1) Characterization of the Human Cytonegalovirus Transcriptional Repressor, pUL34. B. Biegalke, A. Branda, and E. Lester. Department of Biomedical Sciences.
Human cytomegalovirus encodes several proteins that contribute to viral evasion of the immune system. One immune evasion gene, the US3 gene, encodes a protein of 22 kD that retains MHC class I heavy chains in the endoplasmic reticulum. Following infection, expression of the US3 gene is first activated and then repressed, with repression occurring by 5 hpi. US3 transcription is repressed through the expression of another viral gene, the UL34 gene. The protein encoded by the UL34 gene (pUL34) binds to a DNA element (a pUL34 binding site) in the US3 regulatory region to repress US3 transcription. pUL34 is composed of a basic amino terminal half that contains the DNA-binding domain of the protein and an acidic carboxyl terminal half that is required for transcriptional repression. The cytomegalovirus genome contains several other predicted pUL34 binding sites. pUL34 binds to the predicted binding sites and affects the expression of other viral genes that contain the binding sites. Deletion of the UL34 gene from the viral genome prevents viral replication, demonstrating that UL34 is an essential gene and suggests that pUL34 controls critical steps in viral replication.
2) Energy balance or limb alignment? A look at the trailing and leading limbs of galloping dogs. A. Biknevicius and N.Stevens. Department of Biomedical Sciences.
Limb pairs in galloping animals are distinguished as leading and trailing by their position: although the trailing limb lands first, it occupies a position behind the leading limb when both are on the ground. Alexander, Jays and Ker (1980) posited that animals should make use of tendon elasticity as a passive spring mechanism to reduce the metabolic energy costs of locomotion, and their model predicts trailing limbs to be relatively more accelerative and leading limbs to be relatively more decelerative in order to balance negative and positive work across the limb pair. Subsequent studies have yielded equivocal results. The present study combines force platform data with synchronized high-speed videography for a large sample of dogs utilizing a rotary gallop. Our results suggest that while peak vertical forces and breaking impulses of leading and trailing hindlimbs are largely equivalent, trailing hindlimbs take on a greater role in accelerating forward the center of mass. Because a greater braking role was not observed in the leading limb, the energy balance hypothesis is unsupported. However, a more potent factor driving limb pair function may be maintenance of low bone stresses at high speeds. Hence, animals may act to reduce stresses on hindlimbs by altering the effect of substrate reaction force on bone stress via changes in limb joint angles. This limb alignment model predicts that peak ground reaction forces will more closely align with the long axis of the long bones in both leading and trailing hindlimbs, thereby reducing bending strains experienced in that region. We evaluate this model for the tibia.
3) Is Caffeine an Effective Diuretic. K Binns and J. Howell. Department of Biomedical Sciences.
The purpose of this study was to examine if caffeine had diuretic effects in subjects under normal ambulatory conditions. The hypothesis was that under ambulatory conditions, a diuretic effect will not be seen with caffeine ingestion. Participants refrained from caffeine and alcohol use for a continuous 72 hour period. The first 24 hours was used as a “caffeine wash-out” period. During the second 24 hours – used as the “control” period – subjects measured and recorded all fluid intake and urinary output. The third 24 hour period was used as the “experimental” period, subjects continued to measure and record all fluid intake and urinary output. In addition, subjects took two capsules each containing either placebo or 200 mg of caffeine at the beginning of the experimental period. Data consisted of subjects’ recorded fluid intakes and urine outputs for day two and day three of the experiment.
Increased urinary output was observed in the caffeine group suggesting diuresis following ingestion of caffeine capsules versus placebo. More convincing effects were seen during the eight hour period following the time in which the first capsule was taken.
4) The Virtual Haptic Back. J. Howell*, R. Conatser*, A. Chila**, D. Eland**, R. Williams II***, and M. Srivistava***. Departments of Biomedical Science*, Family Medicine **, and Mechanical Engineering***
The Virtual Haptic Back is a computer simulation of palpation of the human back designed to assist in teaching and testing palpatory diagnostic skills. Two years of software development, supported by 1804 Funds from Ohio University, have produced a unit permitting the operator, with his/her finger inserted into a thimble-like device on the end of a mobile mechanical arm, to “feel” the skin and the underlying bones of the vertebral column. The back of the virtual patient being “felt” (=palpated) appears on a computer graphics screen. By pressing through the skin, which on the graphics screen dimples as it is being pressed, to the transverse process below, the operator can rotate the vertebra in the horizontal plane. With pull down menus, any vertebra can be programmed to be rotated out of position or to be unusually stiff to rotation. The task of the student operator is to locate by feel the dysfunctional vertebra and diagnose the problem. Further development of the Haptic Back will be aimed at increasing the degree of realism of the palpatory experience, and inclusion of soft tissues in the simulation and of multiple dimensions of rotation of the vertebrae.
5) Effect of plasma ionized calcium concentration on neuromuscular
excitability-what happened to surface charge theory? N. Ponder and J.
Howell. Department of Biomedical Sciences.
Surface charge theory was first proposed as an approach to understanding the activities of divalent cations on biological membranes, such as the increased excitability experienced with a decrease in the external calcium concentration bathing an excitable cell. Anchored by the work of Stuart McLaughlin et.al in 1971 (J. Gen Physl vol. 78 pp. 667) concerning the presence of fixed negative charges on the membrane surface and their modification by divalent cations, surface potential theory progressed until the research of Clay Armstrong (J Gen Physl vol 79; pp. 935) challenged it with a theory emphasizing divalent cation binding to ion channels. Employing a literature search based on McLaughlin et. al's 1971 paper on the effects of fixed negative charges on membranes and divalent cations; the progression of surface charge theory, its criticisms, contributions to modern membrane electrostatics, and whether or not it provides a relevant explanation for the physical effects associated with hypocalcemic tetany is examined. Surface potential theory may have its short comings yet the binding and screening mechanisms that it provides to explain divalent cation activity on biological membranes can not be discounted and serve as a relevant explanation for the biological effects associated with hypocalcemic tetany.
6) Characterization of Insertion/Deletion Mutations of the tutF, tutD, and tutG Genes of Thauera aromatica Strain T1. B. Bishop, P. Coschigano, and B. Doehle. Department of Biomedical Sciences.
Thauera aromatica strain T1 was isolated for its ability to use the aromatic hydrocarbon toluene as a sole carbon source under denitrifying conditions. It was previously reported that the tutF, tutD, and tutG genes are part of a single operon whose expression is induced in the presence of toluene. We used a genetic approach to examine the roles of these three gene products in the metabolism of toluene. The tutF, tutD, and tutG genes were individually deleted (in part) from the chromosome and the resulting mutants were unable to metabolize toluene. Plasmids carrying individual in-frame gene deletions failed to complement the corresponding chromosomal deletions but complimented chromosomal deletions downstream of the corresponding in-frame deletion. Results from Western and Northern analyses suggest that the regulation of the tutFDGH operon is complex and may require additional factors. It can be concluded that the tutF, tutD, and tutG genes are each essential for the metabolism of toluene in T. aromatica T1 and likely functions as an oligomeric complex.
7) Isolated, Perfused Working-Heart-Lung Preparation. M. Brauner and R. Klabunde. Department of Biomedical Sciences.
The mortality rate following cardiac arrest is extremely high and is related to cellular damage and multi-organ dysfunction, which result from whole-body ischemia and reperfusion (I-R) injury. Injury is in part mediated by reactive oxygen species (ROS) produced in response to I-R, and may damage many distant otherwise healthy organs. Of particular interest are the independent and dependent roles of the lungs and heart in producing cardiac dysfunction. We will evaluate the hypothesis that ROS, derived from the lungs in response to I-R, travel to the heart via the pulmonary venous circulation and cause cardiac dysfunction. To test this hypothesis we have developed an isolated, perfused working-heart-lung model in order to use the heart to detect ROS produced in the lungs in response to global I-R produced by cardiac arrest and resuscitation in vivo. Using the model, we will be able to physiologically quantify the relative contribution of ROS formed in the heart and lungs on myocardial dysfunction. The model works by creating an ex vivo preparation that functions as if it were in vivo. Rat heart and lungs are surgically removed together with their vasculature and airway intact. The preparation is then hung on a Langendorff apparatus, which facilitates vessel cannulation, ventilation, instrumentation and environmental control. The heart-lung preparation is fully instrumented to record physiological parameters real-time using a data acquisition package. The results of this research may allow us to develop better therapeutic modalities for multi-organ system dysfunction (particularly cardiac dysfunction) associated with cardiac arrest and resuscitation.
8) The Affects of Bradykinin on the Coronary Vasculature of Beating and Potassium-Arrested Hearts. E. Clifford and R. Klabunde, Department of Biomedical Sciences.
The modulatory role of reactive oxygen species (ROS) on coronary vascular smooth muscle responses to bradykinin was studied in isolated, Langendorff-perfused, beating and KCl-arrested rat hearts. We tested the hypothesis that antioxidants would enhance bradykinin-induced vasodilation by scavenging ROS, which deactivates nitric oxide. Hearts were perfused under constant flow conditions with an oxygenated Krebs-Henseleit buffer solution. For beating hearts the buffer solution contained U46619 (10µm) to generate tone. To arrest hearts and generate tone, 40 mM KCl was added to the buffer solution. Each heart, whether beating or arrested was tested using buffer that contained antioxidants (ascorbic acid, TEMPO, or superoxide dismutase/catalase) or control. Coronary responses to bradykinin (10-8 M, 10-7 M, 10-6 M) and sodium nitroprusside (10-6 M) were studied. Infusion of bradykinin to the KCl-arrested hearts produced concentration-dependent vasodilation in the control hearts. Antioxidants, instead of enhancing bradykinin-induced vasodilation as we hypothesized, reversed the vasodilation to vasoconstriction. These results cannot be explained by ROS scavenging nitric oxide, and further suggest that bradykinin-induced vasodilation is not directly caused by nitric oxide in KCl-arrested hearts. We are currently examining the role of antioxidants on bradykinin-induced vasodilation in the beating heart to determine if depolarization by high KCl affects bradykinin responses in the presence and absence of antioxidants.
9) New species of artedidraconid fishes from the Ross Sea, Antarctica. J. Eastman. Department of Biomedical Sciences.
The 24 species of plunderfishes of the notothenioid family Artedidraconidae are an important component of the bottom fauna of the Antarctic shelf and upper slope. During two recent cruises of the R/V Nathaniel B. Palmer in the southwestern Ross Sea, trawling yielded 142 artedidraconids comprising 14.5% of the catch of 979 specimens. The collection includes 10 of the 16 artedidraconid species inhabiting the Ross Sea as well as two new artedidraconid species and a distinctive, but undescribed, color morph of a known species. This information is summarized and illustrated in the poster. The first new species is a large representative of the dorsally spotted group of the genus Pogonophryne. It has a long bicolored barbel, with the epidermis of the terminal expansion folded into brain-like convolutions. The second new species, represented by two specimens, is a member of the robust group of species of the genus Artedidraco. It is most similar to A. orianae, but is distinguished from this species by higher counts for dorsal rays and vertebrae, and lower counts for upper lateral line scales. The specimens were taken in a sponge bed at a depth of 130 m near Franklin Island. Supported by NSF.
10) Two new species of snailfishes genus Paraliparis (Pisces: Liparidae) from the Ross Sea, Antarctica. J. Eastman. Department of Biomedical Sciences.
Two new species of Liparidae (snailfishes) were captured in the southwestern Ross Sea, Antarctica (74-75°S, 165-171°E). Paraliparis macrocephalus and Paraliparis rossi are described and illustrated in the poster. A color photo of a live specimen of Paraliparis macrocephalus documents the extensive subdermal gelatinous tissue, a buoyancy adaptation. Paraliparis macrocephalus has the pigmentation pattern and non-visual sensory specializations typical of a deep living liparid. Paraliparis rossi is similar to Paraliparis macrocephalus in meristic counts, pectoral girdle structure and morphometry but differs in having smaller cephalic pores and nostril, teeth with lateral shoulders, lighter color and shallower habitat. These two snailfishes, as well as two new species of artedidraconids, were taken during 15 hours of bottom trawling in the southwestern Ross Sea. With a total of four new species (9%) among the 46 species collected during this relatively small trawling operation, it is obvious that the Ross Sea fish fauna is not completely described and documented. Paraliparis macrocephalus inhabits the deepest locality in the Ross Sea – the Drygalski Trough, a 1200 m-deep inner-shelf depression. Inner-shelf depressions are trenches eroded by outlet glaciers close to the continental margin of the Antarctic shelf. Video from a camera towed at this station and trawled specimens documented the fauna of these little-studied habitats. Supported by NSF.
11) Development and Validation of a Physician-Specific Diabetes Health Locus of Control Questionnaire. C. Flaugher and J. Shubrook. Department of Family Medicine.
The Diabetes-health locus of control score of the physician affects the metabolic control of the patient through the doctor-patient relationship. This project consisted of the generation and validation of a Physician-Specific Diabetes Health Locus of Control Questionnaire for use in our pilot study. The Diabetes Health Locus of Control Questionnaire was adapted to be administered to physicians rather than to diabetic patients. The questionnaire was then distributed to physicians associated with OU-COM. Sixty were distributed and 42 were returned. The questionnaires were scored and analyzed for internal reliability and consistency using the SAS statistical program. We found that all subscales of the questionnaire demonstrated fair to weak internal consistency and reliability. We identified that if one question was removed we could increase the reliability of the questionnaire but this would limit the use of the instrument for larger trials. Instead, we are currently generating a larger sample size, testing it on a population of physicians who regularly treat diabetes and who come from a variety of backgrounds (private practice, large group, or academic) to determine if our adapted questionnaire can achieve the internal consistency and reliability of the original questionnaire. An interesting result of this study is that the physicians whom we interviewed felt that the patients themselves, rather than “powerful others” or “chance”, have the most influence over their diabetes. This research was sponsored through the OU-COM Research and Scholarly Advancement Fellowship.
12) Cytokine Inducible Antimicrobial Activity in Respiratory Epithelial Cells. K. Goodrum, S. Zwick, and K. Prock. Department of Biomedical Sciences.
This study describes a cytokine-inducible antimicrobial activity in respiratory epithelial cells (EC). Group B streptococci (GBS), a leading cause of neonatal pneumonia, sepsis, and meningitis, survived poorly compared with controls in infected and cytokine -activated EC. A combination of cytokines (IL-1b, TNF-a, and IFNg) were necessary for induction of optimal antibacterial activity. Treatment of EC with recombinant cytokines or with conditioned medium (CM) from GBS-treated human mononuclear cells (MNC) reduced recoverable GBS by 95% (pre-invasion treatment) or by 75% (post-invasion treatment). Cycloheximide inhibition of protein synthesis in EC blocked cytokine induction of the antimicrobial activity. The role of cytokine-induced nitric oxide (NO) in this antibacterial activity was tested by monitoring GBS survival and NO production in cytokine-treated EC that were exposed to L-arginine analog inhibitors (L-NAME) of NO production. Though L-NAME significantly blocked NO induction in A549 cells, reduction in NO did not enhance survival of GBS. These results indicate that innate proinflammatory cytokines released from GBS infected phagocytes can induce an antimicrobial state in epithelial cells. Though cytokine treatment is associated with NO production by EC, the known antimicrobial actions of NO appear unnecessary in this killing mechanism. Such a response may help reduce GBS survival in neonatal pneumonia and reduce transcytosis of the respiratory epithelium by GBS leading to sepsis and meningitis.
13)
Active Transmission of Trypanosoma cruzi between sylvatic animals and
humans in two farming communities of the coast of Ecuador.
Mario J. Grijalva,
Cristina Peñaranda*, Jaime Costales*, Miguel Pinto*, Mauricio Lascano*.
Department of Biomedical
Sciences, *Catholic University, Quito, Ecuador.
Chagas’ disease is caused by Trypanosoma cruzi and affects an estimated 18 million people in Latin America. During the summer of 2001, a multidisciplinary study was conducted in two communities located in Manabi province, Ecuador. Blood samples from humans (n =756), dogs (n = 146) and small rodents and marsupials (n = 26) were collected in Cruz Alta and Pimpiguasi. Seven of the twenty-eight animals captured (25%) were positive by Polymerase Chain Reaction (PCR), using four different sets of primers specific for T. cruzi DNA, hemoculture or direct microscopy. Serology using ELISA showed 8.5% seropositivity in humans (13% and 6%, respectively) and 4.8% seropositivity in dogs. Manual search for kissing bugs was conducted in the domicile and peridomicile. Nymphs and adults of Rhodnius ecuadoriensis and Triatoma dimidiata were found in the peridomicile, mainly associated with palm trees, palm tree leaf debris, opossum burrows and rat nests (Rattus rattus). However, only adult insects were found inside of the domicile. These data indicates that these triatomine species are not reproducing inside of the domicile and that an active peridomiciliary and sylvatic cycles of transmission may be occurring.
14) Bone Morphogenetic Protein 4 (BMP4) inducs overgrowth of the embryonic blood island: signaling pathway from BMP4 to vascular endothelial growth factor (VEGF). C. He, 2Y-S. Li, K. Walker, X. Chen. Department of Biomedical Sciences.
Bone Morphogenetic Protein 4 (BMP4), a member of TGFb superfamily, functions as a morphogen and cytokine and regulates the mesodermal development and the dorsal-ventral patterning during vertebrate embryonic development. Microinjection of the expression constructs of pCIhBMP4 or pCIzBMP4, which expressed human BMP4 or zebrafish BMP4, into zebrafish zygotes persistently led to over growth of the embryonic blood island, which contains the nest of endothelial cell progenitors and differentiating blood cell progenitors, arising from the intermediate mass of mesoderm. Whole-mount In-situ hybridization to those who constantly expressed exogenous BMP4-EGFP, showed that zebrafish vegf gene, VEGF receptor-2 gene (flk-1), an endothelial cell marker, and zebrafish smad1 gene, which is one of the direct signaling transducer of BMP4, were over-expressed on the area of the overgrown blood island, compared to those seudo-injected embryos. It is hypothesized that the expression of VEGF and the over-growth of the blood island area are involved in BMP4 signaling.
15) Cloning and Characterization of Toluene Regulated Genes in Thauera aromatica strain T1. B. Henderson-Dean and P. Coschigano. Department of Biomedical Sciences.
The denitryfing bacterium Thauera aromatica strain T1 is capable of degrading toluene and using it as its sole carbon source. The initial step of this degradative pathway is catalyzed by benzylsuccinate synthase, which adds fumurate to the methyl group of toluene to form benzylsuccinate. Benzylsuccinate is then degraded to benzoyl-CoA through a modified b-oxidation pathway. The genes encoding bezylsuccinate synthase have been cloned from T. aromatica T1 and a similar bacterium T. aromatica K172; however differences in organization exist between these two strains. Genes responsible for b-oxidation of benzylsuccintae (bbs genes) have only been cloned from T. aromatica K172. Experimentation was undertaken to determine if T. aromatica T1 contains similar bbs genes, and if they were present, what differences exist between the two strains. We report the cloning of nine bbs genes from T. aromatica T1. Sequence analysis has shown that T. aromatica K172 and T. aromatica T1 bbs genes are 90-100% identical at the nucleotide level. Northern analysis suggests that the transcriptional organization of the two strains may differ. Current experimentation is focusing on identifying transcriptional start sites. Additionally, we are trying to delete these cloned genes from the chromosome of T. aromatica T1.
16) Role of Aging and Satellite Cells in Exercise-Induced Muscle Damage. L. Hilton-Taylor and R. Hikida. Department of Biomedical Sciences.
Introduction. Recovery from exercise-induced muscle damage is slower in the elderly than young people. Satellite cells, responsible for this recovery, tend to decrease with age. Therefore we investigated whether satellite cells were less active as aging occurred. Methods. Exercise-induced muscle damage was brought about by a one-time running exercise (90 mins, downhill at 11 m/min) on adult (11 month old) and aged (22-23 months old) rats. We examined satellite cell parameters and changes in muscle regulatory factor expression as an indicator of soleus muscle activation, comparing with control rats at different times after exercise. Results. Old animals had a similar fiber type distribution and fewer satellite cells, fiber size, and nuclear domain size than young adults. Also, young and old rats showed a similar time course of expression of myogenic factors (MyoD, myf-5, MRF-4 and myogenin) after exercise. There was no impairment in the function of satellite cells with age. Conclusion. Delay in repair of exercise-induced damage in the elderly is not due to impairment of satellite cells to replicate, mature, or fuse with existing myofibers.
17) Analysis of alternatively spliced Manduca sexta allatotropin mRNAs predicts the synthesis of additional bioactive peptides in a tissue- and developmentally-regulated manner. K.-Y. Lee, M. Chamberlin, and F. Horodyski. Department of Biomedical Sciences.
The study of neuropeptides in insects permits the analysis of the function and action of this class of compounds in an experimentally manipulable organism. We are focusing our studies the neuropeptide, allatotropin (AT), in the tobacco hornworm, an important model for the study of insect endocrinology. AT is best known for its role in stimulating the synthesis of juvenile hormone (JH) by the corpora allata (CA), an endocrine gland connected to the brain. The precise control of JH synthesis is critical because of its pivotal roles in preventing metamorphosis in larvae and stimulating reproductive processes in adults. In addition, we showed that AT inhibits active ion transport across the midgut epithelium, suggesting a role for AT in the control of nutrient absorption. The AT gene is expressed as three alternatively spliced mRNAs predicted to encode distinct precursor proteins. AT can be derived from each precursor, but two of the mRNAs can also encode a total of three additional allatotropin-like (ATL) peptides. The biological activities of ATL peptides overlap with those of AT. We demonstrate that alternative splicing is dynamically regulated in a tissue-specific and developmentally-regulated manner. The level of one of these mRNAs is dramatically increased in response to starvation, parasitism and an ecdysteroid agonist. Thus, alternative splicing might provide a mechanism to increase the potency of the overall physiological response mediated by the expression of a neuropeptide gene, and the increased levels of a specific AT mRNA may be part of the complex response of larvae to nutrient deprivation.
18) Affect and cortisol in older adults: state and trait effects. G. Ice, A Katz-Stein, Department of Social Medicine and R. Kane, University of Minnesota, Minneapolis, MN.
Cortisol has frequently been used as a stress marker and has been shown to be elevated in response to severe stressors and acute laboratory stress. Recent research has also demonstrated that cortisol is associated with daily hassles and affect, measured on a momentary basis. The majority of cortisol and stress research, however, has been conducted on young adults. This study examined the relationship of cortisol to stressors and affect measured both by interview (trait variables) and on a momentary basis (state variables) in a sample of relatively healthy older adults. Forty-eight older adults (Mean age = 76.4 ± 5.8) were interviewed regarding health, stress, affect and social networks. Participants collected saliva every 2 hours over a 3-day period while keeping a record of their emotions and activities. Cortisol was assayed using RIA. Data were analyzed using a random mixed effects model. In univariate models cortisol was associated with age (p<0.0001), time of day (p <0.001), and affective traits including stress level (p = 0.01), positive affect measured in interview (p = 0.005), positive mood measured on a momentary basis (p< 0.0001), negative mood measured on a momentary basis (p = 0.09) and morningness (p = 0.0006). In multivariate models, affect was no longer associated with cortisol. These results indicate that cortisol may not be a good marker for acute changes of affect or stress in older populations.
19) Antimicrobial, Lytic and
Structural Properties of Linear (KL)-Repeat Peptides with Amphipathic
β-Sheet Potential. Y. Jin and J. Blazyk. Department of Biomedical
Sciences.
Linear peptides with the ability to form amphipathic β-sheet structure can possess potent antimicrobial activity and high selectivity between bacterial and human cells [J. Biol. Chem. (2001) 276:27899-27906]. We extended our investigation of this new peptide class by examining several amidated (KL)-repeats containing a single W. Length effects were tested using 18-, 14-, 11- and 10-residue peptides. The influence of tryptophan was studied in a family of (KL)5K-NH2 peptides with W at position 2, 4, 6, 8 or 10. For comparison, we made two other 11-mer peptides containing the same amino acids, one with the ability to form an amphipathic α-helix and the other with no amphipathic potential as either a β-sheet or α-helix. We compared these peptides by measuring antimicrobial and hemolytic activities, their ability to induce leakage in E. coli, and their secondary structures in the presence of lipid vesicles. The longer peptides showed lower antimicrobial and higher hemolytic activity. The placement of W in the (KL)5K-NH2 family was important in determining selectivity. Structural and functional comparisons of these peptides revealed that amphipathic β-sheet peptides as short as 10-residues appear to be promising candidates as potent and selective antimicrobial agents.
20) Acetaldehyde and Glutaraldehyde Effects on Erythrocyte Proteins and Resistance to Oxidatively-induced Hemolysis. R. Dodd, O. Tyulina, V. Prokopieva, A. Boldyrev and P. Johnson. Department of Biomedical Sciences.
The effects of acetaldehyde and glutaraldehyde on erythrocyte hemolysis and protein cross-linking have been studied. When rabbit erythrocytes and their ghosts were treated with acetaldehyde (5 or 50 mM) at pH 8.0, no high molecular weight cross-linked products between membrane proteins and cytoskeletal proteins were detected. However, such treatment prevented the complete extraction of monomeric actin and spectrin molecules from the erythrocyte ghosts. Glutaraldehyde (up to 50 mM) also prevented the extraction of actin and spectrin from erythrocyte ghosts, but contrary to acetaldehyde, induced cross-linking between membrane and cytoskeletal proteins under similar conditions. At pH 8.0, both 5 mM and 50 mM acetaldehyde decreased the hemolytic stability of erythrocyte membranes against NaOCl-induced hemolysis in a dose-dependent manner, whereas 5 and 50 mM glutaraldehyde at pH 8.0 caused a pronounced increase in erythrocyte stability against NaOCl action. These studies show that protein modification by acetaldehyde without cross-linking decreases erythrocyte stability, whereas protein cross-linking caused by glutaraldehyde stabilizes the erythrocyte against hemolysis. It is concluded that the mono- and di-aldehydes have quite different effects on erythrocyte stability although both compounds caused pronounced damage to erythrocyte protein structure. Furthermore, these results indicate that changes in erythrocyte stability to oxidative hemolysis can be caused by different types of modification to erythrocyte proteins.
21)
Role of Reactive Oxygen Species in Hyperglycemia-Induced Arteriolar
Vasoconstriction.
R. Klabunde, K. Prock and
M. Van Hook, Department of Biomedical Sciences.
In uncontrolled diabetic patients, plasma glucose concentrations can rapidly change from 50 to more than 400 mg/dl. These changes in plasma glucose may contribute to vascular dysfunction associated with diabetes. Furthermore, it is known that high glucose levels are associated with increased production of reactive oxygen species (ROS). We investigated the hypothesis that acute hyperglycemia causes arteriolar vasoconstriction by a mechanism involving the formation ROS. Intravital video-microscopy was used to quantify arteriolar diameters in the cheek pouch microcirculation of anesthetized hamsters. The microcirculation was initially equilibrated with 100 mg/dl of glucose dissolved in the buffer solution superfusing the tissue, followed by suddenly increasing the glucose concentration to 500 mg/dl for 60 min. We found that hyperglycemia caused arteriolar diameters to decrease from 25±3 to 15±2 µ (a 37% decrease) after 60 min of exposure. Mannitol, used as a hyperosmolar control, did not alter diameters. To determine the role of ROS, separate groups of animals were pretreated with superoxide dismutase (SOD) plus catalase, or with TEMPO (a cell permeable SOD mimic) prior to exposure to the hyperglycemic superfusate. Pretreatment with these ROS scavengers prevented the hyperglycemia-induced vasoconstriction. Therefore, arteriolar vasoconstriction that results from acute exposure to hyperglycemia appears to be related to the formation of ROS.
22) An extract of Lagerstroemia Speciosa L. has an insulin-like glucose uptake stimulatory activity in 3T3-L1 cells F. Liu, J. Kim, Y. Li, X. Liu, J. Li, and X. Chen. Department of Biomedical Sciences
The effect of the extract isolated from Lagerstroemia Speciosa L. (banaba) on glucose transport in 3T3-L1 cells was investigated. It was found that the banaba hot water extract (BE) and methanol eluent (BME) could mimic insulin to stimulate glucose uptake in 3T3-L1 adipocytes with a similar induction time and dose response. The glucose uptake activity of BE was not non-specific. Furthermore, there were no additive or synergistic effects found between BE and insulin on glucose uptake, and the glucose uptake activity of insulin could be reduced to basal level by adding BE. However, unlike insulin, BE not only could not induce adipocyte differentiation in the presence of 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (DEX), but also could inhibit the adipocyte differentiation of 3T3-L1 cells induced by insulin plus IBMX and DEX in a dose-dependent manner. HPLC separation and cell assay results strongly implied that the glucose uptake stimulatory and adipocyte differentiation inhibitory activities of BE are from the same compound. The final isolation and characterization of the effective compound is in progress. The unique combination of the two activities suggested that BE might be useful for the prevention and treatment of hyperglycemia and obesity in Type II diabetics.
23) Insulin-like
glucose transport-stimulatory activity and an anti-insulin adipocyte
differentiation-inhibitory activity of an extract of Lagerstroemia speciosa
L. X.Liu, Y. Li, F. Liu, J. Kim, J. Li,
and X. Chen. Department of Biomedical Sciences.
The effects of extracts from Lagerstroemia speciosa L. (banaba) on glucose transport and adipocyte differentiation in 3T3-L1 cells were investigated. It was shown that Banaba extract (BE) had an insulin-like activity in stimulating glucose uptake in 3T3-L1 cells; however, unlike insulin, it could not induce differentiation of preadipocyte into adipocyte. In addition, BE inhibited the adipocyte differentiation of 3T3 L1 cell co-induced by insulin, IBMX, and DEX in a dose-dependent manner. The inhibition activity was further demonstrated by reductions of peroxisome proliferator-activated receptor 2 (PPAR2) mRNA and glucose transpoter-4 (GLUT4) protein in preadipocytes cells induced with insulin, IBMX and DEX in the presence of BE. BE’s activities are found to be specific and compounds corresponding to theses activities are being isolated and characterized. BE also inhibited Resistin expression during adipogenesis induced by insulin. This unique combination of BE’s activity may be useful for prevention and treatment of hyperglycemia and obesity in type II diabetics.
24) Studies of Hematophagous Insects and Vector-borne Diseases in the Amazon Basin of Ecuador. M. Neira and W. Romoser. Department of Biomedical Sciences.
In Ecuador, mosquitoes transmit two species of malarial parasites and several arboviruses, including yellow fever, dengue, and human encephalitides. Sandflies, close relatives of mosquitoes, transmit parasites that cause cutaneous leishmaniasis. In collaboration with the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), the Walter Reed Biosystematics Unit, and Dr. R. Leon (Ph.D., Ohio U., 2000), our objectives are to survey the mosquito and sandfly species of the Ecuadorian Amazon Basin, and to test mosquitoes for arboviruses and malarial infection. These vectors and diseases pose serious threats to ecotourists (including those from the U.S.), oil workers, and Ecuadorian citizens in general. In the future, our efforts will hopefully lead to the implementation of prevention and control measures. Through the Tropical Disease Biology Workshop in Ecuador, Ohio University students benefit from exposure to, and participation in, this ongoing disease research project.
25) Porf-1 and 2 Expression in Rat Thyroid Cell Line. K.Nemeth, F. Nowak, L. Kohn, A. Meli and C. Giuliani. Department of Biomedical Sciences.
The preoptic regulatory factors, porf-1 and porf-2, are expressed in the rat brain and testis. Porf-1 is a member of a group of proteins known as selenoproteins. The Type II selenodeiodinase enzyme which catalyzes the conversion of prohormone thyroxine (T4) to the active thyroid hormone Triiodothyronine (T3) is also expressed in the central nervous system and is a selenoprotein along with Porf-1. Porf-1 genomic DNA coding region is situated between the Type II selenodeiodinase coding region and a selenocysteine incorporation signal (SECIS), which is believed to be required for complete translation of the deiodinase gene. This along with the spatial and temporal expression of both Porf-1 and Type II selenodeiodinase enzyme in the brain led us to question whether Porf-1 is regulating the Type II selenodeiodinase gene. To test this we used a rat thyroid cell line known as FRTL-5 to see if we could show them expressing Porf-1 and Porf-2 cDNA by RT-PCR analysis and Northern blotting. Our results did not show expression of Porf-1 in the rat thyroid cell line. The development of a new construct of Porf-1 that contains the ORF for both selenocysteine residues and the SECIS element will be used to test in FRTL-5 cells which contain the Type II selenodeiodinase in future studies. Porf-2 was found to be expressed in these cells. Its expression was greatest in the cells in the presence of hydrocortisone. Further investigation of the regulatory effects hydrocortisone has on Porf-2 will help understand this interaction.
26) Soft-Tissue Influences on Archosaurian Vertebrae: Interpreting Pneumatic and Vascular Features. P. O’Connor. Department of Biomedical Sciences.
Osteological features preserved in fossil archosaur specimens are thought to represent traces of soft-tissue structures such as vasculature and pulmonary air sac diverticula. Inferences concerning physiological processes (e.g., metabolism, ventilation) rely on knowledge of such soft tissue systems. Thus, these inferences require explicit anatomical information regarding the interaction of soft tissues and the skeleton. The goals of this project were 1) to establish sorting criteria that distinguish osteological features indicative of pulmonary pneumatic and other soft-tissue systems, particularly vasculature, in vertebrae of living archosaurs; and 2) to apply these criteria to fossil archosaur groups. Within the context of the Extant Phylogenetic Bracket method, extant archosaur taxa examined included crocodilians and various paleognath and basal neognath birds. Whereas vertebrae of both groups are vascularized, pneumaticity of the postcranial skeleton is limited to living birds. Methods include vascular and pulmonary injections followed by corrosion casting and/or dissection, thus yielding an assessment of causal relationships between skeletal features and specific soft-tissue systems. Fossil archosaur taxa were surveyed for the presence of system-specific osteological correlates identified in extant taxa. Examination of extant taxa reveals that relative foramen size alone does not differentiate between osteological correlates of pulmonary pneumatic and vascular features. However, a profile that combines topography, angle of penetration through the cortical surface, and relative foramen size offers better discrimination between the two systems. Application of these criteria to vertebrae of nonavian theropods (e.g., Majungatholus) reveals the presence of distinct vascular and pneumatic features in this group.
27) Regeneration Following a
Muscle Biopsy. P. Pillitteri and R. Hikida. Department of Biomedical
Sciences.
Introduction. Muscle biopsies, often several, are taken in research human muscle research. Each biopsy removes 60-100 mg of muscle, but it is not known if and how the muscle repairs itself after this procedure. Studying this is not feasible with human subjects, so we designed a rat model. Methods. The gastrocnemius from 20 rats were examined from 3 to 35 days after a biopsy. To study the repair process, we used antibodies to type I collagen to follow connective tissue formation; to developmental myosin to determine the time course of regeneration and maturation of the muscle fibers; to m-cadherin, to assess changes in muscle stem cell population. Serial sections were used to study continuity (if present) across the biopsy site. Results. Collagen, myotubes and myoblasts were prominent to 10 days. By 20 days, regeneration was extensive, with myotubes and extensive collagen crossing the wound site. At 35 days there was less collagen, and regenerated fibers crossed the former gap, forming continuity across the damaged area; however fiber sizes were still significantly smaller than the undamaged fibers. The extensive collagen inhibited regeneration in the surface of the muscle. Satellite cell numbers were higher than controls throughout the study. Myonuclei per fiber and myonuclei per mm fiber were decreased at all time points. Nuclear domain size was the only measure to regain control values by the end of the study. Conclusion. Although regeneration was not completed by 35 days, the muscle was healed from the biopsy. Muscle fibers regained continuity across the wound site.
28) Evidence for Dissemination of Arboviruses via the Mosquito Tracheal System from Studies of Venezuelan Equine Encephalitis and Rift Valley Fever. W. Romoser and M. Neira, L. Wasieloski Jr., G. Ludwig, R. Leon, and P. Pushko. Department of Biomedical Sciences.
Evidence consistent with the dissemination of arboviruses within mosquitoes via the tracheal system is accumulating. In light of the tracheal dissemination hypothesis, we consider results of studies of Venezuelan Equine Encephalitis (VEE) virus using VEE replicon particles, a nucleic acid probe and immunocytochemistry, and results of ultrastructural studies of Rift Valley fever (RVF) virus. Currently we are using a multimethod approach to elucidate VEE pathogenesis in the mosquito Ochlerotatus taeniorhynchus. VEE replicons, which direct the expression of green fluorescent protein (GFP) in infected cells enable us to identify the first tissues and organs infected when virus is injected into the mosquito hemocoel. To detect VEE virus genome, we are using a digoxigenin-labeled (DIG) complementary DNA probe. For detection of VEE antigen, we are using the avidin-biotin peroxidase complex (ABC) immunocytochemical technique with polyclonal antibodies directed against VEE antigens. Based on results with VEE, we have revisited electron micrographs of RVF virus in Culex pipiens. Our results coupled with information from other studies are consistent with the idea of the basal lamina acting as a barrier to arboviruses and the spread of virus from the midgut to other organs and tissues via tracheae/tracheoles.
29)
Rapid and Transient Increase in Connexin 43 Expression in the Deafferented Avian
Cochlear Nucleus. L.
Ross. Department of Biomedical Sciences. J. Hallows, B. Tempel, E. Rubel. V.
Bloedel, University of Washington, Seattle, WA.
Deafferentation of the posthatch avian cochlear nucleus, nucleus magnocellularis (NM), results in the death of 20-40% of the neurons within 2-3 days. We investigated the role of gap junctional communication in cell death by examining deafferentation induced changes in expression of connexins (Cx), the proteins forming the intercellular gap junction channels between adjacent cells. Cochlear removal resulted in a rapid and transient increase in Cx 43-immunolabeling in NM cells. As early as 1 hour after cochlea removal, a few densely labeled NM cells were observed in the NM on the deprived side of the brain, while no labeled cells were observed in the contralateral NM. The number of labeled cells continued to increase unilaterally at 3 hours, with the highest number of labeled cells observed at 6 hours after cochlea removal. Only a few NM cells remained labeled at 24 hours following cochlea removal. In situ hybridization analysis demonstrated that Cx 43 messenger RNA levels rose dramatically in the deafferented NM at 3 hours after cochlea removal. This change may play a role in the degenerative changes caused by loss of synaptic input to NM neurons, or may serve to protect some NM neurons from deafferentation-induced cell death.
30) Inhibition of in vitro intracellular growth of Trypanosoma cruzi by dicationic compounds. E. Heaton, D. Moore-Lai, E. Rowland*, J.Seed, C. Stephens and D. Boykin. *Department of Biomedical Sciences
The development of anti-protozoal chemotheraputic agents has gained interest due to the increasing incidence of Toxoplasmosis and Cryptosporidiosis in immunodepressed individuals. Efforts are being made to develop dicationic compounds, which are derivatives of pentamidine, for use as antiprotozoal drugs. These compounds bind to the minor groove of DNA and are thought to inhibit DNA dependent enzymes and thereby prevent cellular replication by the protozoans. The objective of this study was to test the ability of a group of these compounds to inhibit the intracellular and extracellular reproduction of Trypanosoma cruzi in vitro. At present there are no available drugs capable of inhibiting the intracellular stages of this parasite, therefore, compounds with this ability would be of value. Cultures of mouse fibroblasts were infected and treated with doses of dicationic compounds and the numbers of parasites released at the end of the 5-7 day growth cycle were determined. 6 of the14 compounds tested were found to be effective at inhibiting T. cruzi growth at doses that were not toxic to the host cells. The compound found most effective (DB 709) inhibited parasite release at a concentration of 0.82 ng/ml, justifying further study. These results suggest that dicationic compounds may have potential as chemotherapy for T. cruzi infection.
31) An Examination of the Fascial Continuities of the Iliacus Muscle. D. Eland, K. Russell, and E. Zmuda. Department of Family Medicine.
In Osteopathic Manipulative Medicine, a constellation of symptoms involving the Iliacus muscle are often observed including stress into the sacroiliac joint in the form of anterior innominate rotation, ipsilateral functional short lower extremity, and recurrent backward torsion of the sacrum (Eland, 2001). We also see a depression of the ipsilateral 12th rib and somatic dysfunction at the thoracolumbar junction. This study considers the fascial continuities of the Iliacus muscle in five embalmed cadavers, exploring the underlying mechanism of force transmission through fascia to produce the clinical constellation of symptoms described. The results confirm a continuity of fascia from the level of the iliac crest superiorly to the inferior surface of the diaphragm via the endoabdominal fascia and its reflections as investing fascia of individual muscles of the posterior abdominal wall. Some fibers of both the endoabdominal and investing fascias attach to the periosteum of the iliac crest, but the endoabdominal fascia is continuous through the pelvis and reflects anteriorly to form the transversalis fascia and fascia of the anterior wall. The investing fascia of the Iliacus and Psoas muscles continues into the leg as the muscles pass over the pubic ramus. In the leg, the investing fascia of the Iliopsoas becomes continuous with the investing fascias of the muscles of the anterior compartment of the thigh, and is continuous with unique Iliacus muscle fascicles that descend below the lesser trochanter and attach perpendicularly to the linea aspera. The functional significance of this fascial plane merits further study regarding quantification of force transmission and histological examination; however there is no doubt that it has clinical significance in Osteopathic manipulative diagnosis and treatment.
32) Cephalic Vasculature In Archosauria, With Special Emphasis On The Nasal Region. J. Sedlmayr, Dept. Biol. Sci., L. Witmer, Department of Biomedical Sciences.
The blood vascular system is probably the most poorly understood anatomical system in the heads of archosaurs, which is surprising given that it provides not only a means of nutrient supply but also plays a role in such physiological processes as heat and gas exchange and water economy. Thus, the head vasculature in extant archosaurs is being studied using the extant phylogenetic bracket approach, with an objective of being able to shed light on the physiological functions of extinct archosaurs such as dinosaurs. Vascular anatomy and homologies in extant archosaurs, largely using members of Crocodylia, Paleognathae, and Galloanseromorpha, are determined via a variety of anatomical and developmental techniques. Agreement in vascular patterns among extant taxa comprise the similarity test of homology. Vessels often leave clear osteological correlates (grooves, canals, foramina), and these bony features can be surveyed in the fossil taxa, comprising the congruence test of homology. For example, the subnarial canal (or foramen) of saurischian dinosaurs, passing between maxilla and premaxilla and connecting the narial and palatal regions, has long been a functional enigma, but our research suggests a vascular interpretation. Within the narial region of extant archosaurs, there is an anastomosis between narial and palatal branches of the maxillary vessels passing between the maxilla and premaxilla; the anatomical details pass the similarity test of homology and agree with the relationships observed in saurischians. Congruence testing in other extinct archosaurs is not yet complete, but the vascular hypothesis remains the best supported, perhaps suggesting that narial cavernous tissue may have a broad distribution within archosaurs
33) The Effects of a Structured OMT Curriculum on Osteopathic Exams and OMT for Hospitalized Patients. J. Shubrook. Department of Family Medicine.
Hypothesis: A structured clinical curriculum in OPP/OMT taught to housestaff will significantly increase the use of osteopathic exams and OMT for hospitalized patients.
Methods: In this prospective observational study, the author reviewed the charts of all D.O. FP and IM admissions for the presence of an osteopathic exam and use of OMT during a two-month pre-curriculum period. The author then facilitated a six-month curriculum that included lectures, labs and OMT rounds. At the end of the curriculum a second chart review of all D.O. FP and IM admissions was repeated for the following two-month period. A third chart review was completed one year after the completion of the curriculum. The attending physicians and housestaff were unaware of the chart reviews. Results: The percentage of patients that received osteopathic exams increased from 46.3% to 83.7% (p< 0.0001). One year after the completion of the curriculum the percentage of patients receiving an osteopathic exam was still significantly increased (46.3% to 67.4% p< 0.05). OMT use increased from 0% documented to 4% at the end of the curriculum. One year later documented OMT use decreased to 1.3%. Conclusions: It appears from this study that a structured clinical curriculum in OPP/OMT can have a meaningful and lasting effect on the osteopathic practices of housestaff.
Throughout its history, osteopathic manipulative therapy (OMT) has been a distinctive feature of osteopathic medicine. However, the use of OMT among osteopathic physicians is declining. Today fewer D.O.s use OMT in their practice than ever before (Fry 1996, Aguwa 1999). Those who still use OMT treat fewer patients and less frequently (Fry 1996). Recent evidence has found that this decline begins as early as osteopathic medical school. Many osteopathic students are abandoning the use of OMT even before they graduate from a college of osteopathic medicine (COM) (Fry 1996). Those who wish to include OMT in their clinical practice often express that they have lost confidence in their skills by the time they finish their osteopathic training (Shlapentokh 1991). It appears that the decline in OMT use for student in training is partially explained by the lack of OMT training at the clinical sites. In one study only 30% of third and fourth year students thought there was enough training in OMT at the clinical sites (Fry 1996). These percentages drop to below 20% during internship and residency (Fry 1996). In this study the author examined the level of use of OMT and confidence in the SIRs OMT skills for those who were training at the C.O.R.E. hospitals.
35) A Preliminary Satisfaction Assessment of the Family Practice Residency at Ohio University, College of Osteopathic Medicine. C Simpson, L Presutti, G Moden, A Pheley, Department of Family Medicine
Problem: If we are to increase training and retention in rural areas, we must understand why students and residents choose the programs that they choose. We must be certain that the training programs that we support are meeting the needs of the residents. Methods: We conducted a series of focus groups of family medicine residents, 3rd and fourth year medical students and family medicine faculty at Ohio University. The focus groups discussed satisfaction related to residency training. Topics included: What is important, (personally and professionally), as you consider FM residency training? We conducted a preliminary survey of graduates of the O’Bleness Hospital, Family Medicine Residency. Results: Focus Group: Medical students and residents in a small rural program are satisfied with their training. The location of the program and the needs of their family appear to be more important than academic aspects of the program.
Faculty involved in the residency-teaching program indicate that students come to the residency because of academics. Survey: It appears that this family medicine program is attractive to OUCOM graduates who are married, rooted in the community, and relatively older. AOA approval, rural location and ability to moonlight appear to be positive factors
Conclusions: This is a preliminary investigation, and no final conclusions are available. We intend to revisit the data, refine the survey instrument and conduct a CORE wide survey of family medicine residency programs.
36) Unusual Narial Structures In Dinosaurs & Other Vertebrates. L. Witmer*, S. Sampson**, Department of Biomedical Sciences*, New York College of Osteopathic Medicine**
Several clades of dinosaurs exhibit unusual morphology in the narial regions of their skulls. The most divergent narial anatomy is found in the duck-billed dinosaurs (Hadrosaurinae), horned dinosaurs (Ceratopsidae), and the brontosaurs (Sauropoda), in which the rostralmost portion of the nasal cavity (vestibulum nasi) is apomorphically enormous, far exceeding the size of the rest of the nasal cavity (in most vertebrates the vestibule is the smallest portion of the cavity). The function of these enlarged narial apparatuses remains enigmatic, preventing a comprehensive understanding of skull evolution in Dinosauria. Ideas range from inflatable sacs and fleshy probosces to thermoregulatory devices. Functional hypotheses relating to these unusual narial specializations can be tested, employing a combination of historical and ahistorical approaches that elucidate causal associations between soft tissues and their osteological correlates. The noses of the extant outgroups of these dinosaurs (birds and crocodilians) provide baseline anatomical data. Additionally, modern groups with narial novelties similar to those proposed for dinosaurs (e.g., moose, hooded seals, gharials) serve as analogues or “models” for the dinosaurs. Although in its early phases, this research has already falsified some functional hypotheses while corroborating others.
37)
New Approaches For The Study of Skeletal And Soft-Tissue Anatomy In Modern And
Extinct Vertebrates. L. Witmer*, C. Holliday, P. O'Connor, A. Clifford,
J. Sedlmayr, R. Ridgely, B. Beatty, and T. Hieronymus. Depts. of Biomedical
Sciences* & Biological Sciences
Anatomical investigation requires a variety of approaches, because no single method provides all the data required for biological interpretation. Many animal specimens are rare, and thus several approaches must be applied simultaneously or sequentially to extract as much data as possible. (1) We developed a new stereoangiographic technique whereby blood vessels are injected with a novel medium such that the vessels are radio-opaque yet resilient enough for subsequent dissection; viewing stereoradiographs restores three-dimensionality. (2) Vascular injection using polyester resins, followed by maceration, provides exquisite corrosion casts of the vascular tree. (3) CT scanning allows resolution of anatomical details without destructive dissection. CT permits “virtual dissection,” provides a permanent record of the specimen, and allows digital reconstruction of anatomical components. CT can often “see through” rock, and thus provides a safe means of exploring internal anatomy of fossils. (4) Construction of a hypobaric chamber facilitates the study of the pulmonary system; pressure changes within the chamber, coupled with latex injection and subsequent corrosion, yields high-fidelity casts of the pulmonary system. (5) Molding and casting of skeletal or fleshy elements using silicon rubber and resins provides enduring replicas of anatomical structure. (6) Finally, skeletonization is a typical final step, and involves boiling in enzymes, cold-water maceration, or treatment with flesh-eating beetle larvae.
38) Birth Pangs: A Social History of Obstetric Anesthesia. J. Wolf. Department of Social Medicine.
This history of obstetric anesthesia will answer the following questions: How and why do women’s perceptions of labor pain change over time? How do physicians formulate their perceptions of birth? How do physicians’ perceptions shape obstetric practice? How does physician/patient interaction shape medical decision-making and mold medical practice? How do controversial medical procedures become accepted as standard practice? To answer these questions, I use evidence from women’s letters and diaries, physicians’ personal papers, obstetric logs, hospital records, the papers of birth reform organizations, and articles in medical journals and women’s and general interest magazines. Thus far I have gathered and begun to study thousands of these documents. Preliminary conclusions are: 1. Fear of pain has been the major force shaping the birth experience for American women. 2. Physicians’ and women’s perceptions of labor pain, rather than reality, have played a major role in shaping the administration of obstetric anesthesia. 3. Medical historians have long argued that while birth remained in the home, women controlled the event. I have found, however, that women relied on physicians to define appropriate obstetric treatment even when home birth was still the norm. 4. Between at least 1850 and 1930 American obstetricians were unable to agree on basic procedures in normal labors. Emblematic of this lack of standardization were the vast array of medications and techniques used to alleviate labor pain. 5. Obstetric anesthesia has been instrumental in shaping the obstetric belief system that birth generates pathologies to be averted by standard medical procedures.