Discovery and validation of serum biomarkers expressed over the first twelve weeks of Fasciola hepatica infection in sheep.

Serum biomarkers associated with Fasciola hepatica infection of Corriedale sheep were analysed during the first 12 weeks of infection using surface-enhanced laser desorption ionisation time of flight mass spectrometry (SELDI-TOF MS). In the discovery phase of analysis, pooled sera collected at week 0 and at each week p.i. to week 12 were fractionated by anion-exchange chromatography and the protein mass fingerprints obtained in individual fractions were in the M/z range 1.5-150 kDa. A total of 2302 protein clusters (peaks) were identified that varied between time-points following infection with peaks increasing or decreasing in intensity, or showing transient variation in intensity, during the 12 weeks of parasite challenge. In the validation phase, candidate biomarkers in sera of individual sheep at weeks 3 and 9 p.i. were analysed, identifying 100 protein peaks, many of which are small peptides <10 kDa in size: 54% of these peaks were up-regulated in intensity at week 3 or 9 p.i. Twenty-six biomarkers were chosen for further study, ranging in size from 1832 to 89,823 Da: six biomarkers were up-regulated at weeks 3 and 9 p.i., 16 biomarkers were up-regulated only at week 9 p.i. and four biomarkers were down-regulated at week 9 p.i. Two biomarkers up-regulated at week 9 were identified as transferrin (77.2 kDa) and Apolipoprotein A-IV (44.3 kDa), respectively. The results show that the interaction between the host and F. hepatica is complex, with changes in biomarker patterns beginning within 3 weeks of infection and either persisting to weeks 9-12 or showing transient changes during infection. Identification of biomarkers expressed during ovine fasciolosis may provide insights into mechanisms of pathogenesis and immunity to Fasciola and may assist in the rational development and delivery of vaccines.

Characteristics of prostate-derived growth factors for cells of the osteoblast phenotype.

We examined the characteristics of mitogens extracted from human benign prostatic hyperplasia and prostatic adenocarcinoma tissue. Although mitogens for fetal rat skin fibroblasts as well as for rat calvarial osteoblasts and osterosarcoma cells were found, distinct entities that acted selectively in cells of the osteoblast phenotype could be obtained by sequential reverse-phase high performance liquid chromatography. Two peptides with apparent molecular weights of 10,000 and 13,000 D were derived from hyperplastic tissue, whereas a single moiety of 10,000 D was obtained from malignant tissue. These entities increased cell numbers and alkaline phosphatase activity in osteoblastlike cells consistent with effects on both growth and differentiation. Prostatic peptides did not stimulate adenylate cyclase in osteosarcoma cells. Mitogenic activity selective for osteoblastlike cells was identified in postpubertal but not prepubertal normal prostate. The results demonstrate the existence of osteoblastic growth factors in prostatic tissue whose presence may accompany postpubertal development.

Nucleotide and (derived) amino acid sequence of a novel peptide from a rat (hypercalcemic) Leydig cell tumor.

The nucleotide sequence of a novel peptide from a rat Leydig cell hypercalcemic tumor H-500 was determined. This cDNA encodes a peptide of 93 amino acids and contains a heparin binding domain similar to histone 2-B. Northern blot analysis showed tissue specific expression of this peptide mRNA.

Gait slowing as a predictor of incident dementia: 6-year longitudinal data from the Sydney Older Persons Study.

Current definitions for the preclinical phase of dementia focus predominantly on cognitive measures, with particular emphasis on memory and the prediction of Alzheimer's disease. Incorporation of non-cognitive, clinical markers into preclinical definitions may improve their predictive power. The Sydney Older Persons Study examined 6-year outcomes of 630 community-dwelling participants aged 75 or over at recruitment. At baseline, participants were defined as demented, cognitively intact or having a syndrome possibly representing the preclinical phase of Alzheimer's disease, vascular dementia, an extrapyramidal dementia or various combinations of the three. Those with cognitive impairment in combination with gait and motor slowing were the most likely to dement over the 6-year period (OR 5.6; 95% CI 2.5-12.6). This group was also the most likely to die (OR 3.3; 95% CI 1.6-6.9). White matter indices on MRI scanning were not consistently correlated with gait abnormalities. Simple measures of gait may provide useful clinical tools, assisting in the prediction of dementia. However, the underlying nature of these deficits is not yet known.

Immunocytochemical localization of granulin-1 to mononuclear phagocytic cells of the teleost fish Cyprinus carpio and Carassius auratus.

A new class of low-molecular-weight cysteine-rich regulatory growth factors, designated granulins, has been isolated from hematopoietic tissues of a teleost fish (Cyprinus carpio) and structurally characterized. Granulin-1, the predominant form found in carp spleen, was used to raise polyclonal antibodies in rabbits and to establish a radioimmunoassay. This permitted preliminary tissue distribution studies of granulin-1 to be undertaken in carp (Cyprinus carpio) and goldfish (Carassius auratus). Granulin-1 immunoreactivity was found in the melanomacrophage centers of the spleen and head kidney. Carp tissues anatomically involved in the first line of defense against infection, including skin, gills, gut, and also heart, showed intense granulin-1 immunoreactive staining within presumptive macrophage cells. Granulin-1 immunoreactive macrophages prepared from goldfish spleen and head kidney adhered to glass slides, actively phagocytosed carbon particles, and contained granulin-1 immunoreactivity as well as abundant endogenous peroxidase activity. This study demonstrates that granulin-1 is synthesized and stored in macrophages/monocytes of spleen, head kidney, and peripheral tissues of teleost fish.

Isolation, characterization, and corticotropic activity of rabbit adrenocorticotropin.

Rabbit (r) ACTH was extracted from 600 pituitaries, and 2 forms of immunoreactive ACTH were identified with the least polar form accounting for approximately 90% of the total. Peptide mapping and sequence analysis indicated that three tryptic peptides had retention times identical to those obtained from human (h) ACTH. The least polar tryptic fragment from rACTH had a shorter retention time than the corresponding one from hACTH. Sequence analysis indicated that rACTH differed from hACTH at three different loci, namely, an Asn in place of Asp in position 29; a Val in place of Leu in position 37, and a Val in place of Phe in position 39. Biological activity of the ACTH was compared with synthetic hACTH in 2 bioassays with adrenals from 10-day-old pups, the first using dispersed rabbit adrenal cells and the second using monolayer adrenal cells in culture. The biological potencies of the two ACTH preparations were identical with respect to corticosterone (B) release in the short term bioassay, with an ED50 value of 1.67 X 10(-10) M. The ED50 value for cortisol (F) release for rACTH and hACTH were 1.1 X 10(-10) M and 1.67 X 10(-10) M, respectively, which were not statistically different. The biological potency of rACTH in the monolayer adrenal cell system for both F and B was significantly greater than the hACTH, and the ED50 values were 4.4 X 10(-10) M and 8.9 X 10(-10) M, respectively. There was a progressive decrease in the B/F ratios with increasing concentrations of ACTH in both the bioassay systems suggesting that ACTH stimulated the 17 alpha-hydroxylase activity even when the exposure of cells to ACTH was as short as 2 h.

Infusion of N-proopiomelanocortin-(1-77) increases adrenal weight and messenger ribonucleic acid levels of cytochrome P450 17alpha-hydroxylase in the sheep fetus during late gestation.

In the sheep there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10-15 days gestation (term = 147+/-3 days gestation). In the rat, peptides derived from the N-terminal region of POMC play a role in compensatory adrenal growth and in potentiation of ACTH-induced steroidogenesis. We therefore investigated the effects of infusion of bovine N-POMC-(1-77) and its biosynthetic derivative, N-POMC-(1-49) on adrenal growth and on the expression of adrenal steroidogenic enzymes in the late gestation sheep fetus. Twenty-seven pregnant ewes were used in this study. Fetal vascular catheters were inserted between 116-125 days gestation, and purified bovine N-POMC-(1-77) (2 microg/ml x h), N-POMC-(1-49) (2 microg/ml x h) and saline were each infused for 48 h between 136 and 138 days gestation. Intrafetal infusion of N-POMC-(1-77) resulted in an increased adrenal/fetal body weight ratio (94.6+/-5.7 mg/kg) compared with that in saline-infused (75.6+/-1.8 mg/kg), but not N-POMC-(1-49)-infused (82.7+/-6.1 mg/kg), fetal sheep. The ratio of CYP17 messenger RNA (mRNA) to 18S ribosomal RNA was also significantly higher in fetal adrenals ofthe N-POMC-(1-77)-infused group (49.1+/-4.7) compared with that in either the N-POMC-(1-49)-infused (20.4+/-6.4) or saline-infused (15.2+/-4.4) group. There was no difference, however, in the ratios of adrenal CYP11A1 mRNA/3beta-hydroxysteroid dehydrogenase/delta5,delta4-isomerase mRNA and CYP21A1 mRNA/18S ribosomal RNA among the N-POMC-(1-77)-, N-POMC-(1-49)-, and saline-infused groups. There was also no significant change in either plasma cortisol or ACTH concentrations in response to the infusion of either N-POMC-(1-77) or N-POMC-(1-49). In summary, intrafetal infusion of N-POMC-(1-77) stimulated fetal adrenal growth and resulted in a specific increase in adrenal CYP17 gene expression in late gestation. N-POMC-(1-77) may therefore play a modulatory role in the increase in fetal adrenal growth and steroidogenesis that occurs before birth.

Purification of peptides with parathyroid hormone-like bioactivity from human and rat malignancies associated with hypercalcemia.

We have purified peptides with PTH-like bioactivity from a rat Leydig cell tumor (H-500) and a human squamous cell carcinoma, both associated with a syndrome of humor-induced hypercalcemia. Tumor extracts were shown to be active in an in vitro renal cytochemical bioassay and in an in vitro osteosarcoma cell (UMR 108) adenylate cyclase assay; activity in both assays could be reduced by the PTH antagonist [norleucine-8,18,tyrosine-34]bovine PTH-(3-34)-amide. Partially purified extracts of both tumors and of rat tumor-conditioned culture medium were active in vivo in thyroparathyroidectomized rats in preventing hypocalcemia and increasing fractional phosphorus excretion and cAMP excretion. Ion exchange chromatography demonstrated that active peptides were basic in character. Employing reverse phase HPLC and gel permeation HPLC, active peptides of approximately 9,000 and 9,500 daltons were purified from extracts of the human and rat tumors, respectively, which had similar but not identical compositions. Two additional bioactive peptides were detected in rat tumor extract, and the more active had a mol wt of approximately 28,000. The results demonstrate that peptides that mimic PTH in a variety of in vivo and in vitro bioassays can be extracted from malignancies associated with hypercalcemia, that multiple molecular species may be detected in tumors that demonstrate PTH-like activity, and that at least one of these peptides may be similar in two tumors of highly divergent cell and species origin.

Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D3.

We previously showed that the processing of proparathyroid hormone (proPTH) to PTH was accomplished most efficiently by furin (17). Colocalization studies demonstrated that furin is expressed in the parathyroid, whereas proprotein convertase (PC)1 and PC2 are not. Since that time, another member of the PC family, called PC7, has been identified. Here we show, using coinfection studies, that PC7, as well as furin, can appropriately cleave PTH from proPTH. ProPTH and PTH were purified from cell extracts by reversed-phase HPLC and were identified by Western blot analysis and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization studies, using Northern blot and reverse transcriptase-PCR analyses, showed that PC7 messenger RNA (mRNA) is expressed in the parathyroid gland. Therefore, PC7, like furin, has the potential to be involved in the physiological processing of proPTH to PTH. The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems in which regulation of PTH mRNA levels were clearly apparent, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin or PC7 mRNA levels. This is in contrast to the processing of proinsulin to insulin in the pancreatic beta-cell, which is up-regulated by glucose stimulation of PC1 and PC2 synthesis.

Isolation and characterization of three forms of joining peptide from adult human pituitaries: lack of adrenal androgen-stimulating activity.

Three structural variants of the joining peptide (JP) fragment of POMC have been purified from human pituitaries. Ion exchange and reverse phase tissue extraction procedures were combined with reverse phase HPLC to achieve complete purification of each form of JP. Fragments resulting from tryptic hydrolysis of each form were characterized by amino acid analysis and fast atom bombardment mass spectrometry. The predominant form of human JP, accounting for about 50% of the total purified, was found to be conjugated to glutathione through the lone cysteine residue at position 9. The other two variants were identified as human JP with a free cysteine residue and human JP dimer and accounted for 35% and 15%, respectively, of the total purified. Recently, human JP-(1-18) has been suggested as having adrenal androgen-stimulating activity. None of the three JP variants or their respective 1-20 amino-terminal fragments resulting from tryptic hydrolysis showed any ability to promote the secretion of dehydroepiandrosterone sulfate by cultured human fetal adrenal cells. Similarly, no potentiation of the stimulatory effects of ACTH-(1-39) was observed. The three variants of human JP as well as JP purified from rat, porcine, and bovine pituitaries were tested for their ability to stimulate androgenic steroids from dispersed fetal rabbit adrenal cells. None showed any significant biological activity either in stimulating steroid secretion or in potentiating the action of ACTH-(1-39).

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC1-77) is partially processed in the intermediate lobe of the pituitary to N-POMC1-49 and lys-gamma 3-melanotropin. Two pools of N-POMC1-77 were isolated which were differentially glycosylated at threonine45, while N-POMC1-49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC1-77 may regulate cleavage at the Arg49-Lys50 processing site. We tested this hypothesis by incubating N-POMC1-77 glycoforms with purified proopiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC1-77 and O-glycosylated N-POMC1-77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-gamma 3-melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.

Anti-inflammatory drugs protect against Alzheimer disease at low doses.

CONTEXT: Anti-inflammatory medications have an inverse association with Alzheimer disease (AD). OBJECTIVES: To examine at what doses this anti-inflammatory drug effect occurs and whether other medications and/or International Classification of Diseases, Ninth Revision, Clinical Modification diagnoses affect the association. DESIGN: Subjects 75 years and older from a random population sample were classified by consensus using International Classification of Diseases, Ninth Revision, Clinical Modification diagnoses. Drug associations with different types of dementia with and without the International Classification of Diseases, Ninth Revision, Clinical Modification diagnoses as well as dosage data were analyzed. SETTING: The Centre for Education and Research on Aging, Concord Hospital, Concord, Australia. PATIENTS: The Sydney Older Persons Study recruited 647 subjects (average age, 81 years). A total of 163 patients were given diagnoses placing them in different dementia categories and were compared with 373 control subjects. Of the patients with dementia, 78 had AD without vascular dementia, 45 had vascular dementia (permissive of other dementia diagnoses), and 40 had other dementia diagnoses (without AD or vascular dementia). MAIN OUTCOME MEASURES: Fifty drugs or drug groups were subjected to a 2 (drug used vs drug not used) x 4 (dementia and control groups) chi(2) analysis. Drugs with inverse associations were identified and potential confounders (logistic regression) and dosage data (exact small sample 1-tailed tests) analyzed. RESULTS: As expected, there was an inverse association between nonsteroidal anti-inflammatory drugs and aspirin (and unexpectedly angiotensin-converting enzyme inhibitors) and AD. This association was not observed with vascular dementia or any other diagnoses. Analysis showed no evidence for a dosage effect, ie, responses were equivalent for low and high doses. CONCLUSIONS: This study does not support a high-dose anti-inflammatory action of nonsteroidal anti-inflammatory drugs or aspirin in AD. Potential mechanisms for the beneficial effects of these medications are discussed.

Granulins: the structure and function of an emerging family of growth factors.

The granulin/epithelin motif defines a family of structurally unique proteins, of great evolutionary antiquity, which have been implicated as regulators of cell growth. Recurrent in granulin research are the surprising parallels between the granulin and EGF systems. Both are cysteinerich peptides of approximately 6 kDa that can modify cell growth. They show similar, but not identical, biological activities, although granulin/epithelin peptides do not bind EGF receptors; the three-dimensional folds of granulin and EGF are partially superimposible; and the precursors for mammalian granulin/epithelins and EGF are both organized as multiple repeats of conserved cysteine modules. Given the dissimilarity between amino acid sequences of members of the granulin/epithelin family and EGF-related peptides, the parallelism between the two systems probably represents convergent evolution towards related solutions to common biological problems. The granulin/epithelin precursor gene is expressed throughout the body, but its expression is predominantly in epithelial and haematopoietic cells. There is a great deal of versatility in the means by which cells process and handle the granulin/epithelin precursor. In some instances, the precursor is secreted intact (Zhou et al. 1993), and in others it is stored in a vesicular organelle, such as the sperm acrosome (Baba et al. 1993a). It may be processed into small 6-kDa peptides, which, in the neutrophil, can also be stored in vesicles (Bateman et al. 1990, Couto et al. 1992). The 6-kDa peptide forms, the intact precursor, and related proteins such as TGFe, regulate the growth of epithelial and mesenchymal cells. Epithelial cells express putative receptors for granulin/epithelin peptides and TGFe (Culouscou et al. 1993, Parnell et al. 1995). Thus, although much remains to be clarified, granulin/epithelin polypeptides and related proteins are emerging as widely distributed potential autocrine and paracrine growth modulating factors for epithelial and mesenchymal cells.

Distribution and degradation of two tritium-labelled corticotrophin analogues in the rat.

The distribution and degradation of corticotrophin-(1--24)-tetracosapeptide specifically labelled with tritium at Tyr2, Phe7 or Tyr23 and [D-Ser1, Lys17, Lys18]-corticotrophin-(1--18)-octadecapeptide amide labelled at Tyr2 were studied at various times after intravenous injection into rats. By characterizing the radioactivity in plasma and various tissues, an overall picture of the metabolic handling of the two peptides emerged. The peptides left the circulation rapidly, entering mainly muscle and skin where they were extensively degraded. The D-Ser1-containing analogue was less rapidly degraded and intact peptide persisted in muscle and skin for up to 1 h. This peptide probably returned to the circulation giving rise to the sustained plasma levels observed after injection of the D-Ser1-substituted octadecapeptide but not after injection of the tetracosapeptide. Although initially the kidneys did not clear such large amounts of peptide as did muscle and skin they played an important role by continuously and, on the basis of existing evidence, irreversibly clearing the peptides and peptide fragments from the circulation and degrading them.

Isolation and characterization of a cytostatic histone H2B-like protein from fetal lungs of non-diabetic and diabetic rats.

It was observed in the course of other studies that rat fetal lung extracts inhibited proliferation of fetal lung cells in culture. The purpose of the present study was to isolate and characterize this cytostatic factor. It was found that fetal lungs contained a 16 kDa cytostatic factor and its concentration was twofold greater in fetal lungs of diabetic rats compared with control rats. This fetal lung cytostatic protein (FLCP) was purified by reversed-phase, heparin-affinity and gel filtration high-performance liquid chromatography and SDS-PAGE. The purified protein was electroblotted onto polyvinylidene difluoride membrane and subjected to sequence analysis. The amino-terminal sequence of this fetal lung cytostatic protein was P E P A K S A P A P X K G I G K Q X X K A X X K A ... and showed significant homology with histone H2B; however, the amino acid composition of FLCP suggested that it may be structurally distinct from histone H2B. Ion-spray mass spectrometry suggested that FLCP was made up of at least two species of the protein with molecular weights of 13,776.1 and 14,007.3 and was different from the molecular weight of rat histone H2B predicted by its cDNA sequence. The concentration of FLCP, based on amino acid compositions, was 0.32 nmol/g and 0.83 nmol/g wet fetal lung from non-diabetic and diabetic rats respectively. These findings suggest that the fetal rat lung produces a regulatory factor bearing considerable homology with but possibly different from histone H2B and that fetal lung immaturity during diabetic pregnancy might be contributed to by an increase in this factor.

Renal uptake and metabolism of adrenocorticotrophin analogues in the rat: an autoradiographic study.

Renal resorption of tritiated adrenocorticotrophin analogues was studied in the rat using light microscopic and quantitative electron microscopic autoradiography. The synthetic corticotrophins used were Synacthen (corticotrophin-(1-24)-tetracosapeptide) and C41795-Ba ([D-Ser1,Lys17,Lys18]-corticotrophin-(1-18)-octadecapeptide amide), the tetracosapeptide being tritiated in either the tyrosine residue of position 2 or 23 or the phenylalanine of position 7 and the octadecapeptide in the tyrosine of position 2. Inspection of autoradiographs showed that peptides injected intravenously were resorbed into proximal tubules by endocytosis to produce vesicles whose radiolabel later appeared in lysosomes, a route previously elucidated for other peptides and proteins. The use of two techniques for analysis of electron microscopic autoradiographs, however, suggested that apical tubules also acquire label and are in some way involved in the transfer of resorbed labelled material from endocytotic vesicles to lysosomes. In addition, the autoradiographic analyses revealed that the duration of lysosomal labelling depends upon the position of tritium in the chain. Thus, when the CO2H-terminus of Synacthen was labelled, silver grains were more transiently associated with lysosomes than was the case when the NH2-terminal or core regions were tritiated, indicating a greater resistance of these portions of the peptide to attack by intracellular peptidase. The label from the chemically protected C 41795-Ba was also less readily expelled from the lysosomes of the proximal tubules.

Fate of human corticotrophin immediately after intravenous administration to the rat.

The distribution and degradation of tritium-labelled human 1-39 corticotrophin have been studied after intravenous administration to the rat. Within 5 min of injection of single major metabolite, 3-39 corticotrophin, appears in the circulation. This metabolite, however, has only 3.6% of the steroidogenic potency of 1-39 corticotrophin and the evidence suggests that it is formed in muscle and skin. By 5 min, extensive degradation had occurred in all the major tissues in the body (muscle, skin, liver, kidney, gut).

Hypothalamic processing of beta-endorphin in female C57BL/6J mice is altered at middle age.

beta-Endorphin (beta-endo) (1-31) is the active opioid peptide product of pro-opiomelanocortin processing. Further post-translational modifications of beta-endo(1-31) yield beta-endo(1-27), (1-26) and their acetylated forms which are considered to be opiate receptor antagonists. Mechanistically, alteration in opiatergic properties is likely to result in the loss of a number of physiological functions including reproductive capacity. The purpose of this study was to determine whether there are changes in the way beta-endo neurones process the peptide with age in female C57BL/6J mice. Pooled extracts of arcuate nucleus (ARC) and preoptic area (POA) of 3- to 4-month-old normally cycling (4-5 days at dioestrus), 12- to 13-month-old irregularly cycling (5-7 days at dioestrus), 23- to 24-month-old acyclic (in persistent dioestrus) animals were subjected to reversed-phase HPLC (n = 4 experiments). Column fractions were assayed for beta-endo-like-immunoreactivity by sequence-specific RIAs. The opiate receptor active as well as opiate receptor antagonist forms of beta-endo were present in both ARC and POA at all three age groups although their ratios varied. beta-Endo(1-31), the active opiate, was the predominant form in young animals. At middle age there was a threefold (P < 0.05, ANOVA) increase in the antagonist forms of beta-endo and this was associated with a significant (P < 0.05, ANOVA) increase in the ratio of antagonist to active forms. This was accompanied by a trend toward an increase in acetylated forms of beta-endo in middle-aged mice. HPLC profiles from hypothalami of old animals more closely resembled those of young females. The increase in the antagonist forms of beta-endo at middle age may contribute to a decline of opiatergic influences in the female C57BL/6J mouse and suggest a mechanism whereby alterations in opiate influence over gonadotrophin control may occur.

Estrogen, the ovary, and neutotransmitters: factors associated with aging.

Our studies in the C57BL/6J mouse have been designed to examine the interactions of aging and the ovary, and their mutual effects on neuroendocrine function. In the pituitary, ovarian status and not age determines responsiveness to gonadotropin hormone releasing hormone (GnRH), but estrogen (E2) is an important mediator in CNS changes, and removal of the ovary (OVX) is deleterious to the neuroendocrine hypothalamus. OVX for just six days in young animals results in synaptic loss between noradrenergic terminals and gonadotropin hormone releasing hormone (GnRH) neurons. Long-term OVX, hypothesized to protect against neuroendocrine aging, fails to guard against any studied age-related changes. Some age-related changes occur as early as midlife. Although neuron number remains constant at middle age, opiatergic neurons undergo significant functional changes by producing opiate antagonist peptides. This change appears to be caused by alterations in the prohormone convertases, which cleave propeptide to peptide. Altered peptides may trigger the loss of reproductive capacity. The midlife shift in opiate peptide production is a component of natural developmental processes that begin in the neonate and continue through old age. In the cholinergic system, E2 mediates numbers of cholinergic receptors, cholinergic neurons, and cholinergic-modulated memory systems in both young and old animals. Regardless of age, ovarian steroids, if present at physiologic levels, are beneficial to the neuroendocrine CNS, and long-term deprivation from ovarian-produced factors is deleterious in the systems we have examined. Our studies have shown that deprivation from ovarian steroid hormones in the female appears to be a major factor in the health of the CNS and in events associated with aging.

Peptide hormone precursor processing: getting sorted?

Processing of proproteins to biologically active peptides and, in the case of peptide hormones and neuropeptides, their sorting to granules of the regulated secretory pathway, requires the concerted action of a cascade of enzymes and chaperones. The purpose of this review is to summarize the recent emerging knowledge of how these molecules affect specific endocrine systems. This has come about through the study of gene knockout mice as well as endocrinopathies resulting from mutated genes in humans.