Zoonotic filarial infections have been reported from most parts of the world. However, the range of species involved, host-parasite interactions and morphologic criteria for the specific identification of the species is still poorly known and understood. Species of Dirofilaria are the most common agents of human infection and the most studied group of zoonotic filariae. Several other groups, including species of Brugia and Onchocerca are being reported with increasing frequency. Loaina uniformis, a common parasite of rabbits in the southeastern United States, represents yet another species recovered from human tissues, although extremely rare. This parasite is of additional interest because its apparent morphologic and possible biologic similarity to Loa loa a common filaria of humans in West and Central Africa. The present study was undertaken to characterize the morphologic features of Loaina uniformis to enable its recognition when encountered in human tissues. This objective additionally permitted comparison of L. uniformis to Loa loa at both, gross and microscopic levels to determine, to whatever extent possible, the relationship of the two parasites. L. uniformis adult worms both living and dead were collected from the tissues of the natural host, the rabbit Sylvilagus floridanus. These were processed for gross examination and histologic study by light microscopy of serially sectioned material. Detailed microscopic observations were recorded for worms of both sexes and full anatomic characterizations were prepared. Emphasis was placed on the structure of the body and detailed features of the cuticle, hypodermis and musculature. The structural features of the digestive and reproductive systems as well as their arrangement within the pseudocoelom have been characterized. Observations indicate that L. uniformis can be distinguished from all other known species of zoonotic filariae available for study on the basis of their microanatomical features. Equivalent material of L. Loa obtained from experimental definitive hosts was compared with L. uniformis. The anatomical features of L. uniformis and L. loa are similar in many respects and they share many biological features suggesting that they are taxonomically closely related / acase@tulane.edu

The pineal hormone, melatonin has repeatedly been shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 human breast cancer cells. Previous reports have suggested that the actions of melatonin can be mediated either through G protein-coupled membrane receptors, or via retinoid orphan receptors (RORalpha), which constitutively activate gene transcription through their corresponding response elements termed ROREs. Transient transfection assays using an RORE-luciferase reporter construct have shown that the endogenous (RORalpha), receptors expressed in MCF-7 breast cancer cells exhibited a high basal level of transcriptional activity, which was further stimulated by serum. In the presence of serum, both the transcriptional activity and DNA binding ability of (RORalpha), were repressed by melatonin treatment, even though melatonin had no effect on RORalpha protein levels. Although RORalphas were originally proposed as nuclear receptors for melatonin, the direct binding of melatonin to these receptors can not be repeated. Therefore, it is not clear how melatonin can modulate (RORalpha), functions in MCF-7 cells. Consistent with the results from other systems, RORalpha transcriptional activity in MCF-7 cells was found to be responsive to intracellular calcium concentration ([Ca2+]i), calmodulin (CaM), and Ca2+/CaM-dependent protein kinases. Meanwhile, melatonin not only affected CaM subcellular distribution, but also modulated [Ca2+]i change induced by another known calcium stimulator, ATP, indicating that melatonin may regulate RORalpha transcriptional activity via its modulation of the Ca2+/CaM signaling pathway in MCF-7 cells. In exploring the biological functions of (RORalpha), receptors, we found that the decrease in RORalpha protein levels by an (RORalpha), antisense oligonucleotide con-elated with growth inhibition in MCF-7 cells. Our data suggest that RORalpha receptors may play a role in stimulating the proliferation of MCF-7 cells, and thus, the repressive effect of melatonin on these receptors may, at least in part, mediates melatonin's antiproliferative effects on breast cancer cells / acase@tulane.edu

Proenkephalin is one of the three genes known to encode endogenous opioid peptides. An antibody directed against a unique peptide sequence within proenkephalin that is not found in any other known peptide sequence was characterized and compared to a methionine enkephalin antibody. This antibody was found to be specific for enkephalinergic cell bodies, fibers and terminals. The development of proenkephalin mRNA in the neostriatum and its derivative peptides in the globus pallidus were found to parallel neurogenetic gradients. Expression was also found early in the central nucleus of the amygdala, late in the nucleus accumbens and olfactory tubercle, and transiently in clusters within the neostriatum. Dopaminergic innervation was found to precede proenkephalin expression in the rostral neostriatum and to follow proenkephalin expression in the caudal neostriatum. Neostriatal cells becoming postmitotic on embryonic day 14 (E14) moved twice during the course of their development: first ventrolaterally and then later dorsomedially in clusters. In contrast, neurons born at E19 were found to migrate only ventrolaterally. Clusters of neurons born at E14 corresponded to substance P immunoreactive patches. At progressively more rostral levels, progressively fewer E14-generated cells were detected in the neostriatum. The development of the two main peptidergic projection neurons of the neostriatum, enkephalin and substance P, began from opposite poles of the neostriatum and adjacent structures of the extended amygdala. During the perinatal period (from E16 to the day of birth), the distribution of neurons expressing proenkephalin spread primarily dorsomedially and rostrally. In contrast, neurons expressing substance P spread primarily dorsomedially and caudally. By the day of birth, there was extensive overlap in the distributions of proenkephalin and substance P in the neostriatum, but little to no coexpression of both peptides was ever found. Chronic opiate exposure in utero resulted in a selective increase in proenkephalin mRNA expression in the patch compartment of the neostriatum. Similarities in the anatomical connections, neurochemistry and development of the extended amygdala and the neostriatal patch compartment suggest that they may form a fundamental forebrain circuitry involved in motivated behavior and fight or flight responses / acase@tulane.edu

acase@tulane.edu

Luteinizing hormone releasing hormone (LHRH), a decapeptide, is synthesized in the central nervous system, released into the portal blood, and transported to the anterior pituitary, where it regulates the secretion of luteinizing hormone (LW) and follicle-stimulating hormone (FSH). Therefore, LHRH is a key integrator between the neural and endocrine systems and plays a pivotal role in promoting reproductive functions. The synthesis and secretion of LHRH are under the exquisite control of different neurotransmitters and steroid hormones, including glucocorticoids and androgens. In this dissertation, the effect of glucocorticoid treatment on the content of LHRH neurons was examined first. It was shown in male, but not in female rats, that corticosterone treatment not only increased the number of detectable LHRH neurons in the region around the organum vasculosum of the lamina terminalis (OVLT), but also increased the average size of the neurons in this region. Double-labeling immunocytochemistry demonstrated that LHRH neurons do not have androgen receptors. Further studies revealed that a certain number of tyrosine hydroxylase (TH) neurons in the hypothalamic periventricular nucleus and P-endorphin neurons in the arcuate nucleus colocalize with androgen receptors, suggesting that both TH neurons and P-endorphin neurons might be involved in the mediation of the effects of androgens on LHRH neurons. Androgenic-anabolic steroid (AAS) treatment increased the colocalization rate of TH neurons with androgen receptors in the periventricular nucleus. In the arcuate nucleus, the colocalization of TH neurons with androgen receptors was significantly greater in the dorsomedial than in ventrolateral portions of the nucleus. Finally, it was demonstrated that about 70% of somatostatin neurons in the periventricular nucleus contain androgen receptors. This result provides an anatomical basis for the possible direct action of androgens on these somostatin neurons. Contrary to what was expected, AAS treatment had no effect on the colocalization rate, suggesting that a subpopulation of somatostatin neurons may not express androgen receptors at all / acase@tulane.edu

Adrenal corticosteroids have diverse effects on central nervous system (CNS) function. These include regulation of the activity of neurotransmitters-modulators, neuro- and gliogenesis, learning and behavior and neuronal and glial survival. Many of these actions are thought to be mediated at least in part, by intracellular corticosteroid receptors. A Type I receptor, which acts as a mineralocorticoid receptor in the peripheral tissues, is selective for endogenous glucocorticoids in most regions of the CNS. A Type II receptor, also known as the classical glucocorticoid receptor has a low affinity and high capacity for endogenous glucocorticoids in the CNS. We mapped corticosteroid targets in the rat CNS using immunocytochemical detection of corticosteroid receptors. Contrary to previous reports of a restricted distribution of Type I receptors, we found that both types of corticosteroid receptors show widespread expression in neurons, and may indeed be coexpressed by some neurons. However, most glia expressed only Type II receptors. The intracellular location of corticosteroid receptors was regulated by corticosterone and aldosterone, the principle glucocorticoid and mineralocorticoid in rats. In the presence of these steroids, both Type I and II receptors showed a predominantly nuclear location, although cytoplasmic receptor was often present. This suggests a largely genomic role for these receptors in the CNS. We also demonstrated regulation of nuclear Type II receptor immunoreactivity (ir) by progesterone and androgenic-anabolic steroids. Our findings corroborated recent reports on cross-regulation of the Type II receptor by these steroids, based largely on binding studies Using double-labeling immunocytochemical techniques, we demonstrated for the first time, colocalization of Type II receptors by subpopulations of GABA-ir cerebellar Purkinje cells and LHRH-neurons. These cells regulate motor and neuroendocrine processes which are affected by circulating glucocorticoids. Most investigators have suggested that effects of glucocorticoids on these systems are largely indirect. By showing that some Purkinje and LHRH cells are potential direct targets for glucocorticoids, we have broadened the scope of possible mechanisms underlying the effects of glucocorticoids. Finally we examined the regulation expression of preproenkephalin (PPE) gene in the forebrain by glucocorticoids. PPE gene has been used as model, especially in vitro, to study direct regulation by glucocorticoids (i.e. corticosteroid receptor-mediated). We extended this concept in vivo, by examining glucocorticoid regulation of PPE gene expression in forebrain regions, which show an overlap in the distribution of PPE and corticosteroid receptors. In all regions analyzed, we found that circulating glucocorticoids were required for basal expression of PPE. Chronically elevated levels increased expression in selected regions, notably the striatum. We suggest that as in in vitro systems, PPE expression in vivo is regulated directly, at least in part, by glucocorticoids / acase@tulane.edu

No description available.

No description available.

MEQC (mvc embryonic quail cardiomyocytes) is a permanent cell line derived from cardiac tumors produced by infection of 3-day quail with the MC29 myelocytoma virus, which contains the v-myc proto-oncogene. This cell line can be induced to differentiate as evidenced by expression of muscle specific markers upon co-culture with NIH 3T3 fibroblasts. When MEQC are treated with low concentrations of BrdU before co-culture, they can no longer be induced to express phenotypic markers. In the current study, I have isolated BrdU-sensitive transcripts from MEQC cells by subtractive hybridization and investigated their distribution in developing chick embryos. In all, 29 transcripts were isolated, 14 of which could be identified by sequence comparison with the Genbank data base. Complete sequences were obtained for two of the remaining transcripts, pX19 and pX27. pX19 encodes a protein of 23 kDa that contains an amphipathic alpha-helix previously described only in plant seed embryos. By in situ hybridization pX19 was identified mainly in hemopoietic tissues; it was also found in cardiac cushion mesenchyme by PCR. The clone pX27 was identified as the avian homologue of mammalian glypican core proteins and was localized in early stages of development to the cephalic regions of the neural folds, rostral paraxial mesoderm, and newly formed somites. Later, glypican transcripts were found in the apical epidermal ridge of the limb buds, mantle zone of the telencephalon, and endocardial cushions of the atrioventricular canal and aortopulmonary outflow tract. An antibody raised against the glypican core protein was localized to the cell membrane in MEQC cells. Furthermore, upon withdraw of serum from cultures of MEQC expression of glypican transcripts was enhanced and the cells tended to clump. Administration of a glypican antisense oligonucleotide prevented cell clumping and blocked the migration of endocardial endothelial cells over collegen gel. Taken together, these results suggest that MEQC express transcripts unique to either myocardium or endocardium and provide a useful system from which transcripts expressed during development can be isolated.

Embryonic trichloroethylene (TCE) exposure was previously shown to be associated with an increased incidence of cardiac birth defects. Although embryo data are lacking exposure studies on adult animals show an association between halogenated hydrocarbon exposure and modifications in gene expression. The present study was undertaken to identify embryonic mRNA transcripts differentially expressed following TCE or metabolite exposure. This study identified numerous differentially regulated transcripts following halogenated hydrocarbon exposure. Examples of upregulated transcripts include stress responsive genes (Hsp 70, Hsp 70 cognate), a Ca²⁺-ATPase, calreticulin and serum response factor while downregulated transcripts include Midkine (RARP), numerous ribosomal proteins (8s, 18s, 24s), p137 and vimentin. p137 was a candidate sequence marked for further study to determine whether this sequence could be utilized as a molecular marker of TCE exposure. p137 showed a correlation between increased levels of maternal TCE exposure and decreased levels of transcript expressed in E11 fetal tissue. Immunohistochemical staining using an affinity purified antibody to p137 demonstrates widespread expression in rat E11 and chicken St. 17 embryos. p137 protein is broadly expressed in chicken St. 13 through St. 22 heart, but by St. 29 becomes more restricted in the ventricular myocardium with continued endocardial expression. At stages between St. 13 and St. 17 in chick embryos the ectodermal epithelium, yolk sac epithelium, dermatome, developing optic vesicle and neural tube express p137 protein. To explore potential function of p137, atrioventricular explants were exposed to affinity purified p137 antibody. Results show that p137 antibody treatment blocks epithelial-mesenchymal transformation of endothelial cells in-vitro. This study shows that p137 is expressed during rat and chicken mid-gestation in heart and other epithelial tissue derivatives and appears to play a role in the epithelial-mesenchymal transformation of the cardiac atrioventricular cushions. p137 is identified as a useful marker of developmental exposure to halogenated hydrocarbons and its altered expression may contribute to the phenotype of the affected heart.