Douglas F. Paulsen

Latex beads as probes of cell surface-extracellular matrix interactions during chondrogenesis: evidence for a role for amino-terminal heparin-binding domain of fibronectin

Fibronectin-rich mesenchymal condensations form at sites of incipient chondrogenesis in the developing vertebrate limb, and in cultures of limb bud mesenchyme. We have used 6 microns polystyrene latex beads coated with various substances as probes for adhesive interactions that may mediate the formation of these condensations. Beads coated with heparin, chondroitin sulfate, or poly L-lysine, that were mixed with limb bud mesenchymal cells were centripetally conveyed into fibronectin-rich regions of cell condensation over a period of several days. Beads coated with dextran sulfate remained uniformly dispersed throughout the cultures during the same period. A monoclonal antibody directed against the amino-terminal heparin-binding domain of fibronectin completely inhibited accumulation of heparin-coated beads at condensing foci, but monoclonal antibodies directed against the collagen- or cell-binding domains of fibronectin were not inhibitory. Accumulation of chondroitin sulfate- or poly L-lysine-coated beads at condensing foci was unaffected by the antibody against the fibronectin amino terminus. Peptides with the sequence arg-gly-asp-ser or gly-arg-gly-asp-ser, which inhibit adhesive interactions mediated by the integrin-binding domain of fibronectin, had no effect on conveyance or accumulation of heparin-coated beads, but the peptide with the sequence gly-arg-gly, a repeated motif in the amino-terminal heparin-binding domain was completely inhibitory. These findings indicate that the amino-terminal heparin-binding domain of fibronectin can, within a tissue microenvironment, interact adhesively with heparin-like components on the surfaces of polystyrene beads, and by implication, on mesenchymal cells themselves. This interaction may therefore be a component of the condensation-forming mechanism in chondrogenic mesenchyme.

Anatomy as the Backbone of an Integrated First Year Medical Curriculum: Design and Implementation

Morehouse School of Medicine chose to restructure its first year medical curriculum in 2005. The anatomy faculty had prior experience in integrating courses, stemming from the successful integration of individual anatomical sciences courses into a single course called Human Morphology. The integration process was expanded to include the other first year basic science courses (Biochemistry, Physiology, and Neurobiology) as we progressed toward an integrated curriculum. A team, consisting of the course directors, a curriculum coordinator, and the Associate Dean for Educational and Faculty Affairs, was assembled to build the new curriculum. For the initial phase, the original course titles were retained but the lecture order was reorganized around the Human Morphology topic sequence. The material from all four courses was organized into four sequential units. Other curricular changes included placing laboratories and lectures more consistently in the daily routine, reducing lecture time from 120 to 90 minute blocks, eliminating unnecessary duplication of content, and increasing the amount of independent study time. Examinations were constructed to include questions from all courses on a single test, reducing the number of examination days in each block from three to one. The entire restructuring process took two years to complete, and the revised curriculum was implemented for the students entering in 2007. The outcomes of the restructured curriculum include a reduction in the number of contact hours by 28%, higher or equivalent subject examination average scores, enhanced student satisfaction, and a first year curriculum team better prepared to move forward with future integration. Anat Sci Educ 4:157–169, 2011.

β‐Agonist‐induced alterations in organ weights and protein content: Comparison of racemic clenbuterol and its enantiomers

Clenbuterol is a relatively selective beta2-adrenergic partial agonist that has bronchodilator activity. This drug has been investigated as a potential countermeasure to microgravity- or disuse-induced skeletal muscle atrophy because of presumed anabolic effects. The purpose of this study was to: 1) analyze the anabolic effect of clenbuterols (-)-R and (+)-S enantiomers (0.2 mg/kg) on muscles (cardiac and skeletal) and other organs; and 2) compare responses of enantiomers to the racemate (0.4 mg/kg and 1.0 mg/kg). Male Sprague Dawley rats were treated with: a) racemic clenbuterol (rac-clenbuterol, 0.4 or 1.0 mg/kg); b) enantiomers [clenbuterol (-)-R or (+)-S]; or c) vehicle (1.0 mL/kg buffered saline). Anabolic activity was determined by measuring tissue mass and protein content. HPLC teicoplanin chiral stationary phase was used to directly resolve racemic clenbuterol to its individual enantiomers. In skeletal muscle, both enantiomers had equal anabolic activity, and the effects were muscle- and anatomic region-specific in magnitude. Although the enantiomers did not affect the ventricular mass to body weight ratio, clenbuterol (+)-S induced a small but significant increase in ventricular mass. Both clenbuterol enantiomers produced significant increases in skeletal muscle mass, while being less active in producing cardiac ventricular muscle hypertrophy than the racemic mixture.

Muscle-Specific Effects of Hindlimb Suspension and Clenbuterol in Mature Male Rats

Anabolic agents are useful tools for probing the mechanisms by which muscle fibers perceive and respond to disuse. β2-Adrenergic agonists exert protective, and/or reparative, effects on atrophying muscle tissue. The effects of one such agent, clenbuterol (Cb), were examined on muscle mass, total protein content, and myofibrillar protein content in selected hindlimb muscles [adductor longus (ADL), extensor digitorum longus (EDL), plantaris (PLAN), soleus (SOL)] of mature male rats, under different loading conditions. Pair-fed rats were divided into four experimental groups: vehicle- and Cb-treated nonsuspended, vehicle- and Cb-treated hindlimb suspended (HLS). Experiments lasted 14 days, during which the rats received subcutaneous injections of 1 mg/kg Cb or 1 ml/kg vehicle. HLS induced significant atrophy in all muscles, except the EDL, in a generally fiber type-related pattern. However, myofibrillar protein content was affected in a more regional pattern. Cb treatment of nonsuspended rats induced hypertrophy in all muscles, in a generally uniform pattern. However, myofibrillar protein content was affected in a more fiber type-related pattern. Cb treatment of HLS rats reduced or eliminated HLS-induced atrophy in all muscles, in a muscle-specific pattern. Overall, the ADL and SOL were most susceptible to HLS-induced atrophy. The PLAN had the greatest magnitude of Cb-induced sparing of atrophy. The results show that, in mature male rats, Cb exerts anabolic effects that are load-dependent and muscle-specific. Responses to this drug cannot be reliably predicted by fiber-type composition alone.

Comparative analytical quantitation of clenbuterol in biological matrices using GC-MS and EIA.

A simple and sensitive procedure utilizing GC-MS for the identification and quantitation of clenbuterol in biofluids and tissues is described. This improved method utilizes trimethylboroxine for the derivatization of clenbuterol, requires only 1 mL/g of biological sample, and most importantly does not require an extra cleaning step for urine specimens prior to extraction. Linear quantitative response curves have been generated for derivatized clenbuterol over a concentration range of 5-200 ng/mL. The extraction efficiency at four representative points of the standard curve exceeded 90% in both specimen types (plasma and urine). Linear regression analyses of the standard curve in both specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The Limit of detection (LOD) and Limit of quantitation (LOQ) values for plasma specimens were determined to be 0.5 and 1.5 ng/mL respectively. For urine specimens, LOD and LOQ values were 0.2 and 0.7 ng/microL respectively. Percentage recoveries ranged from 91 to 95% for urine and 89 to 101% for plasma. Precision and accuracy (within-run and between-run) studies reflected a high level of reliability and reproducibility of the method. In addition to its reliability, sensitivity and simplicity, this modified procedure is more efficient and cost effective, requiring less time, only 1 mL of sample, and minimal amounts of extraction solvents. The applicability of the method for the detection and quantitation of clenbuterol in biological tissues of rats treated with the drug was demonstrated successfully. For comparative analysis of clenbuterol in plasma and liver samples, both GC-MS and enzyme immunoassay (EIA) methods are found to be suitable. Due to potential antibody-cross reactivity with EIA, the GC-MS method is the method of choice for most samples because of its specificity. However, the EIA method is considered the method of choice for analysis of clenbuterol found in concentrations below the limits of quantitation by GC-MS due to its sensitivity.

Stable, position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures.

SummaryRetinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA’s effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21–22 and 23–24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA’s effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.

Distribution and Muscle-Sparing Effects of Clenbuterol in Hindlimb-Suspended Rats

Based on their anabolic properties in skeletal muscles, β-adrenergic agonists are of interest as potential countermeasures to microgravity-induced skeletal muscle atrophy. The levels of clenbuterol (Cb), a β2-adrenergic agonist, in both plasma and skeletal muscle were higher in hindlimb-suspended rats than in their nonsuspended Cb-treated controls. Cb treatment was shown to help maintain the body weight in suspended rats, while reducing the amount of mesenteric fat. However, hindlimb suspension attenuated Cb’s lipolytic effects. In skeletal muscle, the magnitude of response to unloading and Cb treatment followed a general regional pattern and was muscle and type specific. The highest magnitude of response to unloading was in predominantly slow-twitch muscles, and the least responsive were the predominately fast-twitch muscles.

Retinoic acid in limb-bud outgrowth: review and hypothesis

Limb outgrowth is arguably the most fundamental aspect of limb development. It begins with the emergence of buds from the embryos lateral body wall. More rapid growth along each buds proximodistal axis than along its anteroposterior or dorsoventral axes yields the limbs basic elongated shape. Many processes that generate refinements of this basic limb form are now being explored at the molecular level. Yet, there remain gaps in our understanding of basic limb outgrowth itself. This review examines the pivotal role of the apical ectodermal ridge in promoting and maintaining limb-bud outgrowth. It discusses the interplay between the apical ectodermal ridge and the subridge limb mesenchyme. It examines evidence that the pattern of limb anomalies in the offspring of mothers exposed to exogenous retinoids such as retinoic acid strongly suggests interference with apical ectodermal ridge function. It covers evidence that cellular retinoic acid-binding protein in the cytoplasm of the cells under the apical ectodermal ridge limits the effects of retinoic acid, a potent retinoid and teratogen, on retinoic acid-driven gene transcription. It explains that retinoic acid generally enhances differentiation in a variety of cell types. On the basis of the information presented, it is suggested that the limb ectoderm promotes cellular retinoic acid-binding protein expression in the subridge mesenchyme and thereby limits the access of retinoic acid to its nuclear receptors in these cells. Cellular-retinoic-acid-binding-protein-mediated, local sequestration or inactivation of free retinoic acid is suggested as a prerequisite not only for the continued responsiveness of the distal mesenchyme to growth promotion by the apical ectodermal ridge, but to the maintenance of the apical ectodermal ridge itself by the subridge mesenchyme.

Changes in lymphoid tissue after treatment with a gonadotropin releasing hormone antagonist in the neonatal marmoset (Callithrix jacchus).

PROBLEM: The effect of neonatal treatment with a gonadotropin releasing hormone (GnRH) antagonist on the morphology and distribution of lymphocytes in lymphoid tissue of the infant marmoset was examined.

Implementation and modification of an anatomy-based integrated curriculum

Morehouse School of Medicine elected to restructure its first‐year medical curriculum by transitioning from a discipline‐based to an integrated program. The anatomy course, with regional dissection at its core, served as the backbone for this integration by weaving the content from prior traditional courses into the curriculum around the anatomy topics. There were four primary goals for this restructuring process. Goal 1: develop new integrated courses. Course boundaries were established at locations where logical breaks in anatomy content occurred. Four new courses were created, each containing integrated subject content. Goal 2: establish a curriculum management team. The team consisted of course directors, subject specialists, and a curriculum director. This team worked together to efficiently manage the new curriculum. Goal 3: launch contemporary examination and question banking methods. An electronic system, in which images could be included, was implemented for examinations and quizzes, and for storing and refining questions. Goal 4: ensure equitable distribution of standardized examinations and course grading systems among all courses. Assessments included quizzes, in‐course examinations, and National Board of Medical Examiners® (NBME®) Subject Examinations. A standard plan assigned the contribution of each to the final course grade. Significant improvement was seen on subject examinations. Once the obstacles and challenges of integration were overcome, a robust and efficient education program was developed. The curriculum is expected to continue evolving and improving, while retaining full regional dissection as a core element. Anat Sci Educ 10: 262–275.