Search

ABSTRACT Deregulation and consolidations have changed the operating environment for electric utilities in the United States. One important aspect of this change that may be overlooked is how utilities manage [...]

This set of experiments investigated the appetitive or motivational processes underlying the performance of maternal behavior. The place preference paradigm was adapted to simultaneously investigate the reinforcing properties of cocaine and pups for maternal, lactating dams. These modifications allowed the authors to assess which stimulus, either a 10 mg/kg sc injection of cocaine or 3 pups, had the strongest reinforcing value. At Postpartum Days 10 and 16, the dams preferred the cocaine cue-associated chamber, whereas the dams tested at Postpartum Day 8 preferred the pup cue-associated chamber. Overall, the data revealed an interaction between the postpartum period at testing and the exhibited preference for cocaine or pups. Further testing will investigate the neural circuitry underlying the appetitive processes of each stimulus.

Purpose: The purpose of this study was to assess the relationship between the walkability/bikeability of college campuses and students’ body mass index (BMI) with student physical activity (PA) attitudes and behaviors as potential mediators.Design: Cross-sectional.Setting: Thirteen university campuses.Participants: A total of 1384 student participants.Measures: Walkability/bikeability environmental score (ES): 12-item audit assessed an average of 44 path segments per campus. Students were measured for height and weight and completed online surveys. Physical activity stage of change/behavior intentions were assessed using the transtheoretical model. The Cognitive Behavioral Physical Activity Questionnaire assessed outcome expectations, self-regulation, and personal barriers. International Physical Activity Questionnaire assessed walking-, moderate-, and vigorous-intensity PA.Analysis: Descriptive statistics, zero-order correlations, and path analysis with maximum likelihood estimation.Results: The overall model fit was good with χ2of 171.388 (df= 18), P< .001, comparative fit index value of .95, and a root mean square of approximation of .079. After controlling for gender, there was a direct negative association between walkability/bikeability ES and BMI (β = −.085) and positive association between personal barriers and BMI (β = .134). Walkability/bikeability ES was positively associated with walking-intensity PA (β = .010). Self-regulation was positively associated with moderate-intensity PA (β = .213), which, in turn, was negatively associated with BMI (β = −.057).Conclusions: The ease of walking and biking on a campus was related to college students’ walking behavior and their BMI. Students’ PA behavioral intentions were associated with moderate PA and lower BMI. These results provide evidence to focus on policies and structural supports for walkable/bikeable environments to supplement and enhance interventions encouraging individual behavior change for PA and weight management.

Traditional machining processes, where material is removed by a cutting tool from a workpiece, can introduce residual stresses at the surface of machined pieces. This paper provides an examination of an alternative machining methodology called modulated tool path machining. The ultimate objective of this research is to determine the effects of modulated tooling path machining processes, as applied to control chip geometry, on the surface stress of selected materials. Residual stresses in machined samples were characterised through the use of X-ray diffraction by comparing the modulated path method with a more traditional material removal technique (i.e. constant surface speed and constant contact).This paper is part of a Themed Issue on Measurement, modelling and mitigation of residual stress.

From Mr J. Morrell Sir: Katie Grant's article (`Democracy: who needs it?', 29 June) on Turkmenistan is being passed around online Central Asian groups with queries as to whether it [...]

The ability of boron and fluoride to migrate from a boron/fluoride rod was investigated over a 15 year period in Douglas-fir pole sections. Both components readily moved through the wood, but boron tended to be present at higher levels over the test reflecting the much higher boron content of the rods. The overall chemical levels suggested that higher dosages would be needed to more fully protect Douglas-fir poles.

Lithium containing AIBIIICVIsemiconductors are being considered as alternative materials for room temperature neutron detection. One of the primary challenges in growing a high quality crystal of such a material is the reactivity of lithium metal. The presence of nitrides, oxides, and a variety of alkali and alkaline earth metal impurities prevent pure synthesis and truncate crystal growth by introducing multiple nucleation centers during growth. Multiple lithium metal purification methods have been investigated which ultimately raised the metal purity to 99.996%. Multi-cycle vacuum distillation removed all but 40 ppm of metal impurities in lithium metal. LiGa(Se/Te)2was then synthesized with the high purity lithium metal by a variety of conditions. Lithium metal reacts violently with many standard crucible materials, and thermodynamic studies were undertaken to insure that an appropriate crucible choice was made, with high purity iron and boron nitride crucibles being the least reactive practical materials. Once conditions were optimized for synthesis of the chalcopyrite, vertical Bridgman crystal growth resulted in red crystals. The optical, electronic, and thermodynamic properties were collected.

AbstractThe ultimate purpose of this study was to investigate the use of a Li-Ca mixture for direct reduction of actinide oxides to actinide metals at temperatures below 1500°C. For such a process to be successful, the products of the reduction reaction, actinide metals, Li2O, and CaO must all be liquid at the reaction temperature so that the resulting actinide metal can coalesce and be recovered as a monolith. Since the established melting temperature of Li2O is in the range of 1427 to 1700°C and the melting temperature of CaO is 2654°C, the Li2O-CaO (lithium oxide-calcium oxide) pseudobinary system was investigated in an attempt to identify the presence of low-melting eutectic compositions.The results of our investigation indicate that there is no evidence of ternary Li-Ca-O phases or solutions melting below 1200°C. In the 1200 to 1500°C range utilizing MgO crucibles, there is some evidence for the formation of a ternary phase; however, it was not possible to determine the phase composition. The results of experiments performed with ZrO2crucibles in the same temperature range did not show the formation of the possible ternary phase seen in the earlier experiment involving MgO crucibles, so it was not possible to confirm the possibility that a ternary Li-Ca-O or Li-Mg-O phase was formed. It appears that the Li2O-CaO materials reacted, to some extent, with all of the container materials, alumina (Al2O3), magnesia (MgO), zirconia (ZrO2), and 95% Pt-5% Au; however, to clarify the situation additional experiments are required.In addition to the primary purpose of this study, the results of this investigation led to the following conclusions. First, the melting temperature of Li2O may be as low as 1250°C, which is considerably lower than the previously published values in the range 1427 to 1700°C. Second, lithium oxide (Li2O) vaporizes congruently. Third, lithium carbonate and Li2O react with 95% Pt-5% Au and also react with pure Pt. Fourth, it is likely that some or all of the past high-temperature phase behavior and vaporization experiments involving Li2O(s) at temperatures above 1250°C have actually involved Li2O(l). If these past measurements were actually measurements performed on Li2O(l) instead of the solid, the thermochemical data for phases and species in the Li-O system will require reevaluation.

Abstract: The potential for using temperature reductions to induce biocide deposition at the end of biocide treatment was assessed using Cyproconazole to treat ponderosa pine sapwood boards. Temperature-induced deposition produced higher biocide loadings and steeper preservative gradients from the surface inward than the more commonly used pressure-induced deposition. The results are discussed in relation to treatment results, process time, and the ability to recover treatment components.

The levels of copper, zinc, and arsenic in soil surrounding Douglas‐fir [Pseudotsuga menziesii(Mirb.) Franco] utility poles treated with ammoniacal copper zinc arsenate (ACZA) were investigated at sites in Florida, Virginia, and New York. Copper levels were elevated near the poles and declined with both horizontal distance away from the pole and depth beneath the soil surface. Zinc levels were also elevated next to the poles, but the levels declined more slowly than did those of copper. Arsenic levels were elevated in soil immediately next to the poles but declined to near background levels farther away. The results indicate that metals can leach from ACZA‐treated poles, but do not migrate far in the soil, and thus the levels decline sharply with distance from the poles.

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

NHS Direct, the 24-hour telephone helpline providing information and advice about health problems, is available throughout England and Wales. It was envisaged as a nurse-led service presenting a new opportunity for the nursing profession. Free text comments from a postal survey of NHS Direct nurses revealed that a large proportion of nurses were happy with working in NHS Direct, and that it presented some nurses with the opportunity of a new and challenging role. However, a minority found the work monotonous and felt that NHS Direct is likely to face the challenge of staff retention.

Abstract: The effects of pressure and temperature on supercritical fluid impregnation of tebuconazole were evaluated on Douglas-fir, western red cedar, red alder, white spruce, and white oak. Higher pressure markedly enhanced both the retention and distribution of tebuconazole in these species. When the rate of pressure release was altered at the ends of treatments of Douglas-fir, results varied. Generally, a higher rate of venting increased the steepness of the preservative gradient inward from the surface. Elevated pressures also affected some wood properties. Western red cedar and white spruce showed collapse, while the other three species were free of such defects. Modulus of Elasticity (MOE) and Modulus of Rupture (MOR) tended to decline with higher pressure in western red cedar and white spruce, but the differences were rarely significant. No significant changes in MOE/MOR occurred with the other 3 species.

Computer-based approaches identified three distinct human 2-hydroxy acid oxidase genes, HAOX1, HAOX2, and HAOX3, that encode proteins with significant sequence similarity to plant glycolate oxidase, a prototypical 2-hydroxy acid oxidase. The products of these genes are targeted to peroxisomes and have 2-hydroxy acid oxidase activities. Each gene displays a distinct tissue-specific pattern of expression, and each enzyme exhibits distinct substrate preferences. HAOX1 is expressed primarily in liver and pancreas and is most active on the two-carbon substrate, glycolate, but is also active on 2-hydroxy fatty acids. HAOX2 is expressed predominantly in liver and kidney and displays highest activity toward 2-hydroxypalmitate. HAOX3 expression was detected only in pancreas, and this enzyme displayed a preference for the medium chain substrate 2-hydroxyoctanoate. These results indicate that all three human 2-hydroxy acid oxidases are involved in the oxidation of 2-hydroxy fatty acids and may also contribute to the general pathway of fatty acid alpha-oxidation. Primary hyperoxaluria type 1 (PH1) is caused by defects in peroxisomal alanine-glyoxylate aminotransferase, the enzyme that normally eliminates intraperoxisomal glyoxylate. The presence of HAOX1 in liver and kidney peroxisomes and the ability of HAOX1 to oxidize glyoxylate to oxalate implicate HAOX1 as a mediator of PH1 pathophysiology.

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

SummaryWe investigated the ability of selected buffers to extract proteins and other materials from the surfaces and subsurfaces of ponderosa pine wafers colonized by selected wood stain fungi and potential bioprotectant bacteria. The effects of extraction conditions on enzyme activity were also analyzed. The addition of Tween 80 to the extraction media markedly enhanced total protein recovery as well as enzyme activity. Increasing the extraction time from 6 to 12 hours failed to increase total protein recovery, indicating that either most of the protein was removed earlier or proteases in the wood had attacked proteins liberated during extraction. Relatively short extractions using sodium acetate buffer amended with Tween 80, therefore, produced the best protein recovery and enzyme activity. These conditions can be used for extracting wood colonized by various combinations of stain fungi and bacterial bioprotectants to study the effects of the interaction on physiologic activities of the target fungi.

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility.A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare.The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitroand in vivoscreening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.

Here we describe the identification and characterization of a novel mouse gene, PDCR, that encodes a peroxisomal Delta(2), Delta(4)-dienoyl-CoA reductase. The mouse PDCR cDNA contains an 892-base pair open reading frame and is predicted to encode a 292-amino acid protein with a deduced molecular mass of 31,298 Da that terminates in a consensus type-1 peroxisomal targeting signal. Purified recombinant PDCR protein was generated from Escherichia coli and catalyzed the NADPH-dependent reduction of Delta(2)-trans, Delta(4)-trans-decadienoyl-CoA with a specific activity of 20 units/mg. Enzymatic characterization followed by high pressure liquid chromatography analysis of the products revealed that PDCR converted Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA to a Delta(3)-enoyl-CoA but not to a Delta(2)-enoyl-CoA. Kinetic analyses demonstrated that PDCR is active on a broad range of Delta(2), Delta(4)-dienoyl-CoAs. Although the observed substrate preference was to Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA, PDCR was also active on a C(22) substrate with multiple unsaturations, a result consistent with the role of peroxisomes in the oxidation of complex, very long chain, polyunsaturated fatty acids. The presence of a type-1 peroxisomal targeting signal Ala-Lys-Leu-COOH at the C terminus of PDCR suggested that this protein may be peroxisomal. We observed that tagged PDCR was efficiently transported to the peroxisome lumen in normal human fibroblasts but not in cells derived from a Zellweger syndrome patient with a specific defect in peroxisomal matrix protein import. We conclude that this protein resides within the peroxisome matrix and therefore represents the first mammalian peroxisomal Delta(2),Delta(4)-dienoyl-CoA reductase to be characterized at the molecular level.

Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO(2). Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role of this enzyme in mammals is unknown. A computer-based search for novel peroxisomal proteins led to the identification of a candidate gene for human MCD, which encodes a protein with a canonical type-1 peroxisomal targeting signal of serine-lysine-leucine(COOH). We observed that recombinant MCD protein has high intrinsic malonyl-CoA decarboxylase activity and that a malonyl-CoA decarboxylase-deficient patient has a severe mutation in the MCD gene (c.947-948delTT), confirming that this gene encodes human MCD. Subcellular fractionation experiments revealed that MCD resides in both the cytoplasm and peroxisomes. Cytoplasmic MCD is positioned to play a role in the regulation of cytoplasmic malonyl-CoA abundance and, thus, of mitochondrial fatty acid uptake and oxidation. This hypothesis is supported by the fact that malonyl-CoA decarboxylase-deficient patients display a number of phenotypes that are reminiscent of mitochondrial fatty acid oxidation disorders. Additional support for this hypothesis comes from our observation that MCD mRNA is most abundant in cardiac and skeletal muscles, tissues in which cytoplasmic malonyl-CoA is a potent inhibitor of mitochondrial fatty acid oxidation and which derive significant amounts of energy from fatty acid oxidation. As for the role of peroxisomal MCD, we propose that this enzyme may be involved in degrading intraperoxisomal malonyl-CoA, which is generated by the peroxisomal beta-oxidation of odd chain-length dicarboxylic fatty acids.

Abstract: In order for many large-scale bulk applications of high-temperature superconducting materials to be realized, the cost/performance of the superconductors needs to be optimized. From a performance standpoint, a long, flexible, single-crystal-like wire is required; from a cost-and-fabrication standpoint, an industrially scalable, low-cost process is required. Both of these critical requirements are met by rolling-assisted biaxially textured substrates, a conductor-fabrication technique that employs simple, scalable, thermomechanical processing techniques to obtain a near-single-crystal-like, flexible metal substrate in arbitrary lengths on which epitaxial oxide buffer layers and superconductors are then deposited.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1‐GFP and PTS2‐GFP to the cytoplasm but did incorporate IPMP‐GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation‐competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically‐oriented C3HC4 zinc binding domain that is essential for its biological activity.

In humans, defects in peroxisome assembly result in the peroxisome biogenesis disorders (PBDs), a group of genetically heterogeneous, lethal recessive diseases. We have identified the human gene PXAAA1 based upon its similarity to PpPAS5, a gene required for peroxisome assembly in the yeast Pichia pastoris. Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD. Consistent with this observation, CG4 patients carry mutations in PXAAA1. The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein. Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase. Furthermore, Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p. We conclude that Pxaaa1p plays a direct role in peroxisomal protein import and is required for PTS1 receptor activity.

Four methods used for the neurohistochemical demonstration of horseradish peroxidase (HRP) were quantitatively compared by counting retrogradely labeled neurons found after each method was used. HRP used as a retrograde marker is an important neuroanatomical tracing method, and maximum sensitivity in its demonstration of retrogradely, labeled neurons is important if these neuroanatomical studies are to completely demonstrate afferent neurons. The four methods compared were a diaminobenzidine (DAB) procedure, a Hanker-Yates procedure using P-phenylenediamine and pyrocatechol, an o-dianisidine procedure, and a tetramethyl benzidine (TMB) procedure. The TMB procedure resulted in a more complete topography of neurons afferent to the HRP application site, and demonstrated many more neurons in all afferent cell groups that either of the three other procedures. Use of the TMB method was especially critical in the cases of small HRP applications, a size useful for neuroanatomical studies, where the other methods demonstrated very few or no retrogradely labeled neurons. Neurons were judged to be retrogradely HRP labeled if they had small granules of the reaction product (the color varying with the chromogen) describing the somal shape, usually extending into the processes, and a clear nucleus. In addition, after the o-dianisidine or the TMB reaction a small number of retrogradely labeled neurons had soma and processes especially well filled with reaction product, giving the appearance of neurons from Golgi preparations. For a sensitive TMB reaction giving good results, exact H2O2 concentration, freshly prepared solutions, minimal postreaction exposure to alcohol, counterstaining, and clean glassware were each found to be important.

The afferent neural connections of the ventromedial nucleus of the rat hypothalamus (VMH) have been studied in detail using three horseradish peroxidase (HRP) application methods: HRP crystal implants, HRP-gel implants, and iontophoretic deposition of the enzyme. Examination of the cases in which the retrograde tracer was best confined to various subdivisions of the nucleus revealed that the septal area projects only to the ventrolateral VMH, and that the medial preoptic area, rostral lateral hypothalamus, and the ventral subiculum project mainly to the ventrolateral VMH. Thus, the subdivision of the VMH that contains the highest density of estradiol-concentrating neurons (Morrell et al. 1986) receives a larger set of inputs than the rostral and central parts of the nucleus. The central subdivision receives a more restricted set of projections than either the medial or the lateral regions. These studies suggest that there may be partial anatomical segregation of neural inputs to the various subdivisions of the VMH.

Estradiol (E2)-concentrating neurons afferent to the ventromedial nucleus of the hypothalamus (VMH) were identified by combining fluorescent retrograde tracing with steroid hormone autoradiography. The majority of E2-concentrating neurons that projected to the VMH were located in the medial preoptic area. In the entire medial preoptic area, 10.0% of the E2-sensitive neurons sent axons that terminated in the VMH. Twenty percent of the E2-concentrating neurons located in the periventricular preoptic area projected to the VMH. Of the E2-concentrating neurons found in the medial preoptic nucleus, 8.0% sent axons directly to the VMH. The bed nucleus of the stria terminalis, septum, and medial amygdala contained very few E2-receptive neurons that projected to the VMH. Preoptic area E2-concentrating neurons that project to the VMH may be part of a neural circuit that influences reproduction.

Summary: The topography and number of estradiol (E)-concentrating cells in the lower lumbar and sacral segments of the spinal cord of the female rat have been examined by the steroid autoradiography method. A nuclear-saturating dose of E was administered by intravenous infusion, which kept blood estrogen at or above proestrus levels for 3.5–4 h, much longer than usual for steroid receptor studies. The cord segments selected for examination are known to receive somatosensory information relevant for estrogen-dependent behavior, and to contain some of the motoneurons for epaxial muscles responsible for this behavior.Small numbers of E-concentrating cells were found in the dorsal portion of the gray matter of L4, L5, L6 and the sacral segments. These cells were found in lamina II, in the midline region which includes lamina X, and the medial portions of laminae III, IV, and V when they cross in the midline. E-concentrating cells were also found in the lateral portions of laminae III, IV, and V, and in lamina VII. Virtually no E-concentrating cells were found in the ventral portion of the gray matter or in the white matter. The spinal cord had few E-concentrating cells compared to the hypothalamus.

The temperature-time-location relationships during steam conditioning and pressure treatment of timber poles using ammoniacal copper arsenate (ACA) have been studied and a new mathematical model that incorporates both the thermal properties of the poles and the parameters of the treatment process is discussed. Prediction equations and charts are presented that show the minimum required steaming time to satisfy the 1982 Rural Electrification Authority (REA) purchase specification, i.e. a center temperature above 150 °F (65.5 °C) for 2 hours. A six hour steaming time, commonly used for ACA treatment, has been found to be too short to bring poles with diameters larger than about 40 cm to the required sterilization conditions. Therefore longer steaming times, predicted using the methods given here, are recommended. The temperature of the preservative used does not appear to be a major factor in determining the maximum temperature achieved at the center of a pole, but it can influence the length of time the pole is above 65.5 °C.

Assisted reproductive techniques make an important contribution to the conservation of endangered primate species. In our laboratories marmoset monkeys (Callithrix jacchus) are used as a model species for developing assisted reproductive technologies for New World primates. The studies require a reliable method for collecting functional sperm from these small animals. For this purpose a minimally invasive procedure, vaginal washing after natural mating, was evaluated for its suitability as a routine method of obtaining ejaculated sperm of high quality. The objective of the first series of tests was to identify a behavioural pattern which was easily discernible by the observer and provided a reliable indication that ejaculation had occurred. In a second series of tests the influence of length of separation of the male prior to copulation on the quality of the ejaculate was evaluated. Six adult ovariectomized and five adult intact females were used for vaginal washing, while their mates served as donors of ejaculates. Matings at specific times were achieved by separating the males from their females for a certain time period and subsequently introducing either their own mates or unfamiliar males to the females. After each observed mating vaginal washing was performed on the unsedated females. The seminal samples obtained were analysed for sperm concentration, the proportion of motile sperm and the proportion of live sperm. The results have shown (a) that ejaculation was indicated with a reliability of 86.1 % by the behaviour of the female, who terminates a copulation by moving away from her partner; (b) that separation of the male for up to six days had no negative effect on the sperm quality; (c) that the sperm samples were of high quality. Vaginal washing after natural matings appears to be a practical, reliable and gentle method for routinely collecting ejaculates from marmoset monkeys and possibly other New World primates.

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.

In situ hybridization provides a method for identifying cells that contain specific nucleic acid sequences. This report outlines an in situ hybridization procedure for mammalian neural tissue. The method maintains morphological quality and produces excellent specificity. Seven tritiated nucleic acid probes were examined: two ribosomal RNA probes, a control pBR322 plasmid probe, two probes encoding portions of the gene for oxytocin, one probe each encoding a portion of vasopressin glycoprotein, and neurophysin. Using cryostat-cut rat brain sections, rRNA probes labeled the cytoplasm of all cells and the nucleoli of larger neurons. The plasmid probe failed to produce a strong signal. Oxytocin and vasopressin probes appropriately labeled the cytoplasm of hypothalamic magnocellular neurons. Vasopressin parvocellular neurons were not identified by the current method, and the shorter length neurophysin probe failed to produce a signal. Methodological variables were examined by counting autoradiographic grains in cells. The longer oxytocin probe produced a stronger signal than the shorter oxytocin and vasopressin probes, and higher probe concentrations resulted in stronger signal. Hybridization could be abolished by tissue pretreatment with RNAse A, and longer exposure time increased signal strength. The outlined fixation steps with fresh-frozen tissue produced a superior signal compared to paraformaldehyde-perfused tissue.

We report here the identification of the Saccharomyces cerevisiae peroxisomal Delta3,Delta2-enoyl-CoA isomerase, an enzyme that is essential for the beta-oxidation of unsaturated fatty acids. The yeast gene YLR284C was identified in an in silico screen for genes that contain an oleate response element, a transcription factor-binding site common to most fatty acid-induced genes. Growth on oleic acid resulted in a significant increase in YLR284C mRNA, demonstrating that it is indeed an oleate-induced gene. The deduced product of YLR284C contains a type 1 peroxisomal targeting signal-like sequence at its C terminus and localizes to the peroxisome in a PEX8-dependent manner. Removal of YLR284C from the S. cerevisiae genome eliminated growth on oleic acid, but had no effect on peroxisome biogenesis, indicating a role for YLR284C in fatty acid metabolism. Cells lacking YLR284C had no detectable Delta3,Delta2-enoyl-CoA isomerase activity, and a bacterially expressed form of this protein catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 16 units/mg. We conclude that YLR284C encodes the yeast peroxisomal Delta3,Delta2-enoyl-CoA isomerase and propose a new name, ECI1, to reflect its enoyl-CoA isomerase activity.

The effect of water deprivation or estrogen treatment on the oxytocin content of rat hypothalamic cells was examined using a quantitative immunohistological technique. Oxytocin-containing cells were visualized using the immunoperoxidase technique of Sternberger and a primary antiserum directed against oxytocin. The optical density of the darkest 3.2 µm diameter spot in the cytoplasm of a cell was used as a measure of the oxytocin content of that cell. Water deprivation produced a significant decrease in anti-oxytocin staining in the anterior commissural nucleus of males and females. There was a similar decrease in the paraventricular nucleus of males, but not in the paraventricular nucleus of females or the supraoptic nucleus of either males or females. Estrogen treatment of ovariectomized female rats produced a fall in anti-oxytocin staining in the anterior commissural, but not paraventricular or supraoptic nuclei.

The ability to use sperm motility parameters, obtained by computer‐assisted sperm motility analysis (CASA), as an aid to selecting sperm samples for artificial insemination (AI) would have considerable benefits for commercial organizations and for the captive breeding of endangered species. In this study the Hobson sperm tracker (HST) was validated for use with spermatozoa from Callithrix jacchus, the common marmoset, by comparing values for straight line velocity by CASA with those obtained by direct measurement of sperm tracks. Using the settings established during validation, ejaculated and epididymal spermatozoa were analysed with the HST. The range of values for velocity parameters were used to establish expected motility profiles for the two types of spermatozoa as follows: for epididymal spermatozoa (concentration 2.2–85.8 × 106spermatozoa/mL) VCL 109.8–155.9, VSL 75.2–141.5, VAP 85.8–142.1, MAD 13.7–40.7, ALH 3.8–7.8, BCF 1.4–4.2, LIN 40.5–91.1% and STR 70.1–97.1%; for ejaculated spermatozoa (concentration 3.2–82.0 × 106spermatozoa/mL) VCL 89.6–136.7, VSL 69.6–110.3, VAP 74.5–121.9, MAD 19.2–29.3, ALH 3.0–9.9, BCF 2.8–5.5., LIN 65.4–85.3% and STR 93.8–97.7%. Epididymal spermatozoa from males which were not sexually active had significantly lower values for VCL, VSL and VAP, while values for MAD were significantly higher than for spermatozoa from sexually active males ( p < 0.031). Sperm concentration affected motility parameters significantly. Although motility parameters differed according to the batch of medium used, the differences were not statistically significant. Epididymal sperm samples had significantly higher VCL, VSL and VAP but lower BCF and LIN than ejaculated sperm samples of the same concentration diluted in the same batch of medium, while MAD, ALH and STR were not different. Urine contamination significantly reduced VCL, VSL and VAP ( p < 0.008, < 0.016 and < 0.008, respectively, sample size = 7) whereas MAD, ALH, BCF, LIN and STR were not affected. Therefore CASA could be useful in screening ejaculates for use in AI to eliminate samples with unusual motility patterns.

A two-dimensional fluorescence analysis of spermatozoa stained with Hoechst 33342 was carried out using an Epics V flow cytometer. This analysis involved the measurement of fluorescence both in the conventional manner at 90° and at a narrow forward angle (nominally 0°) to the direction of the interrogating laser beam. The arrangement provided an optical means of distinguishing the orientation of flattened particles passing through the interrogation system and enabled the verification and reinterpretation of one-dimensional (90°) fluorescence histogram data obtained previously. We now consider that the distinction between live and dead spermatozoa was over simplified and that the observed differences are due partly to an artefact of orientation. It has been confirmed that our earlier use of the low fluorescence peak, consisting of (bull, rabbit, sheep and pig) spermatozoa passing through the interrogation point obliquely to both detectors, as criterion for sorting X- from Y-bearing spermatozoa, is more practical than the use of a very much smaller subpopulation, based on limits imposed by selecting only spermatozoa accurately orientated with their narrow edges to one detector, while measurement of total fluorescence (DNA content) was made simultaneously from a broad face by the other detector. We report marked differences in the rate of staining and in attainment of a staining equilibrium between spermatozoa from different animals or from different ejaculates collected on the same or different days from the same animal. This variation introduces an element of subjectivity into the use of flow cytometry for sorting X- and Y- spermatozoa. Spermatozoa from rabbits, mice and chickens were also examined using the two-dimensional system.

Mouse submandibular glands show an androgen-dependent sexual dimorphism, reflected in higher concentrations in males than in females of bioactive peptides, such as epidermal growth factor (EGF), nerve growth factor, and renin in the cells of the granular convoluted tubules (GCT). Biochemical studies have demonstrated androgen receptors in submandibular gland and other androgen-responsive organs in mouse. We have determined the cellular localization of these receptors using steroid autoradiography. Fifteen adult gonadectomized male mice were injected intravenously with 0.13 microgram or 0.26 microgram [3H]-dihydrotestosterone (SA 135 Ci/mM); some animals were pre-treated with cyclocytidine to stimulate secretion by GCT cells. Animals were killed 15 min, 1, 2, or 3 hr after isotope injection. Steroid autoradiographs were prepared, and some were stained immunocytochemically for EGF. Of the different cell types of submandibular gland, the acinar cells most frequently and intensely concentrated [3H]-DHT; GCT cells also concentrated the hormone, as did a small number of striated duct cells. In the other major salivary glands, the only cells that concentrated the androgen were interlobular striated duct cells in sublingual gland. In prostate, anterior pituitary, and brain a large number of cells concentrated androgen, as has been previously reported. Androgen binding by the GCT cells was a predictable finding, since androgen-induced alterations in composition and form of these cells are well documented. The intense androgen concentration by the acinar cells was an unexpected finding and suggests a hitherto unknown androgen regulation of these cells. An incidental finding was intense concentration of [3H]-DHT in the nuclei of the endothelial cells of the post-capillary venules of the cervical lymph nodes.

These experiments were done to compare quantitatively, on a cell-by-cell basis, estradiol retention by cells in the medial preoptic area, arcuate nucleus, ventrolateral subdivision of the ventromedial nucleus, and the caudal half of the medial nucleus of the amygdala. The steroid autoradiograms were prepared from 2 µ sections of brains from ovariectomized, adrenalectomized adult female rats that had been infused intravenously with [3H] estradiol (E2) in a regimen which kept circulating hormone concentration at or above proestrus levels for 3–4 h. Even in these brain regions, containing the most dense collections of E2-concentrating cells, a maximum of only 27–61% of the cells concentrated E2. Therefore, in these regions only a particular subset of the cells retain hormone; other cells in the region do not retain hormone. Frequency distribution histograms of the number of grains per cell versus the number of cells in each region showed a wide range in the amount of E2 retained per cell, and no modes among E2retaining cells. The data followed a distribution markedly different from that predicted by a simple Poisson distribution, confirming that E2-retention does not result from a random, passive process such as diffusion. The overall quantitative characteristics of the frequency distribution histograms were similar across the four brain areas. Therefore, we propose that the different E2-sensitive functions of these brain areas must depend on differences in the neural connectivity or differences in hormone regulated peptide content of the areas.

The relationship between X chromosome-linked adrenoleukodystrophy and the red/green color pigment gene cluster on Xq28 was investigated in a large kindred. The DNA in a hemizygous male showed altered restriction fragment sizes compatible with at least a deletion extending from the 5' end of the color pigment genes. Segregation analysis using a DNA probe within the color pigment gene cluster showed significant linkage with adrenoleukodystrophy (logarithm of odds score of 3.19 at theta = 0.0). These data demonstrate linkage, rather than association, between a unique molecular rearrangement in the color pigment gene cluster and adrenoleukodystrophy. The DNA changes in this region are thus likely to be helpful for determining the location and identity of the responsible gene.

We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.

TRH-related peptides were extracted from the hypothalamus and prostate gland of the rabbit. The peptides were fractionated by gel exclusion chromatography and located by trypsin digestion and radioimmunoassay with antibodies to TRH amide and TRH–Gly Lys. In the hypothalamus TRH-related peptides containing approximately 16 and 30 residues were observed: in these peptides the extensions to the TRH sequence were exclusively in the C-terminal direction. In addition, the three-residue form of TRH was also present. In the prostate complex, the predominant TRH-related peptide contained approximately 50 residues and the extension to the TRH tripeptide was on the N-terminal side; a three-residue form of immunoreactive TRH was also demonstrated. The same pattern of TRH-related peptides was shown to be present in rabbit semen. The results reveal the existence of a novel TRH-related polypeptide in the prostate and semen which does not occur in the hypothalamus. This peptide appears to undergo secretion.Journal of Endocrinology(1989) 120,31–36