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Biomedical subjects

J C Young

Publications and source records attributed to J C Young.

At least 37 records · Page 2Linked to original sources
PubMed2000

Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters.

A major question in plant physiology is how the large amount of sucrose made in leaves is transported to the rest of the plant. Although physiological, biochemical, and anatomical investigations have been performed in this field, to date there have been very few genetic studies. Using a reverse genetic screen, we have identified mutant Arabidopsis plants containing transferred DNA insertions in the gene encoding a phloem-specific sucrose transporter, SUC2. SUC2 is thought to function in loading sugar from the apoplast into the conducting sieve tubes. In the homozygous state, these mutations resulted in stunted growth, retarded development, and sterility. The source leaves of mutant plants contained a great excess of starch, and radiolabeled sugar failed to be transported efficiently to roots and inflorescences. These data provide genetic proof that apoplastic phloem loading is critical for growth, development, and reproduction in Arabidopsis and that SUC2 is at least partially responsible for this step.

Arabidopsis↗
PubMed2000

Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23.

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.

Adenosine Triphosphatases↗
PubMed2000

Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an activating transcription factor/cAMP-responsive element-binding site.

In this study, we demonstrate that methylation-dependent repression of the Pdha-2 core promoter is mediated regionally through a consensus activating transcription factor/cAMP-responsive element-binding site located between nucleotides -54 and -62 upstream of the major transcriptional start site. Targeting of the CpG dinucleotide within this cis-element significantly disrupts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo. DNase I footprinting experiments indicated that availability of the nuclear factor(s) binding this element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early spermatogenic gene expression. Interestingly, CpG dinucleotides associated with the hypersensitive region flanking the activating transcription factor/cAMP-responsive element-binding site appear to confer some conformational structure on the promoter since mutations at these specific CpG dinucleotides result in elevated basal levels of transcription. This raises the possibility of a potential bifunctional role for CpG dinucleotides in either methylation-dependent or -independent processes. Our data support the notion that hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.

3T3 Cells↗
PubMed2000

Effect of culture acclimation on the kinetics of aldicarb insecticide degradation under methanogenic conditions.

This study reports on the kinetics of aldicarb transformation under methanogenic conditions using batch reactors containing acclimated and unacclimated cultures under controlled conditions. Culture acclimation was accomplished by exposing anaerobic microorganisms maintained in a semi-batch reactor to low concentrations of aldicarb. Results of the kinetic studies showed that in an anaerobic system aldicarb is converted to aldicarb nitrile by the hydrolytic pathway. Analysis of the hydrolysis/dehydration rate constants showed that anaerobic cultures enhanced the rate of conversion of aldicarb by 4-fold for acclimated cultures and by 2-fold for unacclimated cultures compared to the rate of abiotic hydrolysis (p < 0.05). Only the acclimated cultures were able to further mineralize the reaction intermediate aldicarb nitrile. Michaelis-Menten and Monod kinetics adequately defined the aldicarb nitrile degradation.

Acclimatization↗
PubMed2000

AXR2 encodes a member of the Aux/IAA protein family.

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.

Arabidopsis↗
PubMed2000

The incubation period of hantavirus pulmonary syndrome.

In 1993 Sin Nombre virus was recognized as the cause of hantavirus pulmonary syndrome (HPS) and the deer mouse (Peromyscus maniculatus) was identified as the reservoir host. Surveillance by the Centers for Disease Control and Prevention and state health departments includes investigation to determine the likely site(s) and activities that led to infection, an environmental assessment of the home and workplace, and possibly rodent trappings at these sites. As of December 31, 1998, there were 200 confirmed cases from 30 states (43% case-fatality ratio). The national HPS case registry was examined to determine the incubation period of HPS. Review of 11 case-patients with well-defined and isolated exposure to rodents suggests that the incubation period of HPS is 9 to 33 days, with a median of 14-17 days. Case investigations allow a better understanding of the incubation time of HPS and may define high-risk behaviors that can be targeted for intervention.

Adult↗
PubMed1999

Effects of chronic N(omega)-nitro-L-arginine methyl ester administration on glucose tolerance and skeletal muscle glucose transport in the rat.

It has been suggested that nitric oxide (NO) is a key regulator of carbohydrate metabolism in skeletal muscle. The present study was undertaken to examine the effects of chronic in vivo competitive antagonism of NO synthase (NOS) by the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) in the drinking water (1 mg/ml) for 14 days on glucose tolerance and skeletal muscle glucose transport in rats. Oral glucose tolerance tests (OGTT) revealed an impaired glucose tolerance in the L-NAME-treated rats as reflected by the area under the glucose curve (4675 +/- 514 mg% x 120 min (control) vs 6653 +/- 571 mg% x 120 min (L-NAME treated); P < 0.03). While a large rise in plasma insulin concentration was present in the control rats (0.87 +/- 0.34 ng/ml, P < 0.001) during the first 15 min of the OGTT, rises in plasma insulin concentration were absent in the L-NAME-treated rats (0.18 +/- 0.13 ng/ml, P = NS). Intravenous glucose tolerance tests confirmed an impaired insulin secretion in the L-NAME-treated rats. In contrast, insulin-stimulated 2-deoxyglucose transport was enhanced (P < 0.03) by chronic NOS inhibition (5.29 +/- 0.83 nmol/g/min) compared to control rats (2.21 +/- 0.90 nmol/g/min). Plasma sodium concentrations were lower and plasma potassium concentrations were higher in the L-NAME-treated group, indicating an impaired electrolyte status. We conclude that chronic in vivo administration of a NOS inhibitor, while not impairing basal parameters of carbohydrate metabolism, may manifest different responses than acute exposure to the same agent in vitro.

Administration, Oral↗
PubMed1999

CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo.

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

Animals↗
PubMed1999

Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.

Antigens, CD34↗
PubMed1999

Effect of vitamin B-6 supplementation on fuels, catecholamines, and amino acids during exercise in men.

PURPOSE: In two separate but identical studies, the effect of vitamin B-6 supplementation was examined on plasma energy substrates, catecholamines, and 13 amino acid concentrations during exercise. METHODS: Eleven trained men performed two separate exhaustive exercise tests at 71.0+/-4.6% VO2max during two separate 9-d controlled diet periods. Exercise test 1 (T1C) occurred following a control diet, and test 2 (T2B6) occurred following a vitamin B-6 supplemented diet (20 mg PN.d(-1)). Blood was drawn pre, during (60 min), post, and post-60 min of exercise, and plasma was analyzed for glucose, lactate, glycerol, free fatty acids (FFA), catecholamines (N = 5), and amino acids (N = 5). RESULTS: Mean FFA concentrations changed over time in both tests (P < 0.001) and were lower in T2B6 compared to T1C at pre (P = 0.03), during (P = 0.05), and post-60 min (P = 0.04) of exercise. Mean lactate, glycerol, and catecholamine concentrations only changed over time (P < 0.0001). The only significant changes in amino acid concentrations were for lower tyrosine (P = 0.007) and methionine (P = 0.03) concentrations in T2B6 relative to TIC at post-60 min of exercise and postexercise, respectively. No differences were observed in exercise times to exhaustion between TIC (108+32.6 min) and T2B6 (109+51.2 min). CONCLUSIONS: These results indicate that vitamin B-6 supplementation can alter plasma FFA and amino acid concentrations during exhaustive endurance exercise without affecting endurance.

Adult↗
PubMed1998

Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90.

The molecular chaperone hsp90 in the eukaryotic cytosol interacts with a variety of protein cofactors. Several of these cofactors have protein domains containing tetratricopeptide repeat (TPR) motifs, which mediate binding to hsp90. Using a yeast two-hybrid screen, the 12-kDa C-terminal domain of human hsp90alpha (C90) was found to mediate the interaction of hsp90 with TPR-containing sequences from the hsp90 cofactors FKBP51/54 and FKBP52. In addition, the mitochondrial outer membrane protein hTOM34p was identified as a TPR-containing putative partner protein of hsp90. In experiments with purified proteins, the TPR-containing cofactor p60 (Hop) was shown to form stable complexes with hsp90. A deletion mutant of hsp90 lacking the C90 domain was unable to bind p60, whereas deletion of the approximately 25-kDa N-terminal domain of hsp90 did not affect complex formation. Both p60 and FKBP52 bound specifically to the C90 domain fused to glutathione S-transferase and competed with each other for binding. In reticulocyte lysate, the C90 fusion protein recognized the TPR proteins p60, FKBP52, and Cyp40. Thus, our results identify the C90 domain as the specific binding site for a set of hsp90 cofactors having TPR domains.

Animals↗
PubMed1998

A transgene encoding a plasma membrane H+-ATPase that confers acid resistance in Arabidopsis thaliana seedlings.

Proton pumps (H+-ATPases) are the primary active transport systems in the plasma membrane of higher plant cells. These enzymes are encoded by a large gene family expressed throughout the plant, with specific isoforms directed to various specialized cells. While their involvement in membrane energetics has been suggested by a large body of biochemical and physiological studies, a genetic analysis of their role in plants has not yet been performed. We report here that mutant Arabidopsis thaliana plants containing a phloem-specific transgene encoding a plasma membrane H+-ATPase with an altered carboxy terminus show improved growth at low pH during seedling development. These observations provide the first genetic evidence for a role of the plasma membrane H+-ATPase in cytoplasmic pH homeostasis in plants.

Acids↗
PubMed1998

New World hantaviruses.

Since the initial description in 1993 of hantavirus pulmonary syndrome and its novel aetiological agent, Sin Nombre virus, our knowledge of the epidemiology of New World hantaviruses has continued to evolve. After the identifying outbreak in the southwestern US, four hantaviruses have been identified in North America with specific rodent hosts and associated with a number of sporadic cases. This stability of case recognition in North America is in contrast to the multiple outbreaks and endemic cases in South America. Despite a plethora of New World hantaviruses and new evidence of person-to-person transmission, the ecological and personal determinants of this human infection remain a mystery.

Ecology↗
PubMed1997

In vitro evidence that hsp90 contains two independent chaperone sites.

Hsp90 is an abundant and constitutively expressed stress protein and molecular chaperone. Here we dissected human hsp90 into three major domains to identify the putative chaperone site at which hsp90 binds unfolded polypeptide. Surprisingly, both the N-terminal and the C-terminal domain of hsp90 prevent the aggregation of denatured polypeptides. The chaperone activity of the N-domain is inhibited by geldanamycin, a specific inhibitor of hsp90-mediated protein refolding. While both domains suppress protein aggregation, only the C-domain binds an antigenic peptide derived from VSV G. Based on these results, hsp90 may be the first chaperone to contain two independent chaperone sites with differential specificity.

Amino Acid Sequence↗
PubMed1997

Role of dihydropyridine sensitive calcium channels in glucose transport in skeletal muscle.

Glucose transport in skeletal muscle is a carrier-mediated process activated by insulin and by contractile activity. Since previous evidence suggests a role for calcium influx in the activation of this process, the purpose of this study was to determine if glucose transport is mediated by muscle's voltage dependent (dihydropyridine sensitive) calcium channels. Soleus and extensor digitorum longus (EDL) muscles, isolated from rats, were incubated with the calcium channel blocker nifedipine. Basal glucose transport was decreased in both soleus and EDL by nifedipine. Treatment with nifedipine effectively blocked both insulin and contraction stimulated glucose transport in soleus. Conversely, glucose transport in EDL, although reduced, was still significantly increased over basal by both insulin and contraction, due, perhaps, to a relatively greater number of dihydropyridine receptors in EDL. These results provide evidence that contraction stimulated, as well as insulin stimulated, glucose transport is mediated in-part by dihydropyridine receptors in skeletal muscle.

Animals↗
PubMed1997

The use of ergosterol to measure exposure to fungal propagules in indoor air.

This report concerns the development of a method for the measurement of ergosterol in indoor air as a determinant of fungal exposure. Ergosterol was determined in spores of 11 species of Aspergillus, Penicillium, and Cladosporium selected from the most common molds in 400 homes in Ontario. Spore ergosterol content was about 1 microgram/mg, which is the range reported for mycelia, and varied by about 25% for the species tested. Ergosterol was determined in bedroom air samples taken in the winter in homes in southern Ontario. The median ergosterol value corresponded to a total concentration of fungal spores on the order of 10 to 10(2) per m3, in the range for other studies where total and viable propagules were determined by other methods. The sampling of air for ergosterol is a robust method for assessing fungal biomass in air, but provides no information on the species present.

Air Microbiology↗