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 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.

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.

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.

Didemnin B: a new immunosuppressive cyclic peptide with potent activity in vitro and in vivo.

Didemnin B (DB) is a 7-amino-acid, cyclic polypeptide with potent (10(7)-10(10)M) antiproliferative effects in vivo and in vitro against a variety of viruses and tumor cell lines. Because lymphocyte blastogenesis is essential for many immune responses, DB appeared likely to exert immunosuppressive effects as well. Using primary cultures of murine (Balb/c) splenic mononuclear cells to evaluate this possibility, we found that DB was a potent (IC50 = 190 ng/ml) inhibitor of lymphocyte protein synthesis, although RNA synthesis and cell viability were unaffected. However, it markedly inhibited blastogenesis stimulated by concanavalin A (IC50 = 50 pg/ml), lipopolysaccharide (IC50 = less than 100 pg/ml) and alloantigen (IC50 = less than 10 pg/ml) when added to cultures immediately after stimulation. DB added later, at the time of thymidine labeling was much less potent (1/46-1/1430), suggesting that the lymphocyte activation process is particularly sensitive to this agent. Our finding that alloantigen-driven proliferation was exquisitely sensitive to DB (greater than 90% inhibition at 10 pg/ml) led us to test its effects in vivo using the Simonsen parental-to-F1 graft-versus-host reaction (GVHR). Treatment of graft recipients with 0.05, 0.10, and 0.20 mg DB/kg/day for 7 days produced 51%, 40%, and 60% inhibition of splenomegaly induced by the GVHR, and treatment with 0.3 mg/kg/day on days 1, 2, 4, and 6 inhibited 71%. These data show that the in vitro inhibition of alloantigen-driven blastogenesis by DB was reproduced by in vivo treatment as well, even across major histocompatibility differences. This leads us to conclude that DB has potent immunosuppressive activity both in vitro and in vivo.

Didemnin B--an immunosuppressive cyclic peptide that stimulates murine hemagglutinating antibody responses and induces leukocytosis in vivo.

Didemnin B (DB) is a cyclic peptide with potent immunosuppressive activity in vitro and in the murine graft-versus-host-reaction (GVHR), the only measure of in vivo immunity tested in our prior studies. Because continued production of mature leukocytes by bone marrow and an intact antibody response are crucial to defense against infection in immunosuppressed patients, we have evaluated the effects of DB on these processes as well. Anti-sheep red blood cell (SRBC) hemagglutinating antibody (hAb) production was induced by i.p. injection of 5 x 10(7) SRBC in CB6F1 mice (5/group) treated with vehicle or DB once/day for six days. Serum was collected on day 7 and hAb titers measured by SRBC agglutination. Control antibody titers were 1/16, while animals receiving DB doses of 0.025, 0.05, 0.10, and 0.20 mg/kg/day yielded titers of 1/37, 1/74, 1/56, and 1/74, respectively. This stimulation of hAb production (4.6 x control) was confirmed by a second experiment. We then studied DB effects (0.1 mg/kg/day x 6 days) on serum hAb titers in separate groups of five mice at 7, 10, 15, and 20 days postimmunization. Control hAb titers were 1/110 on day 7, then dropped to 1/60 on days 10, 15, and 20. DB-treated animals had titers of 1/130 on day 7, and 1/170 on days 10-20. These data show that DB treatment in vivo causes a persisting increase in anti-SRBC hAb titers. Evaluation of DB effects on proliferation and antibody secretion in vitro by three hybridoma cell lines showed a potent inhibition of cell replication but stimulation of antibody production on a per-cell basis in each clone (+26%-+900%, range), suggesting a direct effect on Ig synthesis. During our first in vivo DB studies (0.1 mg/kg/day x 7 days) in mice, we noted that peripheral blood white counts were elevated on day 8 to 21.3 +/- 2.1 x 10(3)/mm3 compared with control (vehicle only) levels of 13.6 +/- 2.0 x 10(3)/mm3 (P less than .01). Kinetic studies showed that by 24 hr after a single i.p. injection of DB (1.0 mg/kg), blood leukocyte, granulocyte, and lymphocyte counts were elevated by 2.5-, 3-, and 2-fold, respectively, but declined rapidly thereafter. 3H-thymidine incorporation (4 hr) by freshly harvested bone marrow leukocytes from DB-treated mice (0.025, 0.05, and 0.10 mg/kg/day x 7 days) was enhanced up to 40% over control (P less than .05), while bone marrow cellularity was increased 200% (P less than .01).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.