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Biomedical subjects

S L Adams

Publications and source records attributed to S L Adams.

At least 19 recordsLinked to original sources
PubMed2005

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes.

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.

Animals↗
PubMed2001

Smad-Runx interactions during chondrocyte maturation.

BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. CLINICAL RELEVANCE: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.

Animals↗
PubMed2000

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

Aggrecans↗
PubMed1999

The internal chondrocyte-specific promoter of the chick type III collagen gene is activated by AP1 and is repressed in fibroblasts by a complex containing an LBP1-related protein.

The chick type III collagen gene contains an internal promoter in intron 23 in addition to the promoter preceding exon 1. The internal promoter, which is used preferentially in cultured chondrocytes, directs production of an alternative transcript that cannot encode type III collagen. This promoter is used ineffic-iently in skin fibroblasts, which transcribe the gene from the upstream promoter. We show below that the internal promoter is regulated by an activation element containing a potential activator protein 1 (AP1) site and a repressor element containing a potential binding site for leader binding protein 1 (LBP1). Electro-phoretic mobility shift assays indicate that the activation and repressor elements are bound by AP1 and an LBP1-related protein, respectively. Replacement of the AP1 site resulted in substantially decreased promoter activity in both chondrocytes and fibroblasts, indicating that this site is required for promoter function, but the low level of promoter activity in fibro-blasts is not due to loss of functional AP1. In contrast, replacement of the LBP1-like site increased activity only in fibroblasts, suggesting that this site is responsible in part for repression of promoter activity in fibroblasts.

Animals↗
PubMed1999

Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis.

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.

Animals↗
PubMed1999

Evaluation and conservative management of chronic lower extremity arterial disease.

Aging of the population has made atherosclerotic cardiovascular disease one of the most significant health problems in the United States. Primary care providers are often the first contact for persons presenting with symptoms of chronic lower extremity arterial disease. Accurate assessment can prevent erroneous referrals, unnecessary diagnostic studies, and added time and medical expense for patients. Understanding of the natural history and risk factors associated with chronic lower extremity arterial disease provides the foundation for developing a knowledgeable, comprehensive plan of care for patients. Through multidisciplinary collaboration and a strong commitment to patient education and support, care providers can enhance the quality of life for individuals with chronic lower extremity arterial disease.

Arterial Occlusive Diseases↗
PubMed1998

Dichotomous effects of beta-chemokines on HIV replication in monocytes and monocyte-derived macrophages.

The role of beta-chemokines in the pathogenesis of HIV disease remains undefined. Given the potent capacities of these proteins to attract mononuclear cells to inflammatory sites, such as lymph nodes of patients with HIV disease, the effects of exposure of monocytes and monocyte-derived macrophages to beta-chemokines before HIV infection were compared with their effects when added either simultaneously with or after HIV infection. In this system, HIV replication was substantially increased in cells that had been exposed to beta-chemokines before HIV infection. These effects were pertussis toxin sensitive. By contrast, HIV replication was inhibited in cells that had been exposed to beta-chemokines either simultaneously with or after HIV infection. These effects were not pertussis toxin sensitive. In view of this potent capacity of beta-chemokines to stimulate HIV replication, treatment approaches for HIV disease based on the apparent inhibitory activity of these proteins on viral replication should be undertaken with caution.

Cells, Cultured↗
PubMed1998

Predicting patient satisfaction: a study of two emergency departments.

To identify perceptions that predict overall patient (dis)satisfaction with Emergency Department (ED) care, we studied responses to a survey mailed to all discharged patients over a 6-month period (Academic Hospital), and to a telephone interview of a random sample of discharged patients over a 1-year period (Community Hospital). The survey and interview both assessed overall satisfaction, as well as satisfaction with perceived waiting times, information delivery, and expressive quality of physicians, nurses, and staff. Data for 1176 patients (training sample) and 1101 patients (holdout sample) who rated overall satisfaction as either "very good" or "very poor" (Academic Hospital), and for 856 patients (training sample) and 431 patients (holdout sample) who rated overall satisfaction as either "excellent" or "poor" (Community Hospital), were retained for analysis. For both hospitals, nonlinear tree models efficiently achieved overall classification accuracy exceeding 98% in training analysis and 95% in holdout analysis (all p < .0001). The findings suggest that overall patient (dis)satisfaction with care received in the ED is nearly perfectly predictable on the basis of patient-rated expressive qualities of ED staff, particularly physicians and nurses. Interventions designed to reinforce positive (and extinguish negative) expressive health-care provider behaviors may cut the number of extremely dissatisfied patients in half.

Chicago↗
PubMed1998

Ambulatory blood pressure and Holter monitoring of emergency physicians before, during, and after a night shift.

BACKGROUND: Occupational stress may affect measured hemodynamic and electrocardiographic variables. Data describing the physiologic effects of work on the emergency physician (EP) are sparse. OBJECTIVE: To determine whether blood pressure (BP) and heart rate variability (HRV) of the EP are affected during a night shift in the ED. METHODS: This prospective study evaluated BP and HRV in attending EPs at an urban academic medical center for a 24-hour period during which a night shift was scheduled. Participants were fitted with an oscillometric ambulatory BP device and a Holter monitor at 1500 hours on the day of a night shift. The monitors were worn continuously before, during, and after a night shift (2300-0700) in the ED and were removed at 1500. Systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), heart rate (HR), measures of HRV, and occurrence of cardiac dysrhythmias were evaluated. Comparisons were made for ED and non-ED awake periods and non-ED sleep periods. RESULTS: Twelve participants completed the study. Eight (67%) subjects were men and 4 (33%) were women. Age ranged from 28 to 40 years (mean 34.1+/-4.1). Results were analyzed using repeated-measures ANOVA. An elevation of mean DBP (5.5 mm Hg+/-4.37; p < 0.05; 95% CI 1-10) during night shift activity was seen. A trend toward elevation of SBP, MAP, and HR was discernible. HRV measures indicated a significant relative increase in sympathetic vs parasympathetic tone and an increase in HR of prework and work compared with postwork. Dysrhythmias observed included sinus tachycardia, sinus bradycardia, sinus pause, atrial premature beats, atrial couplets and triplets, supraventricular tachycardia, and premature ventricular contractions. CONCLUSIONS: The elevation of DBP during a night shift suggests that these patterns of BP variability are activity- or stress-related rather than a result of a true diurnal variation. HRV analysis suggests that sympathetic tone is heightened both before work and during work. The implications of such findings to the health of the EP warrant further investigation.

Adult↗
PubMed1997

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis.

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.

Chromosomes, Bacterial↗
PubMed1997

T-cell response to HIV in natural infection: optimized culture conditions for detecting responses to gag peptides.

The proliferative responses to four gag peptides were examined in 24 HIV-seropositive patients whose CD4 counts ranged between 500 and 1400 cells/mm3. To overcome some of the limitations imposed by HIV infection on the T-cell proliferative assay, recombinant interleukin 2 (rIL-2) was added to the cultures, and the culture time of the cells was increased from the standard 6 to 8 or 10 days. Four of 24 patients responded to one or more core peptides, aa180-194, 208-217, 267-286, and 287-306 by the standard 6-day culture: this increased to 13 of 24 using the optimized culture approach. The greatest number and magnitude of responses occurred after cells were in culture for 8 days. Eight patients responded to gag 180-194, which has not been identified previously as a TH epitope in humans but has considerable homology with a TH epitope recognized by cloned T cells from macaques immunized with simian immunodeficiency virus (SIV). We have identified four T-cell epitopes on the HIV core protein p24, using synthetic peptides as immunogens. Three of the peptides would not have been considered immunogenic had the standard assay system been used to detect T-cell responsiveness. We have also shown that a region of the core protein encompassed by aa180-194 is recognized by TH cells in humans.

Adult↗
PubMed1997

The chick type III collagen gene contains two promoters that are preferentially expressed in different cell types and are separated by over 20 kb of DNA containing 23 exons.

Type III collagen is present in prechondrogenic mesenchyme, but not in cartilages formed during endochondral ossification. However, cultured chick chondrocytes contain an unusual transcript of the type III collagen gene in which exons 1-23 are replaced with a previously undescribed exon, 23A; this alternative transcript does not encode type III collagen. This observation suggested that, although production of type III collagen mRNA is repressed in chondrocytes, transcription of the type III collagen gene may continue from an alternative promoter. To test this prediction, we isolated and characterized both the upstream and internal promoters of this gene and tested their ability to direct transcription in chondrocytes and skin fibroblasts. The upstream promoter is active in fibroblasts, but inactive in chondrocytes, indicating that repression of type III collagen synthesis during chondrogenesis is transcriptionally mediated. Additionally, sequences in intron 23, preceding exon 23A, function as a highly active promoter in chondrocytes; transcription from this promoter is repressed in fibroblasts. Thus transcriptional control of the type III collagen gene is highly complex, with two promoters separated by at least 20 kb of DNA that are preferentially expressed in different cell types and give rise to RNAs with different structures and functions.

Animals↗
PubMed1997

Annexin V-mediated calcium flux across membranes is dependent on the lipid composition: implications for cartilage mineralization.

Annexin V is a major component of matrix vesicles and has a role in mediating the influx of Ca2+ into these vesicles, thus promoting the initiation of hypertrophic cartilage matrix mineralization. However, the mechanisms and factors regulating annexin V-mediated Ca2+ influx into these vesicles are not well understood. Since the lipid composition of matrix vesicles differs from that of the plasma membrane of chondrocytes and is rich in phosphatidylserine, we asked whether the lipid composition may regulate annexin V function. We prepared liposomes containing different concentrations of phosphatidylserine and determined how the lipid composition affected (a) the interactions between annexin V and liposomes and (b) annexin V-mediated Ca2+ influx into the liposomes. We found that annexin V was able to bind to every liposome tested. However, we observed the most prominent increases in tryptophan 187 emission intensity, a measure of the degree of interactions between annexin V and lipid bilayers, only with liposomes containing a high concentration of phosphatidylserine. In addition, a significant fraction of annexin V associated with phosphatidylserine-rich liposomes was not extractable by EDTA treatment but required a detergent, indicating that annexin V inserts into bilayers of these liposomes. Chemical cross-linking analysis revealed that matrix vesicles and phosphatidylserine-rich liposomes induced the formation of the annexin V hexamer. Interestingly, a significant Ca2+ influx in the presence of annexin V occurred only in liposomes containing a high phosphatidylserine content. Moreover, annexin V-mediated Ca2+ influx into these liposomes was inhibited (i) by anti-annexin V antibodies and (ii) by treatment with zinc and cadmium, indicating the essential role of the protein in Ca2+ influx. The results of this study indicate that phosphatidylserine-rich bilayers induce the formation of a hexameric annexin V, possibly leading to a Ca2+-dependent insertion of annexin V into the bilayer and establishment of annexin V-mediated Ca2+ influx into matrix vesicles or liposomes. The phosphatidylserine-rich membrane of matrix vesicles in vivo may thus offer an ideal specialized environment in which the biological function of annexin V is optimized, leading to rapid Ca2+ influx, intralumenal crystal growth, and cartilage matrix mineralization.

Animals↗
PubMed1997

Role of nuclear cardiology in the evaluation of acute coronary syndromes.

Over the last 20 years, nuclear cardiology has become a mainstay in the evaluation of ischemic heart disease. In the setting of acute coronary syndromes (myocardial infarction or unstable angina), myocardial perfusion imaging has emerged as an important tool in assessing the functional significance of angiographic coronary stenoses, evaluating the efficacy of therapeutic interventions, and risk-stratifying patients in the postinfarction period. Recent literature has demonstrated the diagnostic and prognostic value, as well as the cost-effectiveness, of perfusion imaging in acute chest pain syndromes and the diagnostic superiority of perfusion imaging compared with two-dimensional echocardiography. Acute perfusion imaging is now being included in the algorithm for the triage and management of acute chest pain syndromes. Emergency physicians are increasingly using nuclear cardiac imaging modalities for aid in the evaluation of patients who present with chest pain of uncertain origin.

Acute Disease↗
PubMed1997

Molecular characterization of the prokaryotic efp gene product involved in a peptidyltransferase reaction.

The translation factor EF-P is required for efficient prokaryotic peptide bond synthesis on 70S ribosomes from fMet-tRNAfMet. This protein has been purified from Escherichia coli cells and the gene, efp, encoding it has been cloned and sequenced. We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis. Using these clones, we report the purification, crystallization and initial characterization of the efp gene product. The mechanism by which EF-P stimulates peptide-bond synthesis was studied using several antibiotics that inhibit translocation, peptide-bond synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and occupation of the A site (in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and lincomycin. This observation and the requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome.

Anti-Bacterial Agents↗
PubMed1997

Clinical use of the patellar-pubic percussion sign in hip trauma.

To assess the reliability and validity of osteophony (patellar-pubic percussion [PPP] test) as a physical diagnostic sign in the evaluation of hip trauma, a prospective study was undertaken of 41 consecutive patients presenting to the emergency department with a history of hip trauma necessitating radiographic examination. Fifteen of 19 (78.9%) patients who presented with a history of hip trauma and a fracture on radiograph were found to have had an abnormal PPP sign by at least one of two raters (P < .0001). Only 1 of 22 (4.6%) patients without evidence of fracture (eg, contusion) had an abnormal PPP sign. This patient had diffuse Paget's disease. Nine of 10 (90%) patients who had trochanteric fractures had an abnormal PPP sign (P < .02). Overall reliability of the PPP sign based on two observers was 90.2% (P < .0001). In those patients with radiographic evidence of fracture, interrater reliability was 84.2% (P < .0001). For patients in whom physicians agreed on the PPP sign, the PPP test resulted in a 0% false-positive error and a 25% false-negative error. For patients in whom either physician noted an abnormal PPP sign, the PPP test resulted in a 4.6% false-positive error and a 21.1% false-negative error. The presence of an abnormal PPP sign in the evaluation of hip trauma is associated with evidence of fracture or other bony abnormality on radiograph.

Emergency Service, Hospital↗
PubMed1996

The chick alpha2(I) collagen gene contains two functional promoters, and its expression in chondrocytes is regulated at both transcriptional and post-transcriptional levels.

Embryonic chick cartilages contain transcripts derived from the alpha2(I) collagen gene, although type I collagen is not normally found in these tissues; most of these RNAs are alternative transcripts initiating within intron 2. Use of the internal start site results in replacement of exons 1 and 2 with a previously undescribed exon and a change in the translational reading frame; thus, the alternative transcript cannot encode alpha2(I) collagen. We have demonstrated that production of the alternative transcript is due to activation of an internal promoter in chondrocytes and have identified a 179-base pair domain that is required for its activity. Furthermore, we have shown that the alternative transcript resulting from activation of the internal promoter turns over relatively rapidly; thus, the steady-state level of this transcript is less than predicted based on the transcription rate. The upstream promoter is only partially repressed in chondrocytes, suggesting that the lack of authentic alpha2(I) collagen mRNA may also be due in part to decreased mRNA stability. Thus, repression of alpha2(I) collagen synthesis in cartilage involves both transcriptional and post-transcriptional mechanisms. In contrast, repression of alpha1(I) collagen synthesis appears to be mediated primarily at the level of transcription.

Alternative Splicing↗