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A 34-nucleotide A-U sequence located immediately upstream of the editing sites of the Leishmania tarentolae cytochrome b mRNA induces a mitochondrial extract to insert U nucleotides independent of guide RNA. Insertions are localized to positions immediately 5' and 3' of the A-U sequence. When placed within an unedited mammalian transcript, the A-U sequence is sufficient to induce U-insertions. The sequence has a high degree of similarity with the templating nucleotides of a cytochrome b guide RNA and with a sequence adjacent to the editing sites in ND7 mRNA, the other characterized kinetoplastid mRNA supporting guide RNA-independent U-insertions. At least one protein specifically interacts with the A-U sequence. The reaction is consistent with a mechanism proposed for guide RNA-directed editing.

AbstractWith this essay by Roger Burbach, we return to our series of “anniversary essays“ on the ideas, concepts and events that have been central to our understanding of Latin America over the past 30 years. We inaugurated this celebration of our thirtieth anniversary in September 1996 with an essay on imperialism by Elizabeth Dore and John Weeks, and will conclude it in the next issue with Jean Franco’s essay on feminism.

The reactions γp→K+ Λ and γp→K+ Σ0 have been measured with the multiparticle detector system SAPHIR at ELSA in Bonn. Besides the differential cross sections the Λ polarization and, for the first time, the Σ0 polarization have been determined in a photon induced reaction. All data are presented as functions of the photon energy (from threshold up to 1.47 GeV) and of the kaon production angle (0°–180°). The polarization of both Λ and Σ0 is substantial at all energies and varies strongly with the production angle.

Dissolved pesticide concentrations in 168 water samples collected from two closely‐spaced lakes between early May 1990 and mid‐June 1991 indicated large differences in impacts from watershed nonpoint source inputs. Pesticide levels in Maskenthine Lake, a small impoundment of 34 ha, increased in response to spring and early summer runoff events. Twelve pesticides [atrazine, 2‐chloro‐4‐ethylamino‐6‐isopropylamino‐s‐triazine; alachlor, 2‐chloro‐N‐(2,6‐diethylphenyl)‐N‐(methoxymethyl)acetamide; metolachlor, 2‐chloro‐N‐(2‐ethyl‐6‐methylphenyl)‐N‐(2‐methoxy‐1‐methylethyl)acetamide; cyanazine, 2‐[[4‐chloro‐6‐(ethylamino)‐s‐triazin‐2‐yl]amino]‐2‐methylpropionitrile; EPTC, S‐ethyl dipropylthiocarbamate; butylate, S‐ethyl diisobutylthiocarbamate; propachlor, 2‐chloro‐N‐(1‐methylethyl)‐N‐phenylacetamide; trifluralin, α,α,α‐trifluoro‐2, 6‐dinitro‐N,N‐dipropyl‐p‐toluidine; simazine, 2‐chloro‐4,6‐bis(ethylamino)‐s‐triazine; propazine, 2‐chloro‐4,6‐bis(isopropylamino)‐s‐triazine; fonofos, O‐ethyl S‐phenyl ethylphosphonodithioate; metribuzin, 4‐amino‐6‐(1,1‐dimethylethyl)‐3‐(methylthio)‐1,2,4‐triazin‐5(4H)‐one] and two atrazine metabolites, deethylatrazine, 2‐amino‐4‐chloro‐6‐isopropylamino‐s‐triazine (DEA) and deisopropylatrazine, 2‐amino‐4‐chloro‐6‐ethylamino‐s‐triazine (DIA) were detected. Atrazine, cyanazine, DEA and DIA levels were greater than 1 µg/L. Atrazine remained above the maximum contaminant level for potable water of 3 µg/L throughout the period of investigation. The pesticide response to spring and summer runoff events was much less pronounced at Willow Lake, a 284 ha impoundment. Ten of 12 pesticides and two atrazine metabolites in Maskenthine Lake were also detected in Willow Lake; however, concentrations did not exceed 1 µg/L. After the spring flush of pesticides in May and June, concentrations decreased until the following spring. Ratios of DEA to atrazine and DIA to atrazine remained relatively constant suggesting that appreciable amounts of biodegradates were not produced. Apparent half‐lives were calculated from observed concentration decreases that appeared to follow first‐order kinetics. Atrazine was the most persistent agrichemical (t1/2= 193, t1/2= 124) introduced in the spring runoff events. Extracted pesticide levels were higher hi bottom cores from Maskenthine Lake than from Willow Lake. Differences in pesticide levels in the two lakes were related to watershed slope, soil drainage capacity, landuse, and rainfall.

α‐endorphin and γ‐endorphin, two closely related peptides of the pro‐opiomelanocortin family with characteristics biological activities, were purified to homogeneity from single human pituitary glands and chemically identified. Isolation of the peptides was based on size fractionation by Sephadex G‐75 chromatography followed by two HPLC steps using reverse‐phase and paired‐ion reverse‐phase systems and was monitored by radioimmunoassay. During the isolation procedure α‐and γ‐endorphin‐sized material behaved chromatographically and immunologically indistinguishably from synthetic α‐ and γ‐ endorphin. The amino acid composition and NH2‐terminus of isolated peptides demonstrated their identity as authentic α‐endorphin and γ‐endorphin. Acetylated forms were absent. In addition, evidence is provided that large forms with α‐ and γ‐endorphin immunoreactivity detected during gel filtration are human lipotropin‐(1–74) and ‐(1–75), respectively. The data substantiate that α‐endorphin and γ‐endorphin exist as endogenous peptides in the human pituitary gland.

Neuropeptides are present in brain tissue as well as in cerebrospinal fluid. Studies from a variety of disciplines have demonstrated that neuropeptides have neuromodulatory activities. Such activities are apparent in the involvement of neuropeptides in the regulation of adaptive, autonomic, and endocrine functions of the brain. Neuropeptides exert their neuromodulating influences by acting as neurotransmitters or as neurohormones. The neurotransmitter function of neuropeptides is associated with their synthesis in situin brain and anatomical distribution via neuropeptidergic fibre systems. The presence of such neuropeptides in cerebrospinal fluid may be due primarily to drainage into the cerebrospinal fluid compartments, their levels being a reflection of cellular processes such as biosynthesis, release, and metabolism. On the other hand, neuropeptides functioning as neurohormones may be actively delivered into the cerebrospinal fluid from central or peripheral sources. They employ the pathways of cerebrospinal fluid circulation as avenues of transport, and their cerebrospinal fluid levels may be functionally related to neurohormonal activity. In either case the cerebrospinal fluid levels are informative on the neuropeptide climate in brain; thus their determination is meaningful. The methodology for the sensitive and specific determination of neuropeptides is becoming available. Determination of neuropeptides may well be of diagnostic value in clinical practice.

Nuclear magnetic resonance (NMR) and inelastic neutron scattering techniques (INS) have been applied to study the rotational motions and methyl group tunneling in tetramethylstibonium iodide, [Sb(CH3)4 ] I, over er a wide temperature range. Parameters describing the [Sb(CH3)4]+cation tumbling and the methyl group reorientation at high temperatures and quantum mechanical tunneling of the methyl groups at low temperatures were determined. The results for INS and NMR experiments at low temperatures can be explained in terms of two crystallographically inequivalent methyl groups CH3(1) and CH3(2), which were established earlier by the crystal structure determination. In the INS spectra two tunneling lines at 22.0 μeV for CH3(1) and 1.05 μeV for CH3(2) with inelastic intensities in the ratio 3:1 were observed at T = 4 K. The activation energies derived from proton NMR spin-lattice relaxation time measurements for the thermally activated methyl group rotation are 1.50 kJ/mol for CH3(1) and 3.81 kJ/mol for CH3(2). They are in accordance with the activation energies obtained from neutron fixed-window measurements. The activation energy for [Sb(CH3)4]+cation tumbling is 50.9 kJ/mol as determined from the high temperature spin-lattice relaxation behaviour. Rotational potentials for the methyl groups are derived. For both kinds of inequivalent methyl groups the threefold potential terms dominate; three- and sixfold potential contributions are shifted by 180°

Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.

The key expression of the simian virus 40 (SV40) major late promoter could be repressed by the human TR4 orphan receptor via the +55 region of the SV40 major late promoter (nucleotide numbers 368-389, 5' -GTTA-AGGTTCGTAGGTCATGGA-3'). Using the coupled in vitro transcribed and translated TR4 orphan receptor with a molecular mass of 67.3 kilodaltons, electrophoretic mobility shift assay showed specific binding with a dissociation constant of 1.09 nM between the TR4 orphan receptor and the SV40 +55 oligonucleotides. In addition, chloramphenicol acetyltransferase assay demonstrated that this SV40 +55 region can function as a repressor via the TR4 orphan receptor, suppressing the transcriptional activities of both SV40 early and late promoters. Together, our data suggest that the TR4 orphan receptor may play an important role for the suppression of the SV40 gene expression.

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.

[Cyt 6]AVP-(3–9), an intermediate in the metabolism of AVP-(1–9) in vitro, was used to investigate the mechanism of formation of centrally active AVP metabolites. Exposure of [Cyt 6]AVP-(3–9) to rat brain membranes resulted in formation of [Cyt 6]AVP-(4–9),-(5–9), [pGlu 6,Cyt 6]AVP-(4–9) and AVP-(3–5), which were isolated and chemically identified. Products derived from cleavage of the C-terminus of the substrate were absent. Time-course experiments further indicated that the conversion process is predominantly mediby an aminopeptidase-like mechanism. The conversion of [Cyt 6]AVP-(3–9) by membranes from hippocampus, amygdala and septum was quantitatively and qualitatively similar. The results point to a major role of aminopeptidase activity in the metabolic conversion of AVP and the formation of centrally active AVP metabolites.

A cDNA encoding the murine Ah receptor (Ahb-1 allele for aromatic hydrocarbon responsiveness) has been isolated and characterized. Analysis of the deduced protein sequence revealed a region with similarity to the basic region/helix-loop-helix (BR/HLH) motif found in many transcription factors that undergo dimerization for function. In addition to the BR/HLH domain, the N-terminal domain of the Ah receptor has extensive sequence similarity to the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein and two regulatory proteins of Drosophila, Sim and Per. Photoaffinity labeling and peptide mapping studies indicate that the Ah receptor binds agonist at a domain that lies within this conserved N-terminal domain. The Ah receptor appears to be a ligand-activated transcription factor with a helix-loop-helix motif similar to those found in a variety of DNA-binding proteins, including Myc and MyoD.

The authors describe the activities of the Action Research program, designed to link academic projects and requirements for student research activities with the information needs of community service agencies. While interdisciplinary in focus, this program has been especially helpful in allowing community health nursing students to gain practical experience with real-life research questions and processes. An added benefit is to instill in the student an attitude of service while expanding their orientation to the community and the everyday needs of its agencies

A bovine pineal acid extract displays a vasotocin‐like bioactivity in several bioassays, and is recognized by antibodies against the Pro‐Arg‐Gly‐amide ending common to vasopressin and vasotocin. By using molecular sieve filtration and reversed‐phase HPLC, a vasopressin‐ and oxytocin‐like peptide was isolated from this pineal preparation, while no evidence for a vasotocin‐like peptide was obtained. The isolated neuropeptides contain a modified amino acid at position 2. This structural difference with authentic pituitary vasopressin and oxytocin may alter their biological and immunological properties, which have been interpreted as vasotocin‐like, and thus underlies the controversy concerning the existence of vasotocin in the mammalian pineal gland.

While the TR4 orphan receptor (TR4) is able to repress the expression of its target genes via its interaction with the direct repeat 1-hormone response element (DR1-HRE) and DR2-HRE, we now report that TR4 can also induce the transcriptional activity of the reporter gene containing a DR4-HRE via chloramphenicol acetyltransferase assay. Electrophoretic mobility shift assay and Scatchard analysis reveal a strong binding affinity (dissociation constant = 2 nM) between TR4 and DR4-HRE. The induction mediated by TR4 was detected not only in the synthetic DR4-HRE but also in some genes, such as rat alpha-myosin heavy-chain and S14 genes, containing the DR4 or DR4-like motif, which have been suggested to be the response elements for a thyroid hormone receptor. Our data also demonstrate this TR4-mediated gene induction is TR4 dose- and DR4 sequence-dependent. Together, our data suggest that DR4-HRE can be a positive regulatory element for TR4, which may be able to induce the transcriptional activity of the genes containing such positive HREs.

Chromosome banding patterns of the X chromosomes of Boophilus annulatus (Say) and B. microplus (Canestrini) indicate considerable homology between the two species. All bands of the small X chromosome of B. microplus are represented in the large X chromosome of B. annulatus with the exception of one medially located dark band. A large region of material proximal to the centromere and a small segment located at the telomere of the large X chromosome of B. annulatus are not represented in the small X chromosome of B. microplus. Meiotic abnormalities and the potential differentiation/evolution of the two X chromosomes are discussed. The extensive homology between the two species supports the genic data and linkage analyses previously published for these species.

The peptides that represent the major components with alpha-endorphin- and gamma-endorphin-like immunoreactivity in the rat neurointermediate lobe were purified to homogeneity and chemically characterized. Rat neurointermediate lobes were extracted by boiling and homogenization in acetic acid. Peptide purification was based on gel filtration, followed by two high-pressure liquid chromatography steps. Pools containing peptides with the size and immunochemical properties of alpha- and gamma-endorphins were resolved by reverse-phase high-pressure liquid chromatography into multiple immunoreactive components. The major forms were finally purified by paired-ion high-pressure liquid chromatography. The amino acid compositions of these peptides fitted the beta-endorphin sequences 1-16 and 1-17. Tryptic mapping, aminopeptidase M digestion, chromatographic characterization, and immunoreactivity to an antiserum recognizing the N alpha-acetylated terminus of endorphins showed that these peptides were indistinguishable from N alpha-acetyl-alpha-endorphin (N alpha-acetyl-beta-endorphin 1-16), and N alpha-acetyl-gamma-endorphin (N alpha-acetyl-beta-endorphin 1-17). The NH2-terminal residue of the peptides was identified by mass spectrometry as N alpha-acetyltyrosine, substantiating the identity of the peptides. The results demonstrate the existence of N alpha-acetylated alpha- and gamma-endorphin as endogenous peptides in the neurointermediate lobe of the rat pituitary gland. In view of their occurrence and biological properties they should be considered significant members of the pro-opiomelanocortin family.

Cyclic adenosine 3‘,5‘-monophosphate (cAMP) has been implicated as an intracellular messenger mediating osmotic regulation of expression of the gene encoding the neuropeptide vasopressin (VP) in the hypothalamus. We have used a heterologous transient transfection system to demonstrate that cAMP regulates the bovine VP gene promoter following transfection into CV1 cells. Mutational analysis identified a bovine VP cAMP-responsive element (BVP-CRE) 120-112 base-pairs upstream of the start of transcription. DNase I footprint analysis using nuclear protein extract from CV1 cells showed protection at the site of the BVP-CRE. Protection of the BVP-CRE was also observed using purified AP1 protein, while there was a weak interaction with the BVP-CRE using purified rat CREB protein. Nuclear proteins purified from the rat supraoptic nucleus bind to the BVP-CRE. As transgenic mouse studies have shown that the bovine VP gene is subject to appropriate physiological regulation in the mouse hypothalamus (Ang, H. L., Funkhouser, J., Carter, D. A., Ho, M. Y., and Murphy, D. (1991) Soc. Neurosci. Abstr. 513, 12), these data indicate a role for the BVP-CRE element in mediating VP gene expression in vivo. These data demonstrate that cAMP regulates bovine VP gene expression in vitro via a cis-acting element within the VP promoter, and this activation may be mediated by members of the AP1/ATF/CREB family of transcription factors.

A new solution hybridization assay was established for the measurement of renin mRNA. The assay makes use of a radioactively labelled renin complementary RNA as hybridization probe and allows rapid and sensitive detection of renin mRNA in amounts as low as 0.5 pg. With this assay it was possible to quantify renin mRNA in tissues with low-gene expression such as brain and heart as well as in testis, kidney and the submandibular gland (SMG) of mice. The concentrations in these organs were 0.03, 0.07, 1, 5.2 and 164 pg renin mRNA/µg RNA, respectively. These results are in agreement with Northern blotting experiments. The general applicability and ease of the solution hybridization assay described here should greatly improve the rapidity of mRNA measurements in future functional studies

With the emergence of the Information Age, school leaders have the opportunity to make education a major institution in shaping the future. To succeed, however, this writer believes they will have to be more visionary. He profiles how they can revolutionize their thinking.

The molecular dynamics of tetramethylstibonium hexafluorophosphate, [Sb(CH3)4]PF6, is investigated over a broad temperature range. NMR spin lattice relaxation times T1and the NMR second moments of the 1H and 19F nuclei were determined in the range 8.6 ≦ T/K ≦ 332.3 for polycrystalline [Sb(CH3)4]PF6. The complex cation undergoes isotropic tumbling for T > 260 K and thermally activated methyl group rotation in the temperature range T < 196 K. The activation energies for the transition from methyl group rotation to cation reorientation, as derived from NMR wideline (18.1 kJ/mol) and relaxation (22.7 kJ/mol) measurements, match. At very low temperatures pseudo classical line narrowing is observed, indicating tunneling motions of the methyl groups. The existence of two crystallographically inequivalent methyl groups is found by X-ray structure analysis at room temperature. The space group is P63mc, Z = 2; a = 738.6 pm, c = 1089.3 pm. It is confirmed by two steps in the temperature dependence of the signal intensity of the quasielastic line in neutron fixed window measurements in the temperature range 2 < T/K <148. The low temperature spin lattice relaxation times can be explained qualitatively by contributions of two crystallographically inequivalent methyl groups. Apparent activation energies for the two crystallographically different methyl groups are estimated. The complex anion undergoes isotropic tumbling in the temperature range 95 < T/K < 330. Above 330 K additionally translational motion is activated. Below 95 K the rotational motion of PF-6is freezing in via an uniaxial state in range 40 < T/K <80. Activation energies for both isotropical tumbling (10.5 kJ/mol) and uniaxial rotation (5.8 kJ/mol) have been derived from 19F NMR spin lattice relaxation

Amino acid sequence analysis indicates that the human TR4 orphan receptor (TR4) is a member of the estrogen/thyroid receptor subfamily of the steroid/thyroid receptor superfamily and recognizes the AGGTCA direct repeat (DR) of the hormone response element. Here we demonstrate using the electrophoretic mobility shift assay that TR4 binds specifically to DR with a spacing of 1 and 5 base pairs (DR1 and DR5), which are the response elements for retinoic acid receptor (RAR) and retinoid X receptor (RXR), respectively. A reporter gene assay using chloramphenicol acetyltransferase demonstrated that TR4 repressed RA-induced transactivation in a TR4 dose-dependent manner. Inhibition of the retinoid signal pathway also occurs through natural response elements found in CRBPII and RARbeta genes. Our data suggest that the mechanism of repression may not involve the formation of functionally inactive heterodimers between TR4 and RAR or RXR. Instead, we show that TR4 may compete for hormone response elements with RAR and RXR due to its higher binding affinity. Furthermore, treatment of F9 murine teratocarcinoma (F9) cells with 10(-6) M all-trans-retinoic acid increased TR4 mRNA levels, and this change was accompanied by an increased amount of endogenous TR4 protein that can bind to RXRE in electrophoretic mobility shift assay. Our data therefore strongly suggest that the retinoid signal pathway can be regulated by TR4 in a negative feedback control mechanism, which may restrict retinoic acid signaling to certain elements in a cell-specific fashion.

The possible role of cyclic AMP (cAMP) in the regulation of the vasopressin (VP) gene was tested in two cellular expression systems: one cell line with endogenous VP expression and the other which was transiently with a VP promoter-luciferase fusion gene. 8, Bromo-cAMP stimulated the VP mRNA content about 4-fold in the human VP-expressing small cell lung carcinoma cell line GLC-8. The luciferase activity in P19 embryonal carcinoma cells which were transiently transfected with − 174 to +44 of the 5'-flanking region of the human VP gene linked to the firefly luciferase gene, was stimulated about 2-fold by the cAMP analogue. The results indicate that cAMP plays a role in the upregulation of the VP gene and hence point to several putative nucleotide motives in the promoter functionally conferring this response.

The biotransformation of adrenocorticotropin (ACTH-(1-39)) by brain synaptic membranes has been studied. Peptide fragments of ACTH-(1-39) which were formed during in vitro incubation of the peptide with membrane preparations were isolated by high pressure liquid chromatography and characterized by determination of amino acid composition and NH2- terminal residue. At pH 7.4, ACTH-(1-38) was found as the main metabolite, together with ACTH-(7-21) and ACTH-(7-20). In addition, a series of secondary products was identified. At pH 6.2, ACTH-(1-38), ACTH-(1-37), and ACTH-(1-36) were exclusively formed, while at pH 8.5, ACTH-(1-39) was converted into ACTH-(1-16), ACTH-(17-39), ACTH-(22-39), and ACTH-(3-15). Time course experiments demonstrated the action of a carboxypeptidase activity and a trypsin-like endopeptidase on ACTH-(1-39) as predominant proteolytic events. The carboxypeptidase was optimally active at pH values of 5.7 or below. These enzymes play an essential role in the stepwise conversion of ACTH-(1-39) in brain. It is suggested that they are involved in the modulation of the central activities of ACTH fragments in the brain.

The increasing incidence of NO3contamination in municipal wells is a growing concern in the Midwest. While leachates from N fertilizers applied to agricultural lands can impact the water quality in municipal wells, there is also the potential for leachates from turfgrass fertilizers to be groundwater contaminants. In this study, five plots were treated with ammonium nitrate (34‐0‐0,N‐P‐K) at rates of 0, 1.0, 1.5, 2.0, and 2.4 kg N/100 m2and irrigated‐ The turf received approximately 640 mm of water during the 34‐d study. Analysis of soil water from 15, 6‐m continuous cores showed that as much as 95% of the NO3applied in late August leached below the turfgrass root zone. Average NO3concentrations in the pulse ranged from 34 to 70 mg/L NO3‐N. Thirty‐four days after fertilization the center of the pulse was at ≈1.2 m with the leading edge at 2 to 2.5 m. This vertical rate of movement is similar to that predicted by the one‐dimensional CMLS model. The relatively high uniform NO3concentrations in the unfertilized plot indicated that with excessive irrigation the water alone supplies NO3in excess of the turfgrass need.

The influence of adrenalectomy on the level of immunoreactive 18–24 ACTH extracted from hypothalamus, hippocampus and pituitary gland of rats was investigated. Brain ACTH was further characterized by fractionation by gel-permeation chromatography. Porcine 1–39 ACTH was exposed to synaptic plasma membranes in vitroin order to evaluate the role of metabolic conversion in changes of brain ACTH content.Removal of the adrenals, when compared with sham-adrenalectomy, resulted in a transient depletion of ACTH content in the anterior pituitary gland and the hippocampus, but not in the hypothalamus and the neurointermediate lobe. However, sham-adrenalectomy caused a transient reduction in levels of ACTH when compared with levels before operation in all tissues studied.The effects of adrenalectomy on hippocampal ACTH content persisted in hypophysectomized rats. Treatment of adrenalectomized rats with corticosterone failed to restore the reduced ACTH content when it was administered in doses that completely suppressed the release of pituitary ACTH. Adrenal steroids, however, may exert a direct effect on the metabolism of ACTH in the brain as judged from the in-vitro studies with porcine 1–39 ACTH exposed to a synaptosomal plasma membrane fraction of hippocampal tissue. The present study suggests that control of brain ACTH occurs independently of the control of pituitary ACTH release.

A single-base deletion in the single-copy vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). It results in the synthesis of an altered vasopressin precursor of which the axonal transport is blocked. Paradoxically, a small number of solitary hypothalamic neurons displays all the immunoreactivities of the wild-type vasopressin precursor (i.e., vasopressin, neurophysin, and a glycopeptide). In the present paper we provide evidence that these neurons have undergone a switch to a genuine heterozygous (di/+) phenotype; i.e., they contain the immunoreactivities of both the wild-type and the mutated vasopressin precursors. In the neural lobe, glycopeptide fibers are also present, showing that axonal transport of the wild-type precursor is restored. Moreover, the number of neurons displaying this di/+ phenotype increases markedly and in a linear way (from 0.1% up to 3% of the vasopressin cells) with age. These findings indicate that after mitotic division has ceased, genomic alterations occur in somatic neurons in vivo. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes.

Thirty clinic-referred children between ages 7 and 16 years and their mothers were interviewed separately by lay examiners, using the Child Assessment Schedule (CAS) and the Schedule for Affective Disorders and Schizophrenia for School-Age Children (K-SADS). Diagnoses were determined by comparing the informants' responses with the relevant DSM-III criteria, with no clinical judgment involved. Good concordance between the CAS and the K-SADS was obtained when diagnoses were based on the parent interviews alone or when based on combinations of the parent and child interviews for three of the four major diagnoses studied, including: Conduct Disorder, Attention Deficit Disorder, and Affective Disorder. Moderate to poor concordance was obtained: (1) when diagnoses were based solely on child interviews: and (2) for Anxiety Disorder, irrespective of the informant source. However, these latter two findings are consistent with the current literature and appear to be related to the diagnostic criteria, test-retest variance, and developmental influences.

Reactive oxygen species (ROS) generated within the corpus luteum (CL) are believed to play an integral role in luteolysis. Unsaturated lipids are susceptible to peroxidation by ROS, leading to the formation of toxic aldehydes such as malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE) that can attack cellular and extracellular proteins. Aldehyde conjugation of proteins can have diverse effects on cell function, including cross-linking of cell surface receptors, alteration of enzyme activities, and modification of lipoproteins. The objectives of the present study were to 1) determine by immunological techniques whether lipid peroxidation-derived protein epitopes (i.e., MDA- and 4-HNE-lysine) could be detected in situ during the natural life span of the CL, and 2) determine the temporal and spatial localization of these epitopes. Fresh luteal tissue was collected from young gilts at distinct phases of the estrous cycle: early (Days 4–7), mid (Days 8–12), and late (Days 14–18). Immunocytochemistry was performed on tissue sections using monoclonal antibodies against MDA-lysine and 4-HNE-lysine. Positive immunostaining was evident in porcine CL at all stages of the estrous cycle. Immunoactivity was associated primarily with the steroidogenic cells, most consistently with the large luteal cells, and was most intense in regressing tissue (i.e., late). No staining was observed in sections incubated with nonimmune mouse serum or when anti-4-HNE-lysine was preadsorbed with 4-HNE-protein preparations. Immunoblotting of protein extracts from luteal tissue revealed three major reactive bands. At least two of these bands were closely associated with extracted oxidized human low-density lipoprotein (LDL) and with 4-HNE-conjugated human apoprotein B. These results demonstrate the presence of lipid peroxidation-derived protein epitopes within the porcine CL and show that one or more of these may be an oxidized-LDL moiety. This represents the first direct in situ evidence that protein modification by lipid peroxidation products occurs during the natural luteal life span. The physiological consequences remain to be determined.

[Cyt6]AVP‐(3–9), an intermediate in the metabolism of AVP‐(1–9) in vitro, was used to investigate the mechanism of formation of centrally active AVP metabolites. Exposure of [Cyt6]AVP‐(3–9) to rat brain membranes resulted in formation of [Cyt6]AVP‐(4–9),‐(5–9), [pGlu6,Cyt6]AVP‐(4–9) and AVP‐(3–5), which were isolated and chemically identified. Products derived from cleavage of the C‐terminus of the substrate were absent. Time‐course experiments further indicated that the conversion process is predominantly mediby an aminopeptidase‐like mechanism. The conversion of [Cyt6]AVP‐(3–9) by membranes from hippocampus, amygdala and septum was quantitatively and qualitatively similar. The results point to a major role of aminopeptidase activity in the metabolic conversion of AVP and the formation of centrally active AVP metabolites.

To identify reasons for increasing numbers of pregnant adolescents, questionnaires were distributed to teenagers with unplanned pregnancies to determine the level of their knowledge of contraception. Findings revealed that 94% of the subjects had an adequate knowledge of availability, while only 43% had an adequate overall knowledge of contraception. Subjects reported many misconceptions about contraceptives and a general lack of motivation concerning contraceptive use.

Lavaged pulmonary macrophages of Bio 14.6 myopathic hamsters were compared with those from Bio F1B controls. Enlarged foamy macrophages were prevalent in the dystrophic strain. Lipidosis within this cell population was confirmed by morphologic and chemical analyses. The percentage of lipid-positive cells obtained from Bio 14.6 hamsters was three times greater than from control animals, but the total number of macrophages recovered from the lungs of dystrophic animals was approximately one-third lower compared to controls. Nearly two-thirds of the lipid-positive cells from the dystrophic strain were moderately to excessively engorged, whereas a similar percentage of the positive cells from control animals contained only sparse lipid inclusions. Qualitative ultrastructural differences were not observed between strains, but engorged macrophages of the dystrophic strain typically showed a predominance of lipid droplets with grey homogeneous material, crowded cytoplasmic organelles, and fewer primary lysosomes. Lipid analyses showed an 85% increase of total cellular lipids, a 486% increase of cholesteryl esters in neutral lipids, and increased 18:1 fatty acids in total lipids and the cholesteryl esters in cells from the Bio 14.6 strain. The etiology of the lipid excess has not been determined, but elevated chylomicrons and reduced alpha-lipoprotein values were observed in the serum of the dystrophic strain.

Radioactively labelled cRNA for the rat AVP gene exon C was synthetized from a pSP64-vector and used for solution hybridization measurement of AVP mRNA ("SP6 test"). For comparison hybridization was carried out with a gel-purified radioactively labelled cDNA probe synthetized by primer extension of AVP gene exon C cloned into an M13mp9 phage vector DNA ("S1 test"). Both tests had a comparable sensitivity of up to 0.2 pg AVP mRNA. Under optimal hybridization conditions kinetics were similar in both tests. The fast and easy preparation of large amounts of labelled cRNA probe and simple determination of absolute amounts of mRNA by saturation kinetics without need of a mRNA standard makes the "SP6 test" an attractive alternative to the known "S1 test". The "SP6 test" should be applicable for a wide variety of genes.

Peptides with vasopressin- and oxytocin-immunoreactivity were purified from bovine pineal glands. Three immunoreactive peptides were purified by successive high performance liquid chromatography steps in sufficient quantities for identification by fast atom bombardment-mass spectrometry. Two peptides were characterized as authentic vasopressin and oxytocin. Their identities were in agreement with the observed immunoreactivities, high performance liquid chromatography behavior, and biological activity. The third peptide was identified as N alpha-acetyloxytocin. The presence of the acetyl group was demonstrated by the molecular mass of the peptide, and the N alpha position was shown to be the modified site by the presence of a blocked NH2 terminus. N alpha-Acetylation of oxytocin may be involved in altering the biological properties of the peptide.

Abstract. Dispersed corticotrophic cells of 3 patients with Nelson's syndrome were studied in tissue culture for up to 25 days. During this culture period a parallel decrease with time was seen in ACTH and β-endorphin-like immunoreactivity (LIR) release. A concomitant decline was observed for intracellular hormones. The time course of hormone release showed a parallel secretion of ACTH and β-endorphin-LIR up to 8 h.Both the release of ACTH and β-endorphin LIR were stimulated by 0.1 μmlysine vasopressin (LVP) in all three adenoma cell cultures. Dexamethasone (0.1 and 1 μm) suppressed basal hormone secretion for 4 h. Synthetic ovine corticotrophin-releasing factor (CRF) at 10 and 100 nmstimulated the secretion of ACTH and β-endorphin LIR maximally. This stimulation was higher than observed with maximal stimulative concentration of LVP (0.3 μm). The CRF-mediated hormone secretion was calcium-dependent. Dexamethasone (0.1 μm) blocked the stimulating effect of 10 nmCRF completely.Gel-filtration chromatography demonstrated the cells to secrete both β-lipotrophin (β-LPH) and β-endorphin. The ratio of β-LPH to β-endorphin released remained constant upon stimulation by LVP and CRF. HPLC studies demonstrate the possibility that several β-endorphin fragments, including α-endorphin and γ-endorphin, were secreted by cells from a Nelson tumour. CRF caused a simultaneous parallel stimulation of the release of these peptides.

Nuclear hormone receptors comprise a superfamily of over 40 transcription factors. About half of them are classical receptors for lipophilic ligands such as steroids and vitamins. Almost all of these true receptors are present in the brain, where they transduce chemical signals from endocrine organs or signals of nutritional origin into cellular responses. The other members resemble the classical receptors in structure, but have no known ligands, and are hence called ‘orphan receptors’. The issue of whether ligands for nuclear orphan receptors exist is controversial. Evidence is emerging that orphan receptors might be activated by signal transduction pathways or might be constitutive enhancers or repressors that interact with the classical receptors. Thus, nuclear orphan receptors are placed in strategic positions in the regulation of gene expression in the nervous system.

Regulation of co-expression of three neuropeptide genes, i.e. genes encoding enkephalin, cholecystokinin, and gastrin-releasing peptide, was studied in human neuroepithelioma cells. In nondifferentiated state, the continuous cell line SK-N-MC displayed an equally high level of expression of the enkephalin, cholecystokinin, and gastrin-releasing peptide genes. By culturing in medium containing endothelial cell growth supplement the SK-N-MC cells differentiated morphologically into a cell type with neurite-like processes. After 3 days the expression of the enkephalin gene in endothelial cell growth supplement-differentiated cells was significantly reduced by 75% as compared to the nondifferentiated cells, while there was no change in the expression of the cholecystokinin and gastrin-releasing peptide genes during differentiation. The results show that the enkephalin gene is selectively down-regulated during differentiation of neuroepithelioma cells. It is suggested that the down-regulation is related to the transient expression of the enkephalin gene in developing brain and other organs. Thus the neuroepithelioma cell line may provide a cellular model to study the underlying molecular mechanism.

Abstract. Vasopressin and oxytocin exert pronounced effects on behaviour by a direct action on the brain. A single injection of vasopressin results in a long-term inhibition of extinction of a conditioned avoidance response suggesting that vasopressin triggers a long-term effect on the maintenance of a learned response, probably by facilitation of memory processes. In addition vasopressin improves passive avoidance behaviour, delays extinction of appetitive discrimination tasks, affects approach behaviour to an imprinting stimulus in ducklings, improves copulation rewarded behaviour of male rats in a T-maze, prevents or reverses amnesia induced by electroconvulsive shoch. CO2inhalation, pentylenetetrazol or puromycin. The majority of these effects of vasopressin in the various and sometimes relatively complex tasks may be explained by stimulatory influences of this neuropeptide on memory processes. Generally oxytocin exerts effects which are opposite to those of vasopressin and it has been suggested that oxytocin may be an amnesic neuropeptide. Various limbic system structures seem to act as the anatomical substrate for the bevavioural effects of vasopressin. In particular the amygdala, the dentate gyrus of the hippocampal complex, the ventral hippocampus and the dorsal septum seem to be involved. Evidence has been obtained from experiments with homozygous diabetes insipidus rats and from experiments in which antisera were applied that endogenous vasopressin and oxytocin play a physiological role in brain processes related to memory. It appears that highly active fragments can be generated from vasopressin and experiments in which a fragment of vasopressin ([pGlu4, Cyt6]AVP-(4–8)) as well as an AVP-antagonist were used, reveal that the vasopressin receptors mediating the behavioural effects are situated in the brain and differ in specificity from the peripheral (blood pressure) vasopressin receptors. Generally the clinical data obtained so far with vasopressin treatment are in agreement with the results from animal experiments and they support the notion on the involvement of vasopressin in memory function. The sometimes reported conflicting results on vasopressin effects in certain patients (Korsakoff or Alzheimer) may have to do with the wide-spread pathology in these diseases.

This study was designed to determine the source of tumor necrosis factor (TNF) α within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNFα, anti-porcine macrophage-specific antigen, or anti-α-actin (marker of pericyte and smooth muscle cells). Dolichos bifloruslectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNFα was apparent in porcine CL throughout the estrous cycle. TNFα immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNFα in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNFα secretion by each subpopulation was determined by measuring bioactive TNFα in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNFα (up to 400 pg/106cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/106cells) of bioactive TNFα. Large luteal cells secreted slightly greater amounts of TNFα (10–15 pg/106cells). Local macrophages appear to be the primary source of TNFα in the porcine CL.