Anand S. Srivastava

mRNA expression of pancreatic enzyme precursors and estimation of protein digestibility in first feeding larvae of the Japanese flounder, Paralichthys olivaceus

An understanding of digestibility in marine fish larvae is required to formulate a diet to replace zooplankton. Using flounder, this study was aimed at determining which digestive enzymes are synthesized in the larval pancreas, and how the proteins are cleaved in the digestive canal. Whole mount in situ hybridization indicated that the mRNA of all digestive enzyme precursors examined, including trypsin, chymotrypsin, elastase, carboxypeptidase A and B, and lipase, was expressed in the pancreas of first feeding larvae at 3 days post-fertilization. In the larvae before differentiation of the stomach, protein digestion in the digestive canal mainly depends on pancreatic proteases. So, to evaluate protein digestibility in the larval digestive canal, the digestion of proteins by pancreatic extract was monitored by gel electrophoresis. It was indicated that thyroglobulin, albumin and lactate dehydrogenase were rapidly cleaved to polypeptide fragments, but ferritin and catalase exerted resistance to proteolysis, suggesting that digestibility in the larval digestive canal differs depending on protein species.

Experimental induction of jaw, gill and pectoral fin malformations in Japanese flounder, Paralichthys olivaceus, larvae

Abstract The phenotypes of skeletal malformations induced in pharyngeal arches and pectoral fins of Japanese flounder larvae by retinoic acid (RA), disulfiram, 2,2′-dipyridyl and azetidine-2-carboxylic acid were characterized, RA controls gene expression essential for pharyngeal and pectoral fin development; disulfiram is an inhibitor of RA synthase, 2,2′-dipyridyl and azetidine-2-carboxylic acid are inhibitors of collagen synthesis. In larvae exposed to RA at shield stage for 1 h, the Meckels cartilage did not form in mandible arch. Exposure to RA from hatching period shifted the growth direction of the pharyngeal cartilages posteriorly. Disulfiram did not affect the cartilage formation when given before hatching, even though it shortened the trunk. However, disulfiram exerted teratogenic effects when given after hatching time, inducing bending pharyngeal cartilages and S-shaped pectoral fin plate. 2,2′-Dipyridyl given from hatching also caused bending of pharyngeal cartilages and S-shaped pectoral fin plates. Azetidine-2-carboxylic acid reduced the size of cartilages, without causing remarkable malformation. Thus, it was demonstrated that both RA and inhibitor of its synthesis, and inhibitors of collagen synthesis exert specific teratogenic effects on both pharyngeal and pectoral skeletons of flounder larvae.

cDNA cloning and phylogenetic analysis of pancreatic serine proteases from Japanese flounder, Paralichthys olivaceus

To better understand the digestive physiology and phylogeny of the pancreatic serine proteases of teleosts, we cloned trypsin, chymotrypsin and elastase from flounder (Paralichthys olivaceus). Fifty phage plaques randomly chosen from a flounder pancreatic cDNA library were found to contain three species of trypsin, two species of chymotrypsin and four species of elastase. cDNAs of two species of carboxypeptidase A, one carboxypeptidase B and lipase were also obtained. In total, 23 out of 24 digestive enzyme cDNAs were those of proteolytic enzymes. Such a high ratio of proteolytic enzyme cDNA in the pancreas may reflect the carnivorous feeding habits of flounder. A phylogenetic comparison of the peptide sequences of flounder enzymes with those of other teleosts and mammals suggested that duplication of trypsin, chymotrypsin and elastase occurred before the divergence of the ray finned fish. It is also hypothesized that functional descendants of both duplicated genes of elastase exist in the teleosts and mammals, whereas only one of the genes of trypsin and chymotrypsin gave rise to the functional descendants in the teleosts but not in the mammals.

Transplanted Embryonic Stem Cells Successfully Survive, Proliferate, and Migrate to Damaged Regions of the Mouse Brain

An understanding of feasibility of implanting embryonic stem cells (ESCs), their behavior of migration in response to lesions induced in brain tissues, and the mechanism of their in vivo differentiation into neighboring neural cells is essential for developing and refining ESC transplantation strategies for repairing damages in the nervous system, as well as for understanding the molecular mechanism underlying neurogenesis. We hypothesized that damaged neural tissues offer a niche to which injected ESCs can migrate and differentiate into the neural cells. We inflicted damage in the murine (C57BL/6) brain by injecting phosphate‐buffered saline into the left frontal and right caudal regions and confirmed neural damage by histochemistry. Enhanced yellow fluorescent protein‐expressing ESCs were injected into the nondamaged left caudal portion of the brain. Using immunohistochemistry and fluorescent microscopy, we observed migration of ESCs from the injection site (left caudal) to the damaged site (right caudal and left frontal). Survival of the injected ESCs was confirmed by the real‐time polymerase chain reaction analysis of stemness genes such as Oct4, Sox2, and FGF4. The portions of the damaged neural tissues containing ESCs demonstrated a fourfold increase in expression of these genes after 1 week of injection in comparison with the noninjected ESC murine brain, suggesting proliferation. An increased level of platelet‐derived growth factor receptor demonstrated that ESCs responded to damaged neural tissues, migrated to the damaged site of the brain, and proliferated. These results demonstrate that undifferentiated ESCs migrate to the damaged regions of brain tissue, engraft, and proliferate. Thus, damaged brain tissue provides a niche that attracts ESCs to migrate and proliferate.

Thrombopoietin Enhances Generation of CD34+ Cells from Human Embryonic Stem Cells

The role of thrombopoietin (TPO) in adult hematopoiesis is well‐established. A recent report suggests that TPO and vascular endothelial growth factor (VEGF) play a role in promoting formation of early erythropoietic progenitors in a nonhuman primate embryonic stem cell (ES) model. No such report exists for human ES cells as yet. Because TPO may become an important factor promoting human ES cell‐derived hematopoiesis, we sought to investigate whether TPO in combination with VEGF can enhance human ES‐derived hematopoiesis in an EB‐derived culture system. The emphasis of this work was to demonstrate the molecular mechanisms involved in this process, specifically the role of c‐mpl and its ligand TPO. Human ES cells were cultured to the EB state, and EB‐derived secondary cultures supporting hematopoietic differentiation were established: condition 1, control (stem cell factor [SCF] and Flt3 ligand [Flt3L]); condition 2, SCF, Flt3L, and TPO; and condition 3, SCF, Flt3L, TPO, and VEGF. Cells were harvested daily, starting at day 2 and continuing until day 8, for reverse transcription‐polymerase chain reaction and Western blot. There was no evidence of expression of c‐mpl and VEGF receptor on the gene or protein level until day 8, when the formation of well‐established hematopoietic colonies began. This correlated with the formation of CD34+/CD31− negative progenitors, mostly found in blast‐forming units‐erythroid‐like colonies. We concluded that TPO and VEGF play an important synergistic role in the formation of early ES‐derived hematopoietic progenitors that occurs through the c‐mpl and VEGF receptors.

Alloreactivity following in utero transplantation of cytokine-stimulated hematopoietic stem cells: The role of recipient CD4− cells

OBJECTIVE We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca(+)/lin(-)) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13). MATERIALS AND METHODS Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)-purified sca(+)/lin(-) cells of C57BL/6 (H-2b, 1E(+)) background were injected into MHC-mismatched BALB/c (H-2d, 1E(-)) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR). Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance. RESULTS When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca(+)/lin(-) cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells. CONCLUSION These data demonstrate that transplantation of cytokine-stimulated sca(+)/lin(-) allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.

Tolerance and immunity following in utero transplantation of allogeneic fetal liver cells: the cytokine shift.

Although in utero transplantation (IUT) has resulted in donor-specific tolerance to postnatal solid organ transplantation, the mechanisms of this tolerance remain poorly understood. Our recent findings demonstrate that under specific conditions prenatal injection of allogeneic cells may lead to allosensitization instead of tolerance. These laboratory observations were supported by clinical findings as well, and therefore suggested that, depending on the conditions of prenatal transplantation, tolerance or immunity may develop. The present study explored the role of CD4 cells, cytokines, and I-E superantigen in developing tolerance vs. immunity after in utero transplantation. Sixteen animals survived IUT (40–60% survival rate) and were free from any signs of graft-versus-host disease (GVHD). Mice were considered tolerant when their antidonor and antihost CTL responses were similar, sensitized when antidonor responses were significantly higher than antihost and anti-third-party responses, and nontolerant when antidonor responses in transplanted and control mice were similar. The TH1 → TH2 shift was associated with tolerance and TH2 → TH1 shift with allosensitization. Our results showed that tolerant BALB/c (H-2d, I-E+) → C57BL/6 (H-2b, I-E–) (2/7) mice showed higher IL-4 (p < 0.05) in antidonor MLR, and partial deletion of recipient I-E-reactive T cells (CD3Vβ11) (p < 0.045). On the other hand, nontolerant animals (5/7) demonstrated high production of IFN-γ (p < 0.05) without deletion of CD3Vβ11 T cells. In C57CBL/6 (H-2b, I-E–) → C3H (H-2k, I-E+) mice CD3Vβ11 T cells do not play any role in tolerance induction because they are deleted in the C3H background. Tolerant mice (4/9) showed an overproduction of IL-4 (p < 0.05) in antidonor MLR whereas allosensitized animals (5/9) demonstrated high level of IFN-γ (p < 0.05). Suppressor cells seem to play no role in tolerant C57BL/6 → C3H as demonstrated by suppressor assay. Hence, a shift from TH1 → TH2 or TH2 → TH1 cytokines may determine whether tolerance or immunity develops.

Chimerism and tolerance post-in utero transplantation with embryonic stem cells

Background. Clinical application of in utero transplantation (IUT) in human fetuses with intact immune systems resulted in a very low level of donor chimerism. In this study, we examined whether the fetal immune system early in the second trimester of pregnancy (13.5 dpc) can initiate immune tolerance for major histocompatibility complex (MHC)-mismatched embryonic stem (ES) cells. We also examined whether immune tolerance mechanisms respond differently to ontogenetically different stem cells. Methods. MHC-mismatched ES, fetal liver (FL), and bone-marrow (BM) cells (H-2kb) at 1×109 cells/kg fetal body weight were injected intraperitoneally into 13.5 dpc BALB/c fetuses (H-2Kd). Peripheral chimerism was determined in blood by flow cytometry (sensitivity≤0.1%) at monthly intervals. Donor-specific immune responses were determined by cytotoxic lymphocyte (CTL) assay, mixed lymphocyte reaction, and T helper (Th)1 and Th2 cytokine assays. Chimeric mice at the age of 9 months received postnatal boosts (PB) with minimal conditioning of 200 cGy by intravenous injection of 1×109 of the corresponding cells/kg body weight. Results. After IUT with ES, FL, or BM cells, the level of peripheral chimerism within the first 9 months of life was 0% to 0.4%. PB with 1×109/kg of corresponding cells resulted in a decrease in the peripheral chimerism to 0% within 2 weeks of PB. CTL and cytokine assays before and after PB demonstrated a shift toward immunity. Conclusions. Immunologic tolerance was not achieved after IUT of murine fetuses at 13.5 dpc with MHC-mismatched ES cells, and only a low level chimerism was achieved.

In utero detection of T7 phage after systemic administration to pregnant mice

The phage is used as a scaffold to display recombinant libraries of peptides, which provides the means to rescue and amplify peptides that bind target macromolecules. Many reports showed that the T7 phage display method can be used to obtain a ligand-binding peptidefor tissue-targeted therapies in adult animals. In utero tissue targeting of fetal tissues may help in the correction of many genetic and metabolic diseases. Here we demonstrate the distribution and detection of T7 phage displaying the C-X7-C peptide library in mouse fetal tissues after systemic injection of T7 phage into pregnant mouse tail vein. T7 phage was recovered from fetal tissues 15 min after injection of T7 phage. Our results suggest that T7 phage may be a useful tool in selecting the tissue-specific ligand-binding peptide for fetal tissues. This approach may be helpful in designing in utero tissue-targeted therapies.

Induction of Bent Cartilaginous Skeletons and Undulating Notochord in Flounder Embryos by Disulfiram and α, α'-Dipyridyl

Abstract Disulfiram causes undulation of the notochord and bending of pharyngeal cartilages in fish embryos. Using flounder embryos, this study aimed to elucidate which process of pharyngeal arch development was affected by the drug. Since disulfiram is known to block the synthesis of retinoic acid (RA) in vivo, we first examined whether the drug suppresses the expression of Hoxd-4 and shh, RA responsive genes related with the pharyngeal arch development. Disulfiram at a concentration which induces undulation of the notochord and bending of cartilage elements did not affect the expression of these genes. On the other hand, similar phenotypes of anomalies were found to be reproduced both in the notochord and pharyngeal cartilages by α, α′-dipyridyl which reduces the mechanical stability of collagen. Thus, we suppose that disulfiram causes anomalies by decreasing the mechanical stability of collagen and not by suppressing the expression of RA-responsive genes. Since disulfiram blocks ascorbate dehydrogenase, it is our hypothesis that this drug inhibits the maturation of collagen by affecting ascorbic acid metabolism.