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Biomedical subjects

J C Young

Publications and source records attributed to J C Young.

At least 55 records · Page 3Linked to original sources
PubMed1997

Gene cloning and expression and characterization of a toxin-sensitive protein phosphatase from the methanogenic archaeon Methanosarcina thermophila TM-1.

With oligonucleotides modelled after conserved regions within the protein-serine/threonine phosphatases (PPs) of the PP1/2A/2B superfamily, the gene for the archaeal protein phosphatase PP1-arch2 was identified, cloned, and sequenced from the methanogenic archaeon Methanosarcina thermophila TM-1. The DNA-derived amino acid sequence of PP1-arch2 exhibited a high degree of sequence identity, 27 to 31%, with members of the PP1/2A/2B superfamily such as PP1-arch1 from Sulfolobus solfataricus, PP1alpha from rats, PP2A from Saccharomyces cerevisiae, and PP2B from humans. The activity of the recombinant PP1-arch2 was sensitive to several naturally occurring microbial toxins known to potently inhibit eucaryal PP1 and PP2A, including microcystin-LR, okadaic acid, tautomycin, and calyculin A.

Acetates↗
PubMed1996

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells.

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy-1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony-forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.

Adult↗
PubMed1996

Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport.

The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome. As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene. Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed. The genes surveyed include members of various gene families involved in signal transduction and ion transport. As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.

Agrobacterium tumefaciens↗
PubMed1996

Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture.

Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin-/dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.

Adult↗
PubMed1996

The signal recognition particle receptor alpha subunit assembles co-translationally on the endoplasmic reticulum membrane during an mRNA-encoded translation pause in vitro.

Many proteins, including the alpha subunit of the signal recognition particle receptor (SR alpha), are targeted within the cell by poorly defined mechanisms. A 140 residue N-terminal domain of SR alpha targets and anchors the polypeptide to the endoplasmic reticulum membrane by a mechanism independent of the pathway involving the signal recognition particle. To investigate the mechanism of membrane anchoring, translation pause sites on the SR alpha mRNA were used to examine the targeting of translation intermediates. A strong pause site at nucleotide 507 of the mRNA open reading frame corresponded with the shortest nascent SR alpha polypeptide able to assemble on membranes. An mRNA sequence at this pause site that resembles a class of viral -1 frameshift sequences caused translation pausing when transferred into another mRNA context. Site-directed mutagenesis of the mRNA greatly reduced translation pausing without altering the polypeptide sequence, demonstrating unambiguously a role for this mRNA sequence in translation pausing. SR alpha polypeptides synthesized from the non-pausing mRNA were impaired in co-translational membrane anchoring. Furthermore, co-translational membrane assembly of SR alpha appears to anchor polysomes translating SR alpha to membranes.

Animals↗
PubMed1996

Hierarchical structure of human megakaryocyte progenitor cells.

Megakaryocytopoiesis is a complex biological process involving a series of cellular events that begins with the pluripotent hematopoietic stem cell and ultimately results in the biogenesis of platelets. A hierarchy of megakaryocyte (MK) progenitor cells has been previously defined based upon studies of in vitro megakaryocytopoiesis. Ontogeny-related changes in MK progenitor cells were analyzed in order to further define this cellular hierarchy. Unifocal colony-forming unit-megakaryocyte (CFU-MK)-derived colonies cloned from fetal bone marrow (FBM) formed after fewer days of in vitro culture and were 2.6-fold larger than those colonies cloned from adult bone marrow (ABM). The frequency of CFU-MK-derived colonies cloned from ABM was significantly greater. MK colonies, however, cloned from FBM morphologically consisted of both pure MK colonies and mixed colonies containing MKs, in which a core of CD41- cells were surrounded by CD41+ MKs. Large colonies resembling the primitive BFU-MK also were assayed from both FBM and ABM. These BFU-MK-derived colonies appeared after fewer days of incubation when FBM was assayed, compared to ABM, but at a significantly lower frequency. In addition, large unifocal MK colonies consisting of >300 cells (300-1000) appeared from cells cloned from fetal, but not adult, marrow. This type of colony represents a unique type of MK progenitor cell, termed the high-proliferative-potential cell-MK. Such colonies represent the progeny of the most primitive human MK progenitor cell identified to date. We also attempted to investigate the process of commitment of stem cells to the MK lineage. We explored the actions of thrombopoietin (TPO) on primitive hematopoietic cells in order to gain an understanding of stem cell commitment. CD34+ Thy-1+ Lin- marrow cells, which are enriched for pluripotent hematopoietic stem cells, were shown to express c-Mpl by the polymerase chain reaction. In addition, TPO alone was capable of inducing CD34+ Thy-1+ Lin- cells after two to three weeks to produce progeny composed entirely of MKs. These studies indicate that TPO has a profound effect on hematopoietic stem cells, and that the hierarchy of MK progenitor cells begins with the pluripotent hematopoietic stem cell.

Adult↗
PubMed1996

The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering.

Flowering in Arabidopsis thaliana is promoted by longday (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 (elf3) mutant of Arabidopsis, which is insensitive to photoperiod with regard to floral initiation has been characterized elf3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY4. However, when elf3 mutants were made deficient for functional phytochrome by the introduction of hy2 mutant alleles, the elf3 hy2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf3 single mutants. In addition, the inflorescences of one allelic combination of elf3 hy2 double mutants form a terminal flower similar to the structure produced by tfk1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.

Arabidopsis↗
PubMed1995

An amino-terminal domain containing hydrophobic and hydrophilic sequences binds the signal recognition particle receptor alpha subunit to the beta subunit on the endoplasmic reticulum membrane.

The signal recognition particle receptor consists of two subunits of 72 kDa (SR alpha) and 30 kDa (SR beta). Assembly of SR alpha on the endoplasmic reticulum membrane can occur independent of the signal recognition particle-mediated translocation pathway. To identify the sequences within SR alpha necessary for membrane binding, a series of amino-terminal and internal deletion mutants was constructed and translated in a cell-free system. In addition, nascent SR alpha polypeptides of varying lengths were generated by cycloheximide treatment of translation reactions. Microsome binding assays performed on these polypeptides revealed a membrane binding domain consisting of the amino-terminal 140 residues of SR alpha. This domain includes the two hydrophobic sequences originally proposed to bind to membranes and a highly charged region not previously implicated in membrane assembly. Furthermore, the domain forms a protease-resistant folding unit that after proteolysis can target and anchor onto microsomes. Extraction of microsomal SR alpha at high pH supplemented with 1 M NaSCN suggests that SR alpha and the membrane binding domain are not integrated in the endoplasmic reticulum membrane. The membrane binding domain is also the major site of tight binding with SR beta, suggesting that SR beta plays a role in the membrane assembly of SR alpha.

Amino Acid Sequence↗
PubMed1995

Meal size and frequency: effect on potentiation of the thermal effect of food by prior exercise.

Prior exercise potentiates the thermic effect of a carbohydrate meal. The purpose of this study was to determine if meal size or feeding pattern influences this response. Two groups of healthy, normal-weight young women exercised for 45 min on a cycle ergometer at 70% of maximal aerobic capacity. Once aerobic capacity returned to pre-exercise baseline, the thermic effect of food (TEF) was determined by indirect calorimetry over a 2-h period. One group of subjects ingested a 2510-kJ meal and the other a 5020-kJ meal. As a control, subjects ingested the test meal without prior exercise. In addition, subjects ingesting the 5020-kJ meal were studied for an additional 2 h. In a separate trial, these subjects ingested a 5020-kJ meal in two equal portions after a bout of exercise, the second portion 120 min after the first. TEF was less for the 2510-kJ meal compared with the 5020-kJ meal for both the control [mean (SE), 76 (17) vs 158 (19) kJ.2h-1, P < 0.01), and prior exercise [124 (23) vs 197 (24) kJ.2h-1, P < 0.01) trials. However, the same increment in TEF resulted from the prior bout of exercise [48 (9) vs 40 (8) kJ.2h-1 for 2510-and 5020-kJ meals, respectively). TEF was 31% lower when the 5020-kJ meal was given in two portions compared with one [281 (30) vs 369 (41) kJ.4h-1, P < 0.05]. No difference in TEF was found between the first and second 2510-kJ portion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Exercise prescription for individuals with metabolic disorders. Practical considerations.

Regular exercise has been recognised as an important component in the management of patients with diabetes mellitus. In addition to acutely lowering blood glucose, exercise training improves glucose tolerance and peripheral insulin sensitivity, contributes to weight loss and reduces several risk factors for cardiovascular disease. When proper precautions are taken to prevent hypoglycaemia, individuals with diabetes can enjoy the same benefits from exercise as nondiabetic healthy individuals. As a guideline, moderate intensity, aerobic endurance activities should be performed for 20 to 40 minutes at least 3 times a week. Blood glucose should be monitored, and insulin dose and carbohydrate intake adjusted based on the blood glucose response to the type and duration of exercise. This review will summarise current understanding of the therapeutic role of exercise in the treatment of diabetes and will present guidelines for prescribing exercise in diabetic patients.

Blood Glucose↗
PubMed1995

The effects of prophylactic knee bracing on running gait.

Although the use of prophylactic knee braces in football players is common, the effectiveness of this practice is questionable. Some studies have actually shown an increase in the incidence of lower extremity injuries in players wearing such braces. The purpose of this study was to determine if wearing a prophylactic knee brace (Omni Anderson Knee Stabler) caused an alteration in a normal running gait pattern that might then increase the risk for injury. Ten subjects with no prior history of knee injury or brace use were filmed in two planes while running on a treadmill at 5 mph. Two-dimensional digital analysis of each subject's running gait with and without the use of knee braces was performed. The duration of the gait cycle and the range of motion and velocity of movements at the knee, hip, and ankle joints were determined in each leg for each condition. No differences were found between braced and nonbraced conditions for any of the measured variables. Therefore, we conclude that the use of the Omni Anderson Knee Stabler does not alter gait pattern of the lower extremities while running on a treadmill.

Journal Article↗
PubMed1995

Strenuous exercise with caloric restriction: effect on luteinizing hormone secretion.

To test whether strenuous exercise with and without caloric restriction alters LH secretion, and whether these changes are apparent in the immediate post-exercise period, LH pulse parameters were studied in four moderately trained eumenorrheic women over three successive menstrual cycles. Blood samples were obtained 5 h before and 5 h after 90 min of running at 74% VO2max. Each test was preceded by a 7-d treatment of controlled diet and exercise (74% VO2max). During CONTROL, subjects were eucaloric on days 1-7, and performed no exercise on days 5-7. During STTI (short-term training increase), subjects were eucaloric and completed 90 min runs on days 5-7. During DIET/STTI, subjects consumed 60% of the calories necessary to maintain weight on days 1-7, and exercised as in STTI. A significant decrease in overall (0700-1830 h) LH pulse frequency during DIET/STTI compared with CONTROL and STTI treatments was observed. No changes were found in mean serum LH levels or peak amplitude. These results suggest that high-volume training combined with caloric restriction may predispose one to exercise-induced changes in LH pulse frequency, while adequate caloric intake may prevent these changes.

Adult↗
PubMed1994

Analysis of Fusarium mycotoxins by gas chromatography--Fourier transform infrared spectroscopy.

The Fourier transform infrared (FTIR) spectra of selected Fusarium mycotoxins of various structure types were determined. Absorptions were observed for the following functionalities: hydroxyl at 3625-65 cm-1 and 3482 cm-1, the latter being associated with a 7 alpha-hydroxyl adjacent to an 8-carbonyl in keto trichothecenes; carbonyl at 1760-6 cm-1 for 5-membered rings and at 1695-8 cm-1 for those conjugated to a single C = C in a six-membered ring; acetate carbonyl at 1765 cm-1 and acetate C-O at 1220-9 cm-1; and C = C at 1680 cm-1. Gas chromatography combined with FTIR and mass spectrometry was applied to the identification of some mycotoxins in a F. roseum liquid culture extract.

Chromatography, Gas↗
PubMed1994

Pdha-2: a model for studying transcriptional regulation in early spermatocytes.

Precise temporal and tissue-specific expression of genes during spermatocyte differentiation is crucial for the formation of functional spermatozoa. However, the mechanisms that regulate gene expression during spermatogenesis are poorly understood. One testis-specific gene, Pdha-2, is beginning to emerge as a potentially important model for the study of these events. This review focuses on our current understanding of the expression and regulation of Pdha-2 during spermatogenesis.

Animals↗
PubMed1994

Mouse testis Pdha-2 promoter upstream sequences confer tissue-and temporal-specific activity in transgenic mice.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex. To elucidate the mechanisms regulating its expression in vivo, we have begun to investigate the Pdha-2 promoter in transgenic mice. In this paper, a construct containing 3.0 kb of promoter and upstream sequences is reported to be sufficient for directing the testis-specific expression of a CAT reporter gene in mice harbouring the transgene. Similarly to the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. However, the 3.0-kb Pdha-2 promoter is not active in somatic tissue suggesting that repressor elements may be present within these sequences.

3T3 Cells↗
PubMed1994

Blunt vascular injury associated with closed mid-shaft femur fracture: a plea for concern.

During a period of six years, 765 consecutive patients were treated by the trauma service at Allegheny General Hospital for closed mid-shaft femur fractures that were a component of their injury complex. Thirty-one patients underwent angiography of the involved extremity for indications including loss of pulses in eleven and large hematomas or deformities of the thigh in the remainder. Ten patients (1.3%) were found to have acute vascular injuries. In nine patients there was an intimal flap of the superficial femoral artery (SFA), and in one, a pseudoaneurysm. Two patients had injuries of the femoral nerve. Three patients had no other associated major injuries (Injury Severity Score range, 10-19). Twelve months after the initial injury, one patient developed an arteriovenous fistula of the SFA. Detailed, repeated physical examinations, early utilization of angiography, and intensive follow-up by the trauma surgeon or orthopedic surgeon of patients with closed mid-shaft femur fractures should lead to early recognition of this potentially serious association.

Adolescent↗
PubMed1994

Delayed rupture of the spleen--myths, facts, and their importance: case reports and literature review.

Over a 1-year period, three patients were seen in our trauma service with delayed bleeding (> or = 7 days) from an initially inapparent splenic injury. This entity was defined as a late occurrence of signs and symptoms attributed to splenic injury not detected by diagnostic computed tomographic (CT) scanning during the initial examination. We believe that this represents an "injury in evolution" minor enough to go undetected on initial CT scans of the abdomen. A high index of suspicion and liberal utilization of imaging techniques are essential for the identification of delayed splenic rupture. Further multicenter studies are required to delineate the true incidence of its occurrence and its clinical significance. We conclude that "delayed rupture" of the spleen is a true clinical entity. The occurrence of a delayed rupture may prove hazardous to patients discharged early from the hospital after blunt abdominal injury. A classification system to assess this type of injury is suggested.

Accidents, Traffic↗