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Interactions between the upper ocean and air-ice-ocean fluxes in the Southern Ocean play a critical role in global climate by impacting the overturning circulation and oceanic heat and carbon uptake. Remote and challenging conditions have led to sparse observational coverage, while ongoing field programmes often fail to collect sufficient information in the right place or at the time-space scales required to constrain the variability occurring in the coupled ocean-atmosphere system. Only within the last 10 years have we been able to directly observe and assess the role of the fine-scale ocean and rapidly evolving atmospheric marine boundary layer on the upper limb of the Southern Ocean's overturning circulation. This review summarizes advances in mechanistic understanding, arising in part from observational programmes using autonomous platforms, of the fine-scale processes (1-100 km, hours-seasons) influencing the Southern Ocean mixed layer and its variability. We also review progress in observing the ocean interior connections and the coupled interactions between the ocean, atmosphere and cryosphere that moderate air-sea fluxes of heat and carbon. Most examples provided are for the ice-free Southern Ocean, while major challenges remain for observing the ice-covered ocean. We attempt to elucidate contemporary research gaps and ongoing/future efforts needed to address them. This article is part of a discussion meeting issue 'Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities'.

The Southern Ocean greatly contributes to the regulation of the global climate by controlling important heat and carbon exchanges between the atmosphere and the ocean. Rates of climate change on decadal timescales are therefore impacted by oceanic processes taking place in the Southern Ocean, yet too little is known about these processes. Limitations come both from the lack of observations in this extreme environment and its inherent sensitivity to intermittent processes at scales that are not well captured in current Earth system models. The Southern Ocean Carbon and Heat Impact on Climate programme was launched to address this knowledge gap, with the overall objective to understand and quantify variability of heat and carbon budgets in the Southern Ocean through an investigation of the key physical processes controlling exchanges between the atmosphere, ocean and sea ice using a combination of observational and modelling approaches. Here, we provide a brief overview of the programme, as well as a summary of some of the scientific progress achieved during its first half. Advances range from new evidence of the importance of specific processes in Southern Ocean ventilation rate (e.g. storm-induced turbulence, sea-ice meltwater fronts, wind-induced gyre circulation, dense shelf water formation and abyssal mixing) to refined descriptions of the physical changes currently ongoing in the Southern Ocean and of their link with global climate. This article is part of a discussion meeting issue 'Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities'.

The subpolar Southern Ocean is a critical region where CO 2 outgassing influences the global mean air-sea CO 2 flux (F CO2 ). However, the processes controlling the outgassing remain elusive. We show, using a multi-glider dataset combining F CO2 and ocean turbulence, that the air-sea gradient of CO 2 (∆pCO 2 ) is modulated by synoptic storm-driven ocean variability (20 µatm, 1-10 days) through two processes. Ekman transport explains 60% of the variability, and entrainment drives strong episodic CO 2 outgassing events of 2-4 mol m -2 yr -1 . Extrapolation across the subpolar Southern Ocean using a process model shows how ocean fronts spatially modulate synoptic variability in ∆pCO 2 (6 µatm 2 average) and how spatial variations in stratification influence synoptic entrainment of deeper carbon into the mixed layer (3.5 mol m -2 yr -1 average). These results not only constrain aliased-driven uncertainties in F CO2 but also the effects of synoptic variability on slower seasonal or longer ocean physics-carbon dynamics., (© 2022. The Author(s).)

The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application.

Chronic infection with hepatitis B virus (HBV) is a major risk for development of hepatocellular carcinoma (HCC), which is the fifth most common cancer and a leading global cause of mortality. Long noncoding RNAs (lncRNAs) are regulators of complex biological processes and their functional disruption is implicated in the etiology of many cancers including HCC. Several lncRNAs have been shown to have oncogenic or tumor suppressive roles and have recently become the focus of intense investigation. However, the contributions of lncRNAs to HBV-related HCC remain to be fully elucidated. In this review we concentrate on the functional roles of various lncRNAs in HBV-associated HCC. Their involvement in viral replication, the specific association of certain lncRNAs with HBV-related HCC, potential utility as therapeutic targets and diagnostic markers are discussed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viral hepatitides, are complex and currently difficult to treat. The maturation of gene therapy has heralded new avenues for developing effective intervention for these diseases. DNA modification using gene therapy is now possible and available technology may be exploited to achieve long term therapeutic benefit. The ability to edit DNA sequences specifically is of paramount importance to advance gene therapy for application to liver diseases. Recent development of technologies that allow for this has resulted in rapid advancement of gene therapy to treat several chronic illnesses. Improvements in application of derivatives of zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), homing endonucleases (HEs) and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated (Cas) systems have been particularly important. These sequence-specific technologies may be used to modify genes permanently and also to alter gene transcription for therapeutic purposes. This review describes progress in development of ZFPs, TALEs, HEs and CRISPR/Cas for application to treating liver diseases.

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The role of the thymus is vital for orchestration of T-cell development and maturation. With increasing age the thymus undergoes a process of involution which results in a reduction in thymic size, function and output. Until relatively recent it was not feasible to accurately measure the magnitude of age-related loss of thymic function. With the discovery of T-cell receptor excision circles (TRECs), which are the stable by-products of the newly generated T-cells, it is now possible to quantitatively measure the extent of thymic output. This review examines the available data on immune function and zinc deficiency and places them in the context of the aims of the ZINCAGE project which include the evaluation of the role played by zinc in maintaining thymic output in healthy elderly individuals.

Separation of small cell lung carcinoma (SCLC) from nonsmall cell lung carcinoma (NSCLC) is a critical distinction to be made in the diagnosis of lung cancer. However, the diagnosis of SCLC is most commonly made on small biopsies and cytologic specimens, and practicing pathologists may not be familiar with all its morphologic guises and frequent combination with NSCLC elements, which may be seen in larger specimens. Following the most recent WHO classification of lung tumors and with the hope of identifying prognostic markers, we examined in detail the histology of 100 surgical biopsies or resections with a diagnosis of SCLC from the AFIP and pathology panel of the International Association for the Study of Lung Cancer (IASLC). Multiple clinical and histologic features were studied by Kaplan-Meier analysis. Neuroendocrine architectural patterns, including nested and trabecular growth, with peripheral palisading and rosette formation were common in SCLC. Necrosis and apoptotic debris was prominent in all cases, but crush artifact was infrequent. Cell size in surgical biopsy specimens appears larger than in bronchoscopic biopsy specimens and occasional cells may show prominent nucleoli and vesicular nuclear chromatin, but this does not preclude the diagnosis of SCLC. A high percentage of cases (28%) showed combinations with NSCLC, with large cell carcinoma the most common, followed by adenocarcinoma and squamous cell carcinoma. Because of the frequency of a few scattered large cells in SCLC, we arbitrarily recommend that at least 10% of the tumor show large cell carcinoma before subclassification as combined SC/LC. However, combined SCLC is easily recognized if the additional component consists of other NSCLC subtypes such as adenocarcinoma or squamous cell carcinoma, so no percentage requirement is needed. Stage remained the only predictor of prognosis.

Overexpression of E2F-1 induces apoptosis by both a p14ARF-p53- and a p73-mediated pathway. p14ARF is the alternate tumor suppressor product of the INK4a/ARF locus that is inactivated frequently in lung carcinogenesis. Because p14ARF stabilizes p53, it has been proposed that the loss of p14ARF is functionally equivalent to a p53 mutation. We have tested this hypothesis by examining the genomic status of the unique exon 1beta of p14ARF in 53 human cell lines and 86 primary non-small cell lung carcinomas and correlated this with previously characterized alterations of p53. Homozygous deletions of p14ARF were detected in 12 of 53 (23%) cell lines and 16 of 86 (19%) primary tumors. A single cell line, but no primary tumors, harbored an intragenic mutation. The deletion of p14ARF was inversely correlated with the loss of p53 in the majority of cell lines (P = 0.02), but this relationship was not maintained among primary tumors (P = 0.5). E2F-1 can also induce p73 via a p53-independent apoptotic pathway. Although we did not observe inactivation of p73 by either mutation or DNA methylation, haploinsufficiency of p73 correlated positively with either p14ARF or p53 mutation or both (P = 0.01) in primary non-small cell lung carcinomas. These data are consistent with the current model of p14ARF and p53 interaction as a complex network rather than a simple linear pathway and indicate a possible role for an E2F-1-mediated failsafe, p53-independent, apoptotic pathway involving p73 in human lung carcinogenesis.

Background: The nosology of neuroendocrine neoplasia has evolved substantially in recent years. The aim of this study was to review the authors' institutional experience and diagnostic accuracy for cytologic specimens of neuroendocrine carcinoma (NEC) and to identify features most suggestive of neuroendocrine differentiation., Methods: The cytologic and histologic findings of 29 archival NEC in which cytology preceded biopsy or resection were compared. The study was comprised of 6 carcinoid tumors, 3 atypical carcinoid tumors, 17 high grade NEC (5 small cell, 9 large cell, and 3 mixed small/large cell), and 3 combined NEC/nonneuroendocrine carcinomas. Cytologic material was derived from 21 fine-needle aspirates (FNA), 6 bronchial brushing/washings, and 2 gastrointestinal tract brushings., Results: Of the 29 cases, the correct cytologic diagnosis was rendered in 11. Two cases were identified as NEC but were graded incorrectly. The remaining 16 cases were interpreted as nonsmall cell carcinoma (8 cases); diagnostic or suspicious of carcinoma, not otherwise specified (7 cases); and atypical, indeterminate for malignancy (1 case). On review, neuroendocrine features were identified in 14 of the latter 16 cases., Conclusions: The cytologic diagnosis of NEC, both high and low grade, can be difficult. Because of acinus-like formations and columnar cell shapes, low grade NEC may be mistaken for adenocarcinoma. Small cell carcinomas, especially in bronchial brush and wash preparations, may be difficult to classify beyond malignant. Large cell NEC may be confused with nonneuroendocrine carcinomas because of abundant cytoplasm and nucleoli. Attention to the presence of loose cell aggregates in a background of singly dispersed cells; feathery patterns created by tumor cells clinging to capillaries; rosette formations; delicate, granular cytoplasm; inconspicuous nucleoli; molding in high grade tumors; and, most important, speckled or dusty chromatin patterns are useful in identifying neuroendocrine differentiation in cytologic specimens.

Objective: Our goal was to assess the sensitivity of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) for the detection of mucinous carcinoma and to determine the histologic features of these tumors that may affect lesion detectability., Materials and Methods: A retrospective review of all patients with mucinous carcinoma who had undergone FDG PET at our institution from 1995 through 1998 identified 25 patients with new or recurrent mucinous carcinoma at the time of PET. In 22 of these patients, tissue specimens available from either core biopsy or surgical resection allowed detailed histologic analysis., Results: FDG PET revealed mucinous carcinoma in only 13 (59%) of 22 patients, resulting in an unusually high percentage (41%) of false-negative results. Two histologic features were found to be predictive of FDG PET results: tumor cellularity (p = 0.011) and the amount of mucin within the tumor mass (p = 0.009). There was a positive correlation between tumor FDG uptake and cellularity but a negative correlation with the amount of mucin., Conclusion: FDG PET is limited in the evaluation of mucinous tumors, particularly in hypocellular lesions with abundant mucin.

CD31 has been shown to be a sensitive and specific marker for endothelial differentiation among epithelioid and spindled-pleomorphic human neoplasms. However, the role of this marker in the evaluation of small round cell tumors has not been evaluated. Formalin-fixed, paraffin-embedded tissue sections from 276 small round cell tumors, including 85 Ewing's sarcoma/primitive neuroectodermal tumors (ES/PNET), 52 rhabdomyosarcomas, 10 extraabdominal polyphenotypic small cell tumors, six desmoplastic small cell tumors, 11 neuroblastomas, 23 Wilms' tumors, 20 retinoblastomas, 13 esthesioneuroblastomas, and 56 small cell malignant lymphomas were stained with CD31 (JC/70A, 1:40), using a modified avidinbiotin-peroxidase complex technique, after citrate buffer microwave epitope retrieval. Among nonlymphoid small round cell tumors, four of 85 ES/PNET were at least focally reactive. No other lesion in this group was positive. In contrast, the majority of well-differentiated (11 of 17), intermediately differentiated (two of three), and lymphoblastic lymphomas (three of three) were positive. Small cleaved lymphomas (three of 13 follicular, one of 13 diffuse) were less often reactive, whereas small noncleaved lesions were negative. Although reactivity for CD31 in ES/PNET is uncommon, the presence of platelet/endothelial cell adhesion molecule in a small cell neoplasm should not in isolation be taken as evidence of hematopoietic origin. These results further define the utility of CD31 in the evaluation of human neoplasms.

Although it is classically a deep soft-tissue tumor of childhood, primitive neuroectodermal tumor (PNET) can occur at any age and may occasionally involve cutaneous sites. Merkel cell carcinoma (MCC) and basaloid neoplasms of cutaneous adnexa are the principal diagnostic alternatives to that tumor. The common expression of CD99 in PNET and cytokeratin-20 (CK20) in MCC suggests that these markers may be of value in this diagnostic setting, but they have not been rigorously examined in other small-cell and basaloid lesions of the skin. Accordingly, we evaluated CD99 and CK20 reactivity in formalin-fixed, paraffin-embedded sections of 30 MCC, five cutaneous metastases of pulmonary small-cell neuroendocrine carcinomas, 10 primary cutaneous adnexal carcinomas with basaloid features, 18 benign basaloid adnexal neoplasms of the skin (nine spiradenomas and nine cylindromas), and two cutaneous PNETs, using a standard immunohistologic technique and microwave-mediated epitope retrieval. Of the 30 MCC, 12 showed crisp membrane staining for CD99. Among the remaining tumors, only the two PNETs were positive for that marker. Although the majority of MCCs did not label for CD99, the pattern of reactivity in positive cases was indistinguishable from that observed in PNETs. Eighteen of 27 MCCs that were stained for CK20 were reactive for that protein, in contrast to metastatic small cell carcinomas, cutaneous PNETs, and appendageal skin tumors, which were uniformly negative for this marker. However, a subset of nine tumors, which were most consistent with MCC on clinical grounds, was CD99 positive and CK20 negative. Hence, reliance on CD99 alone as a diagnostic marker for PNET in this context cannot be recommended. Rather, careful assessment of the clinical presentation, together with extended immunophenotyping that includes other lineage markers and, when possible, cytogenetic analysis for characteristic chromosomal aberrations, remains the best means of separating MCC from PNET. Finally, the lack of CD99 reactivity in basaloid adnexal neoplasms of the skin suggests a utility in their differential diagnosis from cutaneous tumors with neuroendocrine or neuroectodermal differentiation.

We report on an uncommon entity, the so-called "chest wall chondromatous hamartoma" or "mesenchymal hamartoma of the chest wall" (MHCW), diagnosed by fine-needle aspiration (FNA) cytology in a 6-mo-old boy. Radiologic features were those of an aggressive lesion with rib expansion and destruction, that contrasted with aspirate smears showing bland cartilage and spindled mesenchymal elements. The clinicoradiographic features together with the FNA yield of mixed cellular elements aided in the correct diagnosis of MHCW., (Copyright 2000 Wiley-Liss, Inc.)

Cell culture of Taxus cuspidata represents an alternative to whole plant extraction as a source of taxol and related taxanes. Feeding phenylalanine to callus cultures was previously shown to result in increased taxol yields, probably due to the involvement of this amino acid as a precursor for the N-benzoylphenylisoserine side chain of taxol. Inthis study, we have examined the effect of various concentrations of phenylalanine, benzoic acid, N-benzoylglycine, serine, glycine, alanine, and 3-amino-3-phenyl-propionic acid on taxol accumulation in 2-year-old cell suspensions of Taxus cuspidata, cell line FCL1F, and in developing callus cultures of T. cuspidata. All compounds tested were included in media at stationary phase (suspensions) or after the period of fastest growth (calli). Alanine and 3-amino-3-phenyl-propionicacid were tested only in callus cultures and did not affect taxol accumulation. Significant increases or trends toward increases in taxol accumulationin callus and suspensions were observed in the presence of phenylalanine, benzoic acid, N-benzoylglycine, serine, and glycine. The greatest increases in taxol accumulation were observed in the presence of various concentrations of phenylalanine (1 mM for callus; 0.05, 0.1, and 0.2 mM for suspensions) and benzoic acid (0.2 and 1 mM for callus and 0.05, 0.1, and 0.2 mM for suspensions). Increases in taxol yields of cell suspensions in the presence of the most effective precursors brought taxol amounts at stationary phase from 2 mug . g(-1) to approximately 10 mug . g(-1) of the extracted dry weight. The results are discussed in termsof possible implications to taxol biosynthesis and in terms of practical applications to large-scale cell culture systems for the production ofthis drug. (c) 1994 John Wiley & Sons, Inc.

Abstract Peptide fragments of rat corticotrophin-releasing factor-41 (CRF-41) containing amino-acid residues 21-33 antagonized the 5 nmol/l CRF-41-stimulated adrenocorticotrophin secretion from the adult rat pituitary gland in vitro. The CRF 6-33 sequence had antagonistic effects at equimolar (5 nmol/l) concentrations which were not observed at high (50 nmol/l) concentrations whilst the CRF 21-41 sequence had effects only at high (50 nmol/l) concentrations. Similar effects were observed with CRF 6-33 on basal release of adrenocorticotrophin. Peptide fragments elevated radioimmunoassay measurement of CRF-41 whilst inhibiting measurement of CRF-41 in a two-site enzyme amplified immunometric assay. The inhibitory effects of peptide fragments in the enzyme amplified immunometric assay could be removed by dilution to within the lower end of the standard curve or by increasing the concentration of antibody bound to the solid phase. These inhibitory effects mimic those of peptide fragments on basal adrenocorticotrophin release seen in a rat pituitary gland in vitro bioassay indicating that such two-site immunoassay determinations bear closer relation to bioactivity than those obtained using radioimmunoassay.

In-situ hybridization with synthetic oligonucleotide probes was used to determine the mRNA content of corticotrophin-releasing factor (CRF) and proenkephalin A mRNA in the paraventricular nucleus, and of pro-opiomelanocortin (POMC) mRNA in the anterior pituitary gland of male rats immediately after, and during recovery from, chronic high-dose prednisolone treatment. Levels of transcripts for mRNA for both CRF and POMC were markedly reduced after the treatment, but there was a rapid return to control values for CRF mRNA within 18 h of steroid withdrawal. In untreated animals, the stressful stimulus of i.p. hypertonic saline increased CRF and proenkephalin A mRNA within 4 h with no significant difference in response seen whether the tissues were removed at 13.00 or 20.00 h. The increase in POMC mRNA did not reach statistical significance in these animals. Although prednisolone resulted in a marked reduction of basal CRF mRNA, the stress-induced increment of CRF mRNA remained comparable with that found in untreated animals. On the day following cessation of prednisolone treatment at 09.00 h, basal and stress levels of CRF mRNA were significantly higher in rats killed at 20.00 h than at 13.00 h. Proenkephalin A mRNA transcripts were below quantifiable levels of detection in control or non-stressed prednisolone-treated animals at all the time-points studied. Stress, however, resulted in the accumulation of proenkephalin A mRNA in control animals. This response was inhibited by prednisolone treatment and only returned 18 h after withdrawal. Prednisolone treatment reduced POMC mRNA below the levels detected in untreated animals, with no detectable response to stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine gamma-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7.1 and 6.8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Fragments of rat anterior pituitary glands incubated in vitro and challenged with either of two ACTH secretagogues were used to investigate the extent to which the acute, biphasic, feedback-like inhibitory effects on hormone secretion exerted by the synthetic glucocorticoid dexamethasone were related to alterations in second messenger responses. Addition of dexamethasone was shown to cause both an immediate inhibition (fast inhibition) of the release of ACTH-like immunoreactivity induced by arginine vasopressin (AVP) or corticotrophin-releasing factor (CRF-41), and also an inhibition that occurred after removal of the steroid and was maximal 90 min after its introduction (early delayed inhibition). The accumulation of adenosine 3',5'-monophosphate (cAMP) in the tissue was enhanced in a dose-related manner by CRF-41, as was that of phosphate esters of inositol (inositol phosphates) by AVP. The dose-response curve for the effect of CRF-41 on cAMP production was markedly shifted to the right by dexamethasone acting in the time-domain of fast inhibition (i.e. the response was attenuated, but not abolished). Application of the steroid during the same time-period reduced significantly the inositol phosphate response induced by the higher concentration of AVP tested (3000 nmol/l), but had no effect on the action of a lower concentration (30 nmol/l). In contrast, the cAMP and inositol phosphate dose-response curves to CRF-41 and AVP respectively were unaffected by the glucocorticoid when it was applied at the time which generated early delayed inhibition of ACTH release.(ABSTRACT TRUNCATED AT 250 WORDS)

The occurrence and nature of corticosteroid inhibition of ACTH secretion at the rat anterior pituitary gland was investigated using three experimental models: animals bearing lesions of the basal hypothalamus, and two preparations of the gland incubated in vitro; these were tissue segments and collagenase-dispersed cells. Release of ACTH in the experiments was provoked using one of three distinct stimuli: acid extracts of whole hypothalami, corticotrophin releasing activity released by serotonin from hypothalami incubated in vitro and synthetic ovine corticotrophin releasing factor. Irrespective of whether ACTH was measured directly by radioimmunoassay (in the experiments in vitro) or indirectly in terms of corticosterone production (in the lesioned animals), its stimulated release from the anterior pituitary gland was inhibited by corticosterone. Two phases of inhibition were observed; these had some of the characteristics inferred previously from experiments with intact animals and designated fast feedback and delayed feedback. However, the fast feedback demonstrable in lesioned animals did not show the rate-sensitivity shown previously in intact animals. 11-Deoxycortisol (or 11-deoxycorticosterone) and prednisolone proved to be agonists of corticosterone in provoking fast feedback in lesioned animals, whereas they had been shown respectively to act as an antagonist or to have no effect in intact rats. Several steroids were able to cause delayed feedback in lesioned rats, but beclomethasone dipropionate (shown to be an agonist of corticosterone in intact rats) proved to have no inhibitory effect at the anterior pituitary gland of lesioned animals. It is concluded that the dynamics of corticosteroid feedback mechanisms at the anterior pituitary gland, as indicated by experiments in lesioned animals, differ from those operative in the intact animals. Other work suggests that a more important site for such inhibitory mechanisms in vivo is the hypothalamus.

In our colony of female rats (220-320 g body weight) undergoing regular 4-day oestrous cycles there were significant, marked rises in concentrations of LH, FSH and prolactin between 09.00 and 19.00 h on pro-oestrus. The i.p. injection of difluoromethylornithine (DFMO; 40-400 mg/kg), a specific inhibitor of the activity of ornithine decarboxylase, at 15.00 h on pro-oestrus had a differential effect on the rise in plasma concentrations of the various hormones thereafter. The drug produced a significant, partial, dose-related suppression of the rise in plasma concentrations of LH and prolactin, but had no significant effect on the rise in FSH. For time-course studies, 120 mg DFMO/kg were injected at 13.00, 15.00 or 17.00 h and groups of animals killed at 19.00 h. Only the injection at 15.00 h was effective in causing a significant reduction in plasma concentrations of LH and prolactin at 19.00 h. Pituitary content of the hormones was found to be unaffected by the administration of DFMO at the times and doses tested. These results suggest that DFMO has a selective inhibitory effect on enhanced LH and prolactin secretion on the afternoon of pro-oestrus in the rat, whilst not affecting FSH release. There seems to be a limited time (after 13.00 but before 17.00 h) during which its administration is effective.

Adrenocorticotrophin levels, measured by a cytochemical bioassay, were determined in the plasma and cerebrospinal fluid (CSF) of adult female rhesus monkeys which were ovariectomized and receiving oestrogen replacement therapy. In control monkeys, ACTH bioactivity was found in both CSF (10.2 +/- 1.8 ng/l) and plasma (186 +/- 51 ng/l) in samples taken at 14.00 h (lights on: 07.00-19.00 h). Dexamethasone treatment (0.2 mg/kg) twice daily for 4 days suppressed plasma ACTH levels (52.8 +/- 25.2 ng/l) but had no effect on CSF levels (7.6 +/- 2.7 ng/l). Raising plasma ACTH, either by daily injections of a long-acting preparation of ACTH (1-24) for 6 days or by bilateral adrenalectomy (and subsequently with-drawing cortisol replacement therapy) also resulted in no detectable changes in ACTH levels in the CSF. A regression analysis between ACTH in the plasma and CSF from samples taken throughout the experiments showed no correlation. In contrast, measurement of ACTH by radioimmunoassay, whilst satisfactory for determination of this peptide in plasma, could not identify authentic ACTH in the CSF. It is concluded that bioactive ACTH does not enter the CSF in detectable quantities from either the peripheral vascular compartment or from the animal's own pituitary gland, and that reducing ACTH secretion from the pituitary also has no effect on levels of ACTH in the CSF. This is in marked contrast to other pituitary peptide hormones, including prolactin, which is secreted together with ACTH during 'stress' but which, unlike ACTH, enters the CSF relatively easily.

Two chemically characterized peptides, arginine vasopressin (AVP) and corticotrophin-releasing factor-41 (CRF-41), known to stimulate ACTH secretion by interaction with their respective specific receptors on the corticotroph, were shown to cause the accumulation of phosphate esters of inositol (IP) and adenosine 3',5'-monophosphate (cAMP) respectively when added to rat anterior pituitary fragments incubated in vitro. The former 'second messenger' response (IP production) was unaffected in tissues removed from animals treated with prednisolone in the drinking water (1035 mumol/1) for 14 days. On the other hand, the cAMP response, whilst still present, was inhibited by some 50% in tissues taken from such animals. In contrast, pituitary glands from steroid-treated rats failed to respond to challenge with a variety of substances expected to cause the release of ACTH by mimicking or provoking the production of IP or cAMP. Indeed, of the wide range of ACTH secretagogues tested, only the phospholipase A2 activator melittin was able to cause attenuated ACTH release from tissues removed from treated rats. The failure to provoke ACTH release from tissues removed from steroid-treated animals was also seen when submaximal concentrations of CRF-41 or AVP, or hypothalamic extract or 48 mmol K+/1 were used as the stimuli. The staged recovery of the ACTH secretory response and IP and cAMP accumulation in vitro following the withdrawal of prednisolone treatment was also investigated. A cAMP response that did not differ significantly from that of control tissue and a normal ACTH response to K+ and to melittin were all recovered by 3 days after withdrawal, and the response to cholera toxin showed a partial recovery. Responses to all stimuli of ACTH secretion which cause their effect by entering the corticotrophs were normal by 5 days after withdrawal, when the response to CRF-41 was still significantly, and that to AVP still slightly, reduced compared with controls. Surprisingly, restoration of the ACTH response was most delayed when the expectedly most potent extracellular stimulus (hypothalamic extract) was used. In this case, release was still significantly impaired 7 days after steroid withdrawal. The results show that the glucocorticoid acts to compromise several distinct steps in the process whereby extracellular signals such as CRF-41 and AVP cause the secretion of ACTH. The only step that appears to be spared is the generation of IP by AVP.(ABSTRACT TRUNCATED AT 400 WORDS)

Male Wistar-derived rats (200-250 g) were treated for 14 days with prednisolone 21-sodium succinate at a concentration of 1035 mumol/l in their drinking water. The drug was then replaced with normal tap water and groups of animals were killed at various times during recovery, trunk blood being collected after decapitation. At the same time, hypothalamic slices, anterior pituitary gland fragments and adrenals were removed and their responsiveness assessed by exposure to appropriate stimuli in vitro. Tissues were also extracted to measure changes in content of hormones during recovery. Treatment with prednisolone produced marked reductions in body weight gain, adrenal weight and pituitary ACTH content, but no significant change in hypothalamic corticotrophin-releasing factor (CRF) bio- or immunoreactivity. The ACTH content was restored by 5 days after withdrawal but adrenal weight remained significantly reduced after 9 days of recovery. The responsiveness of the hypothalamus to acetylcholine in vitro was markedly inhibited and was still significantly reduced 7 days after withdrawal. The responsiveness of the anterior pituitary gland to synthetic CRF or arginine vasopressin and that of the adrenal gland to ACTH added in vitro were restored simultaneously after 7 days of withdrawal. In vivo, recovery was assessed by measurement of the response to laparotomy stress. Treatment with prednisolone prevented the increase in the plasma concentrations of ACTH and corticosterone produced by stress, and these responses recovered by 5 days (corticosterone) and 7 days (ACTH) after withdrawal. The abolition of the circadian rhythms of ACTH and corticosterone by treatment was also reversed by 5 days after withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraperitoneal injection of caffeine (12.5-100 mg/kg) into rats caused a significant, dose-related increase in plasma corticosterone 2 h later, when the greatest response was measured. The corticosterone response to laparotomy stress or i.v. injection of ACTH(1-24) was unaffected by prior injection of caffeine. The response to stress or caffeine was unaffected by adrenal enucleation 28 days previously. In vitro, 10 mmol caffeine/l stimulated basal release of corticosterone from adrenal quarters and potentiated the response to a sub-maximal stimulatory concentration of cyclic AMP (cAMP). The drug had no effect on release stimulated by a sub-maximal concentration of ACTH(1-24). Release of ACTH from pituitary fragments incubated in vitro was stimulated in a dose-related manner by caffeine (0.01-10 mmol/l), and the responses to hypothalamic extract and sub-maximal concentrations of corticotrophin-releasing factor (CRF-41) or arginine vasopressin (AVP), but not cAMP, were significantly enhanced by 10 mmol caffeine/l. Release of immunoreactive CRF-41 (but not AVP) was significantly increased by caffeine (0.01-10 mmol/l) added to hypothalami incubated in vitro. The response to injection of caffeine in vivo was completely prevented by pharmacological blockade of endogenous CRF release. Taken together, these results show that caffeine at high concentrations can stimulate directly the release of the hormones of the hypothalamo-pituitary-adrenocortical axis in vitro, but the fact that these concentrations are unlikely to be reached after administration in vivo suggests that the effect of caffeine may be mediated centrally.

The effect of six hypothalamic peptides on the basal release of ACTH and that induced by arginine vasopressin (AVP) or by ovine corticotrophin releasing factor (oCRF) from fragments of the rat anterior pituitary gland incubated in vitro was investigated. Dose-response curves to AVP and to oCRF were obtained, and the response to a low dose of oCRF was potentiated by a low dose of AVP. Basal release of ACTH was not affected by any of the peptides in concentrations in the range 10(-12) to 10(-6) mol/l, and only substance P (SP) and somatostatin (SRIF) inhibited significantly the response to oCRF in a dose-related manner. The responses to a range of doses of oCRF or AVP were reduced by 10(-8) and 10(-6) mol SP or SRIF/l, and to a greater extent by the higher dose. Except in the case of 10(-6) mol SRIF/l on the response to AVP, the response was not further diminished by preincubation of the tissue with the peptide before the stimulating agent was added. The inhibition of the responses to AVP or oCRF by 10(-9) mol SP/l was not potentiated by its combination with either 5 X 10(-10) or 10(-8) mol SRIF/l; the inhibitory effects were merely additive. The results suggest that although SRIF and SP are able to modulate the release of ACTH from the anterior pituitary gland, they do so only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.

Female Wistar-derived rats with regular oestrous cycles were injected s.c. at 15.00 h on pro-oestrus with difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase. The drug (10-100 mg/rat) caused a dose-related reduction in the concentration of LH in plasma taken at 19.00 h (the time of the peak of the LH surge in this colony). There was also a dose-related reduction in the pituitary content of total polyamines. The reduction in the plasma concentration of LH was not due to the shifting of the time of the peak of the surge, as concentrations were significantly lower than control from 17.00 to 21.00 h, the overall reduction in total LH release being approximately 50%. The number of ova in the oviducts at 06.00 h next morning was significantly reduced by treatment with 50 mg DFMO/rat, by an average of 70%. Injection of DFMO enhanced the fall in plasma oestradiol concentrations seen between 15.00 and 19.00 h, in a dose-related manner. It also prevented the rise in progesterone concentrations seen in control animals during this period. The ability of DFMO to prevent the rise in plasma concentrations of LH was not secondary to the effects of the drug on ovarian steroid production because DFMO also significantly reduced the LH surge in animals ovariectomized on dioestrus and given appropriate replacement injections of oestradiol and progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma corticotrophin (ACTH), cortisol, prolactin and growth hormone (GH) responses to insulin-induced hypoglycaemia were measured in normal healthy subjects of both sexes before and after three weeks' treatment with sodium valproate (Epilim, 200 mg three times a day). The drug had no effect on fasting plasma glucose levels, or the extent of hypoglycaemia induced by insulin (0.15 U/kg). There was no significant difference between pre- and post-treatment values for basal or stress-induced concentrations of ACTH and cortisol (n = 12), prolactin (n = 7) or GH (n = 9). The results suggest that treatment of normal subjects with sodium valproate has no effect on the response of the hypothalamo-pituitary-adrenocortical axis to hypoglycaemia, which is in contrast to its inhibitory effects on ACTH secretion in patients suffering from Nelson's syndrome. This implies that in the disease state, there may be a unique sensitivity to GABA-ergic manipulation.