Browsing by Author "Caceci, Thomas"

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    Advanced Studies in Veterinary Anatomy: Angiogenesis in Caprine Reproductive Organs of Non-Pregnant and Pregnant Normal and Swainsonine-Treated Does
    Hafez, Shireen Abdelgawad (Virginia Tech, 2005-04-20)
    The female reproductive organs are among the few adult tissues in which periodic angiogenesis normally occurs. Pathological angiogenesis can occur in various conditions, such as solid tumors. Vascular endothelial growth factor (VEGF) signaling often represents a critical rate-limiting step in physiological and pathological angiogenesis. This study utilizes development of utero-ovarian vasculature during pregnancy in goats as a model of physiological angiogenesis. Non-pregnant does and does at 4, 7, 10, 13, 16, and 18 weeks of gestation were used. Arteries of the reproductive tract were injected in situ with Microfil®. The tracts were fixed, dehydrated, and rendered transparent to reveal the paths of arteries. The ovarian artery was tortuous and lay in close apposition to the uterine tributary of the ovarian vein in all specimens studied. In non-pregnant does, this arrangement may serve as a local utero-ovarian pathway for the corpus luteum (CL) luteolysis at the end of non-fertile estrous cycle. During pregnancy, this arterio-venous arrangement may transfer luteotropic substances from uterus to ovary, which may serve in maternal recognition of pregnancy and fit the fact that the goat is CL dependent throughout gestation. In some cases of triplets, the size of the uterine branch of the ovarian artery was equal to or even larger than that of its parent artery and/or the ipsilateral uterine artery; and the vaginal artery contributed a connecting branch to the uterine artery. These physiological adaptations of the ovarian and/or vaginal arteries correlate well with the increasing nutrient demands of the growing multiple fetuses. In a second experiment, the vasculature of the uterus and ovaries was injected in situ with a mixture of Batson's No.17® and methyl methacrylate and then processed for observation by SEM. The microvasculature differed between non-pregnant and pregnant does, and with advancing gestation. We concluded that goats possess a multivillous type placenta. Capillary sinusoids and crypts on the fetal surface of the caruncle may compensate for the negative effect of the increased interhemal distance. Intussusceptive angiogenesis should be considered as equally possible and important mechanism as sprouting angiogenesis during placental development. Capillary diameters increased significantly during pregnancy especially after 4 weeks. Capillary density index was 66.8, 68.7, 55.5, 63.5, 70.1, 70.4, 64.5 percent in non-pregnant, 4, 7, 10, 13, 16, and 18 weeks of pregnancy, respectively. In the ovary, coiling of the ovarian branch of the ovarian artery around the ovarian tributary of the ovarian vein was observed. This may represent a local channel required for product transport from ovarian vein to ovarian artery and might have a role in regulating blood pressure to various ovarian structures. Immunolocalization of VEGF was performed as a third experiment. Immunostaining was observed in cyto- trophoblasts, maternal epithelial tissues, and vascular endothelium and smooth muscle, but not in binucleate giant cells or connective tissue. No apparent differences were observed in intensity and pattern of VEGF staining associated with advancing gestation. Luteal and follicular cells, and endothelium and smooth muscles of the ovarian vasculature positively stained. Patterns and intensity of staining of VEGF suggest that the fetus is directing its own survival by producing growth factors affecting fetal and maternal tissues. VEGF may have a role in growth and differentiation of cytotrophoblasts, as well as, development and maintenance of CL. In the fourth experiment, the sequential expression of VEGF and its receptors (fms-like tyrosine kinase, Flt-1 and kinase-insert domain-containing receptor, KDR) was measured using real-time quantitative PCR. Targets were detected in all studied tissues; however, levels of expression differed according to the stage of pregnancy. Expression of VEGF and its receptor mRNAs increased with advancing pregnancy, which correlates with the expansion of vasculature during pregnancy. Differences in the time-courses of the expression of Flt-1 and KDR mRNAs during pregnancy suggest that each receptor plays a different role in the angiogenic process. As an application of our model of angiogenesis, we tested the effect of swainsonine (active compound of locoweed and a potential anti-cancer drug) on the process. Does treated with swainsonine were euthanized at 7 and 18 weeks. No significant differences were found in sinusoidal diameters in treated does at 7 weeks, but a decrease in capillary density index was noted. In the ovary, focal avascular areas were observed in the corpus luteum of swainsonine-treated does at 7 weeks of pregnancy. Swainsonine caused great distortion in the uterine and ovarian vasculature at 18 weeks. A decrease in intensity of the immunoreactivity to VEGF antibody was observed in tissues from swainsonine-treated does at 7 and 18 weeks. There was no substantial effect of swainsonine on the expression of VEGF and its receptors' mRNAs in any of the studied tissues (except in the left ovary, where it had an inhibitory effect) at 7 weeks of pregnancy, but it had an inhibitory effect at 18 weeks. Demonstration of swainsonine's potential to negatively affect vascular development and suppress genes likely involved in angiogenesis at critical stages of blood vessel proliferation lends credibility to its potential as anti-cancer drug.
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    Commercial diets do not affect the colonic ultrastructure of normal dogs
    Campbell, Sharon Louise (Virginia Tech, 1993-06-10)
    Commercial and homemade diets are currently used to treat many canine patients with acquired disorders of the colon. Clinically, the efficacy of diets has been found to be unpredictable. Only one study to date has evaluated the effect of diet on the colonic mucosa. This study showed that diet did not observably alter the colonic mucosa of normal dogs, when biopsy samples were evaluated by light microscopy. The effect of diet on colonic ultrastructure in the dog, using transmission electron microscopy, has not previously been investigated. To determine the effect of diet on colonic ultrastructure, cell height, cell area, microvillus height, number of microvilli/apex width and basement membrane width were measured. Ten cells per animal were evaluated. Six dogs were assigned to the control group and fed the control diet for the duration of the study. Six dogs were fed each of the three test diets at four week intervals. The test diets used included a high fiber diet, a hypoallergenic diet and a highly digestible diet. These diets were selected because they are the diets most often recommended for the canine patient with colonic disorders. The value for cell height for the highly digestible group was significantly greater than the other groups, as measured by ANOV A and Duncan's multiple comparison test. No other significant differences were found. The biological relevance of a significantly different value for cell height alone is difficult to evaluate, as other parameters that would indicate an alteration in maturation or proliferation of the colonic epithelial cells did not change. value for cell height alone is difficult to evaluate, as other parameters that would indicate an alteration in maturation or proliferation of the colonic epithelial cells did not change. Therefore, we conclude that commercial diets do not have an effect on the colonic ultrastructure of normal dogs. Although no effect of diet was found, this study does provide morphologic measurements that can be used as a basis for future ultrastructural studies of the colonic mucosa.
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    The effects of congestive heart failure and functional overload on rat skeletal muscle
    Spangenburg, Espen E. (Virginia Tech, 2000-06-23)
    Numerous references have suggested that alterations in exercise capacity during congestive heart failure (CHF) are not simply due to changes in myocardial function. In fact, recent evidence has indicated that reductions in skeletal muscle strength and endurance during CHF significantly impact exercise capacity of the CHF patient. Currently, it is believed that alterations in skeletal muscle phenotype, or more specifically a slow to fast transformation in phenotypic protein isoforms contribute to the reductions in muscle function. However, currently there are few data which directly document this slow to fast transformation of the skeletal muscle. Interestingly, it is well established that exercise training can cause changes in skeletal muscle phenotype, more specifically in the fast to slow direction. This is in direct contrast to what is known to occur during CHF. However, it is unclear if similar adaptations will result from training in a CHF patient. Also, it is not clear if the adaptations are due to alterations in the myocardium or changes directly imposed upon the muscle by the exercise training. Therefore, the purpose of this study was two-fold: 1) to clarify the changes in skeletal muscle myosin heavy chain (MHC) during CHF and 2) to determine if skeletal muscle can adapt to increased activity levels, utilizing functional overload (FO) without significantly improving cardiac function. In the first study the mixed plantaris muscles from rats afflicted with severe CHF demonstrated a significant (p<0.05) increase in fast MHC (e.g. IIb expression at the expense of IIx expression) compared to the control animal (SHAM). The mixed red gastrocnemius, regardless of the severity of CHF, exhibited significant (p<0.05) changes in all of the MHC isoforms. The slow soleus and fast white gastrocnemius did not display any significant changes in MHC expression. The changes in MHC isoform significantly correlated with indicators of disease severity, suggesting there may be an existing relationship between skeletal muscle MHC expression and alterations in myocardial function. In the second study, there were no differences exhibited between CHF and SHAM absolute or specific plantaris mass. There was a significant (p<0.05) 30% increase in both absolute and specific mass of the plantaris in the CHF-FO and SHAM-FO groups compared to the CHF and SHAM groups. There was a significant (p<0.05) 3.5% increase in slow MHC I expression and a significant (p<0.05) 6.5% decrease in fast MHC IIb expression in the CHF-FO group compared to the CHF group. In the SHAM-FO group, there was a significant (p<0.05) 4% increase in MHC I expression and a subsequent 8% decrease in fast MHC IIx+IIb in the SHAM-FO compared to the SHAM groups. There were no differences detected in the rates of Ca²⁺ uptake between the CHF-FO, SHAM, and SHAM-FO. However, Ca²⁺ uptake rates were significantly (p<0.05) elevated by 44% in the CHF group when compared to the other three groups. There were very few changes in plantaris SERCA 1 or 2 protein expression between the four groups. These data suggest that during CHF there are alterations in skeletal muscle isoform expression. However, at least some of the data suggest that changes in function are not always associated with changes in phenotype. Instead, it seems that the changes in Ca²⁺ handling may be due to an alteration in a regulatory mechanism. Also, the data indicate that skeletal muscle is adaptable to increases in activity levels without significantly altering myocardial morphology.
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    The effects of temperature, water quality and culture conditions on the immunology, hematology, and blood chemistry of hybrid striped bass
    Hrubec, Theresa (Virginia Tech, 1994-12-19)
    Sunshine and palmetto bass (different crosses of hybrid striped bass) were used to determine immunologic, hematologic and serum chemistry changes under different culture conditions. The kinetics of the humoral immune response was determined for sunshine bass acclimated to 10, 18, 24, 29°C, and to elevated ammonia (0.15 mg/L NH3) and elevated nitrate (200 mg/L). These conditions are frequently encountered in aquaculture situations. Cooler temperatures decreased both the magnitude and onset of the humoral response, being lowest at 10°C, intermediate at 18°C, the highest at 24 and 29°C. Elevated ammonia did not affect the immune response, while elevated nitrate decreased antibody production to the level of the 18°C response. Hematologic reference intervals were determined for sunshine bass in tanks and recirculating systems, and palmetto bass in tanks. Serum chemistry reference intervals were determined for sunshine bass in tanks, recirculating systems and cage systems. Greater differences were observed in reference intervals between the culture systems, than between the two types of hybrid. To determine if environmental factors influenced the differences seen in the reference, sunshine bass were acclimated to 10, 18, 24, 29°C, elevated ammonia (0.15 mg/L NH3) and elevated nitrate (200 mg/L). The hematology and serum chemistry profiles of these fish were compared with the reference intervals for sunshine bass in tanks. Leukocyte, lymphocyte and monocyte counts at 10°C, and glucose and calcium at 10 and 18°C deviated sufficiently to suggest generating separate reference intervals at these temperatures. In the nitrate treated fish, creatinine levels were elevated and chloride levels were lower than controls and outside the reference interval. These two responses were presumed to be pathologic changes associated with elevated nitrate levels due to the large deviation in the analytes and the mortalities seen in the nitrate treated fish. The remaining analytes for fish in the different environments were within or slightly outside the reference intervals. These slight changes were presumed to be due to individual variation as the reference intervals were determined for fish under relatively uniform conditions and may not be sufficiently broad to cover fish from more varied environments. With minor modification, the reference intervals should apply to sunshine bass in most situations.
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    Evaluation of Diet, Gametogenesis, and Hermaphroditism in Freshwater Mussels (Bivalvia: Unionidae)
    Henley, William F. (Virginia Tech, 2002-05-10)
    To determine the effects of different algal diets on freshwater mussels, tissues of Elliptio complanata were sampled for physiological, somatic, and gametogenic condition from August 1999 to May 2000. Treatments included mussels fed Scenedesmus quadricauda (S), Neochloris oleoabundans (N), a no feed treatment (NF), and a reference group of mussels from the Nottoway River (NR), Virginia. The levels of protein and glucose differed among treatments (p<0.0001), but glycogen and percentage tissue moisture did not (p>0.17). Production of ripe and developing gametes differed significantly among treatments (p=0.001), but stage of gamete development did not (p=0.70). Lipid levels and muscle fiber areas of treatment groups differed significantly (p<0.0001). Results of the feeding trial indicate that S. quadricauda is a suitable feed for E. complanata, but future experiments should identify algal species higher in carbohydrates for a mixed algal diet. To determine sex and stage of gametogenesis, tissue histological sections from gonads of Villosa iris and Utterbackia imbecillis were evaluated. Occurrences of oogenic, spermatogenic, and hermaphroditic tissues were summarized in frequency tables. Visceral sites from which similar tissues were collected from conspecific specimens were evaluated for gametogenic stage. Sex was accurately determined in the central, visceral portion V. iris and female regions of U. imbecillis; and spermatogenic tissue was consistent in the dorsal-anterior areas of U. imbecillis. These areas also provided accurate determination of gamete stage in specimens. Reproductive asynchrony was observed among males and females (p<0.02). Male regions of U. imbecillis showed gamete stage characterized by mature and developing spermatogenic tissue, while 2 groups of mussels were showed oogenic development characterized by mature oocytes and resorption of gametes. Male V. iris showed early gamete development without mature spermatozoa, and 2 groups of female V. iris showed mature and developing gametes and resorption of gametes. Protocols for biopsy tissue collection from selected visceral areas were developed for U. imbecillis and V. iris for sex determination and staging of gametogenesis. The application of this biopsy protocol should be considered population specific, and protocols appropriate for other populations and species should be developed with methods of this study.
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    Gross and Microscopic Observations on the Lingual Structure of the West Indian Manatee (Trichechus manatus latirostris)
    Levin, Milton Jay (Virginia Tech, 2004-03-19)
    The West Indian manatee tongue was examined macroscopically, light microscopically, and electron microscopically (scanning and transmission). The tongue was slender, muscular, and firmly fixed in the oral cavity. Only the cranial tip was free and mobile. Numerous filiform papillae were distributed over the dorsal surface of the rostral lingual region. Caudal to the filiform papillae, multiple raised, round papillae were distributed over the majority of the dorsum. Fungiform papillae were restricted to the lateral margins of the tongue. Caudally, the dorsal and lateral regions showed numerous open fossae and pits. Microscopic examination showed the majority of the lingual dorsum to be covered with a thick stratified squamous epithelium. The caudal dorsal and lateral open pits led to well-developed mucous salivary glands. Foliate papillae, located on the caudal region of the tongue, contained taste buds embedded in the epidermis. Glands within the foliate papillae were mostly mucous, though some seromucous glands were evident. Throughout the tongue, striated muscle was abundant below the epidermis. Blood vessels, lymph channels, and nerve fibers were freely distributed throughout the intermuscular stroma. Nerve fibers reacted positively with neuron specific enolase antibody throughout the lingual structure, including nerve bundles, muscle bundles, glands, and taste buds. Electron microscopy revealed cytoplasmic vacuoles juxtaposed to the nucleus in the stratum spinosum of the foliate papillary region.
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    Identification of platelet activating factor (PAF) receptor in equine spermatozoa and its role in motility, capacitation and the acrosome reaction
    Odeh, Awatef (Virginia Tech, 2002-09-20)
    Platelet activating factor (PAF) is a unique signaling phospholipid that has many biologic properties in addition to platelet activation. PAF roles in reproduction involve ovulation, fertilization, embryo development, implantation and parturition. It may also serve as a biomarker for normal sperm function. The presence of PAF receptor on the spermatozoa of 10 stallions was investigated by immunofluorescence microscopy. Statistical analysis revealed that the fluorescence intensity, FI (Mean+/-SEM), in the post- acrosomal region (FI= 2.60+/-0.15) was significantly higher (P< 0.01) than that in any other region of stallion spermatozoa. The effect of synthetic PAF on stallion spermatozoal motility, capacitation, and the acrosome reaction (AR) were evaluated. Treatment of 10 stallion semen samples with 10 â 4 to 10 â 13 M PAF resulted in statistically significant differences in motility and capacitation (r2 = 0.81 and 0.83 respectively). The concentration of PAF, incubation time and their interaction were highly significant (P< 0.01) for their effect on motility. Concentrations of PAF ranging from 10-9 to10-11 M were able to induce capacitation. Following capacitation in vitro with PAF, and induction of the acrosomal reaction by progesterone, transmission electron microscopy (TEM) was conducted on the spermatozoa of 3 stallions, to detect the true AR. Differences in PAF concentrations were highly significant as indicated by R-square (for intact: 97.2, reacted: 89.8, and for vesiculated: 98.1). Treating spermatozoa from 3 stallions with the PAF antagonist FR-49175 inhibited calcium release and fluorescence intensity with a median inhibitory concentration (IC50) of 10-7.5 M (r2=0.82, P<0.01) and 10-8 M (r2=0.92, P<0.01) respectively, suggesting a receptor mediated process for the mechanism of action of PAF. Although the exact mechanisms of PAF action on equine spermatozoa remain unclear, it is widely reported that PAF acts by a receptor-mediated mechanism and that the PAF receptor is a member of the family of G-protein coupled receptors with phospholipase C as the effector. Since the limited success in equine ART (e.g. IVF) is in part due to lack of efficient treatment of stallion spermatozoa for capacitation, PAF may be useful to help capacitate stallion spermatozoa. Without proper capacitation, spermatozoa are unable to initiate the acrosome reaction which is a prerequisite for fertilization.
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    Mechanisms by which hypoxia augments Leydig cell viability and differentiated cell function in vitro
    Kukucka, Mark A. (Virginia Tech, 1993-04-18)
    The 1980's heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulosa cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture.
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    Mechanisms Controlling Ductal Morphogenesis in the Ruminant Mammary Gland
    Ellis, Steven E. (Virginia Tech, 1998-08-17)
    Basic research into the histology, endocrine control, and local regulation of prepuberal ruminant mammogenesis was conducted to provide a better understanding of this important developmental period. Histologic features of prepuberal ruminant mammary parenchymal morphogenesis were examined in tissue samples taken from ewe lambs at 2 (n = 5), 3 (n = 15), 6 (n = 26), 9 (n = 7), 12 (n = 5), and 13 wk (n = 20), and from Holstein heifers at 4 (n = 1) and 6 mo (n = 2). Examination of approximately 8000 histologic sections revealed that mammary parenchymal morphogenesis in sheep and cattle occurs through the proliferation of highly arborescent ductal structures embedded in a dense stroma. These observations contrast strongly with models of mammogenesis based on murine mammary development. The formation of luminal spaces and the expansion of ducts also differed from murine mammogenesis models. Luminal spaces were shown to develop through a progressive separation of opposing sides in initially solid ductal structures. Likewise, our investigation of prepubertal ovine mammogenesis revealed that parenchymal weight, 3H-thymidine labeling, stromal weight, and parenchymal DNA were all unaffected by ovariectomy (P > 0.05), in marked contrast to the dramatic reduction in mammary development following ovariectomy in rats, mice, and heifers. Responsiveness to exogenous estrogen (0.1 mg/kg) was demonstrated by increased 3H-thymidine labeling (P < 0.05) in both intact and ovariectomized lambs. Three dimensional collagen gel cultures of bovine mammary organoids from the peripheral (OUTER) and medial (INNER) parenchymal zones were used to characterize the proliferative and morphogenetic responses to local-acting growth factors. The proliferation of OUTER cells was 2 to 3 times greater than INNER cells (P < 0.0001) in response to IGF-I stimulation. Dramatic differences in the morphology of INNER and OUTER organoids were also observed. INNER cells grew into smooth-edged colonies when treated with heifer serum but stellate colonies when treated with other mitogens. OUTER cells grew into stellate colonies regardless of mitogen treatment. These investigations highlight the fact that a great deal more research into the basic physiology of prepuberal ruminant mammogenesis is required and that dogma developed in murine model systems may not be applicable to ruminant mammary physiology.
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    Molecular basis of MPTP-induced Parkinson's disease
    Zang, Lun-Yi (Virginia Tech, 1993)
    Self-administration of 1-methyl-4-pheny]-1,2,3,6-tetrahydropyridine (MPTP) has resulted in irreversible symptoms of Parkinson's disease in several young drug abusers. It was found that this neurotoxicant selectively destroys neuronal cells in the substantia nigra of humans and other primates. Although the mechanism of action of MPTP is not fully understood, it is now generally believed that the crucial species for MPTP neurotoxicity is not MPTP itself, but rather some of its metabolites. MPDP⁺, an intermediate in the metabolism of the neurotoxin MPTP, was found to generate superoxide radical (⋅O₂⁻) during its autoxidation process. The generation of ⋅O₂⁻ was detected by their ability to reduce ferricytochrome c. Superoxide dismutase (SOD) inhibited this reduction in a dosedependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP⁺, but also on the pH of the system. Thus, the rate of autoxidation of MPDP⁺ and the sensitivity of this autoxidation to superoxide dismutase inhibitable ferricytochrome c reduction were both augmented as the pH was raised from 7.0 to 10.5. The rate constant (kc) for the reaction of superoxide radical with ferricytochrome c to form ferrocytochrome c was found to be 3.48 x 10⁵ M⁻¹S⁻¹. The rate constant (kMPDP⁺) for the reaction of MPDP⁺ with ferricytochrome c was found to be 4.86 M⁻¹S⁻¹. The generation of ⋅O₂⁻ was further confirmed by spin-trapping in combination with EPR techniques using 5, 5-dimethyl-1-pyrrolonine-N-oxide (DMPO) as the spin trapping agent. The rate of formation of spin adduct (DMPO-O₂⁻) was dependent not only on the concentrations of MPDP⁺ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP⁺, and of SOD to effectively compete with this reaction for the available ⋅O₂⁻, was used as a convenient competition reaction to quantitatively determine various kinetic parameters. Using this technique, the rate constant for scavenging of superoxide radicals by superoxide dismutase was found to be 7.56 x 10⁹ M⁻¹S⁻¹. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP⁺ was found to be 4.48 x 10⁻¹⁰ M⋅S⁻¹. The rate constant (k₁) for MPDP⁺ making superoxide radical was found to be 3.97 x 10⁻⁶ Sec⁻¹. The second order rate constant (kDMPO) for DMPO trapping superoxide radicals was found to be 10.2 M⁻¹S⁻¹. The life time of superoxide radical at pH 10.0 was calculated to be 1.25 seconds. These data indicate that superoxide radicals are produced during spontaneous oxidation of MPDP⁺ and that EPR spin trapping techniques can be used to determine the rate constants and life time of free radicals generated in aqueous solution. Monoamine oxidase type B (MAO-B), an enzyme present in mitochondrial membranes, is known to metabolize MPTP to MPDP⁺, which then spontaneously oxidizes to MPP⁺. In the studies of MAO-B catalyzed oxidation of MPTP, the neurotoxicant was found to generate reactive oxygen species during its interaction with the enzyme. The kinetic parameters, Km and Vmax, for MAO-B catalyzed oxidation of MPTP to the corresponding species MPDP⁺ were found to be 0.194 mM and 0.335 µM/min, respectively. The generation of ⋅O₂⁻ and hydroxyl (⋅OH) radicals was detected as the DMPO spin adduct by spin trapping in combination with EPR techniques. Addition of Fe²⁺ (10 µM) to this system caused a 5-fold enhancement in EPR signal intensity of the DMPO-OH adduct. Catalase, a scavenger of hydrogen peroxide (H₂O₂), inhibited the DMPO-OH spin adduct formation in a dose-dependent manner, indicating that H₂O₂ is produced in the MAO-B catalyzed oxidation of MPTP. Ethanol, a well known scavenger of hydroxy] radical, rapidly produced an alpha-hydroxyethyl radical signal. SOD inhibited the formation of DMPO-O₂⁻ and DMPO-OH spin adducts in a dose-dependent fashion. These data suggest that ⋅O₂⁻ are produced during the oxidation of MPTP by MAO-B and that the generation of H₂O₂ and ⋅OH was secondary to the production of ⋅O₂⁻. MPTP and its metabolites, MPDP⁺ and MPP⁺, were found to inhibit the activity of acetylcholinesterase (AChE). The kinetic parameter, Km for the substrate (acetylthiocholine), was found to be 0.216 mM and Ki values for MPTP, MPDP⁺ and MPP⁺ to inactivate AChE were found to be 2.14, 0.265 and 0.197 mM, respectively. The inactivation of AChE by these neurotoxicants was found to be dose-dependent. It was found that MPTP, MPDP⁺ and MPP⁺ are neither substrates of AChE nor the time-dependent inactivators. The studies of reaction kinetics indicate that the inactivation of ACHE by these inactivators is via a mixed-type inhibition. The dilution of the enzyme-inhibitor complex completely reversed the MPTP inhibition but only partially reversed the MPDP+ and MPP+ inhibition. These data indicate that MPTP and its metabolites can inactivate AChE and thereby increase ACh level in the basal ganglia of the brain, leading to potential cell dysfunction. These results suggest that once MPTP enters the basal ganglia of the brain, it can be catalyzed by MAO-B to generate a series of reactive species, including ⋅O₂⁻, H₂O₂ and ⋅OH, which are known to destroy cell membranes, enzymes and other important biological molecules. The nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may largely be due to the reactivity of these reactive oxygen species in combination with the inactivation of the AChE enzyme in the brain, leading to potential cell dysfunction.
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    Ontogenic Morphology and Enzyme Activities of the Intestinal Tract of the Nile Tilapia, Oreochromis Niloticus
    Tengjaroenkul, Bundit (Virginia Tech, 2000-04-26)
    The gross intestinal configuration of the Nile tilapia intestine changed dramatically from a short, straight intestinal tube at hatch (day 0) to a very complex, coiling pattern first attained at 9 weeks post-hatch. During the developmental period, gut length increased from 90% to 410% of body length. The rate of increase in both intestinal and body lengths took place at an accelerating rate as the fish aged. The great intestinal length provides an advantage to the fish in digestion and absorption of nutrients present in the less energy-efficient herbivorous diet. Formulation of commercial diets to match the development of the fish's intestine may offer commercial advantage. Appearance, localization and distribution of intestinal enzymes were observed in the fish at hatch and at mature stages using enzyme histochemistry. At hatch (day 0), most gut enzymes were already present in the intestinal brush border. As the fish matured, activities of the enzymes were widely distributed along the intestinal tract. The early appearance and broad distribution of activities of all studied intestinal enzymes may be one factor contributing to the rapid growth rate characteristic of tilapia, which differs markedly from other fish species. To investigate the possibility of using alfalfa as a potential protein food replacement in tilapia, the effects of different levels of alfalfa in feeds on growth and intestinal enzyme activities were observed in the fish aged 3-15 weeks. Results demonstrated that replacing 20% and 40% of a commercial diet with alfalfa had an overall negative effect on body and intestinal growth, as well as the intestinal enzyme activities from age 3-9 weeks. Thus, using alfalfa as a food replacement is not optimal for fish of these young ages, but may yet be suitable for older fish.
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    Potential of Stem Cell-Based Therapy for Ischemic Stroke
    Marei, Hany E.; Hasan, A.; Rizzi, R.; Althani, A.; Afifi, N.; Cenciarelli, C.; Caceci, Thomas; Shuaib, Ashfaq (Frontiers, 2018-02-06)
    Ischemic stroke is one of the major health problems worldwide. The only FDA approved anti-thrombotic drug for acute ischemic stroke is the tissue plasminogen activator. Several studies have been devoted to assessing the therapeutic potential of different types of stem cells such as neural stem cells (NSCs), mesenchymal stem cells, embryonic stem cells, and human induced pluripotent stem cell-derived NSCs as treatments for ischemic stroke. The results of these studies are intriguing but many of them have presented conflicting results. Additionally, the mechanism(s) by which engrafted stem/progenitor cells exert their actions are to a large extent unknown. In this review, we will provide a synopsis of different preclinical and clinical studies related to the use of stem cell-based stroke therapy, and explore possible beneficial/detrimental outcomes associated with the use of different types of stem cells. Due to limited/short time window implemented in most of the recorded clinical trials about the use of stem cells as potential therapeutic intervention for stroke, further clinical trials evaluating the efficacy of the intervention in a longer time window after cellular engraftments are still needed.
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    The Roles of Age, Glomerular Location, and Collagen Expression in the Canine Kidney: Analysis of a Lifespan Study
    Pomeroy, Melinda J. (Virginia Tech, 2001-12-05)
    It is well documented that the incidence of renal disease, and therefore renal dysfunction, increases with age in many species of mammals. Such alterations in renal structure and function may significantly affect long-term toxicology studies. The purpose of this study was to assess the temporal evolution of glomerulosclerosis, an important renal lesion, in laboratory housed dogs, an important model system in chronic toxicological studies. We histopathologically examined representative sections of dog kidneys, quantified glomerular lesions (using the 0-5 scale of the World Health Organization classification system) and performed of statistical analysis of the extent and distribution of such changes. The kidney samples were obtained by necropsy, and occasionally biopsy, procedures from a collection of 159 purebred Beagle dogs maintained for their entire lifespan in well-controlled conditions. The lesions were correlated with sex, age, and intra-renal location of affected glomeruli to determine the relationship of each in the development of glomerulosclerosis. All dogs examined had some degree of glomerulosclerosis. In the youngest (up to 2 years of age), this was minimal, but was more advanced by middle age (3-7 years). The condition progressed with further aging and was associated with progressive fibrosis and tubular loss. Location and advancing age were significantly related to the development of glomerulosclerosis such that as age increases, the incidence of glomerulosclerosis increases, with the inner medullary ray and inner cortex demonstrating the highest occurrence. Using immunohistochemical analysis, the percentage of type IV collagen within glomeruli was determined. No significant increase in type IV collagen in glomeruli due to age or location was seen. An increase in type III or type V collagen within glomeruli was not apparent either, upon visual examination. This study indicates that renal lesions, including glomerulosclerosis, occur commonly and progress over the lifetime in a genetically similar population of laboratory Beagle dogs maintained under optimal standard environmental conditions. Such typical, age-related change needs to be taken into consideration when conducting chronic toxicological experiments using such animals.
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    Synthetic antifreeze peptide
    (United States Patent and Trademark Office, 1999-08-03)
    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic gene has been expressed in E. coli. A synthetic insert fragment has been prepared which can be readily inserted into the synthetic gene to alter the number of repeating units and/or amino acid composition in the antifreeze peptide produced.
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    Synthetic antifreeze peptide and synthetic gene coding for its production
    (United States Patent and Trademark Office, 1999-07-20)
    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic gene has been expressed in E. coli. A synthetic insert fragment has been prepared which can be readily inserted into the synthetic gene to alter the number of repeating units and/or amino acid composition in the antifreeze peptide produced.