Vasoactive intestinal peptide (VIP) is a naturally occurring 28-amino acid peptide with a wide range of biological activities. Recent reports suggest that VIP receptors are expressed on a variety of malignant tumor cells and that the receptor density is higher than for somatostatin. Our aims were to label VIP with 99mTc--a generator-produced, inexpensive radionuclide that possesses ideal characteristics for scintigraphic imaging--and to evaluate 99mTc-VIP for bioactivity and its ability to detect experimental tumors. METHODS: VIP28 was modified at the carboxy terminus by the addition of four amino acids that provided an N4 configuration for a strong chelation of 99mTc. To eliminate steric hindrance, 4-aminobutyric acid (Aba) was used as a spacer. VIP28 was labeled with 1251, which served as a control. Biological activity of the modified VIP28 agonist (TP3654) was examined in vitro using a cell-binding assay and an opossum internal anal sphincter (IAS) smooth muscle relaxivity assay. Tissue distribution studies were performed at 4 and 24 h after injection, and receptor-blocking assays were also performed in nude mice bearing human colorectal cancer LS174T. Blood clearance was examined in normal Sprague-Dawley rats. RESULTS: The yield of 99mTc-TP3654 was quantitative, and the yields of 125I-VIP and 1251-TP3654 were >90%. All in vitro data strongly suggested that the biological activity of 99mTc-TP3654 agonist was equivalent to that of VIP28. As the time after injection increased, radioactivity in all tissues decreased, except in the receptor-enriched tumor (P = 0.84) and in the lungs (P = 0.78). The tumor uptake (0.23 percentage injected dose per gram of tissue [%ID/g]) was several-fold higher than 125I-VIP (0.06 %ID/g) at 24 h after injection in the similar system. In mice treated with unlabeled VIP or TP3654, the uptake of 99mTc-TP3654 decreased in all VIP receptor-rich tissues except the kidneys. The blood clearance was biphasic; the alpha half-time was 5 min and the beta half-time was approximately 120 min. CONCLUSION: VIP28 was modified and successfully labeled with 99mTc. The results of all in vitro examinations indicated that the biological activity of TP3654 was equivalent to that of native VIP28 and tumor binding was receptor specific. (+info)
The postnatal development of the alimentary canal in the opossum. I. Oesophagus.
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The oesophageal epithelium of the newborn opossum generally is two to three cells in depth and in some regions appears pseudostratified. By the 9th postnatal day the epithelium shows two distinct strata. Ciliated cells and occasional goblet cells also are observed within the epithelium during this stage and in subsequent stages. Cilia persist in the oesophagus of the adult opossum, but are restricted to the depths of the transverse folds found in the distal part of the organ. The epithelium covering the transverse folds of the adult likewise has an immature appearance. By 4-5 cm (ca. 20 days), the epithelium has assumed a more mature appearance and is of greater depth. This and later stages show three basic strata: a germinal layer, a spinous layer and, adjacent to the lumen, a flattened layer of cells that retain their nuclei. The epithelium throughout the postnatal period and in the adult does not undergo complete keratinization. The oesophageal glands begin as outgrowths from the epithelium just prior to 4-5 cm (ca. 20 days). The glands continue their development throughout the remainder of the postnatal period. The secretory units of the oesophageal glands of the the major portion of the secretory elements, and a light, rounded cell type which is less numerous and which occupies the terminal portions of the secretory units. Secretory material of the former appears complex, consisting of both neutral and acid glycoproteins. The secretory product of the light cell type is unknown at present. Both cell types are encompassed by myoepithelial cells. The relationship of the mitotic sequences to the observations made by microscopic examination of the developing oesophagus is discussed. (+info)
Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH.
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Chronic hypokalemia increases the activity of proximal tubule apical membrane Na+/H+ antiporter NHE3. The present study examined the effect of the incubation of OKP cells (an opossum kidney, clone P cell line) in control medium (K+ concn ([K+]) = 5.4 mM) or low-K+ medium ([K+] = 2.7 mM) on NHE3. The activity of an ethylisopropyl amiloride-resistant Na+/H+ antiporter, whose characteristics were consistent with those of NHE3, was increased in low-K+ cells beginning at 8 h. NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%, respectively, at 24 h but not at 8 h. After incubation in low-K+ medium, intracellular pH (pHi) decreased by 0.27 pH units (maximum at 27 min) and then recovered to the control level. Intracellular acidosis induced by 5 mM sodium propionate increased Na+/H+ antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K+- and sodium propionate-induced activation of the Na+/H+ antiporter at 8 and 24 h. Our results demonstrate that low-K+ medium causes an early decrease in pHi, which leads to an increase in NHE3 activity via a tyrosine kinase pathway. (+info)
Physiological effects and adjuvanticity of recombinant brushtail possum TNF-alpha.
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The present paper describes the physiological properties of recombinant possum TNF-alpha and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-alpha was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-alpha produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-alpha had lower specific activity compared to human TNF-alpha, while in a possum-specific assay, possum TNF-alpha enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-alpha. The physiological effect of the recombinant possum TNF-alpha was investigated in groups of possums administered doses of 6, 30 or 150 micrograms of cytokine. For each dose, TNF-alpha caused profound effects on the numbers of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6-24 h after injection and an initial sharp decrease in lymphocyte numbers. The efficacy of TNF-alpha as an immunological adjuvant was determined in possums administered KLH (125 micrograms) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-alpha (150 micrograms). Serum antibody responses to KLH were monitored by ELISA. The TNF-alpha stimulated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations, respectively. The strongest antibody responses were observed in the group of possums that received KLH formulated in Al(OH)3 with addition of TNF-alpha. (+info)
Cloning and expression of the transferrin and ferritin genes in a marsupial, the brushtail possum (Trichosurus vulpecula).
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Transferrin and ferritin cDNAs have been isolated and characterised from the common brushtail possum (Trichosurus vulpecula), the first marsupial examples of these genes. The transferrin cDNA encodes a 711 amino acid pre-protein which shows high levels of amino acid identity with eutherian transferrins (58-60%) and lactoferrins (54-56%). Phylogenetic analysis suggests that the possum transferrin has evolved independently along a pathway distinct from that of the eutherian transferrins and lactoferrins. Possum H-ferritin is a 182 residue protein which shares 86-94% amino acid identity with mammalian, avian and amphibian sequences. Ferritin mRNA was detected in all tissues tested, whereas transferrin was highly expressed in possum liver and mammary gland, and at lower levels in heart, testis and lung. In the possum mammary gland, ferritin mRNA was expressed throughout lactation with higher levels during the first 30 days which coincides with the high iron concentration of milk at this time. The transferrin gene was differentially expressed during lactation with peak mRNA levels detected during the first 6 days of lactation and after day 106 throughout late lactation. The pattern of transferrin mRNA expression in the mammary gland was identical to that of another whey protein, the late lactation protein, suggesting that the transcription of these genes may be regulated by a similar mechanism in this tissue. (+info)
Two stages of increased IgA transfer during lactation in the marsupial, trichosurus vulpecula (Brushtail possum).
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The polymeric Ig receptor (pIgR) and J chain molecules are involved in the transfer of IgA across the mammary gland epithelia into milk. The J chain binds two IgA molecules to form dimeric IgA, and the pIgR transports this complex through epithelial cells. We report here the cloning of the first marsupial homologues for the pIgR and J chain from the brushtail possum. Marsupial young are born after a short gestation and are less developed than eutherian newborn. The pouch young is completely dependent on milk as its sole source of nutrition during early lactation and this phase can be considered to be equivalent to an external gestation. Two periods of increased expression of pIgR, J chain, and IgA heavy chain mRNAs were observed in the mammary gland during lactation. The first occurs for a brief period after birth of the pouch young and is likely to reflect IgA transfer via the colostrum. The second period of increased expression, which is unique to marsupials, occurs after the early lactation period and just before young exit the pouch. We propose that this represents a second colostral-like phase at the end of the external gestation. (+info)
Molecular mechanism(s) of action of isoproterenol on the expression of the angiotensinogen gene in opossum kidney proximal tubular cells.
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BACKGROUND: beta-adrenoceptors are present in the renal proximal tubules. We have previously reported that isoproterenol stimulates the accumulation of intracellular cAMP and the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta 1-adrenoceptor. We hypothesized that the molecular mechanism(s) of action of isoproterenol on the expression of the ANG gene is mediated via the interaction of the phosphorylated cAMP-responsive element binding protein (CREB) and the cAMP-responsive element (CRE; that is, ANG N-806/-779) in the 5'-flanking region of the rat ANG gene. METHODS: The fusion genes containing the putative ANG-CRE of the rat ANG gene inserted upstream of the rat ANG basal promoter (ANG N-53/+18) fused to a human growth hormone (hGH) gene as reporter were stably cotransfected, with or without the plasmid containing the cDNA for 43 kDa CREB, into the OK cells. The effect of various agonists and antagonists of adrenoceptors on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH secreted into the culture medium. The interactions of OK cellular nuclear protein(s) with the ANG N-806/779 were determined by gel mobility shift assays and by Southwestern and Western blot analysis. RESULTS: The addition of isoproterenol, forskolin, or 8-Bromo-cAMP (8-Br-cAMP) stimulated the expression of pOGH (ANG N-806/-779/-53/+18) by 135, 150, and 160%, respectively, but not mutants of the ANG N-806/-779. The stimulatory effect of isoproterenol was blocked in the presence of propranolol, Rp-cAMP, and atenolol, but not by the presence of stauro-sporine, U73122, and ICI 118,551. Transient transfection of the plasmid containing the cDNA for the catalytic subunit of protein kinase A further enhanced the stimulatory effect of 43 kDa CREB on the expression of the fusion gene. The gel mobility shift assays revealed the the nuclear protein(s) of OK cells binds to the radioactive-labeled ANG N-806/-779. The binding of the labeled ANG N-806/-779 to the OK cell nuclear protein(s) was displaced by unlabeled ANG N-806/-779, but not by the CRE of the somatostatin gene, the CRE of the tyrosine amino-transferase gene, or the mutants of the ANG N-806/-779. Southwestern blot analysis revealed that the labeled ANG N-806/-779 binds to two nuclear species of 43 and 35 kDa proteins. Western blot analysis, however, revealed that rabbit polyclonal antibodies against the 43 kDa CREB interacted with only the 43 kDa molecular species but not with the 35 kDa species. CONCLUSION: These studies demonstrate that the stimulatory effect of isoproterenol on the expression of the ANG gene may be mediated, at least in part, via the interaction of the phosphorylated CREB and the CRE in the 5'-flanking region of the rat ANG gene. The novel 35 kDa nuclear protein that is immunologically different from the 43 kDa CREB may also play a role in the expression of the ANG gene. (+info)
Human munc13 is a diacylglycerol receptor that induces apoptosis and may contribute to renal cell injury in hyperglycemia.
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We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia. (+info)