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ABSTRACTS FOR THE 71st ANNUAL MEETING OF THE COLORADO-WYOMING ACADEMY OF SCIENCE meeting at the UNIVERSITY OF SOUTHERN COLORADO Pueblo, Colorado April 15, 2000.

1. NEURAL GENERATOR OF AUDITORY BRAINSTEM RESPONSE WAVE II IN GUINEA PIGS. R. Steven Ackley, Shay Darnell, Shannon Burns, University of Northern Colorado; Donald J. Nash, Colorado State University.

An electrophysiological test of hearing of laboratory animals is the Auditory Brainstem Response (ABR) procedure. However, reports of precisely what the ABR represents anatomically are contradictory, and indeed the precise neural origin of each component of the Auditory Brainstem Response (ABR) remains obscure. Identifying neural generators of each wave is a mission of all research electrophysiologists, and the ultimate contribution of this effort to clinical science will be in greater diagnostic accuracy of the ABR test. Assignment of wave I to auditory nerve activity is universally accepted and origin of wave V appears to be in the nucleus of the lateral lemniscus in humans (Moller and Janetta, 1984). However, neural origin of the remaining ABR components is controversial, with acceptable candidates for waves II-IV being auditory nerve and pontomedullary nuclei.

The purpose of the present steady was to give experimental evidence of the origin of waves II and III in guinea pigs after destructive procedure. Historically, animal lesion experiments of brainstem nuclei have provided conflicting data regarding ABR neural generators. Surgical precision in lesioning specific brainstem nuclei in laboratory animals is complicated by interrupted blood supply to adjacent nuclei and disturbed cerebrospinal fluid flow throughout the brainstem, thereby causing a more diffuse lesion than is intended. The lesion method described in this study is noninvasive, and, as such, avoids damage to untargeted centers. Lesion was accomplished by acoustic overstimulation to the cochlear base using 122 dB SPL white noise. Prior work on this topic suggested a DCN origin to wave II and inferential assignment of wave III to NLL.

2. CCK-8 INJECTION INHIBITS ALCOHOL INTAKE IN M-HEP AND WISTAR RATS. Karen A. Alvarado(*) and Paul J. Kulkosky(*). Department of Psychology, University of Southern Colorado.

Recently, R.D. Myers and colleagues selectively bred a new strain of Norway rat that exhibits high voluntary alcohol intake and preference: the Myers-High Ethanol Preferring rat (M-HEP rat). CCK-8 (cholecystokinin octapeptide) is an endogenous neuropeptide that is known to reduce alcohol intake and preference in Wistar rats. Since CCK-8 is hypothesized to function as a satiety factor for alcohol intake, it could act differently in M-HEP rats. Water-deprived Wistar and M-HEP rats (N=6) were given access to 10% w/v ethanol for 30 minutes, followed by 30 min access to water, daily. Rats were randomly assigned to receive intraperitoneal injections of either saline (1 ml/kg) or CCK-8 (2 [micro]g/kg) before alcohol access. The experimental data show there is no significant (p [is less than] 0.05) difference between M-HEP and Wistar rats in response to CCK-8. The peptide strongly and equivalently reduced 10% ethanol intake in both strains of rat. The high ethanol consumption of M-HEP rats is not explicable as a lack of sensitivity to the satiety signal of CCK-8.

3. HIGH THROUGHPUT AUTOMATED PLASMID PREPARATION PROCEDURE UTILIZING 96-WELL DNA BINDING FILTERPLATES. Rod Azadan, James Fogleman, and Philip Danielson(*). Department of Biological Sciences, University of Denver.

We have developed a new automated procedure to extract plasmid DNA using a robotic arm and a micro-filtration process based on the procedure developed by Itoh, et al (1997). This simple, automated high-throughput plasmid DNA extraction procedure uses a modified alkaline lysis method in a 96-well DNA Binding Filterplate. In this method, cell harvesting, lysis and plasmid purification are all performed on the filterplate. After washing out RNAs and other contaminants, plasmid DNA is selectively eluted by a low-ion strength solution. Plasmids prepared by this method can subsequently be sequenced or applied to restriction enzyme cleavage. The development of this newly automated process offers many advantages in an educational/research environment. Pre-fabricated DNA plasmid preparation kits based on column purification strategies are available, though are prohibitively expensive, and centrufugal purification strategies can be prohibitively time consuming. In addition, automation of common procedures greatly increases research productivity. Utilizing this automated process, we were able to perform plasmid preparations with greater accuracy, repeatability and speed than traditional methods.

4. EVIDENCE OF Aztlanolagus (Mammalla:Lagomorpha) AT PORCUPINE CAVE, PARK COUNTY, COLORADO. Georgia C. Baxter(1,3), Paul F. Trione(2,3), Colleen N. Ramos(*)(1,3).

Aztlanolagus (Lagomorpha: Leporidae) is currently considered a monotypic genus from the Irvingtonian NALMA. All known specimens of Aztlanolagus are from New Mexico, Texas, and Arizona. Analysis of fossil remains from Porcupine Cave, Park County, Colorado has revealed evidence of another species. Our team measured and analyzed over 200 p3s and jaws. We noted two size classes and found evidence for the presence of a primitive form of Aztlanolagus at Porcupine Cave. This information is helping to clarify the evolutionary dynamics of the Leporidae during the Irvingtonian.

(1) Community College of Aurora, (2) Metropolitan State College of Denver, (3) Denver Museum of Natural History.

5. AN INTEGRATED EIGHT (8) MEDIA CLASSROOM INSTRUCTION SYSTEM. George Becker(*)(1), Daniel Cordova(2), Donald K. Alford(1).

Laptop computers and digital projection systems make it possible to use high technology instruction everyday in any classroom with a large screen and controlled lighting. The system outlined involves eight (8) different media in a tightly integrated classroom instruction program with completely computerized instruction materials and a computerized (double) parallel projection system in the classroom. The media employed were a customized classroom textbook, a study guide/test bank, an oral presentation, colored slides, projected "flash cards," a commercial textbook, examinations and student lecture notes. The classroom presentation centered on an oral presentation, and two remote control systems for projecting "flash cards," colored illustrations, graphs and tables. Continuously running dual and parallel computerized projection systems also provide a wonderful matrix for the addition of many other, high technology classroom instructional methods. The results in the demonstration class were very positive, both for the grade distribution and the reaction of the students to the instruction system. The oral presentation, projected colored illustrations and projected flash cards were selected by 94% of the students as valuable, quite valuable or very valuable to their classroom learning experience, but 86% selected the study guide/test bank as the most valuable media in preparing for examinations!

(1) Department of Biology, MSCD, (2) Tucson Public Library.

6. HARMONIC MOTION REVISITED: THE DE BROGLIE WAVE THEORY. Yardley Beers(*), retired.

The correspondence principle provides the basis of a de Broglie wave theory of the linear harmonic oscillation of a particle of mass m and charge e with a classical amplitude A and angular frequency [Omega] (wavelength [[Lambda].sub.e]). One basic assumption is that the mean de Broglie wavelength [[Lambda].sub.d] is given by substituting the rms velocity into the standard formula for the wavelength, it can be shown that if the oscillating particle is an electron, [[Lambda].sub.d]= ([[Lambda].sub.c][[Lambda].sub.e]) / ([2.sup.1/2][Pi] A), where [[Lambda].sub.c] is the Compton wavelength. A second assumption is that the total distance covered in one oscillation 4A is equal to (n + 1/2) [[Lambda].sub.d], where n is any integer. These assumptions lead to an energy relation identical to that of wave mechanics except for a numerical factor approximately unity. The first power dependence of energy upon frequency evolves naturally from the classical second power dependence. The "1/2" term implies that, contrary the popular belief, energy levels, except for the ground state, are situations of great instability, and an excited system spends most of its time radiating and a negligible time in intermediate excited states.

7. A NON-INVASIVE MODEL FOR ESTIMATING BODY COMPOSITION IN WILD DEER MICE (Peromyscus maniculatus). Michael J. Blair, Matthew E. Granger and Edward T. Unangst, Jr.(*) Department of Biology, United States Air Force Academy, Colorado Springs, CO.

Non-invasive techniques such as EM-SCAN (EM-SCAN, Inc, Springfield, IL) allow researchers to accurately estimate body composition to include measurements of specific individuals over time. The EM-SCAN device creates a 10 MHz field within a chamber and measures the disturbance of this field based of the relative concentration of positively charged cations present in lean tissue versus fat tissue (40:1). We derived a species-specific multiple regression estimation equation using total body length, total wet mass, and the unitless EM-SCAN reading as the independent variables and either lean (fat-free) or lipid (fat) mass as the dependent variable. Resulting regression equations were: fat-free mass = -0.04 + 0.92 (wet mass) + 0.03 (EM2); [r.sup.2] = 0.99 and lipid mass = 0.04 = 0.07 (wet mass) -0.02 (EM2); [r.sup.2] = 0.53, where EM2 = square root of (EM reading x total body length). Both equations demonstrate very strong predictive power for non-invasive estimates of both fat-free and lipid mass (0.29 [+ or -] 0.03 g error), even for animals with very low levels of body fat (3.44 [+ or -] 0.17%).

8. ANALYSIS OF KIN RECOGNITION BY LARVAE OF THE AMAZONIAN DART-POISON FROG, Dendrobates ventrimaculatus (Dendrobatidae). Ryan J. Carr and Donald J. Nash(*), Colorado State University.

Frogs of the genus Dendrobates are well known for the complicated parental behavior displayed by one or both sexes. Eggs are guarded and moistened until hatching. Tadpoles are then transported to a suitable water source. Some species will then lay eggs (fertilized or unfertilized) on the edge of the pool of water containing a tadpole, it has been shown, in several species, that consumption of these eggs is necessary for proper development of the tadpole in the pool. Tadpoles of Dendrobates ventrimaculatus do not require these "food eggs" for proper development, but will occasionally consume available eggs. Cannibalism between tadpoles is also common in this and closely related species. Given their relatively low rate of reproduction, cannibalistic and egg eating behaviors of tadpoles could impose a high cost on the adults. Ability of the tadpoles to recognize and not consume close relatives could present a distinct advantage. This study seeks to determine the extent to which tadpoles of Dendrobates ventrimaculatus recognize closely related tadpoles and eggs. Preliminary data and results will be presented.

9. COMMON SONIC FEATURES OF TREE SWALLOW ALARM, DISTRESS, AND TEE-TEE CALLS. Robert R. Cohen(*). Department of Biology, Metropolitan State College of Denver.

The alarm, distress, raspy, and tee-tee calls of the Tree Swallow (Tachycineta bicolor) all have negative connotations and/or are given in negative or agonistic contexts. The source and function of the tee-tee call, which is given during brief agonistic interactions between adults, are unclear. I tape-recorded Tree Swallows in North-central Colorado from 1983 through 1998 and developed sonagrams of their vocalizations with the PC software program Avisoft Sonagraph Pro. The distress, alarm, and tee-tee calls differ in mean pitch, while the distress and raspy calls have much wider pitch range than do the others. The alarm, distress, and tee-tee calls all tend to have a very rapid zigzag, or saw-tooth, pattern of pitch; this pattern is more distinct in the distress calls of nestlings and juveniles than in those of adults. Although the raspy call, given only by adults in agonistic situations, apparently lacks the zigzag pattern, it is probably derived from the distress call. The zigzag of the tee-tee call suggests that the call is an unusually high-pitched form of alarm call or distress call and supports the hypothesis that it is given by the recipient of aggression, rather than by the aggressor.

10. SONAGRAM COMPARISONS OF TREE SWALLOW AND VIOLET-GREEN SWALLOW CALLS. Robert R. Cohen(*). Metropolitan State College of Denver.

I recorded Tree Swallows (Tachycineta bicolor) (TS) and Violet-green Swallows (T. thalassina) (VS) in Colorado and developed sonagrams with Avisoft-Sonagraph Pro. Only the distress and young-nestling begging calls are very similar in both species. The VS alarm call, a pure note of falling pitch, is similar to only the brief last component of the TS alarm call, and is much more similar to the TS anxiety call, which typically has both falling and rising pitch components but sometimes lacks the rising end; some VS alarm calls have a very brief rising-pitch end. The TS raspy aggression call, given in agonistic interactions, resembles the VS raspy call, given to an intruding nest-site competitor, but only the latter has rising pitch; both resemble the distress call. Compared to the TS pleasure call, the VS pleasure call has fewer notes in its first part, and its last part is much simpler in structure and its notes are given much more slowly. The TS contact call, VS distant-contact call, and nest-site advertising call of both species are obviously similar to the begging calls of their fledglings, but the VS close-contact call instead resembles the contact call of the Cliff Swallow and the Northern Rough-winged Swallow.

11. CREATION OF GFP FUSION PROTEINS AS A SCREENING MARKER FOR TRANSFORMED Escherichia coli. Frank J. Conrad, Lara Vigil, Abdullah Al-Zahrani, Phillip B. Danielson(*). Department of Biological Sciences, University of Denver.

Screening bacterial transformants for antibiotic-resistance has become a commonplace and simple way of identifying colonies containing target vectors. Secondary screening often requires more time-intensive methods, such as PCR or restriction digest analysis, to verify the correct incorporation of target DNA into the bacterial plasmid. A recombinant bacterial expression protein containing Green Fluorescent Protein (GFP) has been constructed in an attempt to produce a novel and instantaneous means of screening colonies. Upon absorption of UV light at 395 nm and 470 nm, colonies transformed with GFP-containing plasmids emit light in the visible range at 509 nm. Any target DNA sequence can modified through primer amplification to be inframe with the expression vector and inserted via a single restriction site at the beginning of the GFP sequence. Upon translation, this should create a functional GFP fusion protein, which expresses the desired insert. Truncated or backward should interrupt the reading frame, resulting in a non-functional fusion protein. The current study investigates the applicability and efficiency of this approach. Successful construction of fusion proteins will offer a less-costly and more time-efficient alternative to traditional methods of colony screens.

13. HOW TO IDENTIFY AN EQUATION OF STATE WHICH IS SUITABLE FOR THE PREDICTION OF LIQUID-PHASE BEHAVIOR. J. G. Eberhart(*), Department of Chemistry, University of Colorado, Colorado Springs, CO.

A wide variety of equations of state have been proposed for the characterization of the p-v-T relationship for real gases. All of these equations have a van der Waals loop in their sub-critical isotherms, suggesting that they can be used to characterize the gaseous and the liquid phases of a fluid, as well as transformations between these two phases. However, not all of these equations have the other features required for liquid-state accuracy, such as (1) a metastable liquid region that can provide negative pressures (or tensile states) at sufficiently low temperature, and (2) a molar volume that approaches a non zero value in the limit of high pressure. Various equations will be compared with these criteria and those that are satisfactory for liquid-phase use will be identified.

14. BEING SENSITIVE TO THE WORLD VIEWS OF OUR CHEMISTRY STUDENTS. J. G. Eberhart(*), Department of Chemistry, University of Colorado, Colorado Springs, CO.

Occasionally topics arise in Chemistry courses that have a religious or philosophical dimension to them in these situations a brief discussion of the various Western World Views and their degree of compatibility with the theory under consideration can be helpful for students. Two examples will be considered: the Big Bang Theory and Just War Theory. The Big Bang Theory is a helpful vehicle for considerations of the abundance of the chemical elements as well as the fundamental particles which make up the atomic nucleus, while a survey of Just War Theory is an appropriate way to end a discussion of nuclear weapons design and the consequences of their use at the end of World War II.

15. PCR BASED EVIDENCE FOR TWO SIMULTANEOUSLY EXPRESSED PROENKEPHALIN GENES IN THE STURGEON FISH (Acipensor transmontanus). Corey P. Gallegos, Erica Blight, and Phillip B. Danielson(*). Department of Biological Sciences, University of Denver.

The expansion of multigene families through gene duplication is a common theme in molecular evolution. It is postulated that redundant copies of existing genes are under relaxed selective pressure and are thus free to diverge and ultimately take on new functions or degenerate into pseudogenes.

An example of this phenomenon can be seen in the opioid/orphanin gene family. It is postulated that an ancestral version of proenkephalin, via a series of gene duplication events, has given rise to proopiomelanocortin, prodynorphin and proorphanin. The possible existence of two proenkephalin genes within sturgeon, however, lends the first tangible support to this hypothesis. To distinctively demonstrate the simultaneous expression of two proenkephalin genes, reverse transcriptase PCR using paralog-specific primers will be employed. It is hypothesized that this second gene has experienced transcriptional down regulation, in order to compensate for increased amounts of peptide hormone produced by the duplicated genes. This is because in the case of neuropeptide hormones such as met-enkephalin and leu-enkephalin (precursor is proenkephalin); increased expression levels may pose a significant physiological challenge. The existence of two expressed proenkephalin genes provides an excellent context in which to investigate the evolution of differential transcriptional regulation. Future studies, therefore, will quantify the relative expression levels of each proenkephalin gene.

16. CALF MUSCLE GIRTH AND PERFORMANCE FOLLOWING KNEE SURGERY IN YOUNG ADULT MALE SUBJECTS. Shelly Hooten and Michael Ross(*). Department of Biology; United States Air Force Academy, Colorado Springs, CO.

Several reports have determined that quadriceps muscles atrophy and weaken following knee surgery. However, no studies have examined calf muscle girth and performance following knee surgery. Therefore, the purpose of this study was to assess calf muscle girth and performance in 10 male subjects (age = 20.55 [+ or -] 1.23 years) that had recently undergone knee surgery (mean time from surgery to testing = 39.20 [+ or -] 9.35 days). The subject's calf girth was assessed circumferentially, and then they performed the standing heel-rise test, which entails performing as many heel-raises as possible on both involved and noninvolved lower extremities. Previous research showed that no significant difference exists between the lower extremities of healthy subjects for calf girth or the number of heel-rise repetitions performed. However, our results revealed that the noninvolved lower extremity performed significantly more repetitions on the standing heel-rise test than the involved lower extremity (28.18 [+ or -] 7.25 versus 20.45 [+ or -] 6.61 repetitions, p=0.008). No significant difference was found between the lower extremities for calf girth measures (p=0.17). We suggest that factors other than girth may be responsible for the calf performance deficits seen in these subjects. To preclude calf weakness, we recommend that calf muscle strengthening exercises be performed in the early stages following knee surgery.

17. INTERACTION OF TRH AND CCK IN THE SATIATION OF ALCOHOL INTAKE. Paul J. Kulkosky(*)(1) and K. Jill Chavez(2)(*).

Thyrotropin releasing hormone (TRH) and cholecystokinin octapeptide (CCK) are endogenous neuropeptides known to inhibit intake of alcohol. Although both peptides are released by alcohol consumption and are hypothesized to satiate alcohol intake, their interaction has not been examined. We deprived ad lib fed male (N=6) and female (N=4) Wistar rats of water for 23 hr and then gave them 30 min access to 5% w/v ethanol, followed by 30 min access to water. After adaptation to this schedule, rats were randomly assigned to receive i.p. injections of either saline + saline, CCK (4 [micro]g/kg) + saline, saline + TRH (10 mg/kg) or CCK + TRH, immediately before alcohol access. Analyses of variance revealed significant (p [is less than] 0.05) effects of sex and CCK, and a significant interaction of CCK and TRH [F(1,9)=16.01] in control of ethanol consumption. CCK reliably reduced alcohol intake, and TRH blocked this satiation effect of CCK. Results indicate an infra-dose-additive interaction of CCK and TRH in satiation of alcohol intake, which may reflect a natural, endogenous neuropeptide interaction in the regulation of caloric intake.

(1) Department of Psychology, University of Southern Colorado; (2) Department of Comparative Environmental Toxicology, Cornell University.

18. FOOD PASSAGE RATES IN LARVAL Bufo boreas. Laura A. Lembke and Lauren J. Livo(*). Department of EPO Biology, University of Colorado, Boulder.

We examined food passage rates in three groups of Bufo boreas tadpoles from Clear Creek County, Colorado. Group 1 was kept in pond water throughout the experiment with no exposure to a dilute carbon solution. Groups 2 and 3 were placed in a dilute carbon solution for 30 minutes to provide a mark in the feces; Group 2 was then kept in pond water while Group 3 was kept in pond water conditioned by a Dytiscus dauricus larva. Every two hours we examined material passed by the tadpoles. After 8 hours, no carbon marks were observed in feces passed by tadpoles in Group 1, indicating that normal metabolic processes do not produce carbon-like marks. There was a highly significant difference between groups 2 and 3 in the number of instances in which carbon was present in feces (13 times for tadpoles in Group 3 versus two times in Group 2; Chi square = 11.12, df= 2, p [is less than] 0.01). Rapid clearance of food might reduce mass sufficiently to allow faster escape during encounters with predators, but chronic increase in food passage rates could affect assimilation efficiency and growth over the course of larval development.

19. THE GENERAL HEALTH STATUS OF YOUNG ADULT MALE SUBJECTS FOLLOWING KNEE SURGERY. Lauren Maher and Michael Ross(*). Department of Biology; United States Air Force Academy, Colorado Springs, CO.

No studies have reported the general health of subjects following knee surgery. Therefore, the purpose of this study was to assess the general health status of male subjects with a recent history of knee surgery (n = 10, age = 20.55 [+ or -] 1.23 years, mean time from surgery to testing = 39.20 [+ or -] 9.35 days) and compare their status to a control group of uninjured male subjects (n=18, age = 20.00 [+ or -] 1.5 years). All subjects were required to complete the Medical Outcome Study 36-item Short-Form Health Survey, which queries subjects about their health over the past four weeks in eight different concept areas: 1) vitality, 2) general health, 3) physical functioning, 4) role limitations due to physical problems, 5) bodily pain, 6) general mental health, 7) role limitations due to emotional problems and 8) social functioning. The results of an independent t-test indicate that subjects in the control group score significantly higher than subjects in the surgery group on the physical functioning, role limitations due to physical problems, bodily pain, role limitations due to emotional problems and social functioning concept areas (p [is less than] 0.05). To enhance the probability of a favorable outcome, we recommend that clinicians pay special attention to these areas when treating patients in the early stages following knee surgery.

20. EFFECTS OF LIPID DEPOSITION SITE ON NON-INVASIVE ESTIMATES OF BODY COMPOSITION. Lance A. Merkley, Brion T. Coleman, and Edward T. Unangst, Jr.(*) United States Air Force Academy, Colorado Springs, CO.

Technologies such as EM-SCAN allow for non-invasive estimation of body composition. Prior studies with EM-SCAN suggest that body fat estimates may vary if fat is deposited differentially. Therefore, we evaluated the effects of lipid location on the output reading of the EM-SCAN Small Mammal Composition Analyzer, Model SA-2 (EM-SCAN, Inc, Springfield. IL). The unitless EM-SCAN output reading is included in a multiple regression equation used to estimate lean and lipid components of body composition non-invasively. We tested 80 g mass and 120 mm total length sausage-shaped molds consisting entirely of 93% ground beef as our control; with experimental groups also being 80 g, 120 mm but with 25% beef fat added to either the head, tail, in the middle, or homogenized throughout the mold. EM-SCAN output readings were: lean 49.75 [+ or -] 0.45; homogenous 24.33 [+ or -] 0.80; head 41.02 [+ or -] 0.50; tail 43.93 [+ or -] 0.76; middle 35.53 [+ or -] 0.40 (mean, se). All pairwise between-group comparisons yielded significant differences at p [is less than or equal to] 0.05. Our results suggest that the lipid deposition site in an organism will affect the reading one gets from EM-SCAN. It is possible that we can develop different regression equations depending upon lipid deposit site and this is the subject of ongoing research by these authors.

21. GENETIC EFFECTS ON LEARNING IN MICE FOLLOWING MK801. Brooke Quigley, Alicia Christensen, Gayle Brosnan-Watters, and Donald J. Nash(*), Colorado State University.

Phencyclidine (PCP) has been found to produce a psychosis indistinguishable from schizophrenia and is known to affect the NMDA receptor site for glutamate. MK-801 also is a non-competitive antagonist at the same receptor site. it has been noted that administration of PCP and MK-801 in mice, rats, and non-human primates has resulted in a learning disability. There is some indication in humans that differences in responses to PCP and MK-801 are due to a genetic predisposition in susceptibility. The present study was designed to examine learning behavior following MK-801 in a strain of mice segregating for the meander tail gene. The meander tail gene is an autosomal recessive mutation that results in a kinked tail and an abnormality in the interior lobe of the cerebellum. Meander tail mice and their normal siblings were trained first to learn a task involving spontaneous alternation of two sides of a T-maze to establish baseline learning scores. Mice that were able to learn the task were subsequently administered 1 mg/kg dose of MK-801 and tested in the maze five hours later. Preliminary results indicate a difference in learning between the meander tail mice and their normal sibs.

22. REGULATION OF CREB PHOSPHORYLATION IN MOUSE SPINAL CORD: POSSIBLE ROLE IN OPIOID TOLERANCE AND PHYSICAL DEPENDENCE. Kirsten M. Raehal(1), Danxin Wang(2), Wolfgang Sadee(2) and Edward J. Bilsky(*)(1).

Phosphorylation of intracellular proteins may regulate the development of opioid tolerance and/or physical dependence in vitro and in vivo. The current studies focus on the phosphorylation of cAMP-responsive element binding protein (CREB) in the lumbar/sacral spinal cords of ICR and 129 SvEv mice. Morphine given i.c.v., i.th. or s.c. produces similar antinociception in each strain of mice. ICR mice develop both antinociceptive tolerance and physical dependence to acute and chronic morphine exposure. In contrast, the 129 SvEv strain does not develop significant tolerance or physical dependence to morphine. Baseline levels of lumbar/sacral spinal pCREB (relative immunoreactivity) were significantly elevated in 129 SvEv mice compared to ICR mice (280 [+ or -] 60 versus 100 [+ or -] 33). Total CREB levels (CREB and pCREB) were no different between the strains. In response to morphine injection (100 mg/kg, s.c., -4hr), pCREB levels decreased significantly (126 [+ or -] 33) in 129 SvEv mice. Spinal pCREB levels did not change in ICR mice treated with morphine. These results suggest that differences in spinal basal pCREB levels, and the subsequent changes in pCREB following morphine administration, may contribute to the unique tolerance/dependence profile seen in 129 SvEv mice.

(1) University of Northern Colorado; (2) University of California at San Francisco.

23. EFFECTS OF THE PONNEQUIN WIND ENERGY STATION, NORTHERN WELD COUNTY, COLORADO, ON SOME BIRDS AND MAMMALS. Ronald A. Ryder(*). Department of Fishery and Wildlife Biology, Colorado State University.

Birds and mammals have been censused on four study sites at the Ponnequin Wind Energy Station of the Public Service Company of Colorado. Two sites have windmills (total 29), and two were reference areas with no windmills. Three types of censuses were used: a strip count under the windmills, point counts for raptors, and plot counts perpendicular to the power tower lines. Counts were made before and after construction. All areas were counted weekly in the summer, twice monthly in spring and fall, and monthly in winter. Avian surveys revealed no endangered, threatened, or species of concern on the sites, and few raptor nests within 5 km of the project area. Horned Larks and McCown's Longspurs (both nesters) accounted for 84.8% of all songbirds observed on the project and reference sites. The remainder were mainly Lark Buntings and Western Meadowlarks (also nesters), although over 20 species have been observed in migration. Three raptor species (Golden Eagle, Swainson's Hawk and Ferruginous Hawk) accounted for 74.9% of the 80 hawk sightings on all sites. Territorial displays and hunting behaviors related to windmill operational heights will be described. To date, 7 bird and 5 bat fatalities related to the wind towers have been noted. Pronghorns and Horned Larks have been attracted by grass seedings under the towers.

24. CHARACTERIZATION OF THE DEVELOPMENTAL EXPRESSION OF MITOCHONDRIAL CYP12B1. Robert J. Shelton and Phillip B. Danielson(*). Department of Biological Sciences, University of Denver.

Cytochrome P450s are involved in the metabolism of xenobiotics and endogenous substrates. Because of their putative role as catalysts in the biosynthesis of insect steroid hormones, mitochondrial insect cytochrome P450s are likely to play an important physiological role in arthropod growth. An understanding of P450 expression during development will further our understanding of the function of these enzymes. Northern blot, quantitative PCR, and Ribonuclease Protection Assays (RPA) are ways to assess mRNA transcription levels. In Drosophila acanthoptera, Northern blot analysis detected expression of CYP12B1 in first instar, which was reduced by half in the second instar. Third instar and pre-pupal stages displayed a six-fold increase in expression levels. In the adult stage, CYP12B1 is expressed at one-half to one-third of peak levels. P450 gene expression profiles obtained by Northern blot analysis often lack the sensitivity and low background that can be achieved with modern RPAs. P450 gene products expressed during development may have low mRNA transcription levels, which are difficult to detect. Another common obstacle with Northerns is the similarity between P450 isoforms that metabolize chemically dissimilar substrates results in cross-hybridization of probes. Efforts to develop Ribonuclease Protection Assays to characterize the developmental expression profile of CYP12B1 will be presented.

25. THE IDENTITY OF THE ISOLATED LOS TUXTLAS POPULATION OF THE FALSE CORAL SNAKE, Pliocercus elapoides, OF SOUTHERN VERACRUZ, MEXICO. (*)Hobart M. Smith(1) and (*)David Chiszar(2).

The Los Tuxtlas uplift in southern Veracruz, Mexico, is a major center of endemism, and is itself a zoogeographic complex with at least two distinct areas of endemism. The pattern differences of specimens of Pliocercus from the Sierra de Santa Marta compared with those from Volcan San Martin Tuxtlas were therefore interpreted by Smith and Chiszar (1996) as evidence of two different taxa, P. wilmarai and P. elapoides aequalis respectively. Material more recently available demonstrates that both pattern types occur on the Sierra de Santa Matra, and accordingly but one taxon of Pliocercus, here regarded as P. elapoides wilmarai, evidently occurs in Los Tuxtlas. It is diagnosed as a subspecies of P. elapoides widely isolated from all others and unique in lacking black rings or saddles except for the nuchal and anal marks, OR, if present, yellow rings usually absent both dorsally and ventrally, it is the most variable subspecies of the species, mimicking four very differently marked taxa of coral snakes (Micrurus).

(1) Department of EPO Biology; (2) Department of Psychology, University of Colorado, Boulder.

26. ELIMINATING VOMERONASAL CHEMORECEPTION IN RATTLESNAKES BY A REVERSIBLE "LESION". C. Patrick Stark(1), David Chiszar(*)(l) and Hobart M. Smith(*)(2).

Several techniques have been used to block vomeronasal chemoreception in snakes, but all have involved more-or-less invasive and traumatic procedures, some irreversible. Our goal was to develop a technique that minimized or eliminated trauma and was completely reversible. Three promising methods failed to meet our criterion of being free of trauma. The fourth method worked best. It involved painting a euthanized mouse with xylocaine, a topical anesthetic for use with mucous membranes, and letting a hungry snake strike this prey item, auto-anesthetizing its vomeronasal organs in the process. Data obtained through the use of this method will be described.

(1) Department of Psychology; (2) Department of EPO Biology, University of Colorado, Boulder.

27. TRAIL-FOLLOWING BEHAVIOR IN COPPERHEADS (Agkistrodon contortrix). Kathryn Stiles(1), David Chiszar(*)(2) and Hobart M. Smith(*)(1).

As in rattlesnakes, copperheads followed trails after striking rodent prey but not after seeing a rodent nearby without an opportunity to strike. This similarity implies that the stereotyped style of ambush predation characteristic of rattlesnakes evolved before this clade branched from its Agkistrodon ancestor. Indeed, it seems probable that the strike-release-trail strategy evolved before the New World pit vipers differentiated from the true vipers of the Old World. Its evolution may have been concomitant with solenoglyph or even endoglyph evolution. Another behavior of interest is that copperheads generally balked after strikes if no cover was available between their starting location and the terminus of the trail. That is, copperheads moved along trails only when cover was available along the trail, a phenomenon seen in some, but not all, rattlesnake species.

(1) Department of EPO Biology; (2) Department of Psychology, University of Colorado, Boulder.

29. EXPRESSION OF NADPH-CYTOCHROME P450 OXIDOREDUCTASE IN Eschericia coli. Lara Vigil, James Fogleman, Phillip Danielson(*), Department of Biology, University of Denver.

NADPH-cytochrome P450 oxidoreductase (P450 reductase) supplies reducing equivalents obtained from NADPH to substrate binding terminal oxidases, (i.e., cytochrome P450 monooxygenases). Cytochrome P450s comprise a superfamily of enzymes, which are responsible for the metabolism of a wide diversity of toxins and endogenous substrates of functional cytochrome P450 systems, therefore it requires the activity of cytochrome P450 reductase. The Drosophila mettleri cytochrome P450 reductase has been cloned and expressed in Eschericia coli as part of studies of P450 mediated xenobiotic metabolism for favorable expression in E. coli. The modified DNA of reductase was ligated downstream of the IPTG inducible tac promoter in the expression plasmid, pCWori+. Induced expression of reductase, with an apparent molecular weight of 76kDa, was detected by SDS-PAGE. The activity of the reductase was assayed by measuring the initial rate of cytochrome c reduction using an extinction coefficient of 19m[M.sup.-1][cm.sup.-1]. As this enzyme is catalytically active in vitro, it will allow for the development and use of reconstituted systems for the assay of the catalytic activity of individual cytochrome P450 isoforms.

30. MOLECULAR CLONING OF CRH FROM Oncorchynchus masou. Katelyn M. Vogelzang and Phillip Danielson. Department of Biological Sciences, University of Denver.

The Hypothalamus Pituitary Axis regulates the communication between the hypothalamus, the anterior pituitary (AP) and the adrenal cortex (AC). In the absence of inhibition from the hippocampus, neurons within the hypothalamus will release corticotropic-releasing hormone (CRH). CRH induces corticotropic cells within the AP to produce adrenocorticotropin (ACTH); ACTH in turn triggers the release of glucocorticoids (GC) from adrenal cortical cells. GC participates in the regulation of its own synthesis by stimulating the hippocampus, which will tonically inhibit CRH neurons in the hypothalamus. In Masu salmon a hypersecretion of cortisol occurs during the beginning of sexual maturation and continues throughout spawning, causing the hypothalamus to deteriorate eventually resulting in death. The question that arises is where within the HPA axis is the regulation loop being compromised. A 3' RACE strategy using a degenerate primer targeting a conserved region illustrated by a sequence alignment of multiple CRH genes was utilized to isolate CRH from Masu salmon. Through a greater understanding of the malfunctions of the HPA axis we will be better able to treat the similar endocrine disorder, Cushing's syndrome.

31. STRIDES TOWARD ENCOURAGING PROFESSIONS IN SCIENCE PROGRAM: A NATIONAL INSTITUTE OF HEALTH BRIDGES TO THE FUTURE GRANT Rosemarie DePoy Walker(*)(1) and Patricia L. Stranahan(*)(2).

The development and implementation of the STEPS program will be presented. The STEPS program is an inter-disciplinary, inter-institutional collaboration with the intended goal of increasing minority participation in Biomedical Research. The following aspects of the program will be discussed:

1. Funding the Program- Grant Proposal Writing & Re-Writing

2. Defining the Roles of the 5 institutions(Aurora Community College, Community College of Denver, Front Range Community College, Metropolitan State College of Denver, and University of Colorado Health Sciences Center) Involved in the Program

3. Designing the Program Activities

4. Recruiting, Retaining, Transferring, and Graduating Students

5. Evaluating and Refining the Program

(1) Department of Chemistry; (2) Department of Biology, Metropolitan State College of Denver.

32. ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL CYTOCHROME P450S USING 3' AND 5' RACE TECHNIQUES. James M. Wallace and Phillip Danielson(*). Department of Biological Sciences, University of Denver.

Mitochondrial P450s have been well documented for their role in vertebrate steroidogenesis. Their likely functions in invertebrates are less apparent. To address questions regarding the possible functions of these enzymes, a complete suite of invertebrate mitochondrial P450s will need to be isolated. The primary amino acid sequence of mitochondrial P450s features two salient characteristics. First, the 25 N terminal amino acids invariably include several charged amino acids that give the amino terminus an amphipathic character unique to mitochondrial P450s. Second there is a conserved region of the meander with the sequence FMPERW. Starting with CYP 12B1 (the first insect mitochondrial P450), northern hybridizations were used to screen size-fractionated mRNAs from different species of Drosophila for the expression of putative CYP12 homologues. 3' RACE reactions employing a degenerate primer targeted to the conserved FMPERW motif of mitochondrial P450s produced two partial cDNA sequences that were designated DU 3 and DU 13, from the fruit fly species Drosophila arizonae. These sequences were found through BLAST searches to have significant sequence homology to vertebrate Vitamin [D.sub.3] 25-hydroxylase. These cDNA sequences were then used to create a gene-specific probe to identify the full-length gene in a heterozygous cDNA pool. Using gene-specific primers and template cDNA with a homopolymeric dGTP tail, 5' RACE reactions can be employed to amplify the remaining 5' end of the gene.

33. THE NEUTRINO: MUCH ADO ABOUT NOTHING? Sallie A. Watkins(*), Department of Physics (Emerita). University of Southern Colorado.

People are looking for them in coalmines and in the ice cap at the South Pole, yet thousands of them are zipping through your body as you read this sentence. "They" are neutrinos, proposed in the 1930s to explain the spread of beta ray energies. This paper will present an overview of the neutrino story--starting with a dance in Zurich, Switzerland, and moving to equally interesting events at other points on Planet Earth. After decades of research, the jury is still out on a fundamental question: Does the neutrino have mass? After hearing the evidence, the audience at this talk will be invited to cast "yes" or "no" votes.

34. STAGES OF OBJECT PERMANENCE ACQUIRED BY Canis familiaris. Jennie K. Willis and Donald J. Nash(*), Department of Biology Colorado State University.

Object permanence is the stage of cognitive development where mental representation of objects first emerges. According to Jean Piaget, human infants acquire this cognitive structure during Substages 4-6 of the sensorimotor period, at about 18 months of age. The work of Piaget has often been applied comparatively to other species, with varying degrees of success. Previous work on dogs failed to control for scent cues during the displacement of the object, thus failing to "displace" the object at all. My research has shown that olfaction is a primary search strategy in dogs, and my present study controls for scent cues appropriately. Preliminary results show (A) Some dogs are able to reach Substage 6b object permanence (B) Some dogs are unable to achieve Substage 4a object permanence and (C) Sharper performance at object permanence problems appears to be correlated with breed.

35. MATERNAL GENETIC EFFECTS ON ETHANOL TERATOGENESIS: WHERE DO WE GO FROM HERE? David M. Gilliam(*) and Paul Gillenwater, Department of Psychology, University of Northern Colorado.

Prenatal alcohol exposure can result in abnormalities in morphology and behavior, collectively known as the Fetal Alcohol Syndrome (FAS). Variations in outcome severity led us to use a mouse model to explore both maternal and fetal genetic influences on susceptibility. Our studies show that genetically distinct mouse stocks differ in susceptibility to prenatal alcohol effects following similar exposure levels. We found no differences in blood ethanol levels or differences in ethanol elimination rates that can account for differences among stocks. Some of the differences in susceptibility are due to the maternal genotype, while other differences are due to the fetal genotype. The maternal genetic effects are not due to sex-linked genes or to cytoplasmic inheritance, but may be due to the different uterine environments provided by each mother.

Supported by the University of Northern Colorado Sponsored Programs & Academic Research Center; Faculty Research and Publications Board; UNC Graduate School.

36. EFFECTS OF QUINKY GENE AND AGE ON LEARNING IN MICE. Donald J. Nash(*)(1) and R. Steven Ackley(*)(2).

The "quinky" gene (Q) is a semidominant gene located on chromosome 8 in the mouse. Homozygous mice die shortly after implantation but heterozygotes are viable and fertile. The gene has effects on the skeleton producing short, kinked tails. Early descriptions of the mutation reported some mice exhibited shaking or circling behavior. The present study examined effects of the quinky gene on learning behavior. A water maze was used to compare learning in congenic strain segregating for the quinky gene. Quinky heterozygotes (Q/+) and normal siblings were tested for three consecutive days in the maze. In order to examine for possible differential age effects on learning, groups of mice were tested at 4 months, 11 months, and 16 months of age. Results indicated an effect of the single gene on learning. At all ages, normal mice had fewer errors than quinky mice did. In addition, although learning was not significantly impaired with aging in normal mice, there was a marked decrement in quinky mice over 11 months of age.

(1) Department of Biology, Colorado State University; (2) Department of Communication Disorders, University of Northern Colorado.

37. THE USE OF RANDOMLY AMPLIFIED POLYMORPHIC DNA TO ASSESS GENETIC VARIATION BETWEEN POPULATIONS OF MAYFLY NYMPHS WITH DIFFERING BEHAVIORAL RESPONSES TO THE RISK OF PREDATION. Lee Anne Martinez(*), Sheila Lehman, Daniel Caprioglio, Felicia Rodriguez, and Yasuko Matsuoka. Department of Biology, University of Southern Colorado.

Studies have shown that stream-dwelling mayfly nymphs (Insecta: Ephemeroptera) perform a number of behaviors that reduce their risk of predation by fish. These behaviors include the restriction of movement to the lower surfaces of benthic rocks and cobble, the restriction of drift (downstream movement in the water column) to cover of night, and the avoidance of surfaces impregnated with the "odor" of fish. While mayflies living in fish-inhabited streams exhibit these behaviors, those from naturally fishless streams do not. it is unclear whether the difference between the mayflies in these two habitats is the result of behavioral plasticity, or whether it is the result of genetically fixed behaviors that vary in frequency between the two populations. Randomly Amplified Polymorphic DNA (RAPD) markers are being used to explore genetic differences between mayflies from fish-inhabited versus nearby fishless streams in Colorado. To date, we have successfully isolated and amplified mayfly DNA. We have also identified one 10-base oligonucleotide primer that amplifies a portion of the mayfly genome. Further work is needed to refine the isolation and marker identification process, and to test additional random sequence primers. This work should reveal genetic variation among the mayfly populations. In combination with controlled laboratory experiments on mayfly avoidance responses, this genetic information will help us to understand the flexibility of insect behavior and evolutionary forces that shape it.

38. HETEROLOGOUS EXPRESSION OF TWO NOVEL INSECT CYTOCHROME P450: CYP4D10 AND CYP28A1. Abdullah AL-Zahrani, Frank Conrad, Philip Danielson(*), James Fogleman. Department of Biological Sciences, University of Denver.

Cytochrome P450s comprise a superfamily of heme-thiolate proteins. Microsomal P450s in the CYP4 hand CYP28 families have been implicated in the metabolism of xenobiotics. Previously, CYP4D10 and CYP28A1 from Drosophila mettleri were shown to be transcriptionally responsive to toxic isoquinoline alkaloids present in Drosophila mettleri's host plant. Since it is virtually impossible to purify individual P450 proteins from multicellular organisms to biochemical homogeneity, heterologous expression of CYP4D10 and CYP28A1 cDNA in Escherichia coli will facilitate purification of individual isoforms for use in metabolism assays. Expression of these proteins is an essential first step in further experiments on the structure and function of CYP4D10 and CYP28A1. Since P450s are microsomal proteins embedded in the endoplasmic reticulum, expression in prokaryotic cells has often been problematic. CYP17[Alpha] (P450c17-hydroxylase), however, is one P450 that has been modified for high level expression in E. coli. In order to facilitate expression of CYP4D10 and CYP28A1, a chimeric cDNA was constructed consisting of the promoter and 5' anchor sequence of CYP17[Alpha] fused to the functional domains of CYP4D10 and CYP28A1. The construct was ligated into [pCWori.sup.+] expression vector and the expression of functional P450 enzyme was assayed based on SDS-PAGE and carbon monoxide difference spectra.

39. THE LIMITATIONS OF A DEGENERATE PCR STRATEGY TO ISOLATE MEMBERS OF A MULTIGENE SUPERFAMILY. Chad Stephen Metcalf and Philip Danielson(*), Department of Biology, University of Denver.

Gene duplication is thought to be the primary mechanism by which new gene function arises and a phenomenon that has driven the evolution of complex life forms. As a result of repeated duplication events throughout evolutionary history, multigene families now account for a substantial portion of protein coding sequences in the genomes of many multicellular organisms. Among the largest of these families are the serine proteases, protein kinases, and cytochrome P450s. The use of degenerate PCR strategies to isolate diverse members of these families is common. PCR-based cloning strategies with moderately degenerate oligonucleotide primers targeted to highly conserved domains in cytochrome P450s, has resulted in the identification of more than 100 novel genes. While degenerate PCR based strategies are extremely powerful, they also have limitations. The efficiency with which transcripts are amplified depends on their abundance. Highly expressed transcripts will outcompete the rarer transcripts. Statistical analyses based on a tag and release model can be used to estimate the diversity of genes that can be isolated using a degenerate PCR approach. Sequences for 500 subcloned cDNAs from a single degenerate 3'RACE reaction were analyzed and provided insight into the utility of a degenerate primer in terms of the maximum number of unique sequences that can be amplified simultaneously.

40. MOLECULAR CLONING OF ACTH FROM Oncorhvnchus masou. Erica Blight, Katelyn M. Vogelzang, Robert M. Dores, and Philip Danielson(*). Department of Biology, University of Denver.

Through secretion of several trophic polypeptide hormones, the hypothalamus and pituitary glands play a key regulatory role ranging from reproductive activity to chronic stress regulation. In the anterior lobe of the pituitary (pars distalis) corticotropic cells express the propiomelanocortin gene (POMC). POMC is the precursor for the peptide hormone, adrenocorticotropin (ACTH). ACTH is a critical link in the hypothalamus/pituitary/adrenal axis, which is a neuroendrocrine network responsible for modulating chronic stress response mechanisms. The hypothalamus induces the secretion of ACTH, which in turn stimulates the adrenal gland to synthesize and release cortisol. Cortisol regulates it's own expression and release by the suppression of ACTH through a negative feedback loop. Failure of the negative feedback loop will increase ACTH, with potentially fatal consequences. ACTH radioimmunoassays on Pacific salmon have demonstrated an increase of ACTH levels upon sexual maturation. Is the increase in ACTH directly related to POMC gene over expression? A 1024-fold degenerate oligonucleotide primer directed to a highly conserved KRYGGFM motif, was designed to isolate cDNA fragments of multiple POMC genes from the pars distalis of Oncorhynchus masou. Isolation of POMC cDNAs will allow POMC gene expression levels to be quantified through Northern Hybridization. In examining expression levels of ACTH a better understanding of the breakdown occurring within the HPA axis of Pacific salmon will be gained.

41. EFFECTS OF THE MICROPHTHALMIC-WHITE GENE ([Mi.sup.wh]) ON CRICKET PREDATION IN MICE. Allan C. Schell and Donald J. Nash(*), Colorado State University.

The Microphthalmic White gene ([Mi.sup.wh]) in mice produces a number of diverse effects including severe ear defects and deafness, and extreme eye anomalies including microphthalmia and cataracts. This study examined effects of these severe sensory deficits on predatory behavior. A congenic inbred strain produced mice identical in genetic background except for the single gene substitution at the microphthalmic locus. The three genotypes tested were [Mi.sup.wh]/[Mi.sup.wh],[Mi.sup.wh]/+ and +/+. Mice were tested for their ability to catch and eat a cricket that was placed in a cage with the mouse. Results indicated that [Mi.sup.wh]/[Mi.sup.wh] mice were not as effective in capturing crickets as were the heterozygous and normal mice.

42. IS YOUR SUNTAN PREPARATION A POTENTIAL REPRODUCTIVE TOXICANT? Moussa M. Diawara, Kathrine J. Chavez, Paul Kulkosky(1)(*), and Patricia B. Hoyer(2). Department of Biology, University of Southern Colorado

The psoralens occur naturally in common grocery fruits and vegetables. Synthetic forms of 5-methoxypsoralen (bergapten) and 8-methoxypsoralen (xanthotoxin) are widely used in skin photochemotherapy and also as ingredients in suntan preparations. Recent studies documented that bergapten and xanthotoxin reduced birthrate in female rats both sexes were dosed. The present investigation determined the direct effect of xanthotoxin on reproductive function in male rats. Male Wistar rats (21 days old) were dosed daily with xanthotoxin (150 mg/kg, p.o.) or the vehicle control and mated with undosed naive females. Males were all sacrificed the same day after all females ware impregnated, and each female was sacrificed two days before parturition. Treated males had smaller (P [is less than] 0.05) pituitary gland and less sperm in the vasa defferentia and in the epididymis than control males. Dosing resulted in elevated (P [is less than] 0.05) levels of circulating testosterone in treated animals. There was a trend for elevated serum luteinizing hormone (P=0.0696) and follicle-stimulating; hormone (P=0.0988) in treated males. Females bred to dosed males required more (P [is less than] 0.05) breeding attempts and took longer time before pregnancy occurred. Fetuses of females bred to treated males were smaller (P [is less than] 0.05) (some discolored and/or malformed) compared with control groups. The study provides strong evidence that xanthotoxin is a reproductive toxicant; the environmental implications of this endocrine disruptive property, are, discussed. Supported by NIEHS grant #ES00288 (to MMD).

(1) Department of Psychology, University of Southern Colorado; (2) Department of Physiology, University of Arizona.

(*) Denotes membership in the Colorado-Wyoming Academy of Science.
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Date:Apr 1, 2000
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