Fishes in a changing world: learning from the past to promote sustainability of fish populations.

Populations of fishes provide valuable services for billions of people, but face diverse and interacting threats that jeopardize their sustainability. Human population growth and intensifying resource use for food, water, energy and goods are compromising fish populations through a variety of mechanisms, including overfishing, habitat degradation and declines in water quality. The important challenges raised by these issues have been recognized and have led to considerable advances over past decades in managing and mitigating threats to fishes worldwide. In this review, we identify the major threats faced by fish populations alongside recent advances that are helping to address these issues. There are very significant efforts worldwide directed towards ensuring a sustainable future for the world's fishes and fisheries and those who rely on them. Although considerable challenges remain, by drawing attention to successful mitigation of threats to fish and fisheries we hope to provide the encouragement and direction that will allow these challenges to be overcome in the future.

Prolactin production by immune cells.

Prolactin (PRL) is a pituitary hormone and a cytokine that plays an important role in rodent and human immune responses, including autoimmune diseases. However, many cells and tissues other than the pituitary make PRL, including immune cells. Here, we will present the evidence demonstrating PRL synthesis by different subtypes of immune cells from humans, mice and rats, describe the regulation of PRL gene expression in human lymphocytes, and discuss the functions of PRL made by immune cells. Finally, we will present evidence for involvement of immune cell PRL in human autoimmune disease and suggest how it might play a unique immunoregulatory role.

Structural and functional effects of high prolactin levels on injured endothelial cells: evidence for an endothelial prolactin receptor.

Stress has been linked to health problems such as atherosclerosis and prolonged wound healing, which involve the responses of injured endothelial cells. Though prolactin (PRL) levels become increased during the physiological response to stress, the significance and effects of these increases are largely unknown. Here we examined the effects of elevated, though physiological, concentrations of PRL on the responses of cultured endothelial cells after mechanical injury to cell monolayers. When treated at the time of injury with PRL levels of 62.5-1000 ng/mL, cells at the wound front became abnormal in shape and had reductions in f-actin staining in comparison to controls that were not PRL-treated. High PRL concentrations also inhibited the adhesion of cells to their growth surface in a dose-dependent manner. Using rhodamine-labeled PRL, we observed specific PRL uptake by these cells that suggested the presence of a PRL receptor. Finally, mRNA for the long form of the PRL receptor was detected by RT-PCR. To our knowledge, this is the first report demonstrating that (1) high PRL concentrations alter the actin cytoskeleton and adhesion of injured endothelial cells and (2) endothelial cells express the transcript for the PRL receptor. Thus, we report novel effects of PRL that may be mediated by activation of an endothelial cell PRL receptor.

Methotrexate causes apoptosis in postmitotic endothelial cells.

Methotrexate (MTX) is a commonly used chemotherapy agent for a variety of cancers. However, therapeutic levels are associated with numerous untoward effects such as central nervous system damage in children with acute lymphoblastic leukemia. The purpose of this study was to determine if MTX caused injury to endothelial cells using cultured bovine pulmonary artery endothelial cells as a model. Light microscopy showed gaps between cells and reduced numbers of endothelial cells after exposure to MTX (10(-9) to 10(-5) M), a range consistent with therapeutic drug levels. Proliferation and viability of subconfluent and confluent MTX-treated endothelial cells were measured by colorimetric (MTS) assay. There was a significant decline in cell numbers in MTX-treated subconfluent (growing) cells cultured after 4 days of MTX exposure compared to controls, as expected. However, there was also an unexpected decline in cell numbers in MTX-treated postmitotic endothelial cells after 1, 3, and 4 days of drug exposure. This suggested that MTX induced endothelial cell death. Fluorescent ApoAlert Enhanced Annexin-V binding demonstrated apoptosis in endothelial cells after 1 day of MTX exposure. Apoptosis was confirmed by a DNA fragment assay. This is apparently the first report of MTX-induced apoptosis of postmitotic, cultured endothelial cells. The findings suggest that apoptosis may be one mechanism of MTX-induced injury to endothelial cells.

Prolactin stimulates phosphorylation of the human T-cell antigen receptor complex and ZAP-70 tyrosine kinase: a potential mechanism for its immunomodulation.

Prolactin (PRL) is an immunomodulatory hormone which promotes T-cell activation and proliferation. However, the intracellular mechanisms of this action in normal lymphocytes are unknown. Because the PRL receptor (PRLR) activates several signals also activated by the T-cell antigen receptor (TCR)/CD3 complex, we evaluated whether signaling "cross-talk" occurs between these distinct receptors. Using human thymocytes, human peripheral blood lymphocytes and the rat Nb2 lymphoma T-cell, we found that PRL induced rapid phosphorylation of multiple, TCR/CD3 complex proteins, an event required for lymphocyte activation. Two of these phosphorylated proteins were identified to be CD3 epsilon and ZAP-70 tyrosine kinase, molecules essential for TCR function. Further, PRL induced tyrosyl phosphorylation of ZAP-70 in each population of T-lymphocytes tested, demonstrating for the first time that ZAP-70 is a target of PRL action. Taken together, our results suggest that the PRLR directly affects T-lymphocyte activation by means of signaling cross-talk with the TCR/CD3 complex.

Prolactin receptor signaling: shared components with the T-cell antigen receptor in Nb2 lymphoma cells.

Previously, we reported that activation of the human prolactin receptor (PRLR) produced a protein phosphorylation pattern strikingly similar to that provoked by Concanavalin A (Con A), an activator of the T-cell antigen receptor (TCR). These results suggested that certain signaling components of the TCR may be shared by the activated PRLR. Additional studies here assessed the levels of TCR expression following PRLR stimulation and the effect of TCR activation on PRL-stimulated proliferation in lactogen-dependent pre-T Nb2-11 lymphoma cells. The results indicated that the TCR was expressed on the surface of approx 4% of exponentially proliferating and prolactin- (PRL) treated cells. In contrast, approx 45% of quiescent cells, cultured in the absence of PRL for 24 h, expressed the TCR at the cell surface, suggesting that lactogen withdrawal may up-regulate TCR cell-surface expression. Moreover, TCR activation with anti-CD3 antibodies attenuated PRL-stimulated Nb2-11 cell proliferation in a concentration-dependent manner. In other experiments, immunoprecipitation and immunoblotting of Nb2-11 lysates revealed that activation of the PRLR resulted in rapid tyrosyl phosphorylation of ZAP-70, a critical TCR-associated tyrosine kinase. In addition, ZAP-70 was found to associate transiently with the putative guanine nucleotide exchange factor and substrate, Vav, in PRL-treated cells. ZAP-70 was also found to associate constitutively with the PRLR; PRL stimulation provoked the transient recruitment of Vav to the complex. These observations suggest that PRL signaling reflects the transient formation of a PRLR-ZAP-70-Vav complex and its immunomodulatory actions involve diverse interactions that affect TCR expression and signaling mechanisms.

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

Human thymocytes express a prolactin-like messenger ribonucleic acid and synthesize bioactive prolactin-like proteins.

Recent evidence has demonstrated an important immunoregulatory role for pituitary PRL. Moreover, PRLs have been identified as products of transformed human lymphocyte cell lines and normal murine lymphocytes, and implicated as regulators of their proliferative responses. However, PRL synthesis by normal human lymphocytes has not yet been reported. Here we demonstrate that human thymocytes and peripheral blood lymphocytes (PBL) synthesize PRL in primary culture. The principal form produced by thymocytes is 24 kilodaltons (kDa), essentially the same size as pituitary PRL, while PBL produced a 27-kDa variant. Size heterogeneity was evident, with products detected ranging from 21-29 kDa in various tissue samples, a phenomenon also found to occur in human pituitary and decidual PRL. Thymocytes and PBLs also synthesized a low mol wt form (11 kDa) that was released into culture supernatants concurrently with the larger PRL. The 24- and 11-kDa forms expressed PRL-like bioactivity in the Nb2 node lymphoma bioassay, further supporting their PRL-like nature. Expression of these PRLs was regulated by mitogen stimulation in thymocytes, but was constitutively produced in PBL. Northern blot analysis of thymocyte RNA using a human PRL cDNA probe detected a single PRL-like mRNA, which was significantly larger than human pituitary PRL mRNA. This was constitutively present in unstimulated thymocytes. Taken together, these data demonstrate that normal human lymphocytes synthesize bioactive PRLs similar in size to those produced by the pituitary. The presence of a single PRL mRNA suggests that the size variation observed in these proteins is probably due to posttranslational modification, such as proteolysis and glycosylation.

Prolactin gene expression in human thymocytes.

Recent evidence suggests that lymphocytes produce prolactin (PRL). Here, we report the cDNA cloning and expression of PRL from normal human thymocytes. Sequence analysis showed that the thymocyte cDNA encodes a 23 kDa protein which is identical to pituitary PRL. RNA blot analysis showed that the thymocyte PRL mRNA is approximately 170 nucleotides larger than the pituitary PRL message. PRL message was also detected in several non-pituitary human cell lines including Jurkat T, HeLa, and JEG cells. Furthermore, PRL gene expression in JEG cells was inhibited by glucocorticoid treatment. Our data support the hypothesis that PRL is a T cell-derived cytokine.

Up-regulation of prolactin gene expression and feedback modulation of lymphocyte proliferation during acute allograft rejection.

BACKGROUND: Although recent evidence suggests that prolactin is important in the immune response, bidirectional communication between prolactin and the immune system has not been demonstrated previously. We examined our hypothesis that this communication exists during mouse skin allograft rejection. METHODS: Serum prolactin levels were measured by bioassay, and pituitary prolactin mRNA was examined by use of Northern blots, in BALB/c mice receiving skin allografts from C57BL mice, on days 2, 4, and 6 after grafting. The feedback effects of prolactin on splenic lymphocytes were assessed in one-way mixed lymphocyte reactions, with or without added interleukin-2 (IL-2) or IL-4. RESULTS: Prolactin mRNA was increased significantly in grafted animals compared with sham animals (2.4-fold by day 4). Serum prolactin bioactivity was also elevated on all days tested. Prolactin treatment resulted in dose-dependent modulation of the mixed lymphocyte reaction with lymphocytes from grafted animals but not from sham animals. These effects depended on the time points and the presence of IL-2 or IL-4; the maximal enhancement occurred with day-4 lymphocytes cultured with IL-4 (80%). CONCLUSIONS: This report is the first to implicate in vivo immune regulation of prolactin gene expression. Our observations indicate that bidirectional interaction exists between prolactin and the immune system and provide a rationale for altering prolactin levels to treat allograft rejection.

Identification of prolactin receptors in hepatic nuclei.

Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturable and specific, high affinity 125I-prolactin binding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes.

A specific binding site in Nb2 node lymphoma cells mediates the effects of didemnin B, an immunosuppressive cyclic peptide.

Didemnin B (DB) is a cyclic depsipeptide with a variety of biologic effects, including potent antiviral, antitumor, and immunosuppressive activities. Although its mechanism of action has been attributed to inhibition of DNA and protein synthesis, the exact cellular site of interaction has not been previously defined. Since DB is strongly antiproliferative in Nb2 node lymphoma cells, we investigated potential DB binding sites in these cells, using [3H]-DB (2.7 mCi/mg) as the radiolabeled ligand. Time course studies with Nb2 cells showed that steady state [3H]-DB binding was attained after 4 h. Scatchard analysis with resting cells yielded a Kd of 180 nM (200 ng/ml), and 7 x 10(6) binding sites/cell. The IC50 of DB inhibition of ongoing protein and DNA synthesis in Nb2 cells, measured 24 h after prolactin (PRL) stimulation, was also in the range of 100 ng/ml. Didemnin analogs, with alterations at critical amino acid residues, inhibited the synthesis of DNA and protein and competed with [3H]-DB binding with the same rank order of potency. This implies that this binding site may mediate the inhibition of macromolecule synthesis. Subcellular fractionation of [3H]-DB labeled Nb2 cells revealed that specific binding occurred predominantly in the 100,000 g cytosolic fraction. Comparison with cyclophilin and the FK506 binding protein, both cytosolic receptors, suggests that the DB binding site may also belong to the family of immunophilins.

Identification of prolactin-like proteins synthesized by normal murine lymphocytes.

Cultured murine lymphoid cells release a PRL-like immunoreactive (IR) protein which may be important in immunity, as anti-PRL antisera inhibit lymphocyte proliferation in vitro. We examined culture supernatants (SNs) and cell lysates from concanavalin A (Con A) activated murine thymocytes to identify these proteins. Western blot analysis of cell lysates revealed three specifically-stained PRL-IRs. A doublet of bands at 35.6 and 33.6 kDa was associated with the particulate fraction of the cell. These PRL-IRs were present in lymphocytes independently of mitogen stimulation. In contrast, a 22 kDa PRL-IR was only produced in mitogen stimulated cells, and was specifically immunoprecipitated with anti-PRL antiserum. In addition, all three PRL-like IRs incorporated 35S-methionine in vitro, indicating that they are synthesized by these cells. Only the 22 kDa PRL-like protein was present in culture medium from stimulated cells, suggesting that this may be the PRL bioactivity previously demonstrated in SNs from murine lymphocytes.

Hepatic protein kinase C: translocation stimulated by prolactin and partial hepatectomy.

Prolactin stimulates a hepatotrophic response similar to that caused by phorbol esters or partial hepatectomy in rats. Since phorbol esters, which activate protein kinase C, mimic prolactin action in liver, the relationship between prolactin administration and subsequent hepatic protein kinase C translocation was assessed. Prolactin administration rapidly stimulated a 4-fold elevation of membrane protein kinase C activity. The effect of prolactin on hepatic protein kinase C was specific for lactogenic hormones but could be duplicated by phorbol esters. Further, an increase in serum prolactin was demonstrated subsequent to partial hepatectomy and preceding hepatic protein kinase C translocation. Therefore, translocation of hepatic protein kinase C appears important for hepatic proliferation in response to prolactin administration and to partial hepatectomy.

Concanavalin A-stimulated murine splenocytes produce a factor with prolactin-like bioactivity and immunoreactivity.

Culture supernatants from concanavalin A activated murine splenic mononuclear cells (splenocytes) were assayed for prolactin-like molecules using the Nb2 node lymphoma bioassay and immunostaining procedures. Con A stimulated splenocytes produced a factor with prolactin-like immunoreactivity that was mitogenic for Nb2 lymphoma cells. This factor first appeared in culture supernatants with the onset of lymphocyte mitogenesis and its activity was proportional to the number of cells cultured. In addition, splenocyte proliferation was inhibited by antiserum to rat prolactin, and this inhibition was partially reversed by added hormone. These findings show that murine splenocytes produce a substance with prolactin-like activity and suggest that this factor is essential for lymphocyte proliferation.