclear-associated membranes called karmellae. In contrast, Hmg2p induces peripheral ER membrane arrays and short nuclear-associated membrane stacks. In spite of their ability to induce different cellular responses, both Hmg1p and Hmg2p have similar structures, including a polytopic membrane domain containing eight predicted transmembrane helices. By examining a series of recombinant HMG-CoA reductase proteins, our laboratory previously demonstrated that the last ER-lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed for proper karmellae assembly. Our goal was to examine the primary sequence requirements within Loop G that were critical for proper function of this signal. To this end, we randomly mutagenized the Loop G sequence, expressed the mutagenized Hmg1p in yeast, and screened for inability to generate karmellae at wild-type levels. Out of approximately 4000 strains with Loop G mutations, we isolated 57 that were unable to induce wild-type levels of karmellae assembly. Twenty-nine of these mutants contained one or more point mutations in the Loop G sequence, including nine single point mutants, four of which had severe defects in karmellae assembly. Comparison of these mutations to single point mutations that did not affect karmellae assembly did not reveal obvious patterns of sequence requirements. For example, both conservative and non-conservative changes were present in both groups and changes that altered the total charge of the Loop G region were observed in both groups. Our hypothesis is that Loop G serves as a karmellae-inducing signal by mediating protein-protein or protein-lipid interactions and that amino acids revealed by this analysis may be important for maintaining the proper secondary structure needed for these interactions.

Alleles↗

PubMed1999

The role of the 3-hydroxy 3-methylglutaryl coenzyme A reductase cytosolic domain in karmellae biogenesis.

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using beta-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

Carbocyanines↗

PubMed1999

Blood pressure responses to acute static and dynamic exercise in three racial groups.

PURPOSE: African Americans have a higher incidence of hypertension than other racial groups. Furthermore, some research suggests that normotensive individuals who exhibit exaggerated blood pressure (BP) responses to exercise may be at risk for future hypertension. This study sought to determine whether normotensive African Americans exhibited exaggerated BP responses to static exercise or dynamic exercise relative to Caucasian Americans and Asian Americans. METHODS: Thirty normotensive subjects participated from each of the three racial groups (15 men and 15 women). Subjects held 30% of maximal voluntary contraction (right knee extension) for 3 min, and BP was recorded during the 3rd min. On a separate occasion, subjects cycled for six min at a power equivalent to 70% of VO2 reserve, and BP was recorded during the 6th min. RESULTS: Static exercise produced large, significant increases in both systolic and diastolic BP (35 +/- 1.5 and 29 +/- 1.3 mm Hg, respectively). Dynamic exercise produced large, significant increases in systolic BP (51 +/- 1.6 mm Hg) and moderate, yet significant, increases in diastolic BP (8 +/- 1.0 mm Hg). There were no significant differences between racial groups in BP response to either static exercise or dynamic exercise. However, during dynamic exercise, men had a higher systolic BP response than did women. CONCLUSIONS: African Americans who are normotensive at rest do not exhibit a greater BP response to static exercise or dynamic exercise than do Caucasian Americans or Asian Americans.

Asian People↗

PubMed1998

Glycaemic regulation of glucose transporter expression and activity in the human placenta.

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.

Adult↗

PubMed1998

Molecular characterization of the fragilysin pathogenicity islet of enterotoxigenic Bacteroides fragilis.

Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.

Amino Acid Sequence↗

PubMed1998

Implicit learning of complex structures: active adaptation and selective processing in acquisition and application.

Subjects exposed to members of a structured domain become sensitive to the general structure of that domain, even when they are unaware that the domain has such structure (e.g., Reber, 1993). Numerous investigators have attempted to characterize this learning as unselective in acquisition and automatic in application. However, we contend that this characterization miscasts the fundamental nature of learning. In a series of experiments, we demonstrate that what subjects learn implicitly about the structure of a domain critically depends on decisions they make about how to organize the structural components. Similarly, the application of knowledge gained implicitly is not stable, but may be selected or even created under the demands of the test task. We conclude that implicit learning, just like explicit learning, proceeds through active organization of the stimulus complex, rather than by passively absorbing any level of structure. We propose a synthesis, in which learning, with and without awareness, is understood through a common set of principles.

Adaptation, Psychological↗

PubMed1997

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis.

The Bacteroides fragilis enterotoxin is an extracellular zinc metalloprotease that has been implicated in diarrheal disease of humans and animals. This toxin causes fluid accumulation in intestinal loops and is cytotoxic for HT-29 cells, an intestinal carcinoma cell line. Here we report the cloning and sequencing of the toxin gene (bftP). bftP is 1191 nucleotides coding for a 397 amino acid protein of 44.4 kDa. The toxin has a signal peptide of 18 amino acids that is typical of many lipoproteins followed by a 379 amino acid protoxin. The portion of the protoxin found in culture filtrates and stools begins at amino acid 212. An additional open reading frame located immediately upstream shows some sequence identity with cobra cytotoxins. If expressed, the ORF protein product could also play a role in the virulence of B. fragilis.

Amino Acid Sequence↗

PubMed1997

Implicit (and explicit) learning: acting adaptively without knowing the consequences.

Subjects exposed to members of a structured domain become sensitive to the general structure of that domain, even when unaware that the domain has such structure. Numerous investigators have taken this as evidence for a mode of learning in which memory passively and unselectively absorbs the structure of the environment. The authors contend that this assumption miscasts the fundamental nature of learning. The authors demonstrate that even when task and stimulus structure are held constant, subjects react to variations in incidental stimulus properties by processing the stimuli in qualitatively different ways. The authors conclude that implicit learning, just like explicit learning, proceeds through active organization of the stimulus complex, rather than through passive absorption of any level of structure. The authors propose a synthesis in which learning, with and without awareness, is understood through a common set of principles.

Adaptation, Physiological↗

PubMed1997

Prediction of VO2peak from submaximal cycle ergometry using 50 versus 80 rpm.

Submaximal cycle ergometry is routinely used for the prediction of mode specific maximal oxygen consumption (VO2peak). Such testing is almost universally performed at a cycling cadence of 50 rpm despite the finding that a cadence of approximately 80 rpm yields greater economy of effort among individuals with cycling experience. We sought to determine if 50 or 80 rpm were superior cadences for predicting VO2peak in individuals with or without cycling experience. Thirty experienced (EXP: 16 male and 14 female) and 28 nonexperienced (NEXP: 15 male and 13 female) subjects between the ages of 18-40 yr completed two incremental exercise tests on a cycle ergometer, one at 50 rpm and one at 80 rpm. in random order. VO2peak was predicted from submaximal data according to the method of the ACSM. There was no difference between actual VO2peak at 50 rpm and at 80 rpm. Correlations between predicted and actual VO2peak were 0.79 at 50 rpm and 0.81 at 80 rpm for all subjects. No significant differences in correlations were observed between groups or between cadences. However, the predicted values of VO2peak overestimated the actual values in both groups. Therefore, we conclude that cadences of 50 and 80 rpm have similar validity in cycle ergometry prediction of VO2peak, but the ACSM protocol overestimates VO2peak.

Adult↗

PubMed1996

Cloning of a potential cytochrome P450 from the archaeon Sulfolobus solfataricus.

Abstract A gene, CYP119, for a potential cytochrome P450 has been isolated and sequenced from the extreme acidothermophilic archaeon Sulfolobus solfataricus. The gene predicts a polypeptide of 368 amino acids containing the consensus heme-binding sequence Phe-Gly-Xaa-Gly-Xaa-His-Xaa-Cys-Xaa-Gly- Xaa3-Ala-Arg-Xaa-Glu. It most closely resembles the cytochrome P450s found in the bacterium Bacillus subtilis, with which it shares 129 identical amino acid residues (35%). This first sequence of a potential archaeal cytochrome P450 represents an important step in tracing the complex evolutionary history of this biologically important enzyme family.

Amino Acid Sequence↗

PubMed1996

Neurotoxin formation from pilot-scale incineration of synthetic ester turbine lubricants with a triaryl phosphate additive.

The high-temperature combustion of synthetic ester turbine engine lubricants has been performed by diluting the lubricant 5, 15, or 25% in diesel fuel and burning the mixture in a pilot-scale boiler facility. The effluent gas from this combustion system was carefully monitored for the formation of a potent neurotoxin, trimethylolpropane phosphate (TMPP). Although TMPP was not detected in the gaseous effluent, elevated levels of the neurotoxin were found in scrapings from the inside of the boiler system. Because of the extreme toxicity of this compound, significant dermal exposure could be a potential risk to incinerator operation and maintenance personnel.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed1996

Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

Endoplasmic Reticulum↗

PubMed1996

The unique proline-rich domain of parotid proline-rich proteins functions in secretory sorting.

When expressed in pituitary AtT-20 cells, parotid proline-rich proteins enter the regulated pathway. Because the short N-terminal domain of a basic proline-rich protein is necessary for efficient export from the ER, it has not been possible to evaluate the role of this polypeptide segment as a sorting signal for regulated secretion. We now show that addition of the six-amino acid propeptide of proparathyroid hormone to the proline-rich protein, and especially to a deletion mutant lacking the N-terminal domain, dramatically accelerates intracellular transport of these polypeptides. Under these conditions the chimeric deletion mutant is stored as effectively as the full-length protein in dense core granules. The propeptide does not function as a sorting signal in AtT-20 cells as it does not reroute a constitutively secreted reporter protein to the regulated pathway. During transit, the propeptide is cleaved from the chimeric polypeptides such that the original structures of the full-length and the deletion mutant proline-rich proteins are reestablished. We have also found that the percentage stimulated secretion of the proline-rich proteins increases incrementally (almost twofold) as their level of expression is elevated. The increase reflects an enrichment of these polypeptides in the granule pool and its incremental nature suggests that sorting of proline-rich proteins involves an aggregation-based process. Because we can now rule out contributions to sorting by both N- and C-terminal segments of the proline-rich protein, we deduce that the unique proline-rich domain is responsible for storage. Thus at least some of the determinants of sorting for regulated secretion are protein-specific rather than universal.

Animals↗

PubMed1995

Identification of the sequences in HMG-CoA reductase required for karmellae assembly.

In all eukaryotic cells that have been examined, specific membrane arrays are induced in response to increased levels of the ER membrane protein, HMG-CoA reductase. Analysis of these inducible membranes has the potential to reveal basic insights into general membrane assembly. Yeast express two HMG-CoA reductase isozymes, and each isozyme induces a morphologically distinct proliferation of the endoplasmic reticulum. The isozyme encoded by HMG1 induces karmellae, which are long stacks of membranes that partially enclose the nucleus. In contrast, the isozyme encoded by HMG2 induces short stacks of membrane that may be associated with the nucleus, but are frequently present at the cell periphery. To understand the molecular nature of the different cellular responses to Hmg1p and Hmg2p, we mapped the region of Hmg1p that is needed for karmellae assembly. For this analysis, a series of exchange alleles was examined in which a portion of the Hmg2p membrane domain was replaced with the corresponding Hmg1p sequences. Results of this analysis indicated that the ER lumenal loop between predicted transmembrane domains 6 and 7 was both necessary and sufficient for karmellae assembly, when present in the context of an HMG-CoA reductase membrane domain. Immunoblotting experiments ruled out the simple possibility that differences in the amounts of the various chimeric HMG-CoA reductase proteins was responsible for the altered cellular responses. Our results are consistent with the hypothesis that each yeast isozyme induces or organizes a qualitatively different organization of ER membrane.

Amino Acid Sequence↗

PubMed1995

The enterotoxin of Bacteroides fragilis is a metalloprotease.

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.

Actins↗

PubMed1995

Differential targeting of recombinant fibronectins in AtT-20 cells based on their efficiency of aggregation.

In pituitary-derived AtT-20 cells, recombinant fibronectin containing the N-terminal matrix assembly domain and the C-terminal half of fibronectin does not follow the regulated secretory pathway but instead concentrates in distinct organelles prior to secretion. These organelles are larger than the dense-core granules and localize to the cell body at sites that differ from lysosomes, endosomes and endoplasmic reticulum. Unlike the dense-core granules, their discharge is not stimulated by 8-bromo-cyclic-AMP or phorbol esters. The kinetics of intracellular transport and secretion of the recombinant fibronectin suggest that it is present in a post-Golgi pool that turns over more slowly than constitutive vesicles. Indeed, the fibronectin-containing organelles disappear with a half-time of 3 hours after inhibiting protein synthesis. Presence of the organelles correlates with intracellular aggregation of dimeric fibronectin polypeptides. The organelles are absent in cells expressing monomeric recombinant fibronectin (lacking C-terminal dimerization sites) or the C-terminal half of fibronectin (which dimerizes but lacks the N-terminal matrix assembly domain), both of which aggregate less efficiently than dimeric fibronectin. Instead, the latter polypeptides enter the dense-core granules. Thus while the formation of the fibronectin-containing organelles may require efficient aggregation, it may not require a specific structural signal. Moreover, efficient aggregation is not necessarily a prerequisite for following the regulated pathway.

Adrenocorticotropic Hormone↗

PubMed1994

Water diffusivity in porcine stratum corneum measured by a thermal gravimetric analysis technique.

Water is a natural constituent of the stratum corneum (sc) affecting its plasticity and modulating its barrier function. Diffusion coefficients (D) were calculated by measuring the desorption rates of water from porcine sc and dermis samples by a thermal gravimetric analysis (TGA) technique at isothermal conditions in the range 30-80 degrees C. Water-loaded samples were exposed to a flow of dry air, and the change of weight and of temperature were monitored with time. Distinct abrupt decreases in rate of desorption marked three different phases of water in the sc, designated as free, bulk, and bound water. Concomitant with the sharp change in evaporation rate, an increase in temperature was observed, in accordance with the absorption of heat accompanied with the water desorption process. Desorption curves were plotted against time and optimized. Values of D were estimated from the ratio of the evaporated water to the initial sc water content, as a function of the square root of time. The "initial slope" and the t1/2 (time for which Mt/Mo = 0.5, where Mo is the equilibrium amount of water absorbed in the membrane and Mt is the amount of water released by the membrane in a time t) methods gave similar results. The water D values of sc at 30 degrees C calculated by the two methods were 3.3 +/- 0.6 x 10(-10) and 2.7 +/- 0.8 x 10(-10) cm2/s, respectively. These values were about two orders of magnitude lower than the calculated D value for water in the dermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Nucleus-basal body connector in Chlamydomonas: evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity.

In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus-basal body connector (NBBC) that contains the calcium-binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903-1912.; Salisbury et al.: Journal of Cell Biology 107:635-642; Huang et al.: Journal of Cell Biology 107:121-131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella-number mutants examined, one, vfl-2, is dramatically defective with respect to the NBBC in that 1) the union between basal bodies and nucleus is very labile, 2) there is no detectible centrin in the NBBC region, and 3) total cellular centrin levels are reduced 75-80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl-2, the mutants vfl-1, vfl-3, uni-1, and bald-2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl-2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.

Animals↗