Oxytocin secretion in response to cholecystokinin and food: differentiation of nausea from satiety.

Administration of cholecystokinin (CCK) to rats caused a dose-dependent increase in plasma levels of the neurohypophyseal hormone oxytocin (OT). The OT secretion was comparable to that found in response to nausea-producing chemical agents that cause learned taste aversions. The effect of CCK on OT secretion was blunted after gastric vagotomy, as was the inhibition of food intake induced by CCK. Food ingestion also led to elevated plasma OT in rats, but CCK and aversive agents caused even greater OT stimulation. Thus, after administration of large doses of CCK, vagally mediated activation of central nausea pathways seems to be predominantly responsible for the subsequent decrease in food intake. Despite their dissimilar affective states, both nausea and satiety may activate a common hypothalamic oxytocinergic pathway that controls the inhibition of ingestion.

Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli.

Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined. Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T. Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7. Expression of human and murine EPO was obtained using constructs based on pGEX-2T. For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions. Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity. Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione. Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive. The pGEX expression system was evaluated as a means of analysing the structure-function relationships of EPO by in vitro mutagenesis. Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins. Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity. The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone.

Oxytocin and vasopressin secretion in response to stimuli producing learned taste aversions in rats.

Administration of lithium chloride, copper sulfate, and apomorphine to rats each stimulated the secretion of oxytocin (OT) and, to a much lesser degree, arginine vasopressin. These agents are assumed to cause visceral illness in rats because of their effectiveness in promoting the acquisition of learned taste aversions. CuSO4 had a greater effect on plasma OT levels when administered ip rather than iv, whereas LiCl did not, results suggesting that LiCl probably stimulates OT secretion by central chemoreceptor activation whereas CuSO4 acts predominantly by local peritoneal irritation. A causal role for circulating OT in the acquisition of learned taste aversions was not found. These and other findings suggest that peripheral levels of OT may represent a quantifiable marker of visceral illness in rats and therefore might be useful in the interpretation of behavioral studies in which learned taste aversions are produced, provided that other stimuli of neurohypophyseal secretion are absent.

Structure-function relationships of the erythropoietin molecule.

The tertiary structure of erythropoietin (EPO) remains to be elucidated by X-ray crystallography. Although the amino acid sequence of EPO is known, the specific features that confer its biological activity are not well understood. In order to study the structure-function relationships of EPO by in vitro mutagenesis, we have used the vector pGEX-2T to express human and murine EPO fused to the carboxyl terminus of glutathione S-transferase (GST) in E. coli. The fusion proteins were the predicted size (46 kDa) by SDS-PAGE. GST-huEPO eluted from glutathione-agarose using reduced glutathione (GSH) was tested by radioimmunoassay and in a mouse spleen cell assay (MSCA). Dose-response curves parallel to recombinant human EPO (rHuEPO) were obtained in both assays. The ratio of immuno- to bioactivity was 4.7:1. Thus the presence of the 26 kDa GST protein at the end terminus of EPO does not abrogate biological activity. GST-mEPO also gave dose-response curves parallel to rHuEPO in the MSCA but not in the RIA. The wild-type murine and three mutant GST-EPO fusion proteins (166 Des-Arg, Glu 159-->Val, and Arg 163-->Glu) were tested in the MSCA and assayed for GST activity. The ratio of bioactivity to enzyme activity for the Arg 163-->Glu mutant was approximately one third of the value obtained for each of the other fusion proteins, indicating that arginine at 163 is functionally important for EPO activity. The availability of these human and murine gene constructs in pGEX should facilitate site-directed mutagenesis and permit detailed studies of the structure-function relationships for the two erythropoietins.

Leukemia-related chromosomal loss detected in hematopoietic progenitor cells of benzene-exposed workers.

Benzene exposure causes acute myeloid leukemia and hematotoxicity, shown as suppression of mature blood and myeloid progenitor cell numbers. As the leukemia-related aneuploidies monosomy 7 and trisomy 8 previously had been detected in the mature peripheral blood cells of exposed workers, we hypothesized that benzene could cause leukemia through the induction of these aneuploidies in hematopoietic stem and progenitor cells. We measured loss and gain of chromosomes 7 and 8 by fluorescence in situ hybridization in interphase colony-forming unit-granulocyte-macrophage (CFU-GM) cells cultured from otherwise healthy benzene-exposed (n=28) and unexposed (n=14) workers. CFU-GM monosomy 7 and 8 levels (but not trisomy) were significantly increased in subjects exposed to benzene overall, compared with levels in the control subjects (P=0.0055 and P=0.0034, respectively). Levels of monosomy 7 and 8 were significantly increased in subjects exposed to <10 p.p.m. (20%, P=0.0419 and 28%, P=0.0056, respectively) and ≥ 10 p.p.m. (48%, P=0.0045 and 32%, 0.0354) benzene, compared with controls, and significant exposure-response trends were detected (P(trend)=0.0033 and 0.0057). These data show that monosomies 7 and 8 are produced in a dose-dependent manner in the blood progenitor cells of workers exposed to benzene, and may be mechanistically relevant biomarkers of early effect for benzene and other leukemogens.

A comparison of the cytogenetic alterations and global DNA hypomethylation induced by the benzene metabolite, hydroquinone, with those induced by melphalan and etoposide.

Specific cytogenetic alterations and changes in DNA methylation are involved in leukemogenesis. Benzene, an established human leukemogen, is known to induce cytogenetic changes through its active metabolites including hydroquinone (HQ), but the specific alterations have not been fully characterized. Global DNA hypomethylation was reported in a population exposed to benzene, but has not been confirmed in vitro. In this study, we examined cytogenetic changes in chromosomes 5, 7, 8, 11 and 21, and global DNA methylation in human TK6 lymphoblastoid cells treated with HQ for 48 h, and compared the HQ-induced alterations with those induced by two well-known leukemogens, melphalan, an alkylating agent, and etoposide, a DNA topoisomerase II inhibitor. We found that rather than inducing cytogenetic alterations distinct from those induced by melphalan and etoposide, HQ induced alterations characteristic of each agent. HQ induced global DNA hypomethylation at a level intermediate to melphalan (no effect) and etoposide (potent effect). These results suggest that HQ may act similar to an alkylating agent and also similar to a DNA topoisomerase II inhibitor in living cells, both of which may be potential mechanisms of benzene toxicity. In addition to cytogenetic changes, global DNA hypomethylation may be another mechanism underlying the leukemogenicity of benzene.

Auditory pathways in the budgerigar. II. Intratelencephalic pathways.

The projections of two telencephalic areas in receipt of projections from the auditory relay nucleus of the thalamus (nucleus ovoidalis) were studied in the budgerigar (Melopsittacus undulatus) with autoradiographic methods. These nuclei are called field 'L' and neostriatum intermedium pars dorsolateralis (NIDL). The results show that neurons in both fields project laterally to a portion of the neostriatum intermedium pars ventrolateralis (NIVL) and rostrally to the rostromedial archistriatum. Horseradish peroxidase experiments confirm these projections and indicate that field 'L', NIDL and NIVL also receive projections from neurons in the hyperstriatum ventrale (HV). The projections of field 'L' and NIDL neurons to the rostromedial archistriatum may act as pathways subserving auditory feedback. Neurons in this portion of the archistriatum project to the contralateral field 'L', NIDL and NIVL. Furthermore, the medial archistriatal projection field of neurons within field 'L', NIDL and NIVL (i.e. rostromedial archistriatum) is located adjacent to a large archistriatal neuronal field projecting to the medulla, including the lateral reticular formation of the medulla. This large archistriatal field includes the nucleus archistriatalis robustus, the telencephalic nucleus identified as the source of projections to the lower motoneurons of the syrinx. Thus, projections from auditory telencephalic areas to the rostromedial archistriatum may serve functions related to processes associated with learning and vocal motor control.

Erythroid gene expression is differentially regulated by erythropoietin, haemin and delta-aminolaevulinic acid in UT-7 cells.

Erythropoietin (Epo) is essential for the later stages of erythropoiesis, acting to promote cell survival and proliferation, but its role in differentiation remains to be defined. The UT-7 cell line exhibits both erythroid and megakaryocytic characteristics and can be induced to differentiate along the erythroid pathway by Epo or the megakaryocytic pathway by phorbol myristic acetate. We have compared the effects of Epo and the chemical inducers, delta-aminolaevulinic acid (delta-ALA) and haemin on the differentiation capacity of UT-7 cells. Epo alone promoted relatively early events in erythroid maturation, without significant changes in haemoglobin production or morphology. GATA-2 and c-myb were down-regulated by Epo, and GATA-2 was further down-modulated by the inducers. Conversely, SCL expression was up-regulated by Epo and further increased by haemin and delta-ALA. Epo caused an increase in the proportion of cells expressing cell surface glycophorin A (GPA) and up-regulated beta- and gamma-globin by several fold. Both haemin and delta-ALA caused a de novo increase in alpha-globin expression as well as enhancing Epo-induced beta-globin expression, leading to a marked increase in haemoglobin production. These results suggest that haemoglobin production in UT-7 cells is limited by a deficiency of erythroid-specific aminolaevulinic acid synthase (ALAS-E) activity or globin synthesis as a consequence of their immaturity as a multipotential cell line.

Auditory pathways in the budgerigar. I. Thalamo-telencephalic projections.

Thalamo-telencephalic auditory pathways in the budgerigar (Melopsittacus undulatus) were studied using horseradish peroxidase (HRP) histochemistry and amino acids autoradiography. The results indicate that in this species the thalamic auditory relay nucleus, n. ovoidalis, projects upon a circumscribed region of the caudal and caudomedial neostriatum including field 'L' and immediately adjacent portions of the neostriatum intermedium, pars dorsolateralis (NIDL). This region of NIDL also receives inputs from another thalamic nucleus, n. dorsolateralis posterior (DLP). In the DLP is in receipt of tectal inputs. Projections of DLP upon NIDL were confirmed with amino acids autoradiography. The results of the HRP experiments indicate that different portions of n. ovoidalis project upon different portions of field 'L' and NIDL. Neurons in the dorsal and lateral portions of the n. ovoidalis project upon more medial portions of field 'L'. Neurons located centrally in the n. ovoidalis project upon central and lateral portions of field 'L'. Neurons in the ventromedial portion of the n. ovoidalis are labeled in all cases in which HRP is placed in either field 'L' or in the DLP projection field immediately adjacent to field 'L' proper. HRP injections placed in NIDL lateral to the projection fields of the n. ovoidalis and DLP label neurons within other diencephalic nuclei including the n. subrotundus. The caudal and intermediate levels of the neostriatum intermedium apparently serve as a complex processing area for many thalamic inputs in this species. The existence of multiple ascending thalamo-telencephalic projections from portions of the thalamus receiving inputs from both the visual (i.e., tectal) and auditory (i.e., n. mesencephalicus lateralis pars dorsalis) portions of the midbrain roof (i.e., from DLP and from n. ovoidalis) suggests the possibility that intermodal associations may take place in these telencephalic fields. Such partially converging pathways may provide a basics for intermodal associations which are important in individual recognition and social signalling systems in this species.

Regulation of erythropoietin gene expression depends on two different oxygen-sensing mechanisms.

Erythropoietin (Epo), a glycoprotein hormone produced principally in the fetal kidney and in the adult liver in response to hypoxia, is the prime regulator of growth and differentiation in erythroid progenitor cells. The regulation of Epo gene expression is not fully understood, but two mechanisms have been proposed. One involves the participation of a heme protein capable of reversible oxygenation and the other depends on the intracellular concentration of reactive oxygen species (ROS), assumed to be a function of pO2. We have investigated the production of Epo in response to three stimuli, hypoxia, cobalt chloride, and the iron chelator desferrioxamine, in Hep3B cells. As expected, hypoxia caused a marked rise in Epo production. When the cells were exposed to the paired stimuli of hypoxia and cobalt no further increase was found. In contrast, chelation of iron under hypoxic conditions markedly enhanced Epo production, suggesting that the two stimuli act by separate pathways. The addition of carbon monoxide inhibited hypoxia-induced Epo production, independent of desferrioxamine concentration. Taken together these data support the concept that pO2 and ROS are sensed independently.