Regulation of aldosterone secretion by mineralocorticoid receptor-mediated signaling.

PubMed

Chong, Cherish; Hamid, Anis; Yao, Tham; Garza, Amanda E; Pojoga, Luminita H; Adler, Gail K; Romero, Jose R; Williams, Gordon H

2017-03-01

We posit the existence of a paracrine/autocrine negative feedback loop, mediated by the mineralocorticoid receptor (MR), regulating aldosterone secretion. To assess this hypothesis, we asked whether altering MR activity in zona glomerulosa (ZG) cells affects aldosterone production. To this end, we studied ex vivo ZG cells isolated from male Wistar rats fed chow containing either high (1.6% Na + (HS)) or low (0.03% Na + (LS)) amount of sodium. Western blot analyses demonstrated that MR was present in both the ZG and zona fasciculata/zona reticularis (ZF/ZR/ZR). In ZG cells isolated from rats on LS chow, MR activation by fludrocortisone produced a 20% and 60% reduction in aldosterone secretion basally and in response to angiotensin II (ANGII) stimulation, respectively. Corticosterone secretion was increased in these cells suggesting that aldosterone synthase activity was being reduced by fludrocortisone. In contrast, canrenoic acid, an MR antagonist, enhanced aldosterone production by up to 30% both basally and in response to ANGII. Similar responses were observed in ZG cells from rats fed HS. Modulating glucocorticoid receptor (GR) activity did not alter aldosterone production by ZG cells; however, altering GR activity did modify corticosterone production from ZF/ZR/ZR cells both basally and in response to adrenocorticotropic hormone (ACTH). Additionally, activating the MR in ZF/ZR/ZR cells strikingly reduced corticosterone secretion. In summary, these data support the hypothesis that negative ultra-short feedback loops regulate adrenal steroidogenesis. In the ZG, aldosterone secretion is regulated by the MR, but not the GR, an effect that appears to be secondary to a change in aldosterone synthase activity. © 2017 Society for Endocrinology.

Dermatopathia pigmentosa reticularis.

PubMed

Brar, Balvinder K; Mehta, Vivek; Kubba, Asha

2007-01-01

Dermatopathia pigmentosa reticularis is a rare inherited pigmentary disorder of the skin characterized by generalized reticulate hyperpigmentation, nonscarring alopecia and onychodystrophy. The reticulate pigmentation occurs at birth or during early childhood. We hereby report a 12-year-old Indian patient with this disorder.

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

PubMed Central

Schnare, M N; Gray, M W

1982-01-01

In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista. Images PMID:7079176

Extensive Variation in Cadmium Tolerance and Accumulation among Populations of Chamaecrista fasciculata

PubMed Central

Henson, Tessa M.; Cory, Wendy; Rutter, Matthew T.

2013-01-01

Plant populations may vary substantially in their tolerance for and accumulation of heavy metals, and assessment of this variability is important when selecting species to use in restoration or phytoremediation projects. We examined the population variation in cadmium tolerance and accumulation in a leguminous pioneer species native to the eastern United States, the partridge pea (Chamaecrista fasciculata). We assayed growth, reproduction and patterns of cadmium accumulation in six populations of C. fasciculata grown on a range of cadmium-contaminated soils. In general, C. fasciculata exhibited tolerance in low to moderate soil cadmium concentrations. Both tolerance and accumulation patterns varied across populations. C. fasciculata exhibited many characteristics of a hyperaccumulator species, with high cadmium uptake in shoots and roots. However, cadmium was excluded from extrafloral nectar. As a legume with tolerance for moderate cadmium contamination, C. fasciculata has potential for phytoremediation. However, our findings also indicate the importance of considering the effects of genetic variation on plant performance when screening plant populations for utilization in remediation and restoration activities. Also, there is potential for cadmium contamination to affect other species through contamination of leaves, fruits, flowers, pollen and root nodules. PMID:23667586

Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus).

PubMed

Ye, Wen-Ling; Wang, Feng-Ling; Wang, Hong-Ju; Wang, Jian-Lin

2017-04-01

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fundamental studies of adrenal retinoid-X-receptor: Protein isoform, tissue expression, subcellular distribution, and ligand availability.

PubMed

Cheng, Behling; Al-Shammari, Fatema H; Ghader, Isra'a A; Sequeira, Fatima; Thakkar, Jitendra; Mathew, Thazhumpal C

2017-07-01

Adrenal gland reportedly expresses many nuclear receptors that are known to heterodimerize with retinoid-X-receptor (RXR) for functions, but the information regarding the glandular RXR is not adequate. Studies of rat adrenal homogenate by Western blotting revealed three RXR proteins: RXRα (55kDa), RXRβ (47kDa) and RXR (56kDa). RXRγ was not detectable. After fractionation, RXRα was almost exclusively localized in the nuclear fraction. In comparison, substantial portions of RXRβ and RXR were found in both nuclear and post-nuclear particle fractions, suggesting genomic and non-genomic functions. Cells immunostained for RXRα were primarily localized in zona fasciculata (ZF) and medulla, although some stained cells were found in zona glomerulosa (ZG) and zona reticularis (ZR). In contrast, cells immunostained for RXRβ were concentrated principally in ZG, although some stained cells were seen in ZR, ZF, and medulla (in descending order, qualitatively). Analysis of adrenal lipid extracts by LC/MS did not detect 9-cis-retinoic acid (a potent RXR-ligand) but identified all-trans retinoic acid. Since C20 and C22 polyunsaturated fatty acids (PUFAs) can also activate RXR, subcellular availabilities of unesterified fatty acids were investigated by GC/MS. As results, arachidonic acid (C20:4), adrenic acid (C22:4), docosapentaenoic acid (C22:5), and cervonic acid (C22:6) were detected in the lipids extracted from each subcellular fraction. Thus, the RXR-agonizing PUFAs are available in all the main subcellular compartments considerably. The present findings not only shed light on the adrenal network of RXRs but also provide baseline information for further investigations of RXR heterodimers in the regulation of adrenal steroidogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Population structure and connectivity in the Mediterranean sponge Ircinia fasciculata are affected by mass mortalities and hybridization

PubMed Central

Riesgo, A; Pérez-Portela, R; Pita, L; Blasco, G; Erwin, P M; López-Legentil, S

2016-01-01

Recent episodes of mass mortalities in the Mediterranean Sea have been reported for the closely related marine sponges Ircinia fasciculata and Ircinia variabilis that live in sympatry. In this context, the assessment of the genetic diversity, bottlenecks and connectivity of these sponges has become urgent in order to evaluate the potential effects of mass mortalities on their latitudinal range. Our study aims to establish (1) the genetic structure, connectivity and signs of bottlenecks across the populations of I. fasciculata and (2) the hybridization levels between I. fasciculata and I. variabilis. To accomplish the first objective, 194 individuals of I. fasciculata from 12 locations across the Mediterranean were genotyped at 14 microsatellite loci. For the second objective, mitochondrial cytochrome c oxidase subunit I sequences of 16 individuals from both species were analyzed along with genotypes at 12 microsatellite loci of 40 individuals coexisting in 3 Mediterranean populations. We detected strong genetic structure along the Mediterranean for I. fasciculata, with high levels of inbreeding in all locations and bottleneck signs in most locations. Oceanographic barriers like the Almeria-Oran front, North-Balearic front and the Ligurian-Thyrrenian barrier seem to be impeding gene flow for I. fasciculata, adding population divergence to the pattern of isolation by distance derived from the low dispersal abilities of sponge larvae. Hybridization between both species occurred in some populations that might be increasing genetic diversity and somewhat palliating the genetic loss caused by population decimation in I. fasciculata. PMID:27599575

Brain stem stimulation and the acetylcholine-evoked inhibition of neurones in the feline nucleus reticularis thalami

PubMed Central

Dingledine, Raymond; Kelly, J. S.

1977-01-01

1. In cats anaesthetized with halothane and nitrous oxide, the responses to iontophoretically applied acetylcholine (ACh) and to high-frequency stimulation of the mid-brain reticular formation (MRF) were tested on spontaneously active neurones in the nucleus reticularis thalami and underlying ventrobasal complex. 2. The initial response to MRF stimulation of 90% of the ACh-inhibited neurones found in the region of the dorsolateral nucleus reticularis was an inhibition. Conversely, the initial response of 82% of the ACh-excited neurones in the ventrobasal complex was an excitation. Neurones in the rostral pole of the nucleus reticularis were inhibited by both ACh and RMF stimulation. 3. The mean latency (and s.e. of mean) for the MRF-evoked inhibition was 13·7 ± 3·2 ms (n = 42) and that for the MRF-evoked excitation, 44.1 ± 4.2 ms (n = 35). 4. The ACh-evoked inhibitions were blocked by iontophoretic atropine, in doses that did not block amino acid-evoked inhibition. In twenty-four ACh-inhibited neurones the effect of iontophoretic atropine was tested on MRF-evoked inhibition. In all twenty-four neurones atropine had no effect on the early phase of MRF-evoked inhibition but weakly antagonized the late phase of inhibition in nine of fourteen neurones. 5. Interspike-interval histograms showed that the firing pattern of neurones in the nucleus reticularis was characterized by periods of prolonged, high-frequency bursting. Both the ACh-evoked inhibitions and the late phase of MRF-evoked inhibitions were accompanied by an increased burst activity. In contrast, iontophoretic atropine tended to suppress burst activity. 6. The possibility is discussed that electrical stimulation of the MRF activates an inhibitory cholinergic projection to the nucleus reticularis. Since neurones of the nucleus reticularis have been shown to inhibit thalamic relay cells, activation of this inhibitory pathway may play a role in MRF-evoked facilitation of thalamo-cortical relay transmission

Disruption of the Crithidia fasciculata RNH1 gene results in the loss of two active forms of ribonuclease H.

PubMed Central

Ray, D S; Hines, J C

1995-01-01

Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments. The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid. The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes. Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted. Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene. Images PMID:7630731

Localization of cytochrome P4501A in liver and extrahepatic organs of the pilot whale, Globiocephala melaena

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stegeman, J.; Miller, C.; Moore, M.

1994-12-31

Marine mammals may be important indicators of effects of contaminants that are globally distributed. Recently the authors described apparent environmental induction of hepatic CYPLA in beluga whales. Here they describe the localization and extent of CYPLA expression in organs of the pilot whale. Tissues from 18 pilot whales stranded on Cape Cod in 1990/91 were frozen in liquid N2 or fixed in formalin and embedded. Liver microsomal EROD activity were comparable to results with other cetaceans. Immunohistochemical analysis showed a periportal localization of CYPLA in liver parenchyma, and staining in the endothelium. Renal staining was strong in brush border andmore » endothelium. Testis, ovary, and spleen showed CYPLA staining only in endothelium. Adrenal zona fasciculata and zona reticularis stained more weakly than did endothelium. In lung there was mild staining of bronchiolar epithelium and strong staining of endothelium. The results indicate that active concentrations of inducer have penetrated throughout the body. CYPLA stained in dermal endothelium, indicating that analysis of skin biopsies could allow nondestructive analysis of CYPLA induction in marine mammals. CYPLA expression in these whales was surprisingly strong, suggesting the possibility of chemical effects related to CYPLA induction.« less

Renin knockout rat: control of adrenal aldosterone and corticosterone synthesis in vitro and adrenal gene expression

PubMed Central

Gehrand, Ashley; Bruder, Eric D.; Hoffman, Matthew J.; Engeland, William C.; Moreno, Carol

2014-01-01

The classic renin-angiotensin system is partly responsible for controlling aldosterone secretion from the adrenal cortex via the peptide angiotensin II (ANG II). In addition, there is a local adrenocortical renin-angiotensin system that may be involved in the control of aldosterone synthesis in the zona glomerulosa (ZG). To characterize the long-term control of adrenal steroidogenesis, we utilized adrenal glands from renin knockout (KO) rats and compared steroidogenesis in vitro and steroidogenic enzyme expression to wild-type (WT) controls (Dahl S rat). Adrenal capsules (ZG; aldosterone production) and subcapsules [zona reticularis/fasciculata (ZFR); corticosterone production] were separately dispersed and studied in vitro. Plasma renin activity and ANG II concentrations were extremely low in the KO rats. Basal and cAMP-stimulated aldosterone production was significantly reduced in renin KO ZG cells, whereas corticosterone production was not different between WT and KO ZFR cells. As expected, adrenal renin mRNA expression was lower in the renin KO compared with the WT rat. Real-time PCR and immunohistochemical analysis showed a significant decrease in P450aldo (Cyp11b2) mRNA and protein expression in the ZG from the renin KO rat. The reduction in aldosterone synthesis in the ZG of the renin KO adrenal seems to be accounted for by a specific decrease in P450aldo and may be due to the absence of chronic stimulation of the ZG by circulating ANG II or to a reduction in locally released ANG II within the adrenal gland. PMID:25394830

NEFM (Neurofilament Medium) Polypeptide, a Marker for Zona Glomerulosa Cells in Human Adrenal, Inhibits D1R (Dopamine D1 Receptor)-Mediated Secretion of Aldosterone.

PubMed

Maniero, Carmela; Garg, Sumedha; Zhao, Wanfeng; Johnson, Timothy Isaac; Zhou, Junhua; Gurnell, Mark; Brown, Morris J

2017-08-01

Heterogeneity among aldosterone-producing adenomas (APAs) has been highlighted by the discovery of somatic mutations. KCNJ5 mutations predominate in large zona fasciculata (ZF)-like APAs; mutations in CACNA1D , ATP1A1, ATP2B3 , and CTNNB1 are more likely to be found in small zona glomerulosa (ZG)-like APAs. Microarray comparison of KCNJ5 mutant versus wild-type APAs revealed significant differences in transcriptomes. NEFM , encoding a neurofilament subunit which is a D1R (dopamine D1 receptor)-interacting protein, was 4-fold upregulated in ZG-like versus ZF-like APAs and 14-fold more highly expressed in normal ZG versus ZF. Immunohistochemistry confirmed selective expression of NEFM (neurofilament medium) polypeptide in ZG and in ZG-like APAs. Silencing NEFM in adrenocortical H295R cells increased basal aldosterone secretion and cell proliferation; silencing also amplified aldosterone stimulation by the D1R agonist, fenoldopam, and inhibition by the D1R antagonist, SCH23390. NEFM coimmunoprecipitated with D1R, and its expression was stimulated by fenoldopam. Immunohistochemistry for D1R was mainly intracellular in ZG-like APAs but membranous in ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZF-like APAs was higher than in cells from ZG-like APAs. Transfection of mutant KCNJ5 caused a large reduction in NEFM expression in H295R cells. We conclude that NEFM is a negative regulator of aldosterone production and cell proliferation, in part by facilitating D1R internalization from the plasma membrane. Downregulation of NEFM in ZF-like APAs may contribute to a D1R/D2R imbalance underlying variable pharmacological responses to dopaminergic drugs among patients with APAs. Finally, taken together, our data point to the possibility that ZF-like APAs are in fact ZG in origin. © 2017 American Heart Association, Inc.

Organization of diencephalic projections from the medullary subnucleus reticularis dorsalis and the adjacent cuneate nucleus: a retrograde and anterograde tracer study in the rat.

PubMed

Villanueva, L; Desbois, C; Le Bars, D; Bernard, J F

1998-01-05

The distribution and organization of diencephalic projections from the subnucleus reticularis dorsalis (SRD) and the neighbouring cuneate nucleus (Cu) were studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin in SRD and Cu and wheat germ agglutinin-apo horseradish peroxidase-gold in some selected thalamic areas. As previously reported, the efferent projections from the Cu were essentially contralateral and terminated mainly in the ventroposterolateral thalamic nucleus. Less dense terminals from the Cu were also observed in the posterior thalamic group, the ventral aspect of the zona incerta and the caudal and dorsal portion of the reuniens area. Retrograde tracer injections in the medial ventroposterolateral thalamic nucleus labeled numerous cells in the contralateral Cu, with a smaller number in the gracile nucleus. From the SRD, terminals were observed in the lateral aspect of the ventromedial thalamic nucleus, the lateral parafascicular area and, to a lesser extent, in the ventral aspect of the zona incerta and the core of the reuniens area. Retrograde tracer injections in the lateral part of the ventromedial thalamic nucleus labeled cells in the caudal medulla, many of which were located in the dorsal-most aspect of the SRD throughout its caudo-rostral extent. The existence of SRD-thalamic connections reinforces the idea that the caudal reticular formation is an important nociceptive relay to the thalamus. Our data shed new light on old hypotheses suggesting that, in addition to spino-thalamic pathways, spino-reticulo-thalamic pathways may play an important role in distributing pain signals to the forebrain.

Inhibitory effects of acetylcholine on neurones in the feline nucleus reticularis thalami.

PubMed

Ben-Ari, Y; Dingledine, R; Kanazawa, I; Kelly, J S

1976-10-01

1. Short iontophoretic pulses of acetylcholine (ACh) inhibited almost every spontaneously active cell encountered in the nucleus reticularis thalami of cats anaesthetized with a mixture of halothane, nitrous oxide and oxygen. On 200 cells the mean current needed to eject an effective inhibitory dose of ACh was 67 +/- 2 nA. When the ACh-evoked inhibition was mimicked by gamma-aminobutyric acid (GABA) or glycine on the same cell, the current required to release ACh was found to be approximately twice as great as that required to release an equally effective dose of GABA or glycine. 2. ACh inhibitions developed with a latency which was very much shorter than that for ACh excitation in cells of the ventrobasal complex. The latency of the ACh-evoked inhibition was as rapid as the onset and offset of the excitation of the same cells glutamate and their inhibition by GABA or glycine. 3. The firing pattern of ACh-inhibited neurones in the nucleus reticularis was characterized by periods of prolonged, high frequency bursts, and their mean firing frequency was 22 Hz. Raster dot displays and interspike interval histograms showed that whereas ACh suppressed the spikes that occurred between bursts much more readily than those that occurred during bursts, all spikes were equally sensitive to the depressant action of GABA and glycine. Large doses of ACh provoked or exaggerated burst activity. 4. ACh-evoked inhibition was extremely sensitive to blockade by short iontophoretic applications of atropine, which had no effect on the inhibitions evoked on the same cell equipotent doses of GABA or glycine. The ACh-evoked inhibitions were also antagonized by dihydro-beta-erythroidine released with slightly larger currents. When tested on the same cell, small iontophoretic applications of picrotoxin and bicuculline methoiodide blocked the inhibition evoked by GABA but had no effect on that evoked by ACh. Iontophoretic strychnine only rarely affected the inhibition evoked by ACh, while

Correlation between concomitant theta waves in nucleus reticularis pontis oralis and in hippocampus, thalamus and neocortex during dreaming in rats.

PubMed

Simões, C A; Valle, A C; Timo-Iaria, C

1996-12-01

Theta waves, which are the main electrophysiological expression of dreaming activity in many brain structures of rats, often undergo specific changes in voltage and frequency according to the oniric patterns. Much is known about their mechanisms but little is known regarding their origin, which has been ascribed to a specific activation of either the reticular formation or the septal nuclei or nucleus reticularis pontis oralis. In the present study, rats were prepared for chronic recording of the electro-oscillograms of cortical areas 10, 3 and 17, of hippocampal CA1 and CA3 fields, of nucleus reticularis thalami, nucleus reticularis pontis oralis and occasionally of nucleus reticularis caudalis. Head, rostrum, eye and forelimb movements were also recorded, so that the oniric behaviors could be precisely identified. The scatter diagrams and the corresponding correlation coefficients (r) of the voltage of concomitant waves were determined for each possible pair of leads. The potentials were analyzed at a frequency of 256 Hz over a period of 1 to 3 sec. A very high degree of correlation was observed between theta waves in nucleus reticularis pontis oralis, hippocampal fields and nucleus reticularis pontis caudalis; sometimes r approached unity. Although these data cannot be taken as proof of nucleus reticularis pontis oralis being the source of theta waves, they are at least compatible with this hypothesis.

Phenolic acids, hydrolyzable tannins, and antioxidant activity of geopropolis from the stingless bee Melipona fasciculata Smith.

PubMed

Dutra, Richard Pereira; Abreu, Bruno Vinicius de Barros; Cunha, Mayara Soares; Batista, Marisa Cristina Aranha; Torres, Luce Maria Brandão; Nascimento, Flavia Raquel Fernandes; Ribeiro, Maria Nilce Sousa; Guerra, Rosane Nassar Meireles

2014-03-26

Geopropolis is a mixture of plant resins, waxes, and soil produced by the stingless bee Melipona fasciculata Smith. This paper describes the antioxidant activity and chemical composition of geopropolis produced by M. fasciculata. The total phenolic content determined with the Folin-Ciocalteu reagent was highest in the ethyl acetate fraction and hydroalcoholic extract. Antioxidant activity was assayed by the in vitro DPPH, ABTS, and FRAP assays. The hydroalcoholic extract and fractions of geopropolis, except for the hexane fraction, exhibited antioxidant activity against DPPH, ABTS, and FRAP. The phenolic compounds were identified by HPLC-DAD-MS on the basis of the evaluation of their UV-vis absorption maxima (λmax) and mass spectral analysis. Eleven compounds belonging to the classes of phenolic acids and hydrolyzable tannins (gallotannins and ellagitannins) were tentatively identified. These compounds are responsible for the antioxidant activity and high phenolic content of geopropolis produced by M. fasciculata.

Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands

PubMed Central

Nishimoto, Koshiro; Tomlins, Scott A.; Kuick, Rork; Cani, Andi K.; Giordano, Thomas J.; Hovelson, Daniel H.; Liu, Chia-Jen; Sanjanwala, Aalok R.; Edwards, Michael A.; Gomez-Sanchez, Celso E.; Nanba, Kazutaka; Rainey, William E.

2015-01-01

Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na+/K+ transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA. PMID:26240369

The nucleus parvocellularis reticularis regulates submandibular-sublingual salivary secretion in the rat: a pharmacological study.

PubMed

Ramos, J M; Puerto, A

1988-09-01

This experiment shows that activation of the nucleus parvocellularis reticularis in the rat brainstem provokes salivary hypersecretion by the submandibular-sublingual glands. The secretory effect is mediated by cholinergic mechanisms, as the administration of atropine blocked the flow of saliva evoked by stimulation of the nucleus parvocellularis. In contrast, injection of dihydroergotamine (an alpha-blocker) and/or propranolol (a beta-blocker) failed to significantly reduce submandibular and sublingual salivary secretion when compared to a control group injected with distilled water. The cholinergic nature of the salivary response suggests that the nucleus parvocellularis reticularis exerts its secretory effect on the salivary glands parasympathetically rather than through mechanisms associated with sympathetic pathways. The area of the brainstem activated in the present study closely overlaps the region in which cell bodies of superior salivatory neurons have recently been identified with retrograde transport of peroxidase. The data presented herein represent functional proof in support of the location of the superior salivatory nucleus within the parvocellularis reticular formation.

Cardiorespiratory responses to stimulation of the nucleus reticularis gigantocellularis.

PubMed

Stremel, R W; Waldrop, T G; Richard, C A; Iwamoto, G A

1990-01-01

The nucleus reticularis gigantocellularis (NGC) has been shown to be involved in somatosensory and somatomotor functions. The purpose of the present study was to determine, in anesthetized cats, the modulatory influence of the portion of the NGC at the ponto-medullary border on respiratory and cardiovascular control. Electrical stimulation (25-100 microA 70 Hz, and 1.0-msec pulse duration) significantly depressed mean arterial pressure, heart rate, breathing frequency, tidal volume and phrenic amplitude. Chemical stimulation of NGC cell bodies (1.0 M L-glutamate or 10(-3) M kainic acid) elicited similar decreases in ventilation, arterial pressure and heart rate. These results show that selective activation of cell bodies in the ponto-medullary NGC can depress, in parallel, respiratory and cardiovascular activity and suggests that the influence of diverse sensory information within this region of the reticular formation must be inhibitory to respiratory and cardiovascular output.

Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demura, T.; Driscoll, W.J.; Lee, Y.C.

1991-01-01

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less

Veluticeps fasciculata sp. nov. (Gloeophyllaceae, Basidiomycota), a close relative to V. berkeleyi

Treesearch

Jiao Yang; Karen K. Nakasone; Shuang-Hui He

2016-01-01

A new brown-rot fungal species, Veluticeps fasciculata, is described from southern China. It is characterized by perennial effused basidiocarps with smooth or tuberculose hymenophore, nodose-septate generative hyphae, fasciculate cystidia (as hyphal pegs in hymenophore) and relatively small basidiospores. It occurs on wood of Cunninghamia...

Dermatopathia pigmentosa reticularis: A rare reticulate pigmentary disorder

PubMed Central

Shanker, Vinay; Gupta, Mudita

2013-01-01

Dermatopathia pigmentosa reticularis is a rare ectodermal dysplasia with a triad of generalized reticulate hyperpigmentation, noncicatricial alopecia, and onychodystrophy. We report a case of a 21 year old woman who had generalized reticulate pigmentation, diffuse noncicatricial alopecia and onychodystrophy of finger and toe nails. Along with this triad she had palmoplantar keratoderma and poorly developed dermatoglyphics. There was no evidence of involvement of other ectodermally derived organ. PMID:23440032

Transcriptome Pathway Analysis of Pathological and Physiological Aldosterone-Producing Human Tissues.

PubMed

Zhou, Junhua; Lam, Brian; Neogi, Sudeshna G; Yeo, Giles S H; Azizan, Elena A B; Brown, Morris J

2016-12-01

Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)-mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary

Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

PubMed

Yao, Junlan; McHedlishvili, David; McIntire, William E; Guagliardo, Nick A; Erisir, Alev; Coburn, Craig A; Santarelli, Vincent P; Bayliss, Douglas A; Barrett, Paula Q

2017-08-01

Ca 2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca 2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K + channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K + channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells. © 2017 American Heart Association, Inc.

Age-related effects of dexamethasone administration in adrenal zona reticularis.

PubMed

Almeida, Henrique; Matos, Liliana; Ferreira, Jorge; Neves, Delminda

2006-05-01

Suppression of adrenocorticotropic hormone results in reduced adrenal steroid output, adrenocortical cell atrophy, and apoptosis in young rats. To verify such effects during aging, dexamethasone was injected into rats for 3 days at five different ages; at day 4, adrenals and blood were collected for morphologic and corticosterone assay. Adrenal structure was similar at all ages, but in dexamethasone-injected animals there were ultrastructural features of apoptosis and a higher percentage of TUNEL and caspase-3-labeled nuclei and cytoplasm; their corticosterone decreased significantly. In both groups, there was age-related decrease in the percentage of apoptotic cells, significant only in dexamethasone-injected rats. The data suggest that aged adrenocortical cells are less susceptible to the lack of adrenocorticotropic hormone (ACTH), possibly as a result of their decreased functional ability.

LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal.

PubMed

Shaikh, Lalarukh Haris; Zhou, Junhua; Teo, Ada E D; Garg, Sumedha; Neogi, Sudeshna Guha; Figg, Nichola; Yeo, Giles S; Yu, Haixiang; Maguire, Janet J; Zhao, Wanfeng; Bennett, Martin R; Azizan, Elena A B; Davenport, Anthony P; McKenzie, Grahame; Brown, Morris J

2015-06-01

Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production. The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover. This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7). Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3). A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured. LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers. LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.

Endocardial fibrosarcoma in a reticulated python (Python reticularis).

PubMed

Gumber, Sanjeev; Nevarez, Javier G; Cho, Doo-Youn

2010-11-01

A female, reticulated python (Python reticularis) of unknown age was presented with a history of lethargy, weakness, and distended coelom. Physical examination revealed severe dystocia and stomatitis. The reticulated python was euthanized due to a poor clinical prognosis. Postmortem examination revealed marked distention of the reproductive tract with 26 eggs (10-12 cm in diameter), pericardial effusion, and a slightly firm, pale tan mass (3-4 cm in diameter) adhered to the endocardium at the base of aorta. Based on histopathologic and transmission electron microscopic findings, the diagnosis of endocardial fibrosarcoma was made.

Antimicrobial activity of honey of stingless bees, tiúba (Melipona fasciculata) and jandaira (Melipona subnitida) compared to the strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa

NASA Astrophysics Data System (ADS)

Tenório, Eleuza Gomes; de Jesus, Natália Rocha; Nascimento, Adenilde Ribeiro; Teles, Amanda Mara

2015-12-01

This study aimed to investigate the antimicrobial activity of honeys of stingless bees produced in Maranhão, tiúba (Melipona fasciculata) and jandaira (Melipona subnitida), opposite the strains of pathogenic bacteria, namely, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The honey samples were collected from different regions of Maranhão. Of the 17 samples collected, twelve samples were honey M. fasciculata and five were honey M. subnitida. We used the Kirby-Bauer method, and the technique of agar disk diffusion through the extent of inhibition in milimetros. Results were negative for all samples from M. fasciculata. However, the tests for M. subnitida demonstrated bacteriostatic halos ranging from 12 to 32,6mm.

Comparative Effects of Angiotensin and ACTH on Cyclic AMP and Steroidogenesis in Isolated Bovine Adrenal Cells

PubMed Central

Peytremann, Andre; Nicholson, Wendell E.; Brown, Ronald D.; Liddle, Grant W.; Hardman, Joel G.

1973-01-01

The comparative effects of angiotensin II and adrenocorticotropic hormone (ACTH) on cyclic AMP and steroidogenesis were investigated employing isolated bovine adrenal cells from the zona fasciculata. Like ACTH, angiotensin produced a prompt increase in cyclic AMP which preceded the increase in corticosteroid production. Although this increase in cyclic AMP was small when compared to that induced by ACTH, it correlated with the amount of steroidogenesis. This observation is consistent with the view that cyclic AMP is the intracellular mediator of the steroidogenic action of angiotensin. Angiotensin acted synergistically with ACTH on cyclic AMP levels. This synergism was not explained by inhibition of phosphodiesterase activity. Unlike ACTH, angiotensin failed to stimulate adenylate cyclase in broken cell preparations. The observations suggest that more than one mechanism may be involved in effects of ACTH and angiotensin on cyclic AMP levels. PMID:4348344

Deficiency of ADA2 mimicking autoimmune lymphoproliferative syndrome in the absence of livedo reticularis and vasculitis.

PubMed

Alsultan, Abdulrahman; Basher, Enas; Alqanatish, Jubran; Mohammed, Reem; Alfadhel, Majid

2018-04-01

Adenosine deaminase-2 (ADA2) deficiency (DADA2) is associated with early onset polyarteritis nodosa and vasculopathy. Classic presentation includes livedo reticularis, vasculitis, and stroke. However, the phenotype and disease severity are variable. We present a 5-year-old female who presented with features that mimicked autoimmune lymphoproliferative syndrome (ALPS) in the absence of classic features of DADA2. Exome sequencing identified a novel homozygous splicing variant in ADA2 c.882-2A > G. Patient responded to anti- tumor necrosis factor medication and is in complete remission. Hematologists should be aware of various hematological presentations of DADA2, including ALPS-like disorder, that might lack vasculitis and livedo reticularis to prevent delay in initiating optimal therapy. © 2017 Wiley Periodicals, Inc.

Electrophysiological study of supraspinal input and spinal output of cat's subnucleus reticularis dorsalis (SRD) neurons.

PubMed

Velo, Patricia; Leiras, Roberto; Canedo, Antonio

2013-01-01

This work addressed the study of subnucleus reticularis dorsalis (SRD) neurons in relation to their supraspinal input and the spinal terminating sites of their descending axons. SRD extracellular unitary recordings from anesthetized cats aimed to specifically test, 1) the rostrocaudal segmental level reached by axons of spinally projecting (SPr) neurons collateralizing or not to or through the ipsilateral nucleus reticularis gigantocellularis (NRGc), 2) whether SPr fibers bifurcate to the thalamus, and 3) the effects exerted on SRD cells by electrically stimulating the locus coeruleus, the periaqueductal grey, the nucleus raphe magnus, and the mesencephalic locomotor region. From a total of 191 SPr fibers tested to cervical 2 (Ce2), thoracic 5 (Th5) and lumbar5 (Lu5) stimulation, 81 ended between Ce2 and Th5 with 39 of them branching to or through the NRGc; 21/49 terminating between Th5 and Lu5 collateralized to or through the same nucleus, as did 34/61 reaching Lu5. The mean antidromic conduction velocity of SPr fibers slowed in the more proximal segments and increased with terminating distance along the cord. None of the 110 axons tested sent collaterals to the thalamus; instead thalamic stimulation induced long-latency polysynaptic responses in most cells but also short-latency, presumed monosynaptic, in 7.9% of the tested neurons (18/227). Antidromic and orthodromic spikes were elicited from the locus coeruleus and nucleus raphe magnus, but exclusively orthodromic responses were observed following stimulation of the periaqueductal gray or mesencephalic locomotor region. The results suggest that information from pain-and-motor-related supraspinal structures converge on SRD cells that through SPr axons having conduction velocities tuned to their length may affect rostral and caudal spinal cord neurons at fixed delays, both directly and in parallel through different descending systems. The SRD will thus play a dual functional role by simultaneously regulating

Electrophysiological Study of Supraspinal Input and Spinal Output of Cat's Subnucleus Reticularis Dorsalis (SRD) Neurons

PubMed Central

Canedo, Antonio

2013-01-01

This work addressed the study of subnucleus reticularis dorsalis (SRD) neurons in relation to their supraspinal input and the spinal terminating sites of their descending axons. SRD extracellular unitary recordings from anesthetized cats aimed to specifically test, 1) the rostrocaudal segmental level reached by axons of spinally projecting (SPr) neurons collateralizing or not to or through the ipsilateral nucleus reticularis gigantocellularis (NRGc), 2) whether SPr fibers bifurcate to the thalamus, and 3) the effects exerted on SRD cells by electrically stimulating the locus coeruleus, the periaqueductal grey, the nucleus raphe magnus, and the mesencephalic locomotor region. From a total of 191 SPr fibers tested to cervical 2 (Ce2), thoracic 5 (Th5) and lumbar5 (Lu5) stimulation, 81 ended between Ce2 and Th5 with 39 of them branching to or through the NRGc; 21/49 terminating between Th5 and Lu5 collateralized to or through the same nucleus, as did 34/61 reaching Lu5. The mean antidromic conduction velocity of SPr fibers slowed in the more proximal segments and increased with terminating distance along the cord. None of the 110 axons tested sent collaterals to the thalamus; instead thalamic stimulation induced long-latency polysynaptic responses in most cells but also short-latency, presumed monosynaptic, in 7.9% of the tested neurons (18/227). Antidromic and orthodromic spikes were elicited from the locus coeruleus and nucleus raphe magnus, but exclusively orthodromic responses were observed following stimulation of the periaqueductal gray or mesencephalic locomotor region. The results suggest that information from pain-and-motor-related supraspinal structures converge on SRD cells that through SPr axons having conduction velocities tuned to their length may affect rostral and caudal spinal cord neurons at fixed delays, both directly and in parallel through different descending systems. The SRD will thus play a dual functional role by simultaneously regulating

Electrophysiological characterization of synaptic connections between layer VI cortical cells and neurons of the nucleus reticularis thalami in juvenile rats.

PubMed

Gentet, Luc J; Ulrich, Daniel

2004-02-01

Corticothalamic (CT) feedback projections to the thalamus outnumber sensory inputs from the periphery by orders of magnitude. However, their functional role remains elusive. CT projections may directly excite thalamic relay cells or indirectly inhibit them via excitation of the nucleus reticularis thalami (nRT), a nuclear formation composed entirely of gamma-aminobutyric acidergic neurons. The relative strengths of these two pathways will ultimately control the effects of CT projections on the output of thalamic relay cells. However, corticoreticular synapses have not yet been fully physiologically characterized. Here, local stimulation of layer VI cells by focal application of K+ or AMPA elicited excitatory postsynaptic potentials in nRT neurons with a mean peak amplitude of 2.4 +/- 0.1 mV (n = 75, mean +/- SEM), a mean rise time (10-90%) of 0.74 +/- 0.03 ms and a weighted decay time constant of 11 +/- 0.3 ms. A pharmacological profile of responses was drawn in both current-clamp and voltage-clamp modes, showing the presence of a small N-methyl-d-aspartate receptor-dependent component at depolarized potentials. In two pairs of synaptically coupled layer VI cell-nRT neuron, moderate rates of transmission failures were observed while the latencies were above 5 ms in both cases. Our results indicate that the corticoreticular pathway fulfills the criteria for 'modulatory' inputs and is temporally restricted. We suggest that it may be involved in coincidence detection of convergent corticoreticular signals.

Screening of the MMV and GSK open access chemical boxes using a viability assay developed against the kinetoplastid Crithidia fasciculata.

PubMed

Kipandula, Wakisa; Young, Simon A; MacNeill, Stuart A; Smith, Terry K

2018-06-01

Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes. The screening assay had an average Z' factor of 0.7 and tolerated a maximum dimethyl sulfoxide concentration of up to 0.5%. We identified from multiple chemical boxes two compound series exhibiting nanomolar potency against C. fasciculata, one centred around a 5-nitrofuran-2-yl scaffold, a well-known moiety in several existing anti-infectives, and another involving a 2-(pyridin-2-yl) pyrimidin-4-amine scaffold which seems to have pan-kinetoplastid activity. This work facilitates the future use of C. fasciculata as a non-pathogenic and inexpensive biological resource to identify mode of action/protein target(s) of potentially pan-trypanocidal potent compounds. This knowledge will aid in the development of new treatments for African sleeping sickness, Chagas disease and leishmaniasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Intracellular recordings of subnucleus reticularis dorsalis neurones revealed novel electrophysiological properties and windup mechanisms

PubMed Central

Soto, Cristina; Canedo, Antonio

2011-01-01

Abstract Aδ- and/or C-fibre nociceptive inputs drive subnucleus reticularis dorsalis (SRD) neurones projecting to a variety of regions including the spinal cord and the nucleus reticularis gigantocellularis (NRGc), but their electrophysiological properties are largely unknown. Here we intracellularly recorded the SRD neuronal responses to injection of polarising current pulses as well as to electrical stimulation of the cervical spinal posterior quadrant (PQ) and the NRGc. Three different classes of neurones with distinct electrophysiological properties were found: type I were characterised by the absence of a fast postspike hyperpolarisation, type II by the presence of a postspike hyperpolarisation followed by a depolarisation resembling low threshold calcium spikes (LTSs), and type III (lacking LTSs) had a fast postspike hyperpolarisation deinactivating A-like potassium channels leading to enlarged interspike intervals. All three classes generated depolarising sags to hyperpolarising current pulses and showed 3–4.5 Hz subthreshold oscillatory activity leading to windup when intracellularly injecting low-frequency repetitive depolarising pulses as well as in response to 0.5–2 Hz NRGc and PQ electrical stimulation. About half of the 132 sampled neurones responded antidromically to NRGc stimulation with more than 65% of the NRGc-antidromic cells, pertaining to all three types, also responding antidromically to PQ stimulation. NRGc stimulation induced exclusively excitatory first-synaptic-responses whilst PQ stimulation induced first-response excitation in most cases, but inhibitory postsynaptic potentials in a few type II and type III neurones not projecting to the spinal cord that also displayed cumulative inhibitory effects (inverse windup). The results show that SRD cells (i) can actively regulate different temporal firing patterns due to their intrinsic electrophysiological properties, (ii) generate windup upon gradual membrane depolarisation produced by

Intracellular recordings of subnucleus reticularis dorsalis neurones revealed novel electrophysiological properties and windup mechanisms.

PubMed

Soto, Cristina; Canedo, Antonio

2011-09-01

Aδ- and/or C-fibre nociceptive inputs drive subnucleus reticularis dorsalis (SRD) neurones projecting to a variety of regions including the spinal cord and the nucleus reticularis gigantocellularis (NRGc), but their electrophysiological properties are largely unknown. Here we intracellularly recorded the SRD neuronal responses to injection of polarising current pulses as well as to electrical stimulation of the cervical spinal posterior quadrant (PQ) and the NRGc. Three different classes of neurones with distinct electrophysiological properties were found: type I were characterised by the absence of a fast postspike hyperpolarisation, type II by the presence of a postspike hyperpolarisation followed by a depolarisation resembling low threshold calcium spikes (LTSs), and type III (lacking LTSs) had a fast postspike hyperpolarisation deinactivating A-like potassium channels leading to enlarged interspike intervals. All three classes generated depolarising sags to hyperpolarising current pulses and showed 3-4.5 Hz subthreshold oscillatory activity leading to windup when intracellularly injecting low-frequency repetitive depolarising pulses as well as in response to 0.5-2 Hz NRGc and PQ electrical stimulation. About half of the 132 sampled neurones responded antidromically to NRGc stimulation with more than 65% of the NRGc-antidromic cells, pertaining to all three types, also responding antidromically to PQ stimulation. NRGc stimulation induced exclusively excitatory first-synaptic-responses whilst PQ stimulation induced first-response excitation in most cases, but inhibitory postsynaptic potentials in a few type II and type III neurones not projecting to the spinal cord that also displayed cumulative inhibitory effects (inverse windup). The results show that SRD cells (i) can actively regulate different temporal firing patterns due to their intrinsic electrophysiological properties, (ii) generate windup upon gradual membrane depolarisation produced by low

Development of the precerebellar nuclei in the rat: IV. The anterior precerebellar extramural migratory stream and the nucleus reticularis tegmenti pontis and the basal pontine gray.

PubMed

Altman, J; Bayer, S A

1987-03-22

Sequential thymidine radiograms from rats injected on days E16, E17, E18, and E19 and killed 2 hours after injection and at daily intervals up to day E22 were used to establish the site of origin, migratory route, and settling patterns of neurons of the nucleus reticularis tegmenti pontis and basal pontine gray. The nucleus reticularis tegmenti pontis neurons, which are produced predominantly on days E15 and E16, derive from the primary precerebellar neuroepithelium. These cells, unlike those of the lateral reticular and external cuneate nuclei, take an anteroventral subpial route, forming the anterior precerebellar extramural migratory stream. This migratory stream reaches the anterior pole of the pons by day E18. In rats injected on day E16 and killed on day E18 some of the cells that reach the pons are unlabeled, indicating that they represent the early component of neurons generated on day E15. The cells labeled on day E16 begin to settle in the pons on day E19, 3 days after their production. These cells, migrating in an orderly temporal sequence, form a posterodorsal-to-anteroventral gradient in the nucleus reticularis tegmenti pontis. Unlike the neurons of all the other precerebellar nuclei, the basal pontine gray neurons derive from the secondary precerebellar neuroepithelium. The secondary precerebellar neuroepithelium forms on day E16 as an outgrowth of the primary precerebellar neuroepithelium, and it remains mitotically active through day E19, spanning the entire period of basal pontine gray neurogenesis. The secondary precerebellar neuroepithelium is surrounded by a horizontal layer of postmitotic cells, representing the head-waters of the anterior precerebellar extramural migratory stream. In rats injected on day E18 and killed on day E19 the cells are labeled in the proximal half of the stream around the medulla but those closer to the pons are unlabeled, indicating an orderly sequence of migration. In rats injected on day E18 and killed on day E20

Detrimental effects of rhizobial inoculum early in the life of partridge pea, Chamaecrista fasciculata.

PubMed

Pain, Rachel E; Shaw, Ruth G; Sheth, Seema N

2018-05-16

Mutualistic relationships with microbes may aid plants in overcoming environmental stressors and increase the range of abiotic environments where plants can persist. Rhizobia, nitrogen-fixing bacteria associated with legumes, often confer fitness benefits to their host plants by increasing access to nitrogen in nitrogen-limited soils, but effects of rhizobia on host fitness under other stresses, such as drought, remain unclear. In this greenhouse study, we varied the application of rhizobia (Bradyrhizobium sp.) inoculum and drought to examine whether the fitness benefits of rhizobia to their host, partridge pea (Chamaecrista fasciculata), would differ between drought and well-watered conditions. Plants were harvested 9 weeks after seeds were sown. Young C. fasciculata plants that had been inoculated had lower biomass, leaf relative growth rate, and stem relative growth rate compared to young uninoculated plants in both drought and well-watered environments. Under the conditions of this study, the rhizobial interaction imposed a net cost to their hosts early in development. Potential reasons for this cost include allocating more carbon to nodule and root development than to aboveground growth and a geographic mismatch between the source populations of host plants and rhizobia. If developing plants incur such costs from rhizobia in nature, they may suffer an early disadvantage relative to other plants, whether conspecifics lacking rhizobia or heterospecifics. © 2018 Botanical Society of America.

Surface alterations of the mouse zona pellucida and ovum following in vivo fertilization: correlation with the cell cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackowski, S.; Dumont, J.N.

1979-01-01

The zona pellucida and cell surface of in vivo fertilized mouse ova exhibit time dependent changes which can be detected with the scanning electron microscope. The periods of ovulation, fertilization and first cleavage in superovulated C3D2/F/sub 1/ hybrids were determined and times corresponding to G/sub 1/, S, G/sub 2/, and M were calculated. The zona of a mature unfertilized ovum has a rough texture with deep furrows; at fertilization and thereafter the zona develops a smoother, ropy and seemingly porous surface. The cell surface of the unfertilized ovum is characterized by uniform microvilli, small blebs and rounded, mound-like elevations. Aftermore » fertilization and development to G/sub 1/, the ovum loses its blebs but retains the mound-like elevations and microvilli which are now less uniform. As the ovum progresses toward S, it loses the mound-like elevations but retains microvilli in the same density as found in G/sub 1/. The ovum in G/sub 2/ exhibits smaller but more numerous microvilli which vary considerably in length. Some appear to bifurcate. The fertilized ovum developing through M and G/sub 1/ of the 2 cell stage exhibits a less dense population of relatively uniform microvilli, periodic blebs and, again, rounded elevations. The data are reminiscent of surface changes associated with the cell cycle in tissue culture cells and indicate a cyclic progression of the in vivo fertilized mouse ovum through the first cleavage division to the 2 cell stage.« less

Fetal programming of adrenal androgen excess: lessons from a nonhuman primate model of polycystic ovary syndrome.

PubMed

Abbott, David H; Zhou, Rao; Bird, Ian M; Dumesic, Daniel A; Conley, Alan J

2008-01-01

Adrenal androgen excess is found in adult female rhesus monkeys previously exposed to androgen treatment during early gestation. In adulthood, such prenatally androgenized female monkeys exhibit elevated basal circulating levels of dehydroepiandrosterone sulfate (DHEAS), typical of polycystic ovary syndrome (PCOS) women with adrenal androgen excess. Further androgen and glucocorticoid abnormalities in PA female monkeys are revealed by acute ACTH stimulation: DHEA, androstenedione and corticosterone responses are all elevated compared to responses in controls. Pioglitazone treatment, however, diminishes circulating DHEAS responses to ACTH in both prenatally androgenized and control female monkeys, while increasing the 17-hydroxyprogesterone response and reducing the DHEA to 17-hydroxyprogesterone ratio. Since 60-min post-ACTH serum values for 17-hydroxyprogesterone correlate negatively with basal serum insulin levels (all female monkeys on pioglitazone and placebo treatment combined), while similar DHEAS values correlate positively with basal serum insulin levels, circulating insulin levels may preferentially support adrenal androgen biosynthesis in both prenatally androgenized and control female rhesus monkeys. Overall, our findings suggest that differentiation of the monkey adrenal cortex in a hyperandrogenic fetal environment may permanently upregulate adult adrenal androgen biosynthesis through specific elevation of 17,20-lyase activity in the zona fasciculata-reticularis. As adult prenatally androgenized female rhesus monkeys closely emulate PCOS-like symptoms, excess fetal androgen programming may contribute to adult adrenal androgen excess in women with PCOS.

Fetal programming of adrenal androgen excess: lessons from a nonhuman primate model of polycystic ovary syndrome

PubMed Central

Abbott, David H; Zhou, Rao; Bird, Ian M; Dumesic, Daniel A; Conley, Alan J

2008-01-01

Adrenal androgen excess is found in adult female rhesus monkeys previously exposed to androgen treatment during early gestation. In adulthood, such prenatally androgenized female monkeys exhibit elevated basal circulating levels of DHEAS, typical of PCOS women with adrenal androgen excess. Further androgen and glucocorticoid abnormalities in PA female monkeys are revealed by acute ACTH stimulation: DHEA, androstenedione and corticosterone responses are all elevated compared to responses in controls. Pioglitazone treatment, however, diminishes circulating DHEAS responses to ACTH in both prenatally androgenized and control female monkeys, while increasing the 17-hydroxyprogesterone response and reducing the DHEA to 17-hydroxyprogesterone ratio. Since 60-min post-ACTH serum values for 17-hydroxyprogesterone correlate negatively with basal serum insulin levels (all female monkeys on pioglitazone and placebo treatment combined), while similar DHEAS values correlate positively with basal serum insulin levels, circulating insulin levels may preferentially support adrenal androgen biosynthesis in both prenatally androgenized and control female rhesus monkeys. Overall, our findings suggest that differentiation of the monkey adrenal cortex in a hyperandrogenic fetal environment may permanently upregulate adult adrenal androgen biosynthesis through specific elevation of 17,20-lyase activity in the zona fasciculata-reticularis. As adult prenatally androgenized female rhesus monkeys closely emulate PCOS-like symptoms, excess fetal androgen programming may contribute to adult adrenal androgen excess in women with PCOS. PMID:18493139

The 28S–18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor

PubMed Central

Schnare, Murray N.; Collings, James C.; Spencer, David F.; Gray, Michael W.

2000-01-01

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from ∼11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an ∼55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A′ pre-rRNA processing sites within the 5′ external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5′ ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C.fasciculata and Trypanosoma brucei involves 3′-terminal addition of three A residues that are not present in the corresponding DNA sequences. PMID:10982863

Gas chromatography-mass spectrometry study of the essential oils of Schinus longifolia (Lindl.) speg., Schinus fasciculata (Griseb.) I. M. Johnst., and Schinus areira L.

PubMed

Murray, Ana P; Frontera, María A; Tomas, María A; Mulet, María C

2005-01-01

The essential oil composition from the aerial parts of three Anacardiaceae growing in Bahía Blanca, Argentina was studied by gas chromatography and gas chromatography-mass spectrometry. The essential oils of S. longifolia and S. fasciculata have been studied for the first time. The major constituents were alpha-pinene (46.5%), beta-pinene (15.1%) and alpha-phellandrene (10.1%) for S. longifolia and limonene (10.9%), beta-phellandrene (6.16%) and alpha-phellandrene (5.6%) for S. fasciculata. The major components of the essential oil of S. areira were limonene (28.6%), alpha-phellandrene (10.1%), sabinene (9.2%) and camphene (9.2%) differing from the literature data. The essential oils from S. areira and S. longifolia exhibited a high biotoxicity in a brine shrimp assay with Artemia persimilis.

[Effects of infrasound on activities of 3beta hydroxysteroid dehydrogenase and acid phosphatase of polygonal cells in adrenal cortex zona fasciculate in mice].

PubMed

Dang, Wei-min; Wang, Sheng; Tian, Shi-xiu; Chen, Bing; Sun, Fei; Li, Wei; Jiao, Yan; He, Li-hua

2007-02-01

To explore the biological effects of infrasound on the polygonal cells in adrenal cortex zona fasciculation in mice. The biological effects of infrasound on the activities of 3beta hydroxysteroid dehydrogenase (3-betaHSDH) and acid phosphatase(ACP) of the polygonal cells in adrenal cortex zona fasciculate were observed when exposure to 8 and 16 Hz infrasound at 80, 90, 100, 110, 120 and 130 dB for 1 day, 7 days and 14 days or 14 days after the exposure. When exposure to 8 Hz infrasound, the enzyme activities of 3-betaHSDH increase as the sound pressure levels increase. Only when the sound pressure levels reach 130 dB, the enzyme activities began to decrease exceptionally. When exposure to 16 Hz, 80 dB infrasound, no significant difference between the treatment and control group in the activities of 3-betaHSDH could be observed, but the injury of the polygonal cells had appeared. When exposure to 16 Hz, 100 dB infrasound, the activities of 3-betaHSDH started to increase. The cell injury still existed. When exposed to 16 Hz, 120 dB infrasound, the local tissue damage represented. Fourteen days after the mice exposure to 8 Hz, 90 dB and 130 dB infrasound for 14 days continuously, the local tissue injury of the adrenal cortex zona fasciculation began to recover at certain extent, but the higher the exposure sound pressure level, the poorer the tissue recovery. The biological effects of infrasound on the polygonal cells in adrenal cortex zona fasciculation response to the frequency of the infrasound are found at certain action strength range, but this characteristic usually is covered by the severe tissue injury. When exposure to infrasound is stopped for a period of time, the local tissue injury of the adrenal cortex zona fasciculation could recovers at certain extent, but the higher the exposure sound pressure level, the more poorer the tissue recovery.

Gaze pursuit responses in nucleus reticularis tegmenti pontis of head-unrestrained macaques.

PubMed

Suzuki, David A; Betelak, Kathleen F; Yee, Robert D

2009-01-01

Eye-head gaze pursuit-related activity was recorded in rostral portions of the nucleus reticularis tegmenti pontis (rNRTP) in alert macaques. The head was unrestrained in the horizontal plane, and macaques were trained to pursue a moving target either with their head, with the eyes stationary in the orbits, or with their eyes, with their head voluntarily held stationary in space. Head-pursuit-related modulations in rNRTP activity were observed with some cells exhibiting increases in firing rate with increases in head-pursuit frequency. For many units, this head-pursuit response appeared to saturate at higher frequencies (>0.6 Hz). The response phase re:peak head-pursuit velocity formed a continuum, containing cells that could encode head-pursuit velocity and those encoding head-pursuit acceleration. The latter cells did not exhibit head position-related activity. Sensitivities were calculated with respect to peak head-pursuit velocity and averaged 1.8 spikes/s/deg/s. Of the cells that were tested for both head- and eye-pursuit-related activity, 86% exhibited responses to both head- and eye-pursuit and therefore carried a putative gaze-pursuit signal. For these gaze-pursuit units, the ratio of head to eye response sensitivities averaged approximately 1.4. Pursuit eccentricity seemed to affect head-pursuit response amplitude even in the absence of a head position response per se. The results indicated that rNRTP is a strong candidate for the source of an active head-pursuit signal that projects to the cerebellum, specifically to the target-velocity and gaze-velocity Purkinje cells that have been observed in vermal lobules VI and VII.

Gaze Pursuit Responses in Nucleus Reticularis Tegmenti Pontis of Head-Unrestrained Macaques

PubMed Central

Suzuki, David A.; Betelak, Kathleen F.; Yee, Robert D.

2009-01-01

Eye-head gaze pursuit–related activity was recorded in rostral portions of the nucleus reticularis tegmenti pontis (rNRTP) in alert macaques. The head was unrestrained in the horizontal plane, and macaques were trained to pursue a moving target either with their head, with the eyes stationary in the orbits, or with their eyes, with their head voluntarily held stationary in space. Head-pursuit–related modulations in rNRTP activity were observed with some cells exhibiting increases in firing rate with increases in head-pursuit frequency. For many units, this head-pursuit response appeared to saturate at higher frequencies (>0.6 Hz). The response phase re:peak head-pursuit velocity formed a continuum, containing cells that could encode head-pursuit velocity and those encoding head-pursuit acceleration. The latter cells did not exhibit head position–related activity. Sensitivities were calculated with respect to peak head-pursuit velocity and averaged 1.8 spikes/s/deg/s. Of the cells that were tested for both head- and eye-pursuit–related activity, 86% exhibited responses to both head- and eye-pursuit and therefore carried a putative gaze-pursuit signal. For these gaze-pursuit units, the ratio of head to eye response sensitivities averaged ∼1.4. Pursuit eccentricity seemed to affect head-pursuit response amplitude even in the absence of a head position response per se. The results indicated that rNRTP is a strong candidate for the source of an active head-pursuit signal that projects to the cerebellum, specifically to the target-velocity and gaze-velocity Purkinje cells that have been observed in vermal lobules VI and VII. PMID:18987125

Inhibition of NMDARs in the Nucleus Reticularis of the Thalamus Produces Delta Frequency Bursting.

PubMed

Zhang, Yuchun; Llinas, Rodolfo R; Lisman, John E

2009-01-01

Injection of NMDAR antagonist into the thalamus can produce delta frequency EEG oscillations in the thalamocortical system. It is surprising that an antagonist of an excitatory neurotransmitter should trigger such activity, and the mechanism is unknown. One hypothesis is that the antagonist blocks excitation of GABAergic cells, thus producing disinhibition. To test this hypothesis, we investigated the effect of NMDAR antagonist (APV) on cells of the nucleus reticularis (nRT) in rat brain slices, a thalamic nucleus that can serve as a pacemaker for thalamocortical delta oscillations and that is composed entirely of GABAergic neurons. We found, unexpectedly, that nRT cells are hyperpolarized by APV. This occurs because these cells have an unusual form of NMDAR (probably NR2C) that contributes inward current at resting potential in response to ambient glutamate. The hyperpolarization produced by APV is sufficient to deinactivate T-type calcium channels, and these trigger rhythmic bursting at delta frequency. The APV-induced delta frequency bursting is abolished by dopamine D2 receptor antagonist, indicating that dopamine and NMDAR antagonist work synergistically to stimulate delta frequency bursting. Our results have significant implications concerning the electrophysiological basis of schizophrenia and bring together the NMDAR hypofunction, dopamine, and GABA theories of the disease. Our results suggest that NMDAR hypofunction and dopamine work synergistically on the GABAergic cells of the nRT to generate the delta frequency EEG oscillations, a thalamocortical dysrhythmia (TCD) in the awake state that is an established abnormality in schizophrenia.

Inhibition of NMDARs in the Nucleus Reticularis of the Thalamus Produces Delta Frequency Bursting

PubMed Central

Zhang, Yuchun; Llinas, Rodolfo R.; Lisman, John E.

2009-01-01

Injection of NMDAR antagonist into the thalamus can produce delta frequency EEG oscillations in the thalamocortical system. It is surprising that an antagonist of an excitatory neurotransmitter should trigger such activity, and the mechanism is unknown. One hypothesis is that the antagonist blocks excitation of GABAergic cells, thus producing disinhibition. To test this hypothesis, we investigated the effect of NMDAR antagonist (APV) on cells of the nucleus reticularis (nRT) in rat brain slices, a thalamic nucleus that can serve as a pacemaker for thalamocortical delta oscillations and that is composed entirely of GABAergic neurons. We found, unexpectedly, that nRT cells are hyperpolarized by APV. This occurs because these cells have an unusual form of NMDAR (probably NR2C) that contributes inward current at resting potential in response to ambient glutamate. The hyperpolarization produced by APV is sufficient to deinactivate T-type calcium channels, and these trigger rhythmic bursting at delta frequency. The APV-induced delta frequency bursting is abolished by dopamine D2 receptor antagonist, indicating that dopamine and NMDAR antagonist work synergistically to stimulate delta frequency bursting. Our results have significant implications concerning the electrophysiological basis of schizophrenia and bring together the NMDAR hypofunction, dopamine, and GABA theories of the disease. Our results suggest that NMDAR hypofunction and dopamine work synergistically on the GABAergic cells of the nRT to generate the delta frequency EEG oscillations, a thalamocortical dysrhythmia (TCD) in the awake state that is an established abnormality in schizophrenia. PMID:20057928

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

PubMed Central

Jin, Mayuko; Fujiwara, Eiji; Kakiuchi, Yasutaka; Okabe, Masaru; Satouh, Yuhkoh; Baba, Shoji A.; Chiba, Kazuyoshi; Hirohashi, Noritaka

2011-01-01

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization. PMID:21383182

GABAergic regulation of REM sleep in reticularis pontis oralis and caudalis in rats.

PubMed

Sanford, Larry D; Tang, Xiangdong; Xiao, Jihua; Ross, Richard J; Morrison, Adrian R

2003-08-01

The nucleus reticularis pontis oralis (RPO) and nucleus reticularis pontis caudalis (RPC) are implicated in the generation of rapid eye movement sleep (REM). Work in cats has indicated that GABA in RPO plays a role in the regulation of REM. We assessed REM after local microinjections into RPO and RPC of the gamma-aminobutyric acid-A (GABA(A)) agonist, muscimol (MUS), and the GABA(A) antagonist, bicuculline (BIC). Rats (90-day-old male Sprague-Dawley) were implanted with electrodes for recording electroencephalographs (EEG) and electromyographs (EMG). Guide cannulae were aimed into RPO (n = 9) and RPC (n = 8) for microinjecting MUS (200, 1,000.0 microM) and BIC (0.056, 0.333, 1.0, 1,000.0, and 10,000.0 microM). Animals received bilateral microinjections of saline, MUS, and BIC (0.2 microl microinjected at 0.1 microl/min) into each region followed by 6-h sleep recordings. In RPO, MUS (1,000.0 microM) suppressed REM and BIC (1,000.0 microM) enhanced REM. In RPC, MUS (200, 1,000.0 microM) suppressed REM, but BIC (1,000.0 microM and less) did not significantly affect REM. Higher concentrations of BIC (10,000.0 microM) injected into RPO (n = 9) and RPC (n = 4) produced wakefulness and escape behavior. The results indicate that GABA in RPO/RPC is involved in the regulation of REM and suggest site-specific differences in this regulation.

Zona-free oocyte fertilized with intracytoplasmic sperm injection and underwent further division: case report and literature review.

PubMed

Hsieh, Y Y; Chang, C C; Tsai, H D

2001-09-01

The zona pellucida (ZP) plays a protective role during fertilization and early embryonic development. It is related to sperm binding, the acrosome reaction, prevention of polyspermic fertilization, and holding blastomeres together before the morular stage. Zona-free oocytes are accidentally encountered. If these oocytes are healthy, they can be fertilized normally by intracytoplasmic sperm injection (ICSI). We reported on a couple with male infertility undergoing oocyte retrieval after ovarian hyperstimulation. Before the ICSI procedure, cumulus cells surrounding the oocytes were removed, which resulted in one oocyte escaping from its ZP. The zona-free oocyte was fertilized normally with ICSI and developed to the 8-cell stage. We observed that the zona-free zygote had the ability to further divide, despite its loose contact. The zona-free embryo was transferred with other zona-intact embryos, but the implantation failed. We conclude that zona-free oocytes can be rescued, fertilized with ICSI, and cultured for further transfer or cryopreservation.

GROWTH AND DIFFERENTIATION OF MITOCHONDRIA IN THE REGENERATING RAT ADRENAL CORTEX

PubMed Central

Yago, Nagasumi; Seki, Masatoshi; Sekiyama, Shigetaka; Kobayashi, Shigeru; Kurokawa, Hiromi; Iwai, Yuko; Sato, Fumiaki; Shiragai, Akihiro

1972-01-01

Diameters of the circular profiles of spherical mitochondria in parenchymal cells of the zona fasciculata in rat adrenal cortex were measured for intact controls and for the regenerating adrenal cortex on electron micrographs recorded at random. The diameter data were then processed by Bach's method which deals with the sphere size distribution. The structural parameters of the mitochondria were computed with the aid of an electronic computer. The total number of mitochondria in all the parenchymal cells of the zona fasciculata were calculated. The surface area of the inner mitochondrial membrane was then determined stereologically. Biochemical parameters were obtained for the protein, the phospholipid, and the cytochrome P-450 content, per averaged mitochondrion. The number of cytochrome P-450 molecules contained in the inner membrane was determined in terms of the unit surface area and of the unit amount of phospholipid. These correlated biochemical and stereological parameters have led to the following conclusions. (a) The genesis of the mitochondria after the adrenal enucleation is almost completed within 10 days. (b) During the period of mitochondrial proliferation, the mitochondria are small in size and also immature both in the structure and in the function of their inner membrane, (c) These small and immature mitochondria grow through an increase of the phospholipid and protein, and this increase is accompanied by expansion of the area of the membrane surface, (d) An enrichment of the inner membrane with cytochrome P-450 molecules occurs, thus indicating the differentiation of adrenocortical mitochondria. The process of membrane differentiation is not tightly coupled with that of membrane growth. PMID:5009515

Axonal trajectories of the nucleus reticularis gigantocellularis neurons in the C2-C3 segments in cats.

PubMed

Sasaki, S; Iwamoto, Y

1999-04-02

Axonal trajectories in the C2-C3 segments of the nucleus reticularis gigantocellularis neurons projecting to the lower cervical cord (C-RSNs) and excited monosynaptically from cortico- and tectofugal fibers were studied by mapping thresholds of antidromic excitation and intra-axonal staining in cats. The C-RSNs descended in various sites of the spinal funiculi, and the projection area of individual cells varied with the funicular location of the stem axon. C-RSNs descending in the ventrolateral funiculus (inRSNs) projected mainly to lamina VIII-IX, those descending in the lateral funiculus (IRSNs) mainly to laminae VI-VIII, and those descending in the contralateral funiculus (coRSNs) chiefly to laminae VIII-IX on that side. It is suggested that inRSNs and coRSNs mediate disynaptic effects from cortico- and tectofugal fibers to dorsal neck motoneurons bilaterally.

Smooth-pursuit eye-movement-related neuronal activity in macaque nucleus reticularis tegmenti pontis.

PubMed

Suzuki, David A; Yamada, Tetsuto; Yee, Robert D

2003-04-01

Neuronal responses that were observed during smooth-pursuit eye movements were recorded from cells in rostral portions of the nucleus reticularis tegmenti pontis (rNRTP). The responses were categorized as smooth-pursuit eye velocity (78%) or eye acceleration (22%). A separate population of rNRTP cells encoded static eye position. The sensitivity to pursuit eye velocity averaged 0.81 spikes/s per degrees /s, whereas the average sensitivity to pursuit eye acceleration was 0.20 spikes/s per degrees /s(2). Of the eye-velocity cells with horizontal preferences for pursuit responses, 56% were optimally responsive to contraversive smooth-pursuit eye movements and 44% preferred ipsiversive pursuit. For cells with vertical pursuit preferences, 61% preferred upward pursuit and 39% preferred downward pursuit. The direction selectivity was broad with 50% of the maximal response amplitude observed for directions of smooth pursuit up to +/-85 degrees away from the optimal direction. The activities of some rNRTP cells were linearly related to eye position with an average sensitivity of 2.1 spikes/s per deg. In some cells, the magnitude of the response during smooth-pursuit eye movements was affected by the position of the eyes even though these cells did not encode eye position. On average, pursuit centered to one side of screen center elicited a response that was 73% of the response amplitude obtained with tracking centered at screen center. For pursuit centered on the opposite side, the average response was 127% of the response obtained at screen center. The results provide a neuronal rationale for the slow, pursuit-like eye movements evoked with rNRTP microstimulation and for the deficits in smooth-pursuit eye movements observed with ibotenic acid injection into rNRTP. More globally, the results support the notion of a frontal and supplementary eye field-rNRTP-cerebellum pathway involved with controlling smooth-pursuit eye movements.

The oculomotor role of the pontine nuclei and the nucleus reticularis tegmenti pontis.

PubMed

Thier, Peter; Möck, Martin

2006-01-01

Cerebral cortex and the cerebellum interact closely in order to facilitate spatial orientation and the generation of motor behavior, including eye movements. This interaction is based on a massive projection system that allows the exchange of signals between the two cortices. This cerebro-cerebellar communication system includes several intercalated brain stem nuclei, whose eminent role in the organization of oculomotor behavior has only recently become apparent. This review focuses on the two major nuclei of this group taking a precerebellar position, the pontine nuclei and the nucleus reticularis tegmenti pontis, both intimately involved in the visual guidance of eye movements.

The formation of zona radiata in Pseudosciaena crocea revealed by light and transmission electron microscopy.

PubMed

Ma, Xiao-Xin; Zhu, Jun-Quan; Zhou, Hong; Yang, Wan-Xi

2012-02-01

The egg envelope is an essential structure occurring during oogenesis. It plays an important role during the process of fertilization in the large yellow croaker Pseudosciaena crocea. Elucidation of egg envelope formation helps us to understand fertilization mechanisms in teleosts. In the present work, we studied the formation of egg envelope in P. crocea by light microscopy, as well as by transmission and scanning electron microscopy. Four layers exist outside the oocyte plasmalemma, i.e., theca cell layer, basal membrane, granulosa cell layer and zona radiata. According to our observation, zona radiata is a multilaminar structure just like the same structure reported in teleosts, but the origin of this structure is a little different. Before it is formed, a peripheral space filled with different density of vesicles is the place where zona radiata is formed. Zona radiata (Z1) is secreted only by oocyte itself, it belongs to the primary envelope; zona radiata 2 (Z2) and zona radiata 3 (Z3) belong to the secondary envelope, because the two layers are formed after granulosa cells appear, and microvilli participate this process. It is very interesting that Z2 and Z3 are situated between Z1 and the granulosa cell first, but they translocate to the other side of Z1. This microanatomy difference may due to the participation of microvilli. The new finding about egg envelope formation in P. crocea will help us to do further investigation on fertilization mechanisms and will make artificial breeding possible which may contribute to the resource recovery of this species. Copyright © 2011 Elsevier Ltd. All rights reserved.

STUDIES ON THE ORIGIN OF CIRCULATING 18-HYDROXYCORTISOL AND 18-OXOCORTISOL IN NORMAL HUMAN SUBJECTS.

PubMed Central

Freel, E Marie; Shakerdi, Loai A; Friel, Elaine C; Wallace, A Michael; Davies, Eleanor; Fraser, Robert; Connell, John MC

2005-01-01

18-hydroxycortisol (18-OHF) and 18-oxocortisol (18oxo-F) are derivatives of cortisol found in Primary Aldosteronism but whose origin and regulation in normal subjects is uncertain. 18-OHF can be synthesised by zona fasciculata 11-β hydroxylase; 18-oxoF can only be produced by zona glomerulosa aldosterone synthase (AS). Stably transfected cell lines expressing either CYP11B1 (11β-hydroxylase) or CYP11B2 (AS) were incubated with cortisol and other substrates over a range of concentrations. Both enzymes could synthesise 18-OHF from cortisol but only AS could synthesise 18-oxoF. AS was more efficient than 11β-hydroxylase at 18-hydroxylation. The apparent Km of AS for cortisol was estimated to be 2.6μM. In 5 patients with adrenal insufficiency maintained on hydrocortisone, urinary free cortisol and cortisone levels were high; 18-oxoF was detectable in all patients and 18-hydroxycortisol in 3. It is likely that the 18-oxygenated steroids were synthesised from circulating cortisol, either in the zona glomerulosa or at extra-adrenal sites. In 8 male volunteers, dexamethasone treatment decreased urinary excretion rates of free cortisol, cortisone, 18-OHF and 18-oxoFl, confirming dependence of 18-oxygenated steroid levels on cortisol availability. In both groups, hydrocortisone administration resulted in detectable levels of 18-OHF and raised levels of 18-oxoF. There was close correlation between 18-oxoF and cortisol excretion during hydrocortisone administration in normal subjects (r=0.86, p<0.001). These data show, for the first time, that 18-OHF and 18oxoF can be synthesised from circulating cortisol. The close correlation between 18-oxoF and cortisol suggests that 18-oxoF is normally produced by the action of aldosterone synthase utilising circulating cortisol as a substrate. Although 18OHF can be synthesized using circulating cortisol as substrate, our data suggest this is normally produced in the zona fasciculata by 11β-hydroxylase from locally available cortisol

[A 65-year-old man with history of claudication, palpable purpura and livedo reticularis].

PubMed

Braun, N; Kimmel, M; Grabner, A; Ott, G; Alscher, M D

2010-04-01

A 65-year-old man was admitted with history of claudication symptoms and painful skin lesions of the lower legs. Physical examination showed palpable purpura of the lower legs and livedo reticularis, most marked at the forefoot and toes. Computed tomography (CT) showed an aortic mass 2 cm above the bifurcation. This was treated after angiography with a covered stent. Biopsy of the skin lesions showed no sign of vasculitis and no cholesterol crystals. The patient was discharged and remained symptom-free for 9 months. He was readmitted at that time with recurrent complaints. CT revealed a subtotal stenosis of the aortic stent. A skin biopsy showed CD31-positive tumor cells in small arteries. Biopsy of a new osteolytic lesion in the ileum confirmed the diagnosis of angiosarcoma of the aorta. The patient decided in favor of palliative care and was discharged from the hospital. Primary tumors of the aorta, although they are rare, should be considered in the presence of an intravascular mass with stenosis to blood flow. A skin biopsy is easy to conduct and often leads to the final diagnosis. Georg Thieme Verlag KG Stuttgart. New York.

[EFFECTS OF ELECTRICAL STIMULATION OF NUCLEUS RETICULARIS PONTIS ORALIS ON THE SLEEP-WAKING STATES IN KRUSHINSKII-MOLODKINA STRAIN RATS].

PubMed

Vataev, S I; Malgina, N A; Oganesyan, G A

2015-07-01

The effects of electrical stimulation of nucleus reticularis pontis oralis on the behavior and brain electrical activity during all phases of the sleep-waking cycle was studied in Krushinskii-Molodkina strain rats, which have an inherited predisposition to audiogenic seizures. Electrical stimulation with 7 Hz frequency in the deep stage of slow-wave sleep cause appearance the fast-wave sleep. Similar stimulation during fast-wave sleep periods did not effects on the electrographic patterns and EEG spectral characteristics of hippocampus, visual, auditory and somatocnen nrnrenc nf the cnrtey ThPe sfimul1stinns did nnt break a fast-wave sleenhut increased almost twice due the duration of these sleep episodes. After electrical stimulation by same frequency during the wakeftlness and superficial slow-wave sleep states, the patterns and spectral characteristics of brain electrical activity in rats showed no significant changes as compared with controls. The results of this study indicate that the state of the animals sleep-waking cycle at the time of stimulation is a critical variable that influences the responses which are induced by electrical stimulation of the nucleus reticularis pontis oralis.

Detection of angiotensin II binding to single adrenal zona glomerulosa cells by confocal Raman microspectroscopy

NASA Astrophysics Data System (ADS)

McCoy, Michael J.; Habermann, Timothy J.; Hanke, Craig J.; Adar, Fran; Campbell, William B.; Nithipatikom, Kasem

1999-04-01

We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep

PubMed Central

Liu, Kai; Kim, Juhyun; Kim, Dong Won; Zhang, Yi Stephanie; Bao, Hechen; Denaxa, Myrto; Lim, Szu-Aun; Kim, Eileen; Liu, Chang; Wickersham, Ian R.; Pachnis, Vassilis; Hattar, Samer; Song, Juan; Brown, Solange P.; Blackshaw, Seth

2017-01-01

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified1. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep2,3. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep–wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep. PMID:28847002

Suspected ontogeny of a recently described hypo-androgenic PCOS-like phenotype with advancing age.

PubMed

Gleicher, Norbert; Kushnir, Vitaly A; Darmon, Sarah K; Wang, Qi; Zhang, Lin; Albertini, David F; Barad, David H

2018-03-01

A recent report described a new PCOS-like phenotype in lean older infertile women, and was characterized by high age-specific anti-Müllerian hormone (AMH) but hypo- rather than the expected hyper-androgenism. The hypo-androgenism was, furthermore, characterized of, likely, adrenal origin and autoimmune etiology. We extracted data on 708 consecutive infertility patients, and separated them into three age-strata, <35, 36-42, and >42 years. In each stratum, we investigated how levels of anti-Müllerian hormone (AMH) and testosterone (T) interrelate between high-AMH (AMH ≥ 75th quantile) and normal AMH (25th-75th quantile) and low-T (total testosterone ≤19.0 ng/dL), normal-T (19.0-29.0 ng/dL) and high-T (>29.0 ng/dL). High-AMH cycles were presumed to reflect PCOS-like patients. Routine in vitro fertilization (IVF) cycle outcomes and clinical phenotypes of patients were then compared between groups with AMH and T as statistical variables. This hypo-androgenic PCOS-like phenotype already exists in age stratum <35 years. It appears to arise from a lean, at very young ages hyper-androgenic PCOS phenotype that develops in comparison to controls (likely autoimmune-induced) insufficiency of the adrenal zona reticularis (low-T and low-DHEAS) and zona fasciculata (low-C), and is characterized by frequent evidence of autoimmunity. A degree of adrenal insufficiency, thus, concomitantly appears to affect adrenal androgen and, to lesser degrees, glucocorticoid production (mineralocorticoids were not investigated). Here investigated new PCOS-like phenotype demonstrates features compatible with what under Rotterdam criteria has been referred to as PCOS phenotype-D. If confirmed, the observation that the ontogeny of this phenotype already at young ages is, likely, driven by adrenal autoimmunity, supports the position of the androgen excess and PCOS society that the etiology of phenotype-D differs from that of classical hyper-androgenic PCOS of mostly ovarian etiology.

GABAB receptor-mediated responses in GABAergic projection neurones of rat nucleus reticularis thalami in vitro.

PubMed

Ulrich, D; Huguenard, J R

1996-06-15

1. Whole-cell voltage-clamp recordings were obtained from GABAergic neurones of rat nucleus reticularis thalami (NRT) in vitro to assess pre- and postsynaptic GABAB receptor-mediated responses. Presynaptic inhibition of GABA release was studied at terminals on local axon collaterals within NRT as well as on projection fibres in the somatosensory relay nuclei. 2. The GABAB receptor agonist (R)-baclofen (10 microM) reduced monosynaptically evoked GABAA-mediated inhibitory postsynaptic currents (IPSCs) in NRT and somatosensory relay cells to 11 and 12% of control, respectively. 3. Action potential-independent miniature IPSCs (mIPSCs) were observed in both cell types. Mean mIPSC amplitude was 20 pA in both NRT and relay cells at a holding potential of 0 mV. The mean mIPSC frequencies were 0.83 and 2.2 Hz in NRT and relay cells, respectively. Baclofen decreased mIPSP frequency by about half in each cell type without affecting amplitude. 4. Paired-burst inhibition of evoked IPSCs was studied in relay and NRT cells by applying pairs of 100 Hz stimulus bursts separated by 600 ms. The mean ratio of second to first peak IPSC amplitudes was 0.77. 5. In NRT cells baclofen induced a linear postsynaptic conductance increase of 0.82 nS with an associated reversal potential of -121 mV. A small (0.14 nS) GABAB component of the evoked IPSC was detected in only a minority of NRT cells (3 of 18). 6. All pre- and postsynaptic effects of baclofen, as well as PBI, were largely reversed by the specific GABAB receptor antagonist CGP 35348 (0.5 mM). 7. We conclude that activation of GABAB receptors in NRT leads to presynaptic autoinhibition of IPSCs in both NRT and relay cells, and to direct activation of a small linear K+ conductance. In addition our experiments suggest that reciprocal connectivity within NRT can be partially mediated by a small GABAB inhibitory event.

Transfer of bovine demi-embryos with and without the zona pellucida.

PubMed

Warfield, S J; Seidel, G E; Elsden, R P

1987-09-01

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.

Thermal effects in laser-assisted pre-embryo zona drilling

NASA Astrophysics Data System (ADS)

Douglas-Hamilton, Diarmaid H.; Conia, Jerome D.

2001-04-01

Diode lasers ((lambda) equals 1480 nm) are used with in vitro fertilization to dissect the zone pellucida (shell) of pre- embryos. A focused laser beam is applied in vitro to form a channel or trench in the zona pellucida. The procedure is used to facilitate biopsy or as a promoter of embryo hatching. We present examples and measurements of zona pellucida ablation using animal models. In using the laser it is vital not to damage pre-embryo cells, e.g., by overheating. In order to define safe regimes we have derived some thermal side effects of zona pellucida removal. The temperature profile in the beam and vicinity is predicted as function of laser pulse duration and power. In a crossed- beam experiment a HeNe laser probe is used to detect the temperature-induced change in the refractive index of an aqueous solution, and estimate local thermal gradient. We find that the diode laser beam produces superheated water approaching 200 degree(s)C on the beam axis. Thermal histories during and following the laser pulse are given for regions in the neighborhood of the beam. We conclude that an optimum regime exists with pulse duration

Nonsalt-losing congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency with normal glomerulosa function.

PubMed

Pang, S; Levine, L S; Stoner, E; Opitz, J M; Pollack, M S; Dupont, B; New, M I

1983-04-01

glucuronide, suggesting the presence of normal peripheral 3 beta-HSD activity. We propose that in these siblings, there is a deficiency of 3 beta-HSD in the adrenal zona fasciculata and zona reticularis, whereas 3 beta-HSD activity is intact in the zona glomerulosa. In addition, in these siblings, 3 beta-HSD deficiency was present in the gonads, while peripheral 3 beta-HSD activity appeared to be intact. These cases demonstrate further the heterogeneity of congenital adrenal hyperplasia due to 3 beta-HSD deficiency.

Response properties of nucleus reticularis lateralis neurons after electroacupuncture stimulation in rats.

PubMed

Moritaka, Kentaro; Zeredo, Jorge L; Kimoto, Mari; Nasution, Fajar H; Hirano, Takafumi; Toda, Kazuo

2010-01-01

A descending inhibitory mechanism from the periaqueductal gray (PAG) to the spinal cord through the nucleus raphe magnus (NRM) is strongly involved in endogenous analgesic system produced by acupuncture stimulation. In addition to the PAG to NRM system which descends in the medial pathway of the brain stem, the nucleus reticularis lateralis (NRL) situated in the lateral part of the brain stem is reported to play an important role in modulating centrifugal antinociceptive action. In the present study, to clarify the role of NRL in acupuncture analgesia, we investigated the response properties of NRL neurons to acupuncture stimulation. The majority of NRM-projecting NRL neurons were inhibited by electroacupuncture stimulation. This effect was antagonized by ionophoretic application of naloxone, indicating that endogenous opioids act directly onto these NRL neurons. By contrast, about half of spinal projecting NRL neurons were excited by electroacupuncture stimulation, suggesting that part of the NRL neurons may modulate pain transmission directly at the spinal level.

Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse.

PubMed

Schimmel, Tim; Cohen, Jacques; Saunders, Helen; Alikani, Mina

2014-12-01

Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 μm and width of the thinned area was 35 μm. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P < 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona

Projections from the nucleus reticularis magnocellularis to the rat cervical cord using electrical stimulation and iontophoretic injection methods.

PubMed

Watanabe, Shigeo; Kitamura, Taiko; Watanabe, Lisa; Sato, Hitoshi; Yamada, Jinzo

2003-03-01

The aim of this study is to clarify the fiber distribution of the nucleus reticularis magnocellularis (NRMC) and adjacent areas in the rat spinal cord. Biotinylated dextran amine was injected iontophoretically through a glass capillary into the areas, in which a single cell responded to noxious electrical stimulation of the sciatic nerve and to a pinch of the thigh skin with multiple spikes. Labeled fibers descended bilaterally through the ventral funiculi of the medulla oblongata and then through the ventral and lateral funiculi of the cervical cord with an ipsilateral predominance, and terminated in the spinal gray (laminae I-X). A single fiber sometimes ran through several laminae while bifurcating many short branches with axon varicosities and terminal buttons in one transverse section, that is, through laminae V, VII and X, through laminae V, IIl-IV and I-II, and through laminae VII to I-II. The present study showed that the wide distribution of a single fiber and a mass of fibers descending from the NRMC and adjacent areas might modulate not only somatic sensory and motor functions but also autonomic functions in the spinal cord.

A Physiological Neural Network for Saccadic Eye Movement Control

DTIC Science & Technology

1994-04-01

cerebellum, substantia nigra, nucleus reticularis tegmenti pontis, the thalamus, the deep layers of the superior colliculus and the oculomotor plant...and pause cells), the vestibular nucleus , abducens nucleus , oculomotor nucleus , cerebellum, substantia nigra, nucleus reticularis tegmenti pontis, the...vestibular nucleus , abducens nucleus , oculomotor nucleus , cerebellum, substantia nigra, nucleus reticularis tegmenti pontis (NRTP), the thalamus, the

Cortical and subcortical afferents to the nucleus reticularis tegmenti pontis and basal pontine nuclei in the macaque monkey.

PubMed

Giolli, R A; Gregory, K M; Suzuki, D A; Blanks, R H; Lui, F; Betelak, K F

2001-01-01

Anatomical findings are presented that identify cortical and subcortical sources of afferents to the nucleus reticularis tegmenti pontis (NRTP) and basal pontine nuclei. Projections from the middle temporal visual area (MT), medial superior temporal visual area (MST), lateral intraparietal area (LIP), and areas 7a and 7b to the basal pontine nuclei were studied using 3H-leucine autoradiography. The results complemented a parallel study of retrograde neuronal labeling attributable to injecting WGA-HRP into NRTP and neighboring pontine nuclei. Small 3H-leucine injections confined to MT, MST, LIP, area 7a, or area 7b, produced multiple patches of pontine terminal label distributed as follows: (1) An injection within MT produced terminal label limited to the dorsolateral and lateral pontine nuclei. (2) Injections restricted to MST or LIP showed patches of terminal label in the dorsal, dorsolateral, lateral, and peduncular pontine nuclei. (3) Area 7a targets the dorsal, dorsolateral, lateral, peduncular, and ventral pontine nuclei, whereas area 7b projects, additionally, to the dorsomedial and paramedian pontine nuclei. Notably, no projections were seen to NRTP from any of these cortical areas. In contrast, injections made by other investigators into cortical areas anterior to the central sulcus revealed cerebrocortical afferents to NRTP, in addition to nuclei of the basal pontine gray. With our pontine WGA-HRP injections, retrograde neuronal labeling was observed over a large extent of the frontal cortex continuing onto the medial surface which included the lining of the cingulate sulcus and cingulate gyrus. Significant subcortical sources for afferents to the NRTP and basal pontine nuclei were the zona incerta, ventral mesencephalic tegmentum, dorsomedial hypothalamic area, rostral interstitial nucleus of the medial longitudinal fasciculus, red nucleus, and subthalamic nucleus. The combined anterograde and retrograde labeling data indicated that visuo-motor cortico

Fine structure and morphogenesis of spironolactone bodies in the zona glomerulosa of the human adrenal cortex

PubMed Central

Kovacs, K.; Horvath, E.; Singer, W.

1973-01-01

Numerous spironolactone bodies have been detected in the zona glomerulosa cells of the adrenal cortex of a 36-year-old spironolactone-treated woman whose non-tumorous right adrenal gland was removed surgically because of primary hyperaldosteronism. Electron microscopy revealed spherical laminated whorls which consisted of a central core composed of an amorphous electron-dense material surrounded by numerous smooth-walled concentric membranes. Continuous with and deriving from the endoplasmic reticulum, they were present in viable cells and were not associated with ultrastructural features indicating cellular injury. Cytoplasmic inclusions similar to spironolactone bodies can be detected in other organs after the administration of various compounds. Thus, they can be regarded as neither specific to spironolactone treatment nor exclusively inducible in the zona glomerulosa of the adrenal cortex. Images PMID:4131694

Mechanics of sperm-egg interaction at the zona pellucida.

PubMed Central

Baltz, J M; Katz, D F; Cone, R A

1988-01-01

Mammalian sperm traverse several layers of egg vestments before fertilization can occur. The innermost vestment, the zona pellucida, is a glycoprotein shell, which captures and tethers the sperm before they penetrate it. We report here direct measurements of the force required to tether a motile human sperm as well as independent calculations of this force using flagellar beat parameters observed for sperm of several species on their homologous zonae. We have compared these sperm-generated forces with the calculated tensile strength of sperm-zona bonds, and found that a motile sperm can be tethered, at least temporarily, by a single bond. Therefore, sperm can be captured by the first bond formed and tethered permanently by a few. The sperm cannot subsequently penetrate the zona unless the bonds are first eliminated. However, premature elimination would simply allow the sperm to escape. Therefore, not only must the bonds be eliminated, but the timing of this must be regulated so that the sperm is already oriented toward the egg and beginning to penetrate as the bonds are broken. Images FIGURE 6 PMID:3224150

Interactions between rewarding lateral hypothalamic and aversive nucleus reticularis gigantocellularis stimulation.

PubMed

Diotte, M; Miguelez, M; Miliaressis, E; Bielajew, C

2000-12-05

The interaction between rewarding and aversive consequences of brain stimulation were assessed in two studies. In the first, the frequency threshold for 300 ms trains of combined lateral hypothalamic (LH) and nucleus reticularis gigantocellularis (Gi) stimulation, in which each LH pulse was followed 2 ms later by the Gi one, was determined for one month. Compared to the threshold for trains of single LH pulses, combined LH-Gi stimulation initially increased the frequency threshold; however, this effect reversed within one session and was subsequently maintained for the duration of the study. The aversion produced by Gi stimulation, as measured by latency to escape, was abolished following a single session of LH-Gi pairs. In the second study, a subset of animals received both presentations of combined pulses, LH followed by Gi, and the reverse; the interval between pulses was varied from 0.2 to 6.4 ms. The effectiveness of combined stimulation, determined by the ratio of LH frequency thresholds to that of the LH-Gi ranged from 0 to 50% across animals but the individual effectiveness functions within animals did not vary with different intervals. In addition, the order of presentation of pulses was of no consequence. Thus, not only did exposure to LH stimulation appear to obliterate Gi aversion, but the combination of LH and Gi pulses added to the rewarding effect produced by LH stimulation alone.

Effects of acrolein on aldosterone release from zona glomerulosa cells in male rats.

PubMed

Wang, Kai-Lee; Huang, Wen-Ching; Chou, Jou-Chun; Weng, Ting-Chun; Hu, Sindy; Lieu, Fu-Kong; Lai, Wei-Ho; Idova, Galina; Wang, Paulus S; Wang, Shyi-Wu

2016-07-01

A positive correlation between smoking and hypertension has been well established. Acrolein is a major toxic volatile compound found in cigarette smoke. Human exposure to low levels of acrolein is unavoidable due to its production in daily activities, such as smoke from industrial, hot oil cooking vapors, and exhaust fumes from vehicles. The toxicity and the action mechanism of acrolein to induce apoptosis have been extensively studied, but the effects of acrolein on hypertension are still unknown. The present study aimed to examine the effects of acrolein on aldosterone release both in vivo and in vitro. Male rats were divided into three groups, and intraperitoneally injected with normal saline, or acrolein (2mg/kg) for 1 (group A-1) or 3 (group A-3) days, respectively. After sacrificing, rat blood samples were obtained to measure plasma aldosterone and angiotensin II (Ang II) levels. Zona glomerulosa (ZG) cells were prepared from rat adrenal cortex, and were incubated with or without stimulants. We found that the serum aldosterone was increased by 1.2-fold (p<0.05) in A-3 group as compared to control group. Basal aldosterone release from ZG cells in A-3 group was also increased significantly. Moreover, acrolein enhanced the stimulatory effects of Ang II and 8-bromo-cyclic AMP on aldosterone secretion from ZG cells prepared in both A-1 and A-3 groups. Furthermore, the enzyme activity of P450scc, the rate-limiting step of aldosterone synthesis, was elevated after acrolein injection. Plasma level of Ang II was increased in both A-1 and A-3 groups. These results suggested that acrolein exposure increased aldosterone production, at least in part, through elevating the level of plasma Ang II and stimulating steroidogenesis pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

PubMed

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

The anatomy of the caudal zona incerta in rodents and primates

PubMed Central

Watson, Charles; Lind, Christopher R P; Thomas, Meghan G

2014-01-01

The caudal zona incerta is the target of a recent modification of established procedures for deep brain stimulation (DBS) for Parkinson's disease and tremor. The caudal zona incerta contains a number of neuronal populations that are distinct in terms of their cytoarchitecture, connections, and pattern of immunomarkers and is located at a position where a number of major tracts converge before turning toward their final destination in the forebrain. However, it is not clear which of the anatomical features of the region are related to its value as a target for DBS. This paper has tried to identify features that distinguish the caudal zona incerta of rodents (mouse and rat) and primates (marmoset, rhesus monkey, and human) from the remainder of the zona incerta. We studied cytoarchitecture, anatomical relationships, the pattern of immunomarkers, and gene expression in both of these areas. We found that the caudal zona incerta has a number of histological and gene expression characteristics that distinguish it from the other subdivisions of the zona incerta. Of particular note are the sparse population of GABA neurons and the small but distinctive population of calbindin neurons. We hope that a clearer appreciation of the anatomy of the region will in the end assist the interpretation of cases in which DBS is used in human patients. PMID:24138151

Distinct effect of orphanin FQ in nucleus raphe magnus and nucleus reticularis gigantocellularis on the rat tail flick reflex.

PubMed

Yang, Z; Zhang, Y; Wu, G

2001-06-22

The aim of the present study is to investigate the effects of orphanin FQ (OFQ) microinjected into the nucleus raphe magnus (NRM) and the nucleus reticularis gigantocellularis (NGC) on pain modulation. The tail-flick latency (TFL) was used as a behavioral index of nociceptive responsiveness. The result showed microinjection of OFQ into the NRM significantly increased the TFL, whereas microinjection of OFQ into the NGC decreased the TFL, suggesting the analgesic effect of OFQ in the NRM and the hyperalgesic effect of OFQ in the NGC. As there are three classes of putative pain modulating neurons in the rostral ventromedial medulla (RVM), the hyperalgesic or analgesic effect of OFQ in the RVM might depend upon the different class of the neurons being acted.

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction.

PubMed

Morales, P; Llanos, M; Yovich, J L; Cummins, J M; Vigil, P

1993-01-01

Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml-1 of pentoxifylline at 37 degrees C, 5% CO2 for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 x 10(6) cells ml-1. One hundred microlitres of each sperm suspension was then deposited under oil and 30-40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.

Antimicrobial activity of honey of africanized bee (Apis mellifera) and stingless bee, tiuba (Melipona fasciculata) against strains of Escherichia coli, Pseudomona aeruginosa and Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Tenório, Eleuza Gomes; Alves, Natália Furtado; Mendes, Bianca Evanita Pimenta

2017-11-01

The objective of this study was to investigate the antimicrobial activity of honey of Africanized bees (Apis mellifera) and stingless bees (Melipona fasciculata), produced under the same flowering conditions, in municipalities of Baixada Maranhese, Brazil, against strains of pathogenic bacteria, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In each municipality, the apiary and meliponario were less than 150 meters away from each other. The Kirby-Bauer method, and the diffusion technique of the agar plate through the extension of the inhibition in millimeters were used. The test results were negative for all samples, which did not demonstrate antimicrobial activity in any of the microorganisms tested.

Processing noxious information at the subnucleus reticularis dorsalis (SRD) of anesthetized cats: wind-up mechanisms.

PubMed

Soto, Cristina; Martín-Cora, Francisco J; Leiras, Roberto; Velo, Patricia; Canedo, Antonio

2008-11-15

With the exception of one monkey's study, where wind-up was not reported, electrophysiological data from SRD neurons were obtained in rodents where they show wind-up. This work was designed to examine the response properties of SRD neurons in anesthetized cats to study how general the data from rats may be. Since cat's SRD cells showed wind-up, its underlying mechanisms were approached, an issue not previously addressed at supraspinal level. Electrical stimulation, extracellular (combined with microiontophoresis) and intracellular techniques revealed that A delta information reaches the SRD via the ventrolateral cord, whereas C information preferentially follows a dorsal route. Wind-up was usually generated by spinal and peripheral stimulation, but it was also evoked either by stimulating the nucleus reticularis gigantocellularis (NRGc), even after spinal cord section and bilateral full thickness removal of the cerebral cortex, or by applying microiontophoretic pulses of l-glutamate at 0.3-1 Hz. Wind-up relied on afferent repetitive activity gradually depolarizing the SRD neurons leading 3-4.5 Hz subthreshold membrane rhythmic activity to threshold. Riluzole retarded wind-up generation and decreased the number of spikes per stimulus during wind-up. GABA or glycine abolished spontaneous and sensory-evoked activity and bicuculline, but not strychnine, increased spontaneous and stimulus-evoked activity. These results demonstrate that wind-up at the SRD is not merely the reflection of spinal wind-up, but (i) can be locally generated, (ii) is partially dependent upon persistent sodium currents, and (iii) is under the modulation of a tonic GABAa-dependent inhibition decreasing SRD excitability. Injury and/or inflammation producing tonic C-fiber activation will surpass tonic inhibition generating wind-up.

Laser assisted zona hatching does not lead to immediate impairment in human embryo quality and metabolism.

PubMed

Uppangala, Shubhashree; D'Souza, Fiona; Pudakalakatti, Shivanand; Atreya, Hanudatta S; Raval, Keyur; Kalthur, Guruprasad; Adiga, Satish Kumar

2016-12-01

Laser assisted zona hatching (LAH) is a routinely used therapeutic intervention in assisted reproductive technology for patients with poor prognosis. However, results are not conclusive in demonstrating the benefits of zona hatching in improving the pregnancy rate. Recent observations on LAH induced genetic instability in animal embryos prompted us to look into the effects of laser assisted zona hatching on the human preimplantation embryo quality and metabolic uptake using high resolution nuclear magnetic resonance (NMR) technology. This experimental prospective study included fifty embryos from twenty-five patients undergoing intra cytoplasmic sperm injection. Embryo quality assessment followed by profiling of spent media for the non-invasive evaluation of metabolites was performed using NMR spectroscopy 24 hours after laser treatment and compared with that of non-treated sibling embryos. Both cell number and embryo quality on day 3 of development did not vary significantly between the two groups at 24 hours post laser treatment interval. Time lapse monitoring of the embryos for 24 hours did not reveal blastomere fragmentation adjacent to the point of laser treatment. Similarly, principal component analysis of metabolites did not demonstrate any variation across the groups. These results suggest that laser assisted zona hatching does not affect human preimplantation embryo morphology and metabolism at least until 24 hours post laser assisted zona hatching. However, studies are required to elucidate laser induced metabolic and developmental changes at extended time periods. AH: assisted hatching; ART: assisted reproductive technology; DNA: deoxy-ribo nucleic acid; LAH: laser assisted hatching; MHz: megahertz; NMR: nuclear magnetic resonance; PCA: principal component analysis; PGD: preimplantation genetic diagnosis; TLM: time lapse monitoring.

Axon collaterals projection from nucleus reticularis tegmenti pontis onto the cerebellar paramedian lobule in the rabbit: a fluorescent double labelling study.

PubMed

Mierzejewska-Krzyzowska, B

1999-01-01

Double labelling method with retrograde transport of fluorescent tracers (Fast Blue; FB and Diamidino Yellow; DY) was employed in the rabbit to investigate whether neurones of the nucleus reticularis tegmenti pontis (NRTP) give off axon collaterals to the cerebellar paramedian lobule (PML) of both sides. Following injections to various regions of the homotopic or heterotopic sublobules of the left (FB) and right (DY) PML cortex, distribution of double labelled neurones within NRTP was analyzed. NRTP of the rabbit consists of a medial principal part (the nucleus papillioformis: PLF) and smaller lateral part (the processus tegmentosus lateralis: PTL). Within PLF three subdivisions are to be distinguished: the dorsomedial part -- zone A, the main part -- zone B and the ventrolateral part -- zone C. The present study in the rabbit indicated collateral projections from neurones in some NRTP regions to the both PML. The cells of origin of these projections were located prominently through the rostrocaudal extent of zone B. Projections from zone A were sparse and those from zone C were absent. Moreover, a weak projection arose mainly from the caudal aspect of PTL. It is concluded that the rostral (e and f) and middle (c and d) sublobules are the main targets for the NRTP-PML branching projections.

Antimicrobial activity against oral pathogens and immunomodulatory effects and toxicity of geopropolis produced by the stingless bee Melipona fasciculata Smith

PubMed Central

2011-01-01

Background Native bees of the tribe Meliponini produce a distinct kind of propolis called geopropolis. Although many pharmacological activities of propolis have already been demonstrated, little is known about geopropolis, particularly regarding its antimicrobial activity against oral pathogens. The present study aimed at investigating the antimicrobial activity of M. fasciculata geopropolis against oral pathogens, its effects on S. mutans biofilms, and the chemical contents of the extracts. A gel prepared with a geopropolis extract was also analyzed for its activity on S. mutans and its immunotoxicological potential. Methods Antimicrobial activities of three hydroalcoholic extracts (HAEs) of geopropolis, and hexane and chloroform fractions of one extract, were evaluated using the agar diffusion method and the broth dilution technique. Ethanol (70%, v/v) and chlorhexidine (0.12%, w/w) were used as negative and positive controls, respectively. Total phenol and flavonoid concentrations were assayed by spectrophotometry. Immunotoxicity was evaluated in mice by topical application in the oral cavity followed by quantification of biochemical and immunological parameters, and macro-microscopic analysis of animal organs. Results Two extracts, HAE-2 and HAE-3, showed inhibition zones ranging from 9 to 13 mm in diameter for S. mutans and C. albicans, but presented no activity against L. acidophilus. The MBCs for HAE-2 and HAE-3 against S. mutans were 6.25 mg/mL and 12.5 mg/mL, respectively. HAE-2 was fractionated, and its chloroform fraction had an MBC of 14.57 mg/mL. HAE-2 also exhibited bactericidal effects on S. mutans biofilms after 3 h of treatment. Significant differences (p < 0.05) in total phenol and flavonoid concentrations were observed among the samples. Signs toxic effects were not observed after application of the geopropolis-based gel, but an increase in the production of IL-4 and IL-10, anti-inflammatory cytokines, was detected. Conclusions In summary

Antimicrobial activity against oral pathogens and immunomodulatory effects and toxicity of geopropolis produced by the stingless bee Melipona fasciculata Smith.

PubMed

Liberio, Silvana A; Pereira, Antônio Luís A; Dutra, Richard P; Reis, Aramys S; Araújo, Maria José A M; Mattar, Nadia S; Silva, Lucilene A; Ribeiro, Maria Nilce S; Nascimento, Flávia Raquel F; Guerra, Rosane N M; Monteiro-Neto, Valério

2011-11-04

Native bees of the tribe Meliponini produce a distinct kind of propolis called geopropolis. Although many pharmacological activities of propolis have already been demonstrated, little is known about geopropolis, particularly regarding its antimicrobial activity against oral pathogens. The present study aimed at investigating the antimicrobial activity of M. fasciculata geopropolis against oral pathogens, its effects on S. mutans biofilms, and the chemical contents of the extracts. A gel prepared with a geopropolis extract was also analyzed for its activity on S. mutans and its immunotoxicological potential. Antimicrobial activities of three hydroalcoholic extracts (HAEs) of geopropolis, and hexane and chloroform fractions of one extract, were evaluated using the agar diffusion method and the broth dilution technique. Ethanol (70%, v/v) and chlorhexidine (0.12%, w/w) were used as negative and positive controls, respectively. Total phenol and flavonoid concentrations were assayed by spectrophotometry. Immunotoxicity was evaluated in mice by topical application in the oral cavity followed by quantification of biochemical and immunological parameters, and macro-microscopic analysis of animal organs. Two extracts, HAE-2 and HAE-3, showed inhibition zones ranging from 9 to 13 mm in diameter for S. mutans and C. albicans, but presented no activity against L. acidophilus. The MBCs for HAE-2 and HAE-3 against S. mutans were 6.25 mg/mL and 12.5 mg/mL, respectively. HAE-2 was fractionated, and its chloroform fraction had an MBC of 14.57 mg/mL. HAE-2 also exhibited bactericidal effects on S. mutans biofilms after 3 h of treatment. Significant differences (p < 0.05) in total phenol and flavonoid concentrations were observed among the samples. Signs toxic effects were not observed after application of the geopropolis-based gel, but an increase in the production of IL-4 and IL-10, anti-inflammatory cytokines, was detected. In summary, geopropolis produced by M. fasciculata can

Targeting the zona pellucida for immunocontraception: a minireview.

PubMed

Tesarik, J

1995-12-01

This minireview summarizes the main data relevant to the development of contraceptive vaccines based on zona pellucida (ZP) antigens, as well as the pros and the cons of this immunocontraceptive strategy. Even though the antifertility efficacy of anti-ZP antibodies in humans is not corroborated by a clear relationship between spontaneous autoimmunization against the ZP and infertility, passive and active immunization studies in laboratory animals have provided convincing results. The contraceptive action of anti-ZP antibodies, targeting events situated upstream of gamete fusion, is devoid of potential ethical concerns related to the destruction of early embryos. The high protein content of the mammalian ZP, knowledge of the complete amino acid sequence of the major ZP proteins, and the high degree of sequence homology between individual species all favour the rapid advancement of anti-ZP vaccine projects. However, certain sequences of ZP proteins, when incorporated into the vaccine construct, activate CD4+ T cells of the recipient organism to direct a cellular immune attack (autoimmune oophoritis) to other functionally relevant ovarian components (primordial follicles, steroidogenic cells). The search for the optimal combination of B cell and T cell epitopes in the vaccine construct will hopefully overcome this problem.

Cat's medullary reticulospinal and subnucleus reticularis dorsalis noxious neurons form a coupled neural circuit through collaterals of descending axons

PubMed Central

Leiras, Roberto; Martín-Cora, Francisco; Velo, Patricia; Liste, Tania

2015-01-01

Animals and human beings sense and react to real/potential dangerous stimuli. However, the supraspinal mechanisms relating noxious sensing and nocifensive behavior are mostly unknown. The collateralization and spatial organization of interrelated neurons are important determinants of coordinated network function. Here we electrophysiologically studied medial medullary reticulospinal neurons (mMRF-RSNs) antidromically identified from the cervical cord of anesthetized cats and found that 1) more than 40% (79/183) of the sampled mMRF-RSNs emitted bifurcating axons running within the dorsolateral (DLF) and ventromedial (VMF) ipsilateral fascicles; 2) more than 50% (78/151) of the tested mMRF-RSNs with axons running in the VMF collateralized to the subnucleus reticularis dorsalis (SRD) that also sent ipsilateral descending fibers bifurcating within the DLF and the VMF. This percentage of mMRF collateralization to the SRD increased to more than 81% (53/65) when considering the subpopulation of mMRF-RSNs responsive to noxiously heating the skin; 3) reciprocal monosynaptic excitatory relationships were electrophysiologically demonstrated between noxious sensitive mMRF-RSNs and SRD cells; and 4) injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin evidenced mMRF to SRD and SRD to mMRF projections contacting the soma and proximal dendrites. The data demonstrated a SRD-mMRF network interconnected mainly through collaterals of descending axons running within the VMF, with the subset of noxious sensitive cells forming a reverberating circuit probably amplifying mutual outputs simultaneously regulating motor activity and spinal noxious afferent input. The results provide evidence that noxious stimulation positively engages a reticular SRD-mMRF-SRD network involved in pain-sensory-to-motor transformation and modulation. PMID:26581870

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei.

PubMed

Verner, Zdeněk; Cermáková, Petra; Skodová, Ingrid; Kováčová, Bianka; Lukeš, Julius; Horváth, Anton

2014-01-01

Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites. Copyright © 2014 Elsevier B.V. All rights reserved.

Differences between antigenic determinants of pig and cat zona pellucida proteins.

PubMed

Jewgenow, K; Rohleder, M; Wegner, I

2000-05-01

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Direct connection of the nucleus reticularis gigantocellularis neurons with neck motoneurons in cats.

PubMed

Sasaki, S

1999-10-01

Functional connections of single reticulospinal neurons (RSNs) in the nucleus reticularis gigantocellularis (NRG) with ipsilateral dorsal neck motoneurons were examined with the spike-triggered averaging technique. Extracellular spikes of single NRG-RSNs activated antidromically from the C6, but not from the L1 segment (C-RSNs) were used as the trigger. These neurons were monosynaptically activated from the superior colliculus and the cerebral peduncle. Single-RSN PSPs were recorded in 43 dorsal neck motoneurons [biventer cervicis and complexus (BCC) and splenius (SPL)] for 21 NRG-RSNs and 135 motoneurons tested. All synaptic potentials were EPSPs, and most of their latencies, measured from the triggering spikes, were 0.8-1.5 ms, which is in a monosynaptic range. The amplitudes of single-RSN EPSPs were 10-360 microV. Spike-triggered averaging revealed single-RSN EPSPs in multiple motoneurons of the same species (SPL or BCC), their locations extending up to nearly 1 mm rostrocaudally. Synaptic connections of single RSNs with both SPL and BCC motoneurons were also found with some predominance for one of them. The results provide direct evidence that NRG-RSNs make monosynaptic excitatory connections with SPL and BCC motoneurons. It appears that some NRG-RSNs connect predominantly with SPL motoneurons and others with BCC motoneurons.

Functional Zonation of the Adult Mammalian Adrenal Cortex

PubMed Central

Vinson, Gavin P.

2016-01-01

The standard model of adrenocortical zonation holds that the three main zones, glomerulosa, fasciculata, and reticularis each have a distinct function, producing mineralocorticoids (in fact just aldosterone), glucocorticoids, and androgens respectively. Moreover, each zone has its specific mechanism of regulation, though ACTH has actions throughout. Finally, the cells of the cortex originate from a stem cell population in the outer cortex or capsule, and migrate centripetally, changing their phenotype as they progress through the zones. Recent progress in understanding the development of the gland and the distribution of steroidogenic enzymes, trophic hormone receptors, and other factors suggests that this model needs refinement. Firstly, proliferation can take place throughout the gland, and although the stem cells are certainly located in the periphery, zonal replenishment can take place within zones. Perhaps more importantly, neither the distribution of enzymes nor receptors suggest that the individual zones are necessarily autonomous in their production of steroid. This is particularly true of the glomerulosa, which does not seem to have the full suite of enzymes required for aldosterone biosynthesis. Nor, in the rat anyway, does it express MC2R to account for the response of aldosterone to ACTH. It is known that in development, recruitment of stem cells is stimulated by signals from within the glomerulosa. Furthermore, throughout the cortex local regulatory factors, including cytokines, catecholamines and the tissue renin-angiotensin system, modify and refine the effects of the systemic trophic factors. In these and other ways it more and more appears that the functions of the gland should be viewed as an integrated whole, greater than the sum of its component parts. PMID:27378832

Cortical afferents onto the nucleus Reticularis thalami promote plasticity of low-threshold excitability through GluN2C-NMDARs.

PubMed

Fernandez, Laura M J; Pellegrini, Chiara; Vantomme, Gil; Béard, Elidie; Lüthi, Anita; Astori, Simone

2017-09-25

Thalamus and cortex represent a highly integrated processing unit that elaborates sensory representations. Interposed between cortex and thalamus, the nucleus Reticularis thalami (nRt) receives strong cortical glutamatergic input and mediates top-down inhibitory feedback to thalamus. Despite growing appreciation that the nRt is integral for thalamocortical functions from sleep to attentional wakefulness, we still face considerable gaps in the synaptic bases for cortico-nRt communication and plastic regulation. Here, we examined modulation of nRt excitability by cortical synaptic drive in Ntsr1-Cre x ChR2 tg/+ mice expressing Channelrhodopsin2 in layer 6 corticothalamic cells. We found that cortico-nRt synapses express a major portion of NMDA receptors containing the GluN2C subunit (GluN2C-NMDARs). Upon repetitive photoactivation (10 Hz trains), GluN2C-NMDARs induced a long-term increase in nRt excitability involving a potentiated recruitment of T-type Ca 2+ channels. In anaesthetized mice, analogous stimulation of cortical afferents onto nRt produced long-lasting changes in cortical local field potentials (LFPs), with delta oscillations being augmented at the expense of slow oscillations. This shift in LFP spectral composition was sensitive to NMDAR blockade in the nRt. Our data reveal a novel mechanism involving plastic modification of synaptically recruited T-type Ca 2+ channels and nRt bursting and indicate a critical role for GluN2C-NMDARs in thalamocortical rhythmogenesis.

Multimodal sensory responses of nucleus reticularis gigantocellularis and the responses' relation to cortical and motor activation.

PubMed

Martin, Eugene M; Pavlides, Constantine; Pfaff, Donald

2010-05-01

The connectivity of large neurons of the nucleus reticularis gigantocellularis (NRGc) in the medullary reticular formation potentially allows both for the integration of stimuli, in several modalities, that would demand immediate action, and for coordinated activation of cortical and motoric activity. We have simultaneously recorded cortical local field potentials, neck muscle electromyograph (EMG), and the neural activity of medullary NRGc neurons in unrestrained, unanesthetized rats to determine whether the activity of the NRGc is consistent with the modulation of general arousal. We observed excitatory responses of individual NRGc neurons to all modalities tested: tactile, visual, auditory, vestibular, and olfactory. Excitation was directly linked to increases in neck muscle EMG amplitude and corresponded with increases in the power of fast oscillations (30 to 80 Hz) of cortical activity and decreases in the power of slow oscillations (2 to 8 Hz). Because these reticular formation neurons can respond to broad ranges of stimuli with increased firing rates associated with the initiation of behavioral responses, we infer that they are part of an elementary "first responder" CNS arousal mechanism.

Multimodal Sensory Responses of Nucleus Reticularis Gigantocellularis and the Responses' Relation to Cortical and Motor Activation

PubMed Central

Pavlides, Constantine; Pfaff, Donald

2010-01-01

The connectivity of large neurons of the nucleus reticularis gigantocellularis (NRGc) in the medullary reticular formation potentially allows both for the integration of stimuli, in several modalities, that would demand immediate action, and for coordinated activation of cortical and motoric activity. We have simultaneously recorded cortical local field potentials, neck muscle electromyograph (EMG), and the neural activity of medullary NRGc neurons in unrestrained, unanesthetized rats to determine whether the activity of the NRGc is consistent with the modulation of general arousal. We observed excitatory responses of individual NRGc neurons to all modalities tested: tactile, visual, auditory, vestibular, and olfactory. Excitation was directly linked to increases in neck muscle EMG amplitude and corresponded with increases in the power of fast oscillations (30 to 80 Hz) of cortical activity and decreases in the power of slow oscillations (2 to 8 Hz). Because these reticular formation neurons can respond to broad ranges of stimuli with increased firing rates associated with the initiation of behavioral responses, we infer that they are part of an elementary “first responder” CNS arousal mechanism. PMID:20181730

The nucleus reticularis gigantocellularis modulates the cardiopulmonary responses to central and peripheral drives related to exercise.

PubMed

Richard, C A; Waldrop, T G; Bauer, R M; Mitchell, J H; Stremel, R W

1989-03-13

It is known that muscle afferents and the hypothalamic locomotor region (HLR) both project to the nucleus reticularis gigantocellularis (NGC) and that the NGC is capable of influencing cardiovascular and respiratory variables. Therefore, the role of NGC in the cardiovascular and respiratory response to exercise-related signals was investigated in anesthetized cats. These signals were generated by stimulation of: (1) spinal ventral roots to induce hindlimb muscle contraction (MC) and (2) the HLR. Bilateral electrolytic lesion of the NGC at the pontomedullary border caused tidal volume, respiratory frequency and heart rate responses to HLR stimulation to be greater than the responses recorded prior to lesioning. Lesioning had no effect on the ventilatory or cardiovascular responses to MC but did decrease phrenic responsiveness; lesion had no effect on any resting values. In this preparation, the pontomedullary NGC acts as an inhibitory influence on tidal volume, breathing frequency and heart rate responses to the central command for exercise. In addition, NGC modulation of ventilation would appear to be selective for certain respiratory muscle groups.

Discharge of Monkey Nucleus Reticularis Tegmenti Pontis Neurons Changes During Saccade Adaptation

PubMed Central

Takeichi, N.; Kaneko, C.R.S.; Fuchs, A. F.

2006-01-01

Saccade accuracy is maintained by adaptive mechanisms that continually modify saccade amplitude to reduce dysmetria. Previous studies suggest that adaptation occurs upstream of the caudal fastigial nucleus (CFN), the output of the oculomotor cerebellar vermis but downstream from the superior colliculus (SC). The nucleus reticularis tegmenti pontis (NRTP) is a major source of afferents to both the oculomotor vermis and the CFN and in turn receives direct input from the SC. Here we examine the activity of NRTP neurons in four rhesus monkeys during behaviorally induced changes in saccade amplitude to assess whether their discharge might reveal adaptation mechanisms that mediate changes in saccade amplitude. During amplitude decrease adaptation (average, 22%), the gradual reduction of saccade amplitude was accompanied by an increase in the number of spikes in the burst of 19/34 neurons (56%) and no change for 15 neurons (44%). For the neurons that increased their discharge, the additional spikes were added at the beginning of the saccadic burst and adaptation also delayed the peak-firing rate in some neurons. Moreover, after amplitude reduction, the movement fields changed shape in all 15 open field neurons tested. Our data show that saccadic amplitude reduction affects the number of spikes in the burst of more than half of NRTP neurons tested, primarily by increasing burst duration not frequency. Therefore adaptive changes in saccade amplitude are reflected already at a major input to the oculomotor cerebellum. PMID:15917328

Discharge of monkey nucleus reticularis tegmenti pontis neurons changes during saccade adaptation.

PubMed

Takeichi, N; Kaneko, C R S; Fuchs, A F

2005-09-01

Saccade accuracy is maintained by adaptive mechanisms that continually modify saccade amplitude to reduce dysmetria. Previous studies suggest that adaptation occurs upstream of the caudal fastigial nucleus (CFN), the output of the oculomotor cerebellar vermis but downstream from the superior colliculus (SC). The nucleus reticularis tegmenti pontis (NRTP) is a major source of afferents to both the oculomotor vermis and the CFN and in turn receives direct input from the SC. Here we examine the activity of NRTP neurons in four rhesus monkeys during behaviorally induced changes in saccade amplitude to assess whether their discharge might reveal adaptation mechanisms that mediate changes in saccade amplitude. During amplitude decrease adaptation (average, 22%), the gradual reduction of saccade amplitude was accompanied by an increase in the number of spikes in the burst of 19/34 neurons (56%) and no change for 15 neurons (44%). For the neurons that increased their discharge, the additional spikes were added at the beginning of the saccadic burst and adaptation also delayed the peak-firing rate in some neurons. Moreover, after amplitude reduction, the movement fields changed shape in all 15 open field neurons tested. Our data show that saccadic amplitude reduction affects the number of spikes in the burst of more than half of NRTP neurons tested, primarily by increasing burst duration not frequency. Therefore adaptive changes in saccade amplitude are reflected already at a major input to the oculomotor cerebellum.

Evaluation of the glucocorticoid, mineralocorticoid, and adrenal androgen secretion dynamics in a large cohort of patients aged 6-18 years with transfusion-dependent β-thalassemia major, with an emphasis on the impact of cardiac iron load.

PubMed

Uçar, Ahmet; Öner, Nergiz; Özek, Gülcihan; Çetinçakmak, Mehmet Güli; Abuhandan, Mahmut; Yıldırım, Ali; Kaya, Cemil; Ünverdi, Sena; Emeksiz, Hamdi Cihan; Yılmaz, Yasin; Yetim, Aylin

2016-07-01

The variable presence of adrenal insufficiency (AI) due to hypocortisolemia (HC) in patients with thalassemia is well established; however, the prevalence of adrenocortical hypofunction (ACH) in the zona glomerulosa and zona reticularis of the adrenal cortex is unknown. To establish the prevalence of ACH, we examined the cortisol response to 1-µg and 250-µg ACTH tests, plasma aldosterone (A)/plasma renin activity (PRA) ratio, and serum dehydroepiandrosterone sulfate (DHEAS) levels in a large cohort of patients with thalassemia, and to investigate the impact of total body iron load (TBIL) on adrenocortical function. The setting used was University hospital and government-based tertiary care center. One hundred twenty-one (52 females) patients with β-thalassemia major (β-TM) and 72 healthy peers (38 females) were enrolled. The patients underwent a 250-µg cosyntropin test if their peak cortisol was <500 nmol/L in a 1-µg cosyntropin test. Magnetic resonance imaging (MRI) was performed to assess the MRI-based liver iron content and cardiac MRI T2* iron. The associations between ACH and TBIL were investigated. The patients with thalassemia had lower ACTH, cortisol, DHEAS, and A/PRA values compared with the controls (p < 0.001). Thirty-nine patients (32.2 %) had HC [primary (n = 1), central (n = 36), combined (n = 2)], and 47 (38.8 %) patients had reduced DHEAS levels; 29 (24.0 %) patients had reduced A/PRA ratios. Forty-six (38.0 %) patients had hypofunction in one of the adrenal zones, 26 (21.5 %) had hypofunction in two adrenal zones, and 9 (7.4 %) had hypofunction in all three zones. Patient age and TBIL surrogates were significant independent parameters associated with ACH. Cardiac MRI T2* iron was the only significant parameter that predicted the severity of ACH at a cut-off of 20.6 ms, with 81 % sensitivity and 78 % specificity. Patients with thalassemia have a high prevalence of AI due to HC and zona glomerulosa and zona reticularis

Responses to vertical vestibular stimulation of neurons in the nucleus reticularis gigantocellularis in rabbits.

PubMed

Fagerson, M H; Barmack, N H

1995-06-01

1. Because the nucleus reticularis gigantocellularis (NRGc) receives a substantial descending projection from the caudal vestibular nuclei, we used extracellular single-unit recording combined with natural vestibular stimulation to examine the possible peripheral origins of the vestibularly modulated activity of caudal NRGc neurons located within 500 microns of the midline. Chloralose-urethan anesthetized rabbits were stimulated with an exponential "step" and/or static head-tilt stimulus, as well as sinusoidal rotation about the longitudinal or interaural axes providing various combinations of roll or pitch, respectively. Recording sites were reconstructed from electrolytic lesions confirmed histologically. 2. More than 85% of the 151 neurons, in the medial aspect of the caudal NRGc, responded to vertical vestibular stimulation. Ninety-six percent of these responded to rotation onto the contralateral side (beta responses). Only a few also responded to horizontal stimulation. Seventy-eight percent of the neurons that responded to vestibular stimulation responded during static roll-tilt. One-half of these neurons also responded transiently to the change in head position during exponential "step" stimulation, suggesting input mediated by otolith and semicircular canal receptors or tonic-phasic otolith neurons. 3. Seventy-five percent of the responsive neurons had a "null plane." The planes of stimulation resulting in maximal responses, for cells that responded to static stimulation, were distributed throughout 150 degrees in both roll and pitch quadrants. Five of these cells responded only transiently during exponential "step" stimulation and responded maximally when stimulated in the plane of one of the vertical semicircular canals. 4. The phase of the response of the 25% of medial NRGc neurons that lacked "null planes" gradually shifted approximately 180 degrees during sinusoidal vestibular stimulation as the plane of stimulation was shifted about the vertical axis

Death of adrenocortical cells during murine acute T. cruzi infection is not associated with TNF-R1 signaling but mostly with the type II pathway of Fas-mediated apoptosis.

PubMed

Pérez, Ana R; Lambertucci, Flavia; González, Florencia B; Roggero, Eduardo A; Bottasso, Oscar A; de Meis, Juliana; Ronco, Maria T; Villar, Silvina R

2017-10-01

Earlier studies from our laboratory demonstrated that acute experimental Trypanosoma cruzi infection promotes an intense inflammation along with a sepsis-like dysregulated adrenal response characterized by normal levels of ACTH with raised glucocorticoid secretion. Inflammation was also known to result in adrenal cell apoptosis, which in turn may influence HPA axis uncoupling. To explore factors and pathways which may be involved in the apoptosis of adrenal cells, together with its impact on the functionality of the gland, we carried out a series of studies in mice lacking death receptors, such as TNF-R1 (C57BL/6- Tnfrsf1a tm1Imx or TNF-R1 -/- ) or Fas ligand (C57BL/6 Fas-deficient lpr mice), undergoing acute T. cruzi infection. Here we demonstrate that the late hypercorticosterolism seen in C57BL/6 mice during acute T. cruzi infection coexists with and hyperplasia and hypertrophy of zona fasciculata, paralleled by increased number of apoptotic cells. Apoptosis seems to be mediated mainly by the type II pathway of Fas-mediated apoptosis, which engages the mitochondrial pathway of apoptosis triggering the cytochrome c release to increase caspase-3 activation. Fas-induced apoptosis of adrenocortical cells is also related with an exacerbated production of intra-adrenal cytokines that probably maintain the late supply of adrenal hormones during host response. Present results shed light on the molecular mechanisms dealing with these phenomena which are crucial not only for the development of interventions attempting to avoid adrenal dysfunction, but also for its wide occurrence in other infectious-based critical illnesses. Copyright © 2017 Elsevier Inc. All rights reserved.

Smooth pursuitlike eye movements evoked by microstimulation in macaque nucleus reticularis tegmenti pontis.

PubMed

Yamada, T; Suzuki, D A; Yee, R D

1996-11-01

1. Smooth pursuitlike eye movements were evoked with low current microstimulation delivered to rostral portions of the nucleus reticularis tegmenti pontis (rNRTP) in alert macaques. Microstimulation sites were selected by the observation of modulations in single-cell firing rates that were correlated with periodic smoothpursuit eye movements. Current intensities ranged from 10 to 120 microA and were routinely < 40 microA. Microstimulation was delivered either in the dark with no fixation, 100 ms after a fixation target was extinguished, or during maintained fixation of a stationary or moving target. Evoked eye movements also were studied under open-loop conditions with the target image stabilized on the retina. 2. Eye movements evoked in the absence of a target rapidly accelerated to a constant velocity that was maintained for the duration of the microstimulation. Evoked eye speeds ranged from 3.7 to 23 deg/s and averaged 11 deg/s. Evoked eye speed appeared to be linearly related to initial eye position with a sensitivity to initial eye position that averaged 0.23 deg.s-1.deg-1. While some horizontal and oblique smooth eye movements were elicited, microstimulation resulted in upward eye movements in 89% of the sites. 3. Evoked eye speed was found to be dependent on microstimulation pulse frequency and current intensity. Within limits, evoked eye speed increased with increases in stimulation frequency or current intensity. For stimulation frequencies < 300-400 Hz, only smooth pursuit-like eye movements were evoked. At higher stimulation frequencies, accompanying saccades consistently were elicited. 4. Feedback of retinal image motion interacted with the evoked eye movements to decrease eye speed if the visual motion was in the opposite direction as the evoked, pursuit-like eye movements. 5. The results implicate rNRTP as part of the neuronal substrate that controls smooth-pursuit eye movements. NRTP appears to be divided functionally into a rostral, pursuit

Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

East, I.J.; Keenan, A.M.; Larson, S.M.

1984-08-31

The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.

Determination of the reactivity of cytotoxic immune cells with preimplantation mouse embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ewoldsen, M.A.

1987-01-01

Cytotoxic immune cells were used in an assay, MELIA (mixed embryo leukocyte interaction assay) to test the ability of the cells to kill blastocyst stage embryos. The cytotoxic immune cells generated for use in this study, cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and lymphokine activated killer (LAK) cells were shown to have phenotypic and cytolytic characteristics similar to those reported by other investigators. The lysis of the blastocysts in the MELIA was determined by measuring the inhibition of blastocoel retention and/or by the inhibition of incorporation of tritiated thymidine (/sup 3/H-TdR) into embryonic DNA. Blastocysts which possess ormore » lack their zonae pellucidae were tested to determine whether the zona pellucida plays an immunoprotective role in preimplantation development. The results indicated that CTLs only lysed embryonic cells when the zona pellucida was absent, but NK and LAK cells lysed embryonic cells whether the zona pellucida was present or absent. The results suggest that the zona pellucida may protect the preimplantation mouse embryo from lysis by CTLs but what protects the embryo from lysis by NK and LAK cells is unclear.« less

Expression of the IGF and the aromatase/estrogen receptor systems in human adrenal tissues from early infancy to late puberty: implications for the development of adrenarche.

PubMed

Belgorosky, Alicia; Baquedano, María Sonia; Guercio, Gabriela; Rivarola, Marco A

2009-03-01

Adrenarche is a process of postnatal sexual maturation occurring in higher primates, in which there is an increase in the secretion of adrenal androgens. It is the consequence of a process of postnatal organogenesis characterized by the development of a new zone in the adrenal cortex, the zona reticularis (ZR). The mechanism of this phenomenon remains poorly understood, suggesting that it might be a multifactorial event. A relationship between circulating IGF-I, insulin sensitivity, and adrenal androgens has been postulated. Boys and girls have different patterns of changes in insulin sensitivity at puberty, perhaps secondary to differences in the estrogen milieu. Estrogen effects may also play a role in premature adrenarche. Peripheral or local IGF-1 actions could regulate adrenal progenitor cell proliferation and migration. Since adrenal progenitor cells as well as IGF-I and the IGF-R1 are located in the outer zone of the adrenal cortex during childhood and adolescence, this peripheral cell layer, below the capsule, may contain undifferentiated progenitor cells. Therefore, the IGF-R1 signaling pathway might positively modulate the proliferation and migration of adrenal progenitor cell to stimulate the development of adrenal zones, including ZR. However, no evidence of a direct action of IGF-I on ZR was found. In addition, a role for estrogens in the ontogenesis of ZR is suggested by the presence of aromatase (CYP19) in the subcapsular zona glomerulosa and in the adrenal medulla. Estrogens produced locally could act on ZR by interacting with estrogen receptor beta (ERbeta), but not alpha, and membrane estrogen receptor GPR-30. An estradiol-induced increase in DHEA/cortisol ratio was indeed seen in cultures of adrenocortical cells from post-adrenarche adrenals. In summary, several lines of evidence point to the action of multiple factors, such as local adrenal maturational changes and peripheral metabolic signals, on postnatal human adrenal gland ZR formation.

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

PubMed

Tao, T; Reichelt, B; Niemann, H

1995-07-01

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

PubMed

Moro, L N; Jarazo, J; Buemo, C; Hiriart, M I; Sestelo, A; Salamone, D F

2015-10-01

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation. © 2015 Blackwell Verlag GmbH.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

PubMed

Wong, Christopher Yee; Mills, James K

2017-03-01

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Axonal ramification of neurons in the nucleus reticularis tegmenti pontis projecting to the paramedian lobule in the rabbit cerebellum.

PubMed

Bukowska, Dorota; Mierzejewska-Krzyzowska, Barbara; Zguczyński, Leszek

2005-01-01

Projections of the nucleus reticularis tegmenti pontis (NRTP) to the cerebellar paramedian lobule were examined in the rabbit by means of the double fluorescent retrograde tract-tracing method. The rabbit NRTP is composed of a medial, large part comprising zones A (dorsomedial), B (central) and C (lateral), and of a lateral, small part (the processus tegmentosus lateralis; PTL). Following unilateral injections of Fast Blue (FB) into the rostral part of the paramedian lobule (rPML) and of Diamidino Yellow (DY) into the caudal part (cPML), known to receive spinal inputs from forelimb and hindlimb, respectively, substantial numbers of single labeled neurons were found in all bilateral NRTP divisions, apart from the zone C. Most projection neurons to the PML were located in the medial and medioventral regions of the zone B. Smaller numbers of projection neurons were located in the PTL, zone A and outside the zone B among fibers of the medial lemniscus. The pattern of FB and DY labeling suggested that neurons projecting to the rPML and cPML originated in common rather than separate regions within the NRTP. In addition, a small percentage (mean 1.3%) of double FB+DY labeled neurons were detected with a clear contralateral preponderance, among single labeled FB or DY cells. In spite of the rarity, all the NRTP neurons giving rise to intralobular collateral projections can be regarded as potential sources of simultaneous modulating influences upon two functional different forelimb (rPML) and hindlimb (cPML) regions. The findings have been discussed in relation to earlier studies in other species and commented on with respect to the possible functional meaning of these projections.

Evidence for electrical synapses between neurons of the nucleus reticularis thalami in the adult brain in vitro.

PubMed

Blethyn, Kate L; Hughes, Stuart W; Crunelli, Vincenzo

2008-03-01

It has been conclusively demonstrated in juvenile rodents that the inhibitory neurons of the nucleus reticularis thalami (NRT) communicate with each other via connexin 36 (Cx36)-based electrical synapses. However, whether functional electrical synapses persist into adulthood is not fully known. Here we show that in the presence of the metabotropic glutamate receptor (mGluR) agonists, trans-ACPD (100 muM) or DHPG (100 muM), 15% of neurons in slices of the adult cat NRT maintained in vitro exhibit stereotypical spikelets with several properties that indicate that they reflect action potentials that have been communicated through an electrical synapse. In particular, these spikelets, i) display a conserved all-or-nothing waveform with a pronounced after-hyperpolarization (AHP), ii) exhibit an amplitude and time to peak that are unaffected by changes in membrane potential, iii) always occur rhythmically with the precise frequency increasing with depolarization, and iv) are resistant to blockers of conventional, fast chemical synaptic transmission. Thus, these results indicate that functional electrical synapses in the NRT persist into adulthood where they are likely to serve as an effective synchronizing mechanism for the wide variety of physiological and pathological rhythmic activities displayed by this nucleus.

Projections of the basilar pontine nuclei and nucleus reticularis tegmenti pontis to the cerebellar nuclei of the rat.

PubMed

Parenti, Rosalba; Zappalà, Agata; Serapide, Maria Francesca; Pantò, Maria Rosita; Cicirata, Federico

2002-10-14

This study showed the precise projection pattern of the basilar pontine nuclei (BPN) and the nucleus reticularis tegmenti pontis (NRTP) to the cerebellar nuclei (CN), as well as the different anatomic features of BPN and NRTP projections. The staining of BPN or NRTP with biotinylated dextran labeled projection fibers to complementary topographic areas in the CN. In fact, BPN principally project to a rostrocaudally oriented column of the nucleus lateralis (NL), which at the midcentral level shifts to the lateroventral part of the nucleus, as well as to the caudolateral part of the nucleus interpositus posterioris. The NRTP projects to a rostrocaudal column of the NL, which at the midcentral level shifts medially, as well as to the nucleus interpositalis and to the caudal part of the nucleus medialis. BPN axons in the CN usually branch into short collaterals of simple morphology that involve small terminal areas, whereas NRTP axons branch into longer collaterals of complex morphology involving terminal areas of different sizes. Each site of injection is at the origin of a set of terminal areas in the CN. The set of projections from different BPN or NRTP areas were partially, but never completely, overlapping. Thus, the set of terminal areas in the CN was specific for each area of both BPN and NRTP. Injection of tetramethyl-rhodamine-dextran-amine into the CN stained cell bodies of BPN and NRTP with different repartition on the two sides. The study showed that CN are innervated by the contralateral BPN and not very much by the ipsilateral BPN, whereas they are innervated by NRTP bilaterally, even if with a contralateral prevalence. In conclusion, this study supports the hypothesis that both BPN and NRTP are concerned in the central program for skilled movements, even if they are probably involved in different functional roles. Copyright 2002 Wiley-Liss, Inc.

Abolished perineuronal nets and altered parvalbumin-immunoreactivity in the nucleus reticularis thalami of wildtype and 3xTg mice after experimental stroke.

PubMed

Härtig, Wolfgang; Appel, Simon; Suttkus, Anne; Grosche, Jens; Michalski, Dominik

2016-11-19

Treatment strategies for ischemic stroke are still limited, since numerous attempts were successful only in preclinical research but failed under clinical condition. To overcome this translational roadblock, clinical relevant stroke models should consider co-morbidities, age-related effects and the complex neurovascular unit (NVU) concept. The NVU includes neurons, vessels and glial cells with astrocytic endfeet in close relation to the extracellular matrix (ECM). However, the role of the ECM after stroke-related tissue damage is poorly understood and mostly neglected for treatment strategies. This study is focused on alterations of perineuronal nets (PNs) as ECM constituents and parvalbumin-containing GABAergic neurons in mice with emphasis on the nucleus reticularis thalami (NRT) in close proximity to the ischemic lesion as induced by a filament-based stroke model. One day after ischemia onset, immunofluorescence-based quantitative analyses revealed drastically declined PNs in the ischemia-affected NRT from 3- and 12-month-old wildtype and co-morbid triple-transgenic (3xTg) mice with Alzheimer-like alterations. Parvalbumin-positive cells decreased numerically in the ischemia-affected NRT, while staining intensity did not differ between the affected and non-affected hemisphere. Additional qualitative analyses demonstrated ischemia-induced loss of PNs and allocated neuropil ECM immunoreactive for aggrecan and neurocan, and impaired immunoreactivity for calbindin, the potassium channel subunit Kv3.1b and the glutamate decarboxylase isoforms GAD65 and GAD67 in the NRT. In conclusion, these data confirm PNs as highly sensitive constituents of the ECM along with impaired neuronal integrity of GABAergic neurons. Therefore, specific targeting of ECM components might appear as a promising strategy for future treatment strategies in stroke. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

PubMed

Morales, P; Vantman, D; Barros, C; Vigil, P

1991-03-01

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.

Naegeli-Franceschetti-Jadassohn Syndrome and Dermatopathia Pigmentosa Reticularis: Two Allelic Ectodermal Dysplasias Caused by Dominant Mutations in KRT14

PubMed Central

Lugassy, Jennie; Itin, Peter; Ishida-Yamamoto, Akemi; Holland, Kristen; Huson, Susan; Geiger, Dan; Hennies, Hans Christian; Indelman, Margarita; Bercovich, Dani; Uitto, Jouni; Bergman, Reuven; McGrath, John A.; Richard, Gabriele; Sprecher, Eli

2006-01-01

Naegeli-Franceschetti-Jadassohn syndrome (NFJS) and dermatopathia pigmentosa reticularis (DPR) are two closely related autosomal dominant ectodermal dysplasia syndromes that clinically share complete absence of dermatoglyphics (fingerprint lines), a reticulate pattern of skin hyperpigmentation, thickening of the palms and soles (palmoplantar keratoderma), abnormal sweating, and other subtle developmental anomalies of the teeth, hair, and skin. To decipher the molecular basis of these disorders, we studied one family with DPR and four families with NFJS. We initially reassessed linkage of NFJS/DPR to a previously established locus on 17q11.2-q21. Combined multipoint analysis generated a maximal LOD score of 8.3 at marker D17S800 at a recombination fraction of 0. The disease interval was found to harbor 230 genes, including a large cluster of keratin genes. Heterozygous nonsense or frameshift mutations in KRT14 were found to segregate with the disease trait in all five families. In contrast with KRT14 mutations affecting the central α-helical rod domain of keratin 14, which are known to cause epidermolysis bullosa simplex, NFJS/DPR-associated mutations were found in a region of the gene encoding the nonhelical head (E1/V1) domain and are predicted to result in very early termination of translation. These data suggest that KRT14 plays an important role during ontogenesis of dermatoglyphics and sweat glands. Among other functions, the N-terminal part of keratin molecules has been shown to confer protection against proapoptotic signals. Ultrastructural examination of patient skin biopsy specimens provided evidence for increased apoptotic activity in the basal cell layer where KRT14 is expressed, suggesting that apoptosis is an important mechanism in the pathogenesis of NFJS/DPR. PMID:16960809

Evidence for electrical synapses between neurons of the nucleus reticularis thalami in the adult brain in vitro

PubMed Central

Blethyn, Kate L.; Hughes, Stuart W.; Crunelli, Vincenzo

2008-01-01

It has been conclusively demonstrated in juvenile rodents that the inhibitory neurons of the nucleus reticularis thalami (NRT) communicate with each other via connexin 36 (Cx36)-based electrical synapses. However, whether functional electrical synapses persist into adulthood is not fully known. Here we show that in the presence of the metabotropic glutamate receptor (mGluR) agonists, trans-ACPD (100 μM) or DHPG (100 μM), 15% of neurons in slices of the adult cat NRT maintained in vitro exhibit stereotypical spikelets with several properties that indicate that they reflect action potentials that have been communicated through an electrical synapse. In particular, these spikelets, i) display a conserved all-or-nothing waveform with a pronounced after-hyperpolarization (AHP), ii) exhibit an amplitude and time to peak that are unaffected by changes in membrane potential, iii) always occur rhythmically with the precise frequency increasing with depolarization, and iv) are resistant to blockers of conventional, fast chemical synaptic transmission. Thus, these results indicate that functional electrical synapses in the NRT persist into adulthood where they are likely to serve as an effective synchronizing mechanism for the wide variety of physiological and pathological rhythmic activities displayed by this nucleus. PMID:18701937

Eye movements and abducens motoneuron behavior after cholinergic activation of the nucleus reticularis pontis caudalis.

PubMed

Márquez-Ruiz, Javier; Escudero, Miguel

2010-11-01

the aim of this work was to characterize eye movements and abducens (ABD) motoneuron behavior after cholinergic activation of the nucleus reticularis pontis caudalis (NRPC). six female adult cats were prepared for chronic recording of eye movements (using the scleral search-coil technique), electroencephalography, electromyography, ponto-geniculo-occipital (PGO) waves in the lateral geniculate nucleus, and ABD motoneuron activities after microinjections of the cholinergic agonist carbachol into the NRPC. unilateral microinjections of carbachol in the NRPC induced tonic and phasic phenomena in the oculomotor system. Tonic effects consisted of ipsiversive rotation to the injected side, convergence, and downward rotation of the eyes. Phasic effects consisted of bursts of rhythmic rapid eye movements directed contralaterally to the injected side along with PGO-like waves in the lateral geniculate and ABD nuclei. Although tonic effects were dependent on the level of drowsiness, phasic effects were always present and appeared along with normal saccades when the animal was vigilant. ABD motoneurons showed phasic activities associated with ABD PGO-like waves during bursts of rapid eye movements, and tonic and phasic activities related to eye position and velocity during alertness. the cholinergic activation of the NRPC induces oculomotor phenomena that are somewhat similar to those described during REM sleep. A precise comparison of the dynamics and timing of the eye movements further suggests that a temporal organization of both NRPCs is needed to reproduce the complexity of the oculomotor behavior during REM sleep.

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

PubMed Central

Baibakov, Boris; Boggs, Nathan A.; Yauger, Belinda; Baibakov, Galina

2012-01-01

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1–3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm–egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy. PMID:22734000

Assessment of Mouse Germinal Vesicle Stage Oocyte Quality by Evaluating the Cumulus Layer, Zona Pellucida, and Perivitelline Space

PubMed Central

Liu, Ying-Lei; Chen, Ying; Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Liang, Cheng-Guang

2014-01-01

To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications. PMID:25144310

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

PubMed Central

Zimmerman, Shawn W.; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K.; Sutovsky, Miriam; Odhiambo, John F.; Powell, Michael D.; Miller, David J.; Sutovsky, Peter

2011-01-01

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

Expression of a functional hyperpolarization-activated current (Ih) in the mouse nucleus reticularis thalami.

PubMed

Rateau, Y; Ropert, N

2006-05-01

The GABAergic neurons of the nucleus reticularis thalami (nRT) express the type 2 hyperpolarization-activated cAMP-sensitive (HCN2) subunit mRNA, but surprisingly, they were reported to lack the hyperpolarization-activated (Ih) current carried by this subunit. Using the voltage-clamp recordings in the thalamocortical slice preparation of the newborn and juvenile mice (P6-P23), we demonstrate that, in the presence of 1 mM barium (Ba2+), the nRT neurons express a slow hyperpolarization-activated inward current, suggesting that the Ih is present but masked in control conditions by K+ leak currents. We investigate the identity of the hyperpolarization-activated current in the nRT by studying its physiological and pharmacological profile in presence of Ba2+. We show that it has voltage- and time-dependent properties typical of the Ih, that it is blocked by cesium and ZD7288, two blockers of the Ih, and that it is carried both by the K+ and Na+ ions. We could also alter the gating characteristics of the hyperpolarization-activated current in the nRT by adding a nonhydrolysable analogue of cAMP to the pipette solution. Finally, using the current-clamp recording, we showed that blocking the hyperpolarization-activated current induced an hyperpolarization correlated with an increase of the R(in) of the nRT neurons. In conclusion, our results demonstrate that the nRT neurons express the Ih with slow kinetics similar to those described for the homomeric HCN2 channels, and we show that the Ih of the nRT contributes to the excitability of the nRT neurons in normal conditions.

Chronic valproic acid treatment triggers increased neuropeptide y expression and signaling in rat nucleus reticularis thalami.

PubMed

Brill, Julia; Lee, Michelle; Zhao, Sheng; Fernald, Russell D; Huguenard, John R

2006-06-21

Valproate (VPA) can suppress absence and other seizures, but its precise mechanisms of action are not completely understood. We investigated whether VPA influences the expression of neuropeptide Y (NPY), an endogenous anticonvulsant. Chronic VPA administration to young rats (300-600 mg.kg(-1).d(-1) in divided doses over 4 d) resulted in a 30-50% increase in NPY mRNA and protein expression in the nucleus reticularis thalami (nRt) and hippocampus, but not in the neocortex, as shown by real-time PCR, radioimmunoassay, and immunohistochemistry. No increased expression was observed after a single acute dose of VPA. Chronic treatment with the pharmacologically inactive VPA analog octanoic acid did not elicit changes in NPY expression. No significant expression changes could be shown for the mRNAs of the Y1 receptor or of the neuropeptides somatostatin, vasoactive intestinal polypeptide, and choleocystokinin. Fewer synchronous spontaneous epileptiform oscillations were recorded in thalamic slices from VPA-treated animals, and oscillation duration as well as the period of spontaneous and evoked oscillations were decreased. Application of the Y1 receptor inhibitor N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine-amide (BIBP3226) enhanced thalamic oscillations, indicating that NPY is released during those oscillations and acts to downregulate oscillatory strength. Chronic VPA treatment significantly potentiated the effect of BIBP3226 on oscillation duration but not on oscillation period. These results demonstrate a novel mechanism for the antiepileptic actions of chronic VPA therapy.

The natural history of autoimmune Addison's disease with a non-classical presentation: a case report and review of literature.

PubMed

Manso, Jacopo; Pezzani, Raffaele; Scarpa, Riccardo; Gallo, Nicoletta; Betterle, Corrado

2018-05-24

Autoimmune Addison's disease (AAD) is the most frequent cause of adrenocortical insufficiency. The natural history of AAD usually comprises five consecutive stages with the first stage characterized by the increase of plasma renin consistent with the impairment of pars glomerulosa, which is usually the first affected layer of the adrenal cortex. We describe a 19-year-old female with Hashimoto's thyroiditis (HT) who underwent an autoantibody screening due to having the personal and family history of other autoimmune diseases in the absence of relevant clinical manifestations. She was positive for adrenal cortex autoantibodies (ACA) and steroid 21-hydroxylase autoantibodies (21-OH Ab) at high titers. She had increased basal levels of ACTH with normal basal cortisol not responding to ACTH stimulation, reduced levels of dehydroepiandrosterone-sulfate but normal levels of orthostatic renin and aldosterone. This scenario was consistent with a subclinical AAD presenting with first impairments in pars fasciculata and reticularis and conserved pars glomerulosa function. Only subsequently, progressive deficiency in pars glomerulosa function has become evident. Review of the literature showed that there was only one case, reported to date, with a similar atypical natural history of AAD. The strategies for screening for ACA/21-OH Ab in patients with HT are discussed.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

PubMed

Skrzyszowska, M; Smorag, Z; Katska, L

1997-09-01

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

Differential effects of NMDA antagonists microinjections into the nucleus reticularis pontis caudalis on seizures induced by pentylenetetrazol in the rat.

PubMed

Manjarrez, J; Alvarado, R; Camacho-Arroyo, I

2001-07-01

It has been shown that NMDA antagonists block the tonic but not the clonic component of seizures when they are injected in the oral region of the rat pontine reticular formation (PRF). The participation of the caudal PRF in the effects of NMDA antagonists upon the tonic and the clonic components of generalized seizures induced by pentylenetetrazol (PTZ) is unknown. The aim of the present study was to evaluate the effects of unilateral microinjections of competitive and non-competitive NMDA antagonists, 2-amino-7-phosphonoheptanoic acid (AP-7) and dizocilpine (MK-801), respectively, into the nucleus reticularis pontis caudalis of the rat PRF upon seizures induced by PTZ (70 mg/kg i.p.). MK-801 induced a dose-related decrease both in the incidence of generalized tonic-clonic seizures (GTCS) and in the presence of spikes in the EEG. MK-801 also increased GTCS latency. On the contrary, AP-7 did not have effects on GTCS. Interestingly, it induced ipsilateral circling behavior. These results suggest that in the caudal region of the rat PRF only non-competitive NMDA antagonists should block the generation of tonic and clonic components of generalized seizures.

CB1 receptor mediated analgesia from the Nucleus Reticularis Gigantocellularis pars alpha is activated in an animal model of neuropathic pain.

PubMed

Monhemius, R; Azami, J; Green, D L; Roberts, M H

2001-07-20

Cannabinoids are known to suppress responses to noxious stimulation in animals and man. Recent research has suggested a role for endogenous cannabinoids in the descending inhibition of dorsal horn cells via a supraspinal site of action. We have recently demonstrated [J. Physiol. 506(2) (1998) 459] that the nucleus reticularis gigantocellularis pars alpha (GiA) is a major source of such descending modulation, and importantly, that this system is activated in response to noxious stimulation. We have therefore investigated the role of CB1 receptor activation in mediating the antinociceptive effects of activation of GiA in models of acute and chronic pain. Microinjections (0.5 microl 60% DMSO) of either WIN 55,212-2 (5 microg, selective CB1 agonist), SR141716A (50 microg, competitive CB1 antagonist), both compounds together, or vehicle alone into GiA were performed prior to these tests in a randomised, blind manner. In control animals, WIN 55,212-2 markedly increased withdrawal latencies in the tail flick test and reduced responses to subcutaneous formalin. These effects were blocked by co-administration of SR141716A. These data suggest that activation of cannabinoid CB1 receptor subtypes in GiA leads to behavioural analgesia. In animals with partial sciatic nerve ligation, microinjection of drugs and injection of formalin were performed contralaterally to the site of ligation. Partial sciatic nerve ligation significantly reduced behavioural responses to contralaterally applied formalin. Microinjection of SR141716A to GiA reversed this inhibition of responses to formalin in animals with partial sciatic nerve ligation. These data provide evidence that endogenous CB1 receptor ligands are involved in GiA mediated antinociception, and that this system is important for the modulation of nociceptive transmission in an animal model of chronic neuropathic pain.

ACTH Modulates PTP-PEST Activity and Promotes Its Interaction With Paxillin.

PubMed

Gorostizaga, Alejandra Beatriz; Mori Sequeiros Garcia, M Mercedes; Acquier, Andrea B; Lopez-Costa, Juan J; Mendez, Carlos F; Maloberti, Paula M; Paz, Cristina

2016-09-01

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in-gel PTP assays have shown the activation of a 115-kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP-PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP-PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT-PCR studies revealed PTP-PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP-PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non-ACTH-treated rats increased KM and VM . Finally, in-gel PTP assays of immunoprecipitated paxillin from control and ACTH-treated rats suggested a hormone-mediated increase in paxillin-PTP115 interaction, while PTP-PEST and paxillin co-localize in Y1 cells. Taken together, these data demonstrate PTP-PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH-induced PTP-PEST-paxillin interaction. J. Cell. Biochem. 117: 2170-2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Electrical stimulation of rhesus monkey nucleus reticularis gigantocellularis. I. Characteristics of evoked head movements.

PubMed

Quessy, Stephan; Freedman, Edward G

2004-06-01

The nucleus reticularis gigantocellularis (NRG) receives monosynaptic input from the superior colliculus (SC) and projects directly to neck motor neuron pools. Neurons in NRG are well situated to play a critical role in transforming SC signals into head movement commands. A previous study of movements evoked by NRG stimulation in the primate reported a variety of ipsilateral and contralateral head movements with horizontal, vertical and torsional components. In addition to head movements, it was reported that NRG stimulation could evoke movements of the pinnae, face, upper torso, and co-contraction of neck muscles. In this report, the role of the rhesus monkey NRG in head movement control was investigated using electrical stimulation of the rostral portion of the NRG. The goal was to characterize head movements evoked by NRG stimulation, describe the effects of altering stimulation parameters, and assess the relative movements of the eyes and head. Results indicate that electrical stimulation in the rostral portion of the NRG of the primate can consistently evoke ipsilateral head rotations in the horizontal plane. Head movement amplitude and peak velocity depend upon stimulation parameters (primarily frequency and duration of stimulation trains). During stimulation-induced head movements the eyes counter-rotate (presumably a result of the vestibulo-ocular reflex: VOR). At 46 stimulation sites from two subjects the average gain of this counter-rotation was -0.38 (+/-0.18). After the end of the stimulation train the head generally continued to move. During this epoch, after electrical stimulation ceased, VOR gain remained at this reduced level. In addition, VOR gain was similarly low when electrical stimulation was carried out during active fixation of a visual target. These data extend existing descriptions of head movements evoked by electrical stimulation of the NRG, and add to the understanding of the role of this structure in producing head movements.

Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation.

PubMed

Gadella, B M

2012-01-01

Recent findings have refined our thinking on sperm interactions with the cumulus-oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona-cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm-ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.

Collateral projections of nucleus raphe dorsalis neurones to the caudate-putamen and region around the nucleus raphe magnus and nucleus reticularis gigantocellularis pars alpha in the rat.

PubMed

Li, Y Q; Kaneko, T; Mizuno, N

2001-02-16

It was examined whether or not the nucleus raphe dorsalis (RD) neurons projecting to the caudate-putamen (CPu) might also project to the motor-controlling region around the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (Gia) in the rat. Single RD neurons projecting to the CPu and NRM/Gia by way of axon collaterals were identified by the retrograde double-labeling method with fluorescent dyes, Fast Blue and Diamidino Yellow, which were injected respectively into the CPu and NRM/Gia. Then, serotonin (5-HT)-like immunoreactivity of the double-labeled RD neurons was examined immunohistochemically; approximately 60% of the double-labeled RD neurons showed 5-HT-like immunoreactivity. The results indicated that some of serotonergic and non-serotonergic RD neurons might control motor functions simultaneously at the levels of the CPu and NRM/Gia by way of axon collaterals.

Distribution of CGRP in the minipig brainstem.

PubMed

Lisardo Sánchez, Manuel; Vecino, Elena; Coveñas, Rafael

2014-05-01

For the first time, an in-depth study has been made of the distribution of fibers and cell bodies containing calcitonin gene-related peptide (CGRP) in the minipig brainstem using an indirect immunoperoxidase technique. The animals studied were not treated with colchicine. Cell bodies containing CGRP were found in 20 nuclei/regions of the brainstem. These perikarya were located in somatomotor, brachiomotor and raphae nuclei, nucleus ambiguus, substantia nigra, nucleus reticularis tegmenti pontis, nucleus prepositus hypoglossi, nuclei olivaris inferior and superior, nuclei pontis, formatio reticularis, nucleus dorsalis tegmenti of Gudden, and in the nucleus reticularis lateralis. Fourteen of the 20 brainstem nuclei showed a high density of immunoreactive cell bodies. In comparison with other species, the minipig, together with the rat, show the most widespread distribution of cell bodies containing CGRP in the mammalian brainstem. Immunoreactive fibers were also observed in the brainstem. However, in the minipig brainstem the density of these fibers is low, as in many brainstem nuclei only single immunoreactive fibers were observed. A high density of immunoreactive fibers was only observed in the pars caudalis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis tegmenti of Gudden. According to the observed anatomical distribution of the immunoreactive structures containing CGRP, the peptide could be involved in motor, somatosensory, gustative, and autonomic mechanisms. Copyright © 2014 Wiley Periodicals, Inc.

Neuronal basis of the slow (<1 Hz) oscillation in neurons of the nucleus reticularis thalami in vitro.

PubMed

Blethyn, Kate L; Hughes, Stuart W; Tóth, Tibor I; Cope, David W; Crunelli, Vincenzo

2006-03-01

During deep sleep and anesthesia, the EEG of humans and animals exhibits a distinctive slow (<1 Hz) rhythm. In inhibitory neurons of the nucleus reticularis thalami (NRT), this rhythm is reflected as a slow (<1 Hz) oscillation of the membrane potential comprising stereotypical, recurring "up" and "down" states. Here we show that reducing the leak current through the activation of group I metabotropic glutamate receptors (mGluRs) with either trans-ACPD [(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid] (50-100 microM) or DHPG [(S)-3,5-dihydroxyphenylglycine] (100 microM) instates an intrinsic slow oscillation in NRT neurons in vitro that is qualitatively equivalent to that observed in vivo. A slow oscillation could also be evoked by synaptically activating mGluRs on NRT neurons via the tetanic stimulation of corticothalamic fibers. Through a combination of experiments and computational modeling we show that the up state of the slow oscillation is predominantly generated by the "window" component of the T-type Ca2+ current, with an additional supportive role for a Ca2+-activated nonselective cation current. The slow oscillation is also fundamentally reliant on an Ih current and is extensively shaped by both Ca2+- and Na+-activated K+ currents. In combination with previous work in thalamocortical neurons, this study suggests that the thalamus plays an important and active role in shaping the slow (<1 Hz) rhythm during deep sleep.

Residual effect of a 7-amino metabolite of clonazepam on GABAA receptor function in the nucleus reticularis thalami of the rat.

PubMed

Munakata, Mitsutoshi; Tsuchiya, Shigeru

2008-10-01

A considerable amount of 7-aminoclonazepam (ACZP), a major metabolite of clonazepam (CZP), is present in the brain during CZP treatment, yet the pharmacological properties of ACZP remain unknown. We investigated the effects of ACZP on the GABA(A) receptor-mediated currents (I(GABA)) in neurons from the nucleus reticularis thalami (NRT) of the rat, using a nystatin-perforated patch technique. Neurons in which CZP (10 nM) exerted prominent augmentation (>100% augmentation) of I(GABA), which comprised 32% of the neurons tested, were included for the analysis of ACZP. In these neurons, ACZP augmented I(GABA), which was blocked by 10 microM flumazenil, a benzodiazepine receptor (BZR) antagonist. The half-maximal effective concentration of ACZP was 124 nM, whereas that of CZP was 1.8 nM. The maximal enhancements induced by ACZP and CZP were 38% and 170%, respectively. In neurons from the ventrobasal complex of the thalamus, the effect of ACZP was negligible. Our results suggest that ACZP was a weak partial BZR agonist and that ACZP may competitively modify the effect of CZP, leading to clinical consequences for patients with high levels of ACZP.

Use of Monoclonal Antibodies to Study the Structural Basis of the Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

DTIC Science & Technology

1989-03-16

nucleus robustus archistriatalis 1 1 1 nucleus reticularis gigantocellularis 1 3 3 nucleus reticularis lateralis 1 3 3 nucleus ... reticularis pontis caudalis 1 1 3 nucleus reticularis parvocellularis 1 1 2 nucleus rotundus 1 1 1 nucleus tractus solitarii 1 3 3 nucleus semilunaris...Structure a-bungarotoxin mAb 35 inAb 270 nucleus accumbens 1 1 1 nucleus basalis 1 1 1 nucleus cerebelli intermedium 2 3 3

The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid.

PubMed

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny; Huszar, Gabor

2014-05-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

PubMed Central

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

2014-01-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Lean, A.; Cantin, M.

1986-03-05

The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levelsmore » of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with (/sup 32/P) NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production.« less

Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding.

PubMed

Hou, S T; Ma, A; Jones, R; Hall, L

1996-10-01

The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.

The practical side of immunocontraception: zona proteins and wildlife.

PubMed

Kirkpatrick, J F; Rowan, A; Lamberski, N; Wallace, R; Frank, K; Lyda, R

2009-12-01

With shrinking habitat, the humane control of certain wildlife populations is relevant. The contraceptive vaccine based on native porcine zona pellucida (PZP) has been applied to various wildlife populations for 20 years. Prominent efforts include wild horses, urban deer, zoo animals and African elephants, among others. This approach has been successful in managing entire populations and to date, no significant debilitating short- or long-term health effects have been documented.

Polycystic ovarian disease: the adrenal connection.

PubMed

Marouliss, George B; Triantafillidis, Ioannis K

2006-01-01

Polycystic ovarian disease (PCOD) is characterized by hyperandrogenemia, ovulatory dysfunction and polycystic ovaries (PCO). The increased androgen production in PCOD comes primarily from the ovaries. However, in about 40% of patients there is excessive adrenal androgen production (DHEA, DHEA-Sulfate, Androstenedione, Testosterone and Dihydrotestosterone). The contribution of the adrenal in the PCOD is suggested by the presence of adrenal androgen excess in PCO, the presence of PCO in women with enzymatic adrenal hyperplasia as well as in women with adrenal tumors. However, the cause of adrenal androgen hypersecretion is not yet fully understood but it may include endogenous hypersecretion of the zona reticularis of unclear cause, hypersecretion of cortical-androgen-stimulating hormone (CASH), stress, hyperprolactinemia, adrenal enzymatic defects etc. This short review covers the aspects of adrenal androgen hypersecretion in PCOD.

Effect of pharmacological inactivation of nucleus reticularis tegmenti pontis on saccadic eye movements in the monkey.

PubMed

Kaneko, Chris R S; Fuchs, Albert F

2006-06-01

The superior colliculus (SC) provides signals for the generation of saccades via a direct pathway to the brain stem burst generator (BG). In addition, it sends saccade-related activity to the BG indirectly through the cerebellum via a relay in the nucleus reticularis tegmenti pontis (NRTP). Lesions of the oculomotor vermis, lobules VIc and VII, and inactivation of the caudal fastigial nucleus, the cerebellar output nucleus to which it projects, produce saccade dysmetria but have little effect on saccade peak velocity and duration. We expected similar deficits from inactivation of the NRTP. Instead, injections as small as 80 nl into the NRTP first slowed ipsiversive saccades and then gradually reduced their amplitudes. Postinjection saccades had slower peak velocities and longer durations than preinjection saccades with similar amplitudes. Contraversive saccades retained their normal kinematics. When the gains of ipsiversive saccades to 10 degrees target steps had fallen to their lowest values (0.28 +/- 0.19; mean +/- SD; n = 10 experiments), the gains of contraversive saccades to 10 degrees target steps had decreased very little (0.82 +/- 0.11). Eventually, ipsiversive saccades did not exceed 5 degrees , even to 20 degrees target steps. Moreover, these small remaining saccades apparently were made with considerable difficulty because their latencies increased substantially. When ipsiversive saccade gain was at its lowest, the gain and kinematics of vertical saccades to 10 degrees target steps exhibited inconsistent changes. We argue that our injections did not compromise the direct SC pathway. Therefore these data suggest that the cerebellar saccade pathway does not simply modulate BG activity but is required for horizontal saccades to occur at all.

Effect of Pharmacological Inactivation of Nucleus Reticularis Tegmenti Pontis on Saccadic Eye Movements in the Monkey

PubMed Central

Kaneko, Chris R. S.; Fuchs, Albert F.

2006-01-01

The superior colliculus (SC) provides signals for the generation of saccades via a direct pathway to the brain stem burst generator (BG). In addition, it sends saccade-related activity to the BG indirectly through the cerebellum via a relay in the nucleus reticularis tegmenti pontis (NRTP). Lesions of the oculomotor vermis, lobules VIc and VII, and inactivation of the caudal fastigial nucleus, the cerebellar output nucleus to which it projects, produce saccade dysmetria but have little effect on saccade peak velocity and duration. We expected similar deficits from inactivation of the NRTP. Instead, injections as small as 80 nl into the NRTP first slowed ipsiversive saccades and then gradually reduced their amplitudes. Postinjection saccades had slower peak velocities and longer durations than preinjection saccades with similar amplitudes. Contraversive saccades retained their normal kinematics. When the gains of ipsiversive saccades to 10° target steps had fallen to their lowest values (0.28 ± 0.19; mean ± SD; n = 10 experiments), the gains of contraversive saccades to 10° target steps had decreased very little (0.82 ± 0.11). Eventually, ipsiversive saccades did not exceed 5°, even to 20° target steps. Moreover, these small remaining saccades apparently were made with considerable difficulty because their latencies increased substantially. When ipsiversive saccade gain was at its lowest, the gain and kinematics of vertical saccades to 10° target steps exhibited inconsistent changes. We argue that our injections did not compromise the direct SC pathway. Therefore these data suggest that the cerebellar saccade pathway does not simply modulate BG activity but is required for horizontal saccades to occur at all. PMID:16467420

Organization of cerebellar and area "y" projections to the nucleus reticularis tegmenti pontis in macaque monkeys.

PubMed

Stanton, G B

2001-04-02

Axonal projections to the nucleus reticularis tegmenti pontis (RTP) were studied in 11 macaque monkeys by mapping axonal degeneration from lesions centered in the dentate and interpositus anterior (IA) nuclei and by mapping anterograde transport of tritiated amino acid precursors injected into the dentate nucleus. Projections from the dentate and IA nuclei overlap in central parts of the body of RTP, but the terminal field of dentate axons extends dorsomedial and rostral to the terminal field of IA axons, and IA terminal fields extend more ventrolaterally. A caudal to rostral topography of projections from each nucleus onto dorsal to ventral parts of RTP was seen. Projections from rostral parts of both nuclei terminate in a sublemniscal part of the nucleus. The topography of dentate and IA projections onto central to ventrolateral RTP appears to match somatotopic maps of these cerebellar nuclei with the somatotopic map of projections to RTP from primary motor cortex. Projections from caudal and ventral parts of the dentate nucleus appear to overlap oculomotor inputs to rostral, dorsal, and medial RTP from the frontal and supplementary eye fields, the superior colliculus, and the oculomotor region of the caudal fastigial nucleus. Projections to the paramedian part of RTP from vestibular area "y" were also found in two cases that correlated with projections to vertical oculomotor motoneurons. The maps of dentate and IA projections onto RTP correlate predictably with maps of dentate and IA projections to the ventrolateral thalamus and subnuclei of the red nucleus that were made from these same cases (Stanton [1980b] J. Comp. Neurol. 192:377-385). Copyright 2001 Wiley-Liss, Inc.

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Que, Emily L.; Duncan, Francesca E.; Bayer, Amanda R.

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy,more » transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. Finally, this adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.« less

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

DOE PAGES

Que, Emily L.; Duncan, Francesca E.; Bayer, Amanda R.; ...

2017-01-19

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy,more » transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. Finally, this adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.« less

GABA release in the zona incerta of the sheep in response to the sight and ingestion of food and salt.

PubMed

Kendrick, K M; Hinton, M R; Baldwin, B A

1991-05-31

In order to establish which neurotransmitters may influence the activity of zona incerta neurones in the sheep which respond selectively to the sight or ingestion of food, we have measured the release of amino acid and monoamine neurotransmitters from this region using microdialysis sampling. Co-ordinates for the placement of microdialysis probes in regions of the zona incerta where cells respond to the sight or ingestion of food were first established by making single-unit extracellular recordings. When animals were food-deprived results showed that release of gamma-aminobutyric acid (GABA) was increased in response to the sight and ingestion of food but not of aspartate, glutamate, taurine, noradrenaline, dopamine or serotonin. This release of GABA was absent when the animals were shown non-food objects or saw or ingested salt solutions. When the same animals were physiologically sodium-depleted GABA release was evoked by the sight and ingestion of salt solutions and release following the sight and ingestion of food was significantly reduced. These results provide further evidence that GABA is an important neurotransmitter in neural circuits controlling the regulation of food intake.

Inhibitory effect of the nucleus reticularis pontis oralis on the pontine micturition center and pontine urine storage center in decerebrate cats.

PubMed

Sugaya, Kimio; Nishijima, Saori; Miyazato, Minoru; Oda, Masami; Ogawa, Yoshihide

2006-10-01

The influence of the nucleus reticularis pontis oralis (PoO) on the pontine micturition center (PMC) and pontine urine storage center (PUSC) was examined in decerebrate cats by electrical and chemical stimulations of the PMC, PUSC or PoO. Microinjection of carbachol into the rostral and dorsolateral part of the PoO rapidly inhibited reflex micturition and external urethral sphincter (EUS) activity. After confirming the inhibition of reflex micturition and EUS activity by microinjection of carbachol into the PoO, intravenous injection of atropine sulfate or its microinjection into the PoO recovered both reflex micturition and EUS activity. Microinjection of carbachol into the PMC evoked micturition and then inhibited reflex micturition, but intravenous injection of atropine or its microinjection into the PoO recovered reflex micturition. After confi rming the inhibition of reflex micturition and EUS activity by microinjection of carbachol into the PoO, electrical stimulation of the PUSC enhanced EUS activity, but electrical stimulation of the PMC failed to evoke micturition. However, electrical stimulation of the PMC evoked micturition after microinjection of atropine into the PoO. These results suggest that the PoO strongly inhibits the PMC and less strongly inhibits the PUSC. Therefore, the PoO seems to be the pontine micturition inhibitory area.

Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type.

PubMed

Lagutina, Irina; Lazzari, Giovanna; Duchi, Roberto; Colleoni, Silvia; Ponderato, Nunzia; Turini, Paola; Crotti, Gabriella; Galli, Cesare

2005-10-01

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

The behavioural importance of dynamically activated descending inhibition from the nucleus reticularis gigantocellularis pars alpha.

PubMed

Azami, J; Green, D L; Roberts, M H; Monhemius, R

2001-05-01

We have recently demonstrated (J Physiol 506 (1998) 459) that the dynamic activation of descending inhibition of the nociceptive response of spinal multireceptive cells occurs in the nucleus reticularis gigantocellularis pars alpha (GiA). In the same paper we have shown that Lamina I dorsal horn cells are responsible for activating this inhibition via a pathway which runs in the contralateral dorsolateral funiculus. The effects of dynamically activating this system by noxious stimulation on behavioural responses to noxious stimuli have not been established. Here we demonstrate the effects of GiA on the behavioural response during application of standardized noxious stimuli. As this system is activated in response to noxious stimulation (J Physiol 506 (1998) 459), it is possible that chronic pain states may also activate GiA. We have therefore investigated this possibility in animals following partial sciatic nerve ligation (an animal model of chronic pain; Pain 43 (1990) 205). Male Wistar rats (280-310 g) were anaesthetized with halothane (0.5-2% in O(2)). Guide cannulae for microinjections were stereotaxically placed above GiA. In one group of animals the sciatic nerve was partially ligated. Animals were allowed to recover for 4-6 days. The responses of each animal during the formalin test (Pain 4 (1977) 161) and the tail flick test (Pain 12 (1982) 229) were recorded on different days. Microinjections (0.5 microl) of either gamma-aminobutyric acid (GABA, 200 mM), D-L homocysteic acid (DLH, 25 mM) or 0.9% saline (as control) into GiA were performed during these tests in a randomized, blind manner. In animals without sciatic nerve ligation, microinjection of GABA to GiA did not significantly affect the animal's response during the tail flick test. However microinjection of DLH significantly increased the latency of tail flick from 6.2 +/- 0.8 to 8.4 +/- 0.5 s for up to 15 min (n = 7, P < 0.01, Mann-Whitney U-test). Microinjection of GABA to GiA increased the

Body mass index is not associated with sperm-zona pellucida binding ability in subfertile males.

PubMed

Sermondade, Nathalie; Dupont, Charlotte; Faure, Céline; Boubaya, Marouane; Cédrin-Durnerin, Isabelle; Chavatte-Palmer, Pascale; Sifer, Christophe; Lévy, Rachel

2013-09-01

Lifestyle factors, such as weight and nutritional status may affect male fertility, including sperm fertilization ability. The objective of this retrospective study was to evaluate the association between body mass index (BMI) and sperm-zona pellucida binding ability assessed according to the zona binding (ZB) test, which has been described to be a relevant diagnostic tool for the prediction of in vitro fertilization (IVF) ability. Three hundred and six male patients from couples diagnosed with primary idiopathic or mild male factor infertility were included. Correlations between BMI and semen parameters according to ZB test indices were assessed, together with frequencies of positive and negative tests across the BMI categories. In this selected population, BMI was not related to conventional semen parameters or sperm quality assessed according to the ability of spermatozoa to bind to the zona pellucida. The previously described poor outcomes of IVF procedures in cases of male obesity could be due to other sperm defects, such as alterations of sperm capacitation or acrosome reaction. The link between male BMI and biological outcomes during IVF procedures, such as fertilization rates, should be further evaluated.

Multiple zonal projections of the nucleus reticularis tegmenti pontis to the cerebellar cortex of the rat.

PubMed

Serapide, M F; Parenti, R; Pantò, M R; Zappalà, A; Cicirata, F

2002-06-01

Compartmentalization (alternating labelled and unlabelled stripes) of mossy fibre terminals was found in the cerebellar cortex after iontophoretic injections of biotinylated dextran amine into discrete regions of the nucleus reticularis tegmenti pontis (NRTP). The zonal pattern was only observed when volumes of nuclear tissue ranging from 4.5 x 106 to 17.66 x 106 microm3 were impregnated. Up to nine compartments (i.e. up to five stripes separated by four interstripes) were found in crus I and in vermal lobule VI. Up to seven compartments (four stripes and three interstripes) were found in crus II; up to five compartments (three stripes and two interstripes) were identified in the lobulus simplex, the paraflocculus and vermal lobules IV, V and VII; up to three compartments (two stripes and one interstripe) were identified in the paramedian lobule and, finally, up to two compartments (one stripe and one interstripe) were identified in the copula pyramidis, in the flocculus and in vermal lobules II, III, VIII and IX. The projections of the NRTP are arranged according to a divergent/convergent projection pattern. From single injections in the NRTP, projections were traced to a set of cortical stripes widely distributed over the cerebellar cortex. The set of stripes labelled from different regions of the NRTP partially overlapped but complete overlap was never found. This finding revealed that the topographic combination of the projections of the NRTP to the cerebellar cortex is specific for each region of the NRTP. Finally, the projections to single cortical areas were arranged according to a pattern of compartmentalization that is specific for each cortical area, independent of the site of injection in the NRTP and of the number of stripes evident in the cortex.

Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

PubMed

Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

2013-07-01

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

Phosphorylation of zona occludens-2 by protein kinase C epsilon regulates its nuclear exportation.

PubMed

Chamorro, David; Alarcón, Lourdes; Ponce, Arturo; Tapia, Rocio; González-Aguilar, Héctor; Robles-Flores, Martha; Mejía-Castillo, Teresa; Segovia, José; Bandala, Yamir; Juaristi, Eusebio; González-Mariscal, Lorenza

2009-09-01

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation.

PubMed

Zhang, Xiaobing; van den Pol, Anthony N

2017-05-26

The neuronal substrate for binge eating, which can at times lead to obesity, is not clear. We find that optogenetic stimulation of mouse zona incerta (ZI) γ-aminobutyric acid (GABA) neurons or their axonal projections to paraventricular thalamus (PVT) excitatory neurons immediately (in 2 to 3 seconds) evoked binge-like eating. Minimal intermittent stimulation led to body weight gain; ZI GABA neuron ablation reduced weight. ZI stimulation generated 35% of normal 24-hour food intake in just 10 minutes. The ZI cells were excited by food deprivation and the gut hunger signal ghrelin. In contrast, stimulation of excitatory axons from the parasubthalamic nucleus to PVT or direct stimulation of PVT glutamate neurons reduced food intake. These data suggest an unexpected robust orexigenic potential for the ZI GABA neurons. Copyright © 2017, American Association for the Advancement of Science.

Immunomodulatory/inflammatory effects of geopropolis produced by Melipona fasciculata Smith in combination with doxorubicin on THP-1 cells.

PubMed

Oliveira, Lucas Pires Garcia; Conte, Fernanda Lopes; Cardoso, Eliza de Oliveira; Conti, Bruno José; Santiago, Karina Basso; Golim, Marjorie de Assis; Cruz, Maria Teresa; Sforcin, José Maurício

2016-12-01

Geopropolis (GEO) in combination with doxorubicin (DOX) reduced HEp-2 cells viability compared to GEO and DOX alone. A possible effect of this combination on the innate immunity could take place, and its effects were analysed on THP-1 cell - a human leukaemia monocytic cell line used as a model to study monocyte activity and macrophage activity, assessing cell viability, expression of cell markers and cytokine production. THP-1 cells were incubated with GEO, DOX and their combination. Cell viability was assessed by MTT assay, cell markers expression by flow cytometry and cytokine production by ELISA. GEO + DOX did not affect cell viability. GEO alone or in combination increased TLR-4 and CD80 but not HLA-DR and TLR-2 expression. GEO stimulated TNF-α production while DOX alone or in combination did not affect it. GEO alone or in combination inhibited IL-6 production. GEO exerted a pro-inflammatory profile by increasing TLR-4 and CD80 expression and TNF-α production, favouring the activation of the immune/inflammatory response. GEO + DOX did not affect cell viability and presented an immunomodulatory action. Lower concentrations of DOX combined to GEO could be used in cancer patients, avoiding side effects and benefiting from the biological properties of GEO. © 2016 Royal Pharmaceutical Society.

Smooth-pursuit eye-movement deficits with chemical lesions in macaque nucleus reticularis tegmenti pontis.

PubMed

Suzuki, D A; Yamada, T; Hoedema, R; Yee, R D

1999-09-01

Anatomic and neuronal recordings suggest that the nucleus reticularis tegmenti pontis (NRTP) of macaques may be a major pontine component of a cortico-ponto-cerebellar pathway that subserves the control of smooth-pursuit eye movements. The existence of such a pathway was implicated by the lack of permanent pursuit impairment after bilateral lesions in the dorsolateral pontine nucleus. To provide more direct evidence that NRTP is involved with regulating smooth-pursuit eye movements, chemical lesions were made in macaque NRTP by injecting either lidocaine or ibotenic acid. Injection sites first were identified by the recording of smooth-pursuit-related modulations in neuronal activity. The resulting lesions caused significant deficits in both the maintenance and the initiation of smooth-pursuit eye movements. After lesion formation, the gain of constant-velocity, maintained smooth-pursuit eye movements decreased, on the average, by 44%. Recovery of the ability to maintain smooth-pursuit eye movements occurred over approximately 3 days when maintained pursuit gains attained normal values. The step-ramp, "Rashbass" task was used to investigate the effects of the lesions on the initiation of smooth-pursuit eye movements. Eye accelerations averaged over the initial 80 ms of pursuit initiation were determined and found to be decremented, on the average, by 48% after the administration of ibotenic acid. Impairments in the initiation and maintenance of smooth-pursuit eye movements were directional in nature. Upward pursuit seemed to be the most vulnerable and was impaired in all cases independent of lesioning agent and type of pursuit investigated. Downward smooth pursuit seemed more resistant to the effects of chemical lesions in NRTP. Impairments in horizontal tracking were observed with examples of deficits in ipsilaterally and contralaterally directed pursuit. The results provide behavioral support for the physiologically and anatomic-based conclusion that NRTP is a

Laterally Versus Medially Projecting Spinothalamic Neurons and their Axon Collaterals to the Periaqueductal Gray and Medullary Reticular Formation in the Rat

DTIC Science & Technology

1987-06-30

nucleus reticularis gigantocellularis. No distinct tracts were reported in the brainstem as far rostral as the superior olivary complex. At the level...that stimulation of the PAG 12 activates neurons which project to the MRF, specifically the nucleus reticularis gigantocellularis, nucleus ... reticularis magnocellularis and the nucleus raphe magnus. Neurons in the raphe magnus receive convergent input from the PAG and other MRF regions and, via

Comparison of projection neurons in the pontine nuclei and the nucleus reticularis tegmenti pontis of the rat.

PubMed

Schwarz, C; Thier, P

1996-12-16

Dendritic features of identified projection neurons in two precerebellar nuclei, the pontine nuclei (PN) and the nucleus reticularis tegmenti pontis (NRTP) were established by using a combination of retrograde tracing (injection of fluorogold or rhodamine labelled latex micro-spheres into the cerebellum) with subsequent intracellular filling (lucifer yellow) in fixed slices of pontine brainstem. A multivariate analysis revealed that parameters selected to characterize the dendritic tree such as size of dendritic field, number of branching points, and length of terminal dendrites did not deviate significantly between different regions of the PN and the NRTP. On the other hand, projection neurons in ventral regions of the PN were characterized by an irregular coverage of their distal dendrites by appendages while those in the dorsal PN and the NRTP were virtually devoid of them. The NRTP, dorsal, and medial PN tended to display larger somata and more primary dendrites than ventral regions of the PN. These differences, however, do not allow the differentiation of projection neurons within the PN from those in the NRTP. They rather reflect a dorso-ventral gradient ignoring the border between the nuclei. Accordingly, a cluster analysis did not differentiate distinct types of projection neurons within the total sample. In both nuclei, multiple linear regression analysis revealed that the size of dendritic fields was strongly correlated with the length of terminal dendrites while it did not depend on other parameters of the dendritic field. Thus, larger dendritic fields seem not to be accompanied by a higher complexity but rather may be used to extend the reach of a projection neuron within the arrangement of afferent terminals. We suggest that these similarities within dendritic properties in PN and NRTP projection neurons reflect similar processing of afferent information in both precerebellar nuclei.

Sponge mass mortalities in a warming Mediterranean Sea: are cyanobacteria-harboring species worse off?

PubMed

Cebrian, Emma; Uriz, Maria Jesus; Garrabou, Joaquim; Ballesteros, Enric

2011-01-01

Mass mortality events are increasing dramatically in all coastal marine environments. Determining the underlying causes of mass mortality events has proven difficult in the past because of the lack of prior quantitative data on populations and environmental variables. Four-year surveys of two shallow-water sponge species, Ircinia fasciculata and Sarcotragus spinosulum, were carried out in the western Mediterranean Sea. These surveys provided evidence of two severe sponge die-offs (total mortality ranging from 80 to 95% of specimens) occurring in the summers of 2008 and 2009. These events primarily affected I. fasciculata, which hosts both phototrophic and heterotrophic microsymbionts, while they did not affect S. spinosulum, which harbors only heterotrophic bacteria. We observed a significant positive correlation between the percentage of injured I. fasciculata specimens and exposure time to elevated temperature conditions in all populations, suggesting a key role of temperature in triggering mortality events. A comparative ultrastructural study of injured and healthy I. fasciculata specimens showed that cyanobacteria disappeared from injured specimens, which suggests that cyanobacterial decay could be involved in I. fasciculata mortality. A laboratory experiment confirmed that the cyanobacteria harbored by I. fasciculata displayed a significant reduction in photosynthetic efficiency in the highest temperature treatment. The sponge disease reported here led to a severe decrease in the abundance of the surveyed populations. It represents one of the most dramatic mass mortality events to date in the Mediterranean Sea.

Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl-DDE and o,p'-DDD in Y-1 adrenal cells.

PubMed

Hermansson, Veronica; Asp, Vendela; Bergman, Ake; Bergström, Ulrika; Brandt, Ingvar

2007-11-01

The environmental pollutant 3-MeSO(2)-DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p'-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO(2)-DDE and o,p'-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO(2)-DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p'-DDD reached similar levels of binding as 3-MeSO(2)-DDE. The binding of o,p'-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO(2)-DDE, but not of o,p'-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO(2)-DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO(2)-DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO(2)-DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO(2)-DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO(2)-DDE and o,p'-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice

Tonic suppression of spontaneous baroreceptor reflex by endogenous angiotensins via AT(2) subtype receptors at nucleus reticularis ventrolateralis in the rat.

PubMed

Lin, K; Chan, S H; Chan, J Y

2001-04-01

We evaluated the role of endogenous angiotensins at the rostral nucleus reticularis ventrolateralis (NRVL) in the modulation of spontaneous baroreceptor reflex (BRR) response and the subtype of angiotensin receptors involved using rats anesthetized and maintained with pentobarbital sodium. Bilateral microinjection of angiotensin II (ANG II) or its active metabolite angiotensin III (ANG III) (5, 10, or 20 pmol) into the NRVL significantly suppressed the spontaneous BRR response, as represented by the magnitude of transfer function between systemic arterial pressure and heart rate signals. The inhibitory effect of ANG III (20 pmol) was discernibly reversed by coadministration with its peptide antagonist, [Ile(7)]ANG III (1.6 nmol), or the nonpeptide AT(2) receptor antagonist, PD-123319 (1.6 nmol), but not by the nonpeptide AT(1) receptor antagonist, losartan (1.6 nmol). On the other hand, the peptide antagonist, [Sar(1), Ile(8)]ANG II (1.6 nmol) or both non-peptide antagonists appreciably reversed the suppressive action of ANG II (20 pmol). Whereas losartan produced minimal effect, blocking the endogenous activity of the angiotensins by microinjection into the bilateral NRVL of PD-123319, [Sar(1), Ile(8)]ANG II or [Ile(7)]ANG III elicited significant enhancement of the spontaneous BRR response. We conclude that under physiologic conditions both endogenous ANG II and ANG III may exert a tonic inhibitory modulation on the spontaneous BRR response by acting selectively on the AT(2) subtype receptors at the NRVL. Copyright 2001 Wiley-Liss, Inc.

Distribution of methionine-enkephalin in the minipig brainstem.

PubMed

Sánchez, Manuel Lisardo; Vecino, Elena; Coveñas, Rafael

2013-05-01

We have studied the distribution of immunoreactive cell bodies and axons are containing methionine-enkephalin in the minipig brainstem. Immunoreactive axons were widely distributed, whereas the distribution of perikarya was less widespread. A high or moderate density of axons containing methionine-enkephalin were found from rostral to caudal levels in the substantia nigra, nucleus interpeduncularis, nucleus reticularis tegmenti pontis, nucleus dorsalis raphae, nucleus centralis raphae, nuclei dorsalis and ventralis tegmenti of Gudden, locus ceruleus, nucleus sensorius principalis nervi trigemini, nucleus cuneatus externalis, nucleus tractus solitarius, nuclei vestibularis inferior and medialis, nucleus ambiguus, nucleus olivaris inferior and in the nucleus tractus spinalis nervi trigemini. Immunoreactive perikarya were observed in the nuclei centralis and dorsalis raphae, nucleus motorius nervi trigemini, nucleus centralis superior, nucleus nervi facialis, nuclei parabrachialis medialis and lateralis, nucleus ventralis raphae, nucleus reticularis lateralis and in the formatio reticularis. We have also described the presence of perikarya containing methionine-enkephalin in the nuclei nervi abducens, ruber, nervi oculomotorius and nervi trochlearis. These results suggest that in the minipig the pentapeptide may be involved in many physiological functions (for example, proprioceptive and nociceptive information; motor, respiratory and cardiovascular mechanisms). Copyright © 2013 Elsevier B.V. All rights reserved.

Viscous forces are predominant in the zona pellucida mechanical resistance

NASA Astrophysics Data System (ADS)

Papi, Massimiliano; Maiorana, Alessandro; Douet, Cécile; Maulucci, Giuseppe; Parasassi, Tiziana; Brunelli, Roberto; Goudet, Ghylène; De Spirito, Marco

2013-01-01

The zona pellucida (ZP) is a multilayer glycoprotein spherical shell surrounding mammalian eggs. The ZP's mechanical response plays a crucial role in mammalian fertilization and is a parameter commonly adopted in "in vitro fertilization" to characterize the oocytes quality. While it is assumed that ZP mechanical response is purely elastic, here we prove that dissipative forces cannot be neglected. Physiologically, this evidence implies that an increase in the spermatozoa motility can induce dramatic changes on the ZP reaction force turning ZP shell in an impenetrable barrier leading to fertility impairments.

Use of Both Cumulus Cells’ Transcriptomic Markers and Zona Pellucida Birefringence to Select Developmentally Competent Oocytes in Human Assisted Reproductive Technologies

PubMed Central

2015-01-01

Background Selection of the best oocyte for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in human infertility treatment procedure. Oocyte selection using morphological criteria mainly Zona pellucida (ZP) has been the gold standard method in assisted reproductive technologies (ART) clinics, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs-based approaches have allowed the identification of key molecular markers that quantitatively and non-invasively predict the oocyte quality for higher pregnancy rates and efficient infertility treatment. These biomarkers are a valuable reinforcement of the morphological selection criteria widely used in in vitro fertilization (IVF) clinics. In this context, this study was designed to investigate the relationship between transcriptomic predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality mainly the ZP birefringence. Results Microarray data revealed that 48 and 27 differentially expressed candidate genes in cumulus cells (CCs) were respectively overexpressed and underexpressed in the ZGP (Zona Good Pregnant) versus ZBNP (Zona Bad Non Pregnant) groups. More than 70% of previously reported transcriptomic biomarkers of oocyte developmental competence were confirmed in this study. The analysis of possible association between ZP birefringence versus molecular markers approach showed an absence of correlation between them using the current set of markers. Conclusions This study suggested a new integrative approach that matches morphological and molecular approaches used to select developmentally competent oocytes able to lead to successful pregnancy and the delivery of healthy baby. For each ZP birefringence score, oocytes displayed a particular CCs' gene expression pattern. However, no correlations were found between the 7 gene biomarkers of oocyte developmental

Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements.

PubMed

Florman, H M; First, N L

1988-08-01

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

Electrical stimulation of rhesus monkey nucleus reticularis gigantocellularis. II. Effects on metrics and kinematics of ongoing gaze shifts to visual targets.

PubMed

Freedman, Edward G; Quessy, Stephan

2004-06-01

Saccade kinematics are altered by ongoing head movements. The hypothesis that a head movement command signal, proportional to head velocity, transiently reduces the gain of the saccadic burst generator (Freedman 2001, Biol Cybern 84:453-462) can account for this observation. Using electrical stimulation of the rhesus monkey nucleus reticularis gigantocellularis (NRG) to alter the head contribution to ongoing gaze shifts, two critical predictions of this gaze control hypothesis were tested. First, this hypothesis predicts that activation of the head command pathway will cause a transient reduction in the gain of the saccadic burst generator. This should alter saccade kinematics by initially reducing velocity without altering saccade amplitude. Second, because this hypothesis does not assume that gaze amplitude is controlled via feedback, the added head contribution (produced by NRG stimulation on the side ipsilateral to the direction of an ongoing gaze shift) should lead to hypermetric gaze shifts. At every stimulation site tested, saccade kinematics were systematically altered in a way that was consistent with transient reduction of the gain of the saccadic burst generator. In addition, gaze shifts produced during NRG stimulation were hypermetric compared with control movements. For example, when targets were briefly flashed 30 degrees from an initial fixation location, gaze shifts during NRG stimulation were on average 140% larger than control movements. These data are consistent with the predictions of the tested hypothesis, and may be problematic for gaze control models that rely on feedback control of gaze amplitude, as well as for models that do not posit an interaction between head commands and the saccade burst generator.

Distribution of Neurotensin and Somatostatin-28 (1-12) in the Minipig Brainstem.

PubMed

Sánchez, M L; Vecino, E; Coveñas, R

2016-08-01

Using an indirect immunoperoxidase technique, an in depth study has been carried out for the first time on the distribution of fibres and cell bodies containing neurotensin and somatostatin-28 (1-12) (SOM) in the minipig brainstem. The animals used were not treated with colchicine. The distribution of neurotensin- and SOM-immunoreactive fibres was seen to be quite similar and was moderate in the minipig brainstem: a close anatomical relationship between both neuropeptides was observed. The distribution of cell bodies containing neurotensin or SOM was quite different and restricted. Cell bodies containing neurotensin were found in four brainstem nuclei: nucleus centralis raphae, nucleus dorsalis raphae, in the pars centralis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis raphae. Cell bodies containing SOM were found in six nuclei/regions of the brainstem: nucleus ambiguus, nucleus dorsalis motorius nervi vagus, formatio reticularis, nucleus parabrachialis medialis, nucleus reticularis lateralis and nucleus ventralis raphae. According to the observed anatomical distribution of the immunoreactive structures containing neurotensin or SOM, the peptides could be involved in sleep-waking, nociceptive, gustatory, motor, respiratory and autonomic mechanisms. © 2015 Blackwell Verlag GmbH.

Adrenal androgen secretion and dopaminergic activity in anorexia nervosa.

PubMed

Devesa, J; Pérez-Fernández, R; Bokser, L; Gaudiero, G J; Lima, L; Casanueva, F F

1988-01-01

The aim of the present study was to investigate if the postulated deficient adrenal androgen secretion in Anorexia Nervosa (AN), could be associated with a status of sustained dopaminergic hyperactivity. The adrenal responses to ACTH and PRL response to dopaminergic receptor blockade were studied in seven patients with Anorexia Nervosa and seven regularly menstruating women. AN patients showed lower baseline DHEA-sulphate (DHEA-S), androstenedione (Adione) and prolactin (PRL) levels than controls. The response to ACTH revealed evidences of significantly decreased 17-20 desmolase activity in AN, with apparent predominance of glucocorticoid over androgenic pathways relative to controls. Because dopaminergic receptor blockade with Domperidone (DOM) showed intense dopaminergic hyperactivity in AN, we postulate that the adrenal regression seen in the disease is the consequence of a reduced zona reticularis as a consequence of the lack of trophic support by PRL and/or intermediate lobe proopiomelanocortin (IL-POMC). This is consistent with our previous results in pre-adrenarchal dogs and rabbits.

[Preparation and application of anti-ouabain IgY antibody].

PubMed

Zhang, Ming-juan; Yang, Jun; Duan, Zong-ming; Qiang, Lei

2007-09-01

To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues. Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry. The IgY titer increased rapidly after the second immunization, with the highest titer of 1:10240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation (CV) of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex. Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.

In-vitro developmental potential of individual mouse blastomeres cultured with and without zona pellucida: future implications for human assisted reproduction.

PubMed

Illmensee, K; Kaskar, K; Zavos, P M

2006-08-01

This study was designed to compare the developmental potential of individual blastomeres derived from 2-, 4-, 6- and 8-cell mouse embryos cultured with and without zona pellucida (ZP). In the first series, one, three, five and seven blastomeres were biopsied from 2-, 4-, 6- and 8-cell embryos respectively, and inserted individually into empty ZP recipients, leaving the remaining blastomere within its original ZP. In the second series, the same protocol was used except that the biopsied blastomeres were cultured without ZP and compared with the remaining blastomere within its original ZP. For the first series, individual blastomeres derived from 2-, 4-, 6- and 8-cell embryos cultured with ZP showed blastocyst development of 82.4, 68.6, 44.4 and 23.1% respectively, with corresponding hatching rates of 70.6, 60.0, 25.9 and 7.7%. For the second series, individual blastomeres cultured without ZP progressed with blastocyst development of 73.3, 64.5, 35.7 and 22.7% respectively. Blastocyst multiplication was achieved most efficiently when using individual blastomeres from 4- and 6-cell embryos. This is the first report on comparative in-vitro propagation of single blastomeres derived from various cleavage stages in a mammalian species. Blastomere cloning with its multiple applications may be envisaged for human assisted reproductive technologies.

Time-lapse monitoring of zona pellucida-free embryos obtained through in vitro fertilization: a retrospective case series.

PubMed

Bodri, Daniel; Kato, Ryutaro; Kondo, Masae; Hosomi, Naoko; Katsumata, Yoshinari; Kawachiya, Satoshi; Matsumoto, Tsunekazu

2015-05-01

To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators. Retrospective case series. Private infertility clinic. Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014. Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle. In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns. Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

PubMed Central

Hung, Pei-hsuan; Miller, Marion G.; Meyers, Stuart A.; VandeVoort, Catherine A.

2008-01-01

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 μM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque. PMID:18480469

The basilar pontine nuclei and the nucleus reticularis tegmenti pontis subserve distinct cerebrocerebellar pathways.

PubMed

Cicirata, Federico; Serapide, Maria Francesca; Parenti, Rosalba; Pantò, Maria Rosita; Zappalà, Agata; Nicotra, Annalisa; Cicero, Deborah

2005-01-01

Previous studies often considered the basilar pontine nuclei (BPN) and the nucleus reticularis tegmenti pontis (NRTP) as relays of a single cerebro-(ponto)-cerebellar pathway. Conversely, the different cortical afferences to the BPN and the NRTP, as well as the anatomical and functional features of the cerebellopetal projections from these pontine nuclei, support the different, and for some aspect, complementary arrangement of the cerebrocerebellar pathways relayed by the BPN or NRTP. Both the BPN and the NRTP are innervated from the cerebral cortex, but with regional prevalence. The NRTP is principally innervated from motor or sensori-motor areas while the BPN are principally innervated from sensory, mainly teloceptive, and associative area. Projections from sensory-motor areas were also traced to the BPN. The BPN and NRTP project to all parts of the cerebellar cortex with a similar pattern. In fact, from single areas of them projections were traced to set of sagittal stripes of the cerebellar cortex. In variance to such analogies, the projections to the cerebellar nuclei differed between those traced from the NRTP and from BPN. In fact, BPN and NRTP have private terminal areas in the cerebellar nuclei with relatively little overlaps. The BPN innervated the lateroventral part of the nucleus lateralis and the caudoventral aspect of the nucleus interpositalis posterioris. The NRTP principally innervated the mediodorsal part of the nucleus lateralis, the nucleus interpositalis anterioris, the nucleus medialis. Since the single cerebellar nuclei have their specific targets in the extracerebellar brain areas, it follows that the BPN and the NRTP, passing through their cerebellar nuclei relays, are devoted to control different brain areas and thus likely to play different functional roles. From single pontine regions (of both BPN and NRTP) projections were traced to the cerebellar cortex and to the cerebellar nuclei. In some cases these projections reached areas which

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

PubMed

Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J

2017-05-01

Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in

INDUCTION OF ZONA RADIATA PROTEINS AND VITELLOGENINS IN ESTRADIOL AND NONYLPHENOL EXPOSED MALE SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)

EPA Science Inventory

Knoebl, Iris, Michael J. Hemmer and Nancy D. Denslow. 2004. Induction of Zona Radiata Proteins and Vitellogenins in Estradiol and Nonylphenol Exposed Male Sheepshead Minnows (Cyprinodon variegatus). Mar. Environ. Res. 58(2-5):547-551. (ERL,GB X1059).

Several genes normall...

[Microinjections of heat shock protein 70 kDa into the nucleus reticularis pontis oralis induce inhibition of rapid eye movement sleep in pigeons].

PubMed

Gusel'nikova, E A; Pastukhov, Iu F

2008-03-01

Recently it was indicated that microinjections of heat shock proteins 70 kDa (Hsp70) into the third ventricle of brain in pigeons results in an increase in the duration of slow wave sleep and a decrease in somato-visceral indices. It is suggested that Hsp70 effect may be related to GABA(A) receptors activation in the preoptic area of the hypothalamus. However, what transmitter mechanisms of activation are related to the removal effect (in 2-3 hrs) of rapid eye movement sleep inhibition still remains poorly understood. To solve this problem in the present study, microinjections of Hsp70 into the Nucleus reticularis pontis oralis (NRPO) were done. It is well known that cholinergic neurons of the NRPO are crucial for rapid eye movement sleep generation. The data show that Hsp70 produces more early (for first two hrs) a decrease in number of episodes and total time of rapid eye movement sleep, a diminution of electroencephalogram (EEG) power spectra in the 9-14 Hz band, a decrease in contractile muscle activity and brain temperature. It is suggested that Hsp70 effects are realized due to activation of GABA(A) receptors in the NRPO and induced inhibition of cholinergic mechanisms of rapid eye movement sleep triggering. The microinjections of Hsp70 into the NRPO increase the slow wave sleep total time with long latency (for 8-12 hrs). This effect may be related to influence of Hsp70 on neurons population, which are responsible for slow wave sleep maintenance outside the NRPO.

Strong, reliable and precise synaptic connections between thalamic relay cells and neurones of the nucleus reticularis in juvenile rats.

PubMed

Gentet, Luc J; Ulrich, Daniel

2003-02-01

The thalamic reticular nucleus (nRT) is composed entirely of GABAergic inhibitory neurones that receive input from pyramidal cortical neurones and excitatory relay cells of the ventrobasal complex of the thalamus (VB). It plays a major role in the synchrony of thalamic networks, yet the synaptic connections it receives from VB cells have never been fully physiologically characterised. Here, whole-cell current-clamp recordings were obtained from 22 synaptically connected VB-nRT cell pairs in slices of juvenile (P14-20) rats. At 34-36 degrees C, single presynaptic APs evoked unitary EPSPs in nRT cells with a peak amplitude of 7.4 +/- 1.5 mV (mean +/- S.E.M.) and a decay time constant of 15.1 +/- 0.9 ms. Only four out of 22 pairs showed transmission failures at a mean rate of 6.8 +/- 1.1 %. An NMDA receptor (NMDAR)-mediated component was significant at rest and subsequent EPSPs in a train were depressed. Only one out of 14 pairs tested was reciprocally connected; the observed IPSPs in the VB cell had a peak amplitude of 0.8 mV and were completely abolished in the presence of 10 microM bicuculline. Thus, synaptic connections from VB cells to nRT neurones are mainly 'drivers', while a small subset of cells form closed disynaptic loops.

Strong, reliable and precise synaptic connections between thalamic relay cells and neurones of the nucleus reticularis in juvenile rats

PubMed Central

Gentet, Luc J; Ulrich, Daniel

2003-01-01

The thalamic reticular nucleus (nRT) is composed entirely of GABAergic inhibitory neurones that receive input from pyramidal cortical neurones and excitatory relay cells of the ventrobasal complex of the thalamus (VB). It plays a major role in the synchrony of thalamic networks, yet the synaptic connections it receives from VB cells have never been fully physiologically characterised. Here, whole-cell current-clamp recordings were obtained from 22 synaptically connected VB-nRT cell pairs in slices of juvenile (P14–20) rats. At 34–36 °C, single presynaptic APs evoked unitary EPSPs in nRT cells with a peak amplitude of 7.4 ± 1.5 mV (mean ± s.e.m.) and a decay time constant of 15.1 ± 0.9 ms. Only four out of 22 pairs showed transmission failures at a mean rate of 6.8 ± 1.1 %. An NMDA receptor (NMDAR)-mediated component was significant at rest and subsequent EPSPs in a train were depressed. Only one out of 14 pairs tested was reciprocally connected; the observed IPSPs in the VB cell had a peak amplitude of 0.8 mV and were completely abolished in the presence of 10 μm bicuculline. Thus, synaptic connections from VB cells to nRT neurones are mainly ‘drivers’, while a small subset of cells form closed disynaptic loops. PMID:12563005

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

PubMed Central

Patrat, Catherine; Auer, Jana; Fauque, Patricia; Leandri, Roger L; Jouannet, Pierre; Serres, Catherine

2006-01-01

Background The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes. Results The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. Conclusion The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans. PMID:17147816

Direct and indirect nigrofugal projections to the nucleus reticularis pontis caudalis mediate in the motor execution of the acoustic startle reflex.

PubMed

Hormigo, Sebastian; López, Dolores E; Cardoso, Antonio; Zapata, Gladys; Sepúlveda, Jacqueline; Castellano, Orlando

2018-07-01

The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.

Oocytes with a dark zona pellucida demonstrate lower fertilization, implantation and clinical pregnancy rates in IVF/ICSI cycles.

PubMed

Shi, Wei; Xu, Bo; Wu, Li-Min; Jin, Ren-Tao; Luan, Hong-Bing; Luo, Li-Hua; Zhu, Qing; Johansson, Lars; Liu, Yu-Sheng; Tong, Xian-Hong

2014-01-01

The morphological assessment of oocytes is important for embryologists to identify and select MII oocytes in IVF/ICSI cycles. Dysmorphism of oocytes decreases viability and the developmental potential of oocytes as well as the clinical pregnancy rate. Several reports have suggested that oocytes with a dark zona pellucida (DZP) correlate with the outcome of IVF treatment. However, the effect of DZP on oocyte quality, fertilization, implantation, and pregnancy outcome were not investigated in detail. In this study, a retrospective analysis was performed in 268 infertile patients with fallopian tube obstruction and/or male factor infertility. In 204 of these patients, all oocytes were surrounded by a normal zona pellucida (NZP, control group), whereas 46 patients were found to have part of their retrieved oocytes enclosed by NZP and the other by DZP (Group A). In addition, all oocytes enclosed by DZP were retrieved from 18 patients (Group B). No differences were detected between the control and group A. Compared to the control group, the rates of fertilization, good quality embryos, implantation and clinical pregnancy were significantly decreased in group B. Furthermore, mitochondria in oocytes with a DZP in both of the two study groups (A and B) were severely damaged with several ultrastructural alterations, which were associated with an increased density of the zona pellucida and vacuolization. Briefly, oocytes with a DZP affected the clinical outcome in IVF/ICSI cycles and appeared to contain more ultrastructural alterations. Thus, DZP could be used as a potential selective marker for embryologists during daily laboratory work.

Anxiety-like behavior in transgenic mice with brain expression of neuropeptide Y.

PubMed

Inui, A; Okita, M; Nakajima, M; Momose, K; Ueno, N; Teranishi, A; Miura, M; Hirosue, Y; Sano, K; Sato, M; Watanabe, M; Sakai, T; Watanabe, T; Ishida, K; Silver, J; Baba, S; Kasuga, M

1998-01-01

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in behavior and its disorders. To understand the long-term modulation of neuronal functions by NPY, we raised transgenic mice created with a novel central nervous system (CNS) neuron-specific expression vector of human Thy- gene fragment linked to mouse NPY cDNA. In situ hybridization analysis demonstrated transgene-derived NPY expression in neurons (e.g., in the hippocampus, cerebral cortex, and the arcuate nucleus of the hypothalamus) in the transgenic mice. The modest increase of NPY protein in the brain was demonstrated by semiquantitative immunohistochemical analysis and by radioreceptor assay (115% in transgenic mice compared to control littermates). Double-staining experiments indicated colocalization of the transgene-derived NPY message and NPY protein in the same neurons, such as in the arcuate nucleus. The transgenic mice displayed behavioral signs of anxiety and hypertrophy of adrenal zona fasciculata cells, but no change in food intake was observed. The anxiety-like behavior of transgenic mice was reversed, at least in part, by administration of corticotropin-releasing factor (CRF) antagonists, alpha-helical CRF9-41, into the third cerebral ventricle. These results suggest that NPY has a role in anxiety and behavioral responses to stress partly via the CRF neuronal system. This genetic model may provide a unique opportunity to study human anxiety and emotional disorders.

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

2005-09-01

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell

Electrocortical and behavioral responses elicited by acute electrical stimulation of inferior thalamic peduncle and nucleus reticularis thalami in a patient with major depression disorder.

PubMed

Velasco, Marcos; Velasco, Francisco; Jiménez, Fiacro; Carrillo-Ruiz, José D; Velasco, Ana Luisa; Salín-Pascual, Rafael

2006-02-01

Our aim was to study electrocortical and behavioral responses elicited by 6, 60 and 3/s stimulation of the inferior thalamic peduncle (ITP) and nucleus reticularis thalami (Re) in a patient with of major depression disorder resistant to psychotherapy, pharmacotherapy and electroconvulsive therapy and candidate to be treated by electrical stimulation of the ITP. In this patient, two multicontact electrodes were implanted bilaterally through frontal coronal parasagittal burr-holes with oblique trajectories aiming ITP and Re. Stimulation was performed through externalized systems. Referential scalp electroencephalographic (EEG) recordings were performed and subjective sensations and clinical symptoms reported by patient and changes in responsiveness in single response tasks during stimulation trials were systematically recorded. Unilateral, low (6/s) and high (60/s) frequency stimulation of either ITP or Re produced identical recruiting-like responses or desynchronization-DC shift changes predominant at frontopolar region, bilaterally. Billateral, high intensity 3/s stimulation or either ITP or Re produced electrocortical responses that consisted in generalized 3/s spike-wave complexes predominant at frontopolar, frontocentral and frontotemporal regions. However, while ITP responses were accompanied by all symptoms described for a spontaneous absence attack, Re responses were behaviorly accompanied only by delayed reaction time. These data suggests that in humans as in cats, ITP and Re are both part of a non-specific thalamo-orbitofrontal system normally engaged in cortical synchronization, selective attention and sleep. Under abnormal conditions, ITP and RE may play a role in the physiopathology of typical absence attacks and depression disorders.

Cold laser technique for cell surgery

NASA Astrophysics Data System (ADS)

Palanker, Daniel V.; Ohad, Shoshanit; Lewis, Aaron; Laufer, Neri

1992-08-01

A new cell surgery technique has been developed to produce well-defined alterations in cells and tissues without detectable heating and/or other structural damage in the surroundings. The technique involves the use of a 193 nm argon fluoride excimer laser which is guided through a glass pipette filled with a positive air pressure. To demonstrate the method holes were drilled in the zona pellucida of mouse oocytes. The diameter of the drilled hole was determined by the pipette tip size, and its depth by an energy emitted per pulse and number of pulses. Scanning electron microscopy of the drilled mouse oocytes showed uniform, round, well circumscribed holes with sharp edges. Oocytes that had their zona pellucida drilled with this new method fertilized in vitro and developed to the blastocyst stage in a rate similar to that of control group. These results demonstrate the non-perturbing nature of this cold laser microsurgical procedure. In addition to the extension of our results for clinical in vitro fertilization purposes, such as enhancement of fertilization and embryo biopsy, there are wide ranging possible uses of our method in fundamental and applied investigations that require sub-micron accuracy in cellular alteration.

The roles of NMDA receptor activation and nucleus reticularis gigantocellularis in the time-dependent changes in descending inhibition after inflammation.

PubMed

Terayama, R; Dubner, R; Ren, K

2002-05-01

Previous studies indicate that descending modulation of nociception is progressively increased following persistent inflammation. The present study was designed to further examine the role of supraspinal neurons in descending modulation following persistent inflammation. Constant levels of paw withdrawal (PW) and tail flick (TF) latencies to noxious heat stimuli were achieved in lightly anesthetized rats (pentobarbital sodium 3-10 mg/kg/h, i.v.). Electrical stimulation (ES, 0.1 ms, 100 Hz, 20-200 A) was delivered to the rostral ventromedial medulla (RVM), mainly the nucleus raphe magnus (NRM). ES produced intensity-dependent inhibition of PW and TF. Following a unilateral hindpaw inflammation produced by injection of complete Freund's adjuvant (CFA), ES-produced inhibition underwent time-dependent changes. There was an initial decrease at 3 h after inflammation and a subsequent increase after inflammation in the excitability of RVM neurons and the inhibition of nocifensive responses. These changes were most robust after stimulation of the inflamed paw although similar findings were seen on the non-inflamed paw and tail. The inflammation-induced dynamic changes in descending modulation appeared to be correlated with changes in the activation of the N-methyl--aspartate (NMDA) excitatory amino acid receptor. Microinjection of an NMDA receptor antagonist, AP5 (1 pmol), resulted in an increase in the current intensity required for inhibition of the PW and TF. The effect of AP5 was less at 3 h after inflammation and significantly greater at 11-24 h after inflammation. In a subsequent experiment, ES-produced inhibition of nocifensive responses after inflammation was examined following selective chemical lesions of the nuclei reticularis gigantocellularis (NGC). Compared to vehicle-injected animals, microinjection of a soma-selective excitotoxin, ibotenic acid, enhanced ES-produced inhibition at 3 h but not at 24 h after inflammation. We propose that these time course

Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida.

PubMed

Körschgen, Hagen; Kuske, Michael; Karmilin, Konstantin; Yiallouros, Irene; Balbach, Melanie; Floehr, Julia; Wachten, Dagmar; Jahnen-Dechent, Willi; Stöcker, Walter

2017-09-01

How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. We isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test. Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like

Glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamazaki, T.S.; Nagahama, Y.; Iuchi, I.

1989-05-01

A glycoprotein from the liver, which shares epitopes with chorion (egg envelope or zona pellucida) glycoproteins, is present only in the spawning female fish, Oryzias latipes, under natural conditions. This spawning female-specific (SF) substance is distinct from vitellogenin but closely resembles a major glycoprotein component, ZI-3, of the inner layer (zona radiata interna) of the ovarian egg envelope with respect to some biochemical and immunochemical characteristics. Here we report that the (/sup 125/I)SF substance, injected into the abdominal cavity of the spawning female fish, was rapidly transported by the blood circulation into the ovary and incorporated into the inner layermore » of egg envelope of the growing oocytes. The result strongly suggests that the SF substance from the liver is a precursor substance of the major component, ZI-3, of the inner layer of egg envelope in the fish.« less

Blockade of Opioid Receptors in the Medullary Reticularis Nucleus Dorsalis, but not the Rostral Ventromedial Medulla, Prevents Analgesia Produced by Diffuse Noxious Inhibitory Control in Rats With Muscle Inflammation

PubMed Central

de Resende, Marcos A.; Silva, Luis Felipe S.; Sato, Karina; Arendt-Nielsen, Lars; Sluka, Kathleen A.

2014-01-01

Diffuse Noxious Inhibitory Controls (DNIC) involves application of a noxious stimulus outside the testing site to produce analgesia. In human subjects with a variety of chronic pain conditions, DNIC is less effective; however, in animal studies, DNIC is more effective after tissue injury. While opioids are involved in DNIC analgesia, the pathways involved in this opioid-induced analgesia are not clear. The aim of the present study was to test the effectiveness of DNIC in inflammatory muscle pain, and to study which brainstem sites mediate DNIC- analgesia. Rats were injected with 3% carrageenan into their gastrocnemius muscle and responses to cutaneous and muscle stimuli were assessed before and after inflammation, and before and after DNIC induced by noxious heat applied to the tail (45°C and 47°C). Naloxone was administered systemically, into rostral ventromedial medulla (RVM), or bilaterally into the medullary reticularis nucleus dorsalis (MdD) prior to the DNIC-conditioning stimuli. DNIC produced a similar analgesic effect in both acute and the chronic phases of inflammation reducing both cutaneous and muscle sensitivity in a dose-dependent manner. Naloxone systemically or microinjected into the MdD prevented DNIC-analgesia, while naloxone into the RVM had no effect on DNIC analgesia. Thus, DNIC analgesia involves activation of opioid receptors in the MdD. PMID:21330219

Incentivos para atraer y retener personal de salud de zonas rurales del Perú: un estudio cualitativo

PubMed Central

Huicho, Luis; Canseco, Francisco Díez; Lema, Claudia; Miranda, J. Jaime; Lescano, Andrés G.

2014-01-01

El objetivo fue identificar incentivos de atracción y retención en zonas rurales y distantes de Ayacucho, Perú. Fueron realizadas entrevistas en profundidad con 80 médicos, enfermeras, obstetras y técnicos (20 por grupo) de las zonas más pobres y con 11 funcionarios. No existen políticas sistemáticas de atracción y retención de personal de salud en Ayacucho. Los principales incentivos, en orden de importancia, fueron mejoras salariales, oportunidades de formación y capacitación, estabilidad laboral y nombramiento, mejoras en infraestructura y equipos, e incremento del personal. Se mencionaron también mejoras en la vivienda y alimentación, mayor cercanía con la familia y reconocimiento por el sistema de salud. Existen coincidencias y singularidades entre los distintos grupos sobre los incentivos clave para estimular el trabajo rural, que deben considerarse al diseñar políticas públicas. Las iniciativas del Estado deben comprender procesos rigurosos de monitoreo y evaluación, para asegurar que las mismas tengan el impacto deseado. PMID:22488318

Combinatorial effects of geopropolis produced by Melipona fasciculata Smith with anticancer drugs against human laryngeal epidermoid carcinoma (HEp-2) cells.

PubMed

Bartolomeu, Ariane Rocha; Frión-Herrera, Yahima; da Silva, Livia Matsumoto; Romagnoli, Graziela Gorete; de Oliveira, Deilson Elgui; Sforcin, José Maurício

2016-07-01

The identification of natural products exerting a combined effect with therapeutic agents could be an alternative for cancer treatment, reducing the concentration of the drugs and side effects. Geopropolis (Geo) is produced by some stingless bees from a mixture of vegetable resins, gland secretions of the bees and soil. It has been used popularly as an antiseptic agent and to treat respiratory diseases and dermatosis. To determine whether Geo enhances the anticancer effect of carboplatin, methotrexate and doxorubicin (DOX), human laryngeal epidermoid carcinoma (HEp-2) cells were treated with Geo alone or in combination with each drug. Cell growth, cytotoxicity and apoptosis were evaluated using 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release, and flow cytometry. Scratch assay was used to analyze cell migration and transmission electron microscopy to observe morphologic alterations. The influence of Geo on drug resistance was also investigated assessing P-glycoprotein (P-gp) action. Geo inhibited cell proliferation and migration. The combination Geo+DOX led to the highest cytotoxic activity and induced apoptosis, leading to loss of membrane integrity. Geo had no effect on P-gp-mediated efflux of DOX. Data indicate that Geo combined with DOX could be a potential clinical chemotherapeutic approach for laryngeal cancer treatment. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Sediment and Nutrient Sources as well as Interspecific Competition Control Growth of 2 Common Species of Coral Reef Macroalgae

NASA Astrophysics Data System (ADS)

Moore, T.; Fong, P.; Cuker, B.

2016-02-01

Aquatic communities worldwide are increasingly subjected to multiple anthropogenic stressors that often result in shifts in structure and function. On coral reefs, human impacts have been associated with phase-shifts from coral to algal domination. We hypothesized that the proliferation of these algal communities, especially on fringing reefs, may be facilitated by human alterations in nutrient enrichment and input of sediments from developed watersheds, which may also influence competitive outcomes among dominant algal species. To evaluate how changes in these abiotic stressors as well as competition may affect the growth of 2 common species of calcifying coral reef algae, Galaxaura fasciculata and Padina boryana, we conducted 3 separate 2 factor mesocosm experiments modeling fringing reefs in Moorea, French Polynesia. In the first experiment, we varied sediment source (marine vs. terrestrial) and water column nutrients (ambient vs. enriched) for each species separately and measured growth after 7 days. While both algae grew faster in enriched compared to ambient nutrients, P. boryana performed best with marine sediment (+27% change in biomass) and G. fasciculata with terrestrial sediment (+14% change in biomass). Next, we varied sediment source (as above) as well as sediment nutrients (ambient/enriched) for each species. While P. boryana lost 44% biomass in the eutrophic terrestrial sediment treatment, G. fasciculata performed the best and gained 19% biomass. Finally, we varied competition (alone/together) and terrestrial sediment nutrients (ambient/enriched). Over the 7 day period, P. boryana lost 64% biomass when in competition with G. fasciculata in the enriched treatment while G. fasciculata gained 38% biomass when in competition with P. boryana in the ambient treatment. These results indicate that, while growth of both species of macroalgae was regulated by nutrients, sediments, and competition, each responded uniquely to these controlling factors.

Effects of Nicotine and Nicotinic Antagonists on the Acoustic Startle Response and on Pre-Pulse Inhibition in Rats

DTIC Science & Technology

1996-06-07

the auditory nerve, the ventral cochlear nucleus , nuclei of the lateral lemniscus, nucleus reticularis pontis caudalis, spinal neuron, and lower... nucleus , nuclei of the lateral lemniscus, nucleus reticularis pontis caudalis, hippocampus, and striatum (Davis, et al., 1982; Swerdlow, et aI, 1992...Davis, M. (1985) Cocaine effects on acoustic startle and startle elicited electrically from cochlear nucleus . P§ychQpharmacology, 87, 396-399 James

Pitch Variability in Patients with Parkinson's Disease: Effects of Deep Brain Stimulation of Caudal Zona Incerta and Subthalamic Nucleus

ERIC Educational Resources Information Center

Karlsson, Fredrik; Olofsson, Katarina; Blomstedt, Patric; Linder, Jan; van Doorn, Jan

2013-01-01

Purpose: The purpose of the present study was to examine the effect of deep brain stimulation (DBS) of the subthalamic nucleus (STN) and the caudal zona incerta (cZi) pitch characteristics of connected speech in patients with Parkinson's disease (PD). Method: The authors evaluated 16 patients preoperatively and 12 months after DBS surgery. Eight…

Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus.

PubMed

Cox, C L; Huguenard, J R; Prince, D A

1997-08-05

Detailed information regarding the contribution of individual gamma-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. "Weak" inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, "strong" inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1-3 release sites, whereas stronger inhibition would require simultaneous activation of 5-70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.

Advances in Oculomotor and Vestibular Physiology

DTIC Science & Technology

1980-11-24

and nucleus reticularis tegmenti pontis in serving as an input to the flocculus and in mediating visual-vestibular interactions. Karten showed direct...demonstrated that the region in an around the Interstitial Nucleus of Cajal is important for the generation of torsional or rolling eye movements. 3...demonstrated that activity related to full field motion reaches the flocculus of mammals over mossy fiber Lnpits that arise from cells in nucleus

Activation of NMDA receptors in the brainstem, rostral ventromedial medulla, and nucleus reticularis gigantocellularis mediates mechanical hyperalgesia produced by repeated intramuscular injections of acidic saline in rats.

PubMed

Da Silva, Luis F; Desantana, Josimari M; Sluka, Kathleen A

2010-04-01

Repeated injections of acidic saline into the gastrocnemius muscle induce both muscle and cutaneous hypersensitivity. We have previously shown that microinjection of local anesthetic into either the rostral ventromedial medulla (RVM) or the nucleus reticularis gigantocellularis (NGC) reverses this muscle and cutaneous hypersensitivity. Although prior studies show that NMDA receptors in the RVM play a clear role in mediating visceral and inflammatory hypersensitivity, the role of NMDA receptors in the NGC or in noninflammatory muscle pain is unclear. Therefore, the present study evaluated involvement of the NMDA receptors in the RVM and NGC in muscle and cutaneous hypersensitivity induced by repeated intramuscular injections of acidic saline. Repeated intramuscular injections of acidic saline, 5 days apart, resulted in a bilateral decrease in the withdrawal thresholds of the paw and muscle in all groups 24 hours after the second injection. Microinjection of NMDA receptor antagonists into the RVM reversed both the muscle and cutaneous hypersensitivity. However, microinjection of NMDA receptor antagonists into the NGC only reversed cutaneous but not muscle hypersensitivity. These results suggest that NMDA receptors in the RVM mediate both muscle and cutaneous hypersensitivity, but those in the NGC mediate only cutaneous hypersensitivity after muscle insult. The current study shows that NMDA receptors in supraspinal facilitatory sites maintain noninflammatory muscle pain. Clinical studies in people with chronic widespread, noninflammatory pain, similarly, show alterations in central excitability. Thus, understanding mechanisms in an animal model could lead to improved treatment for patients with chronic muscle pain. Copyright 2010 American Pain Society. Published by Elsevier Inc. All rights reserved.

Enhanced contraception of canine zona pellucida 3 DNA vaccine via targeting DEC-205 in mice.

PubMed

Wang, Ying; Zhang, Beibei; Li, Jinyao; Aipire, Adila; Li, Yijie; Zhang, Fuchun

2018-06-01

Zona pellucida 3 (ZP3) is a potential antigen for the development of contraceptive vaccines to control animal population. In this study, we designed a canine ZP3 (CZP3) DNA vaccine through targeting DEC-205 (named as pcD-scFv-CZP3c) and investigated its contraceptive effect in mice. Female BALB/c mice were intramuscularly immunized 3 times at 2 weeks intervals. After immunization, humoral and cellular immune responses were detected by ELISA and flow cytometry. The results showed that pcD-CZP3 and pcD-scFv-CZP3c induced CZP3-specific antibody (Ab) responses both in serum and vaginal secretions compared to pcDNA3.1. Additionally, compared to pcD-CZP3, pcD-scFv-CZP3c increased the levels of CZP3-specific Abs after a third immunization. Abs induced by these two DNA vaccines could bind with mice and dogs oocytes. Moreover, pcD-scFv-CZP3c enhanced the activation of CD4 + T cells characterized by the increased frequencies of CD4 + CD44 + T cells. Finally, the contraceptive effect was evaluated in the immunized mice. These two DNA vaccines significantly decreased a mean litter size of mice compared to pcDNA3.1, but pcD-scFv-CZP3c group showed the smallest mean litter size. The mean litter size of pcD-scFv-CZP3 were 3.2 ± 0.742 and 4.6 ± 1.118 in two mating tests, which were significantly lower than pcDNA3.1(P < 0.001 and P < 0.05). Our results suggest that the CZP3 DNA vaccine targeted with DEC-205 may be a potential strategy for developing a contraceptive DNA vaccine. Copyright © 2018 Elsevier Inc. All rights reserved.

Distinct Thalamic Reticular Cell Types Differentially Modulate Normal and Pathological Cortical Rhythms.

PubMed

Clemente-Perez, Alexandra; Makinson, Stefanie Ritter; Higashikubo, Bryan; Brovarney, Scott; Cho, Frances S; Urry, Alexander; Holden, Stephanie S; Wimer, Matthew; Dávid, Csaba; Fenno, Lief E; Acsády, László; Deisseroth, Karl; Paz, Jeanne T

2017-06-06

Integrative brain functions depend on widely distributed, rhythmically coordinated computations. Through its long-ranging connections with cortex and most senses, the thalamus orchestrates the flow of cognitive and sensory information. Essential in this process, the nucleus reticularis thalami (nRT) gates different information streams through its extensive inhibition onto other thalamic nuclei, however, we lack an understanding of how different inhibitory neuron subpopulations in nRT function as gatekeepers. We dissociated the connectivity, physiology, and circuit functions of neurons within rodent nRT, based on parvalbumin (PV) and somatostatin (SOM) expression, and validated the existence of such populations in human nRT. We found that PV, but not SOM, cells are rhythmogenic, and that PV and SOM neurons are connected to and modulate distinct thalamocortical circuits. Notably, PV, but not SOM, neurons modulate somatosensory behavior and disrupt seizures. These results provide a conceptual framework for how nRT may gate incoming information to modulate brain-wide rhythms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Projections of the nucleus of the optic tract to the nucleus reticularis tegmenti pontis and prepositus hypoglossi nucleus in the pigmented rat as demonstrated by anterograde and retrograde transport methods.

PubMed

Korp, B G; Blanks, R H; Torigoe, Y

1989-01-01

The visual pathways from the nucleus of the optic tract (NOT) to the nucleus reticularis tegmenti pontis (NRTP) and prepositus hypoglossi nucleus (ph) were studied following injections of tritiated leucine into the NOT of pigmented rats. The cell bodies of origin of the pretectal-NRTP, NRTP-ph, and pretectal-ph projections were determined using retrograde horseradish peroxidase (HRP) technique. The pretectum projects strongly to the rostral two-thirds of the central and pericentral subdivisions of the NRTP and sends a remarkably smaller projection to the ph. Both are entirely ipsilateral. The fibers destined for the ph travel with the NOT-NRTP bundle, pass through the NRTP, traverse the medial longitudinal fasciculus, and are distributed to the rostral one-half of the ph. The retrograde HRP studies confirm these pathways. The pretectal projections to the NRTP arise from neurons in the rostromedial NOT; those to the ph are located primarily in the rostral NOT although small numbers are found within the anterior, posterior, and olivary pretectal nuclei. Of major importance is the fact that the ph injections retrogradely label neurons within the NRTP and the adjacent paramedian pontine reticular formation. This NRTP-ph projection is entirely bilateral and arises from parts of both subdivisions of the nucleus targeted by NOT afferents. Both the direct NOT-ph and indirect NOT-NRTP-ph connections provide the anatomical basis for the relay of visual (optokinetic) information to the perihypoglossal complex and, presumably, by virtue of reciprocal ph-vestibular nuclear connections, to the vestibular nuclei itself. Such pathways confirm previous physiological studies in rat and, in particular, clarify the contrasting effects of electrolytic lesions of NRTP in rat which completely abolishes optokinetic nystagmus (OKN) (Cazin et al., 1980a) vs kainic acid lesions which produce only minor effects on OKN slow velocity (Hess et al., 1988). Given these differential effects, one

Microanatomical diversification of the zona pellucida in aplochelioid killifishes.

PubMed

Thompson, A W; Furness, A I; Stone, C; Rade, C M; Ortí, G

2017-07-01

This study investigates zona pellucida (ZP) ultrastructure in fertilized eggs of annual killifishes (suborder Aplocheiloidei), a group of highly specialized fishes that are able to survive desiccation for several weeks to months before they hatch. Little is known about ZP or chorionic ultrastructure sustaining these life-history modes, so scanning electron microscopy (SEM) was used to describe this trait in a large number of aplocheiloids with a focus on the family Rivulidae and the genus Hypsolebias. New images of ZP ultrastructure for 52 aplocheiloid species are provided, more than doubling the number characterized thus far. The evolution of chorionic structure within this group is studied using these new data. Characters were coded into a morphological matrix and optimized onto a consensus phylogeny to assess phylogenetic signal and reconstruct ancestral character states. Although ZP characters seem highly homoplastic and exhibit a large amount of structural convergence among lineages, aplocheiloid killifishes have evolved a number of unique structures associated with the chorion. Some annual species seem to have lost long filaments because eggs are deposited in the soil instead of being adhered to aquatic plants. © 2017 The Fisheries Society of the British Isles.

Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation.

PubMed

Wei, F; Dubner, R; Ren, K

1999-03-01

Previous findings indicate that the brain stem descending system becomes more active in modulating spinal nociceptive processes during the development of persistent pain. The present study further identified the supraspinal sites that mediate enhanced descending modulation of behavior hyperalgesia and dorsal horn hyperexcitability (as measured by Fos-like immunoreactivity) produced by subcutaneous complete Freund's adjuvant (CFA). Selective chemical lesions were produced in the nucleus raphe magnus (NRM), the nuclei reticularis gigantocellularis (NGC), or the locus coeruleus/subcoeruleus (LC/SC). Compared to vehicle-injected animals with injection of vehicle alone, microinjection of a serotoninergic neurotoxin 5,7-dihydroxytryptamine into the NRM significantly increased thermal hyperalgesia and Fos protein expression in lumbar spinal cord after hindpaw inflammation. In contrast, the selective bilateral destruction of the NGC with a soma-selective excitotoxic neurotoxin, ibotenic acid, led to an attenuation of hyperalgesia and a reduction of inflammation-induced spinal Fos expression. Furthermore, if the NGC lesion was extended to involve the NRM, the behavioral hyperalgesia and CFA-induced Fos expression were similar to that in vehicle-injected rats. Bilateral LC/SC lesions were produced by microinjections of a noradrenergic neurotoxin, DSP-4. There was a significant increase in inflammation-induced spinal Fos expression, especially in the ipsilateral superficial dorsal horn following LC/SC lesions. These results demonstrated that multiple specific brain stem sites are involved in descending modulation of inflammatory hyperalgesia. Both NRM and LC/SC descending pathways are major sources of enhanced inhibitory modulation in inflamed animals. The persistent hyperalgesia and neuronal hyperexcitability may be mediated in part by a descending pain facilitatory system involving NGC. Thus, the intensity of perceived pain and hyperalgesia is fine-tuned by descending

Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the zona pellucida of mouse embryos.

PubMed

Novo, Sergi; Penon, Oriol; Barrios, Leonardo; Nogués, Carme; Santaló, Josep; Durán, Sara; Gómez-Matínez, Rodrigo; Samitier, Josep; Plaza, José Antonio; Pérez-García, Luisa; Ibáñez, Elena

2013-06-01

Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading

Fear-Potential Startle as a Model System for Analyzing Learning and Memory

DTIC Science & Technology

1988-09-21

connection between the central nucleus of the amygdala and the nucleus reticularis pontis caudalis, an obligatory part of the startle pathway. Because we...Miserendino, M and Davis, M. A direct pathway from the central nucleus of the amygdala to the region of the nucleus reticularis pontis caudalis critical for...blocked by drugs that decrease anxiety in humans as well as by lesions of the central nucleus of the amygdala, an area of the brain known to be critical for

Characterization of oocyte retrieval cycles with empty zona pellucida.

PubMed

Oride, Aki; Kanasaki, Haruhiko; Hara, Tomomi; Ohta, Hiroko; Kyo, Satoru

2018-01-01

To identify the factors that characterize cycles with empty zona pellucida (EZP). Thirty-six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP. The mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles. The observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.

Articulatory Closure Proficiency in Patients with Parkinson's Disease Following Deep Brain Stimulation of the Subthalamic Nucleus and Caudal Zona Incerta

ERIC Educational Resources Information Center

Karlsson, Fredrik; Olofsson, Katarina; Blomstedt, Patric; Linder, Jan; Nordh, Erik; van Doorn, Jan

2014-01-01

Purpose: The present study aimed at comparing the effects of deep brain stimulation (DBS) treatment of the subthalamic nucleus (STN) and the caudal zona incerta (cZi) on the proficiency in achieving oral closure and release during plosive production of people with Parkinson's disease. Method: Nineteen patients participated preoperatively and…

[Biological activity tests of chemical constituents from two Brazilian Labiatae plants].

PubMed

Isobe, Takahiko; Doe, Matsumi; Morimoto, Yoshiki; Nagata, Kumiko; Masuoka, Noriyoshi; Ohsaki, Ayumi

2007-02-01

We studied the bioactivities of constituents from two tropical medicinal plants, Cunila spicata and Hyptis fasciculata. These plants found in Brazil belong to the Labiatae family. Four known compounds obtained from these herbs were identified as 3alpha, 24-dihydoxylurs-12-en-28-oic acid, betulinic acid, aurantiamide acetate, and aurantiamide benzoate by spectroscopic means. 3alpha, 24-Dihydoxylurs-12-en-28-oic acid has potent inhibitory activity against Streptococcus salivarius, Streptococcus pneumoniae, Streptococcus pyogenes, and Porphyromomas gingivalis. Aurantiamide benzoate exhibited moderate inhibitory activity against xanthine oxidase. It was clarified that herbs Cunila spicata and Hyptis fasciculata are effective against bronchitis and gout.

Structural Analysis of Peptide-Analogues of Human Zona Pellucida ZP1 Protein with Amyloidogenic Properties: Insights into Mammalian Zona Pellucida Formation

PubMed Central

Louros, Nikolaos N.; Iconomidou, Vassiliki A.; Giannelou, Polina; Hamodrakas, Stavros J.

2013-01-01

Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as ‘aggregation-prone’ by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted ‘aggregation-prone’ parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization. PMID:24069181

Capital social de los padres de escolares de una zona vulnerable.

PubMed

Cornejo, Susana; Herrera, Ariel; Hilas, Elena; Gigena, Y Pablo

2018-01-01

El capital social supone importantes inversiones materiales, simbólicas y de esfuerzos. Conocer el capital social de una comunidad puede facilitar la comprensión del beneficio de las relaciones comunitarias para la promoción de salud. Con el objetivo de reconocer los componentes del capital social de padres de escolares atendiendo una escuela en una zona vulnerable de la ciudad de Córdoba, Argentina, indagamos a través de grupos focales acerca de elementos esenciales del capital social. La asintonía socio-cultural entre los pobladores originarios y los reubicados, la estigmatización policial y el clientelismo político han generado desconfianza del uno hacia el otro en diferentes aspectos convivenciales, siendo la familia la única red de apoyo. La escuela se reconoce como espacio adonde la gente espontáneamente se organiza para invertir en redes sociales, presentándose así con potencialidad para la promoción de conductas saludables, por el lugar simbólico que ocupa para los padres.

Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.

PubMed

Lacham-Kaplan, Orly; Chy, Hun; Trounson, Alan

2006-02-01

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-alpha and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human.

Insidious adrenocortical insufficiency underlies neuroendocrine dysregulation in TIF-2 deficient mice.

PubMed

Patchev, Alexandre V; Fischer, Dieter; Wolf, Siegmund S; Herkenham, Miles; Götz, Franziska; Gehin, Martine; Chambon, Pierre; Patchev, Vladimir K; Almeida, Osborne F X

2007-01-01

The transcription-intermediary-factor-2 (TIF-2) is a coactivator of the glucocorticoid receptor (GR), and its disruption would be expected to influence glucocorticoid-mediated control of the hypothalamo-pituitary-adrenal (HPA) axis. Here, we show that its targeted deletion in mice is associated with altered expression of several glucocorticoid-dependent components of HPA regulation (e.g., corticotropin-releasing hormone, vasopressin, ACTH, glucocorticoid receptors), suggestive of hyperactivity under basal conditions. At the same time, TIF-2(-/-) mice display significantly lower basal corticosterone levels and a sluggish and blunted initial secretory response to brief emotional and prolonged physical stress. Subsequent analysis revealed this discrepancy to result from pronounced aberrations in the structure and function of the adrenal gland, including the cytoarchitectural organization of the zona fasciculata and basal and stress-induced expression of key elements of steroid hormone synthesis, such as the steroidogenic acute regulatory (StAR) protein and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). In addition, altered expression levels of two nuclear receptors, DAX-1 and steroidogenic factor 1 (SF-1), in the adrenal cortex strengthen the view that TIF-2 deletion disrupts adrenocortical development and steroid biosynthesis. Thus, hyperactivity of the hypothalamo-pituitary unit is ascribed to insidious adrenal insufficiency and impaired glucocorticoid feedback.

Blockade of opioid receptors in the medullary reticularis nucleus dorsalis, but not the rostral ventromedial medulla, prevents analgesia produced by diffuse noxious inhibitory control in rats with muscle inflammation.

PubMed

de Resende, Marcos A; Silva, Luis Felipe S; Sato, Karina; Arendt-Nielsen, Lars; Sluka, Kathleen A

2011-06-01

Diffuse Noxious Inhibitory Controls (DNIC) involves application of a noxious stimulus outside the testing site to produce analgesia. In human subjects with a variety of chronic pain conditions, DNIC is less effective; however, in animal studies, DNIC is more effective after tissue injury. While opioids are involved in DNIC analgesia, the pathways involved in this opioid-induced analgesia are not clear. The aim of the present study was to test the effectiveness of DNIC in inflammatory muscle pain, and to study which brainstem sites mediate DNIC- analgesia. Rats were injected with 3% carrageenan into their gastrocnemius muscle and responses to cutaneous and muscle stimuli were assessed before and after inflammation, and before and after DNIC induced by noxious heat applied to the tail (45 °C and 47 °C). Naloxone was administered systemically, into rostral ventromedial medulla (RVM), or bilaterally into the medullary reticularis nucleus dorsalis (MdD) prior to the DNIC-conditioning stimuli. DNIC produced a similar analgesic effect in both acute and the chronic phases of inflammation reducing both cutaneous and muscle sensitivity in a dose-dependent manner. Naloxone systemically or microinjected into the MdD prevented DNIC-analgesia, while naloxone into the RVM had no effect on DNIC analgesia. Thus, DNIC analgesia involves activation of opioid receptors in the MdD. The current study shows that DNIC activates opioid receptors in the MdD, but not the RVM, to produce analgesia. These data are important for understanding clinical studies on DNIC as well as for potential treatment of chronic pain patients. Copyright © 2011 American Pain Society. Published by Elsevier Inc. All rights reserved.

Involvement of 5-HT(2) serotonergic receptors of the nucleus raphe magnus and nucleus reticularis gigantocellularis/paragigantocellularis complex neural networks in the antinociceptive phenomenon that follows the post-ictal immobility syndrome.

PubMed

de Oliveira, Rithiele Cristina; de Oliveira, Ricardo; Ferreira, Célio Marcos Dos Reis; Coimbra, Norberto Cysne

2006-09-01

The post-ictal immobility syndrome is followed by a significant increase in the nociceptive thresholds in animals and men. In this interesting post-ictal behavioral response, endogenous opioid peptides-mediated mechanisms, as well as cholinergic-mediated antinociceptive processes, have been suggested. However, considering that many serotonergic descending pathways have been implicated in antinociceptive reactions, the aim of the present work is to investigate the involvement of 5-HT(2)-serotonergic receptor subfamily in the post-ictal antinociception. The analgesia was measured by the tail-flick test in seven or eight Wistar rats per group. Convulsions were followed by statistically significant increase in the tail-flick latencies (TFL), at least for 120 min of the post-ictal period. Male Wistar rats were submitted to stereotaxic surgery for introduction of a guide-cannula in the rhombencephalon, aiming either the nucleus raphe magnus (NRM) or the gigantocellularis complex. In independent groups of animals, these nuclei were neurochemically lesioned with a unilateral microinjection of ibotenic acid (1.0 microg/0.2 microL). The neuronal damage of either the NRM or nucleus reticularis gigantocellularis/paragigantocellularis complex decreased the post-ictal analgesia. Also, in other independent groups, central administration of ritanserin (5.0 microg/0.2 microL) or physiological saline into each of the reticular formation nuclei studied caused a statistically significant decrease in the TFL of seizing animals, as compared to controls, in all post-ictal periods studied. These results indicate that serotonin input-connected neurons of the pontine and medullarly reticular nuclei may be involved in the post-ictal analgesia.

Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

PubMed

Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

2016-09-01

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. © 2016 American Heart Association, Inc.

Swallowing Quality of Life After Zona Incerta Deep Brain Stimulation.

PubMed

Sundstedt, Stina; Nordh, Erik; Linder, Jan; Hedström, Johanna; Finizia, Caterina; Olofsson, Katarina

2017-02-01

The management of Parkinson's disease (PD) has been improved, but management of signs like swallowing problems is still challenging. Deep brain stimulation (DBS) alleviates the cardinal motor symptoms and improves quality of life, but its effect on swallowing is not fully explored. The purpose of this study was to examine self-reported swallowing-specific quality of life before and after caudal zona incerta DBS (cZI DBS) in comparison with a control group. Nine PD patients (2 women and 7 men) completed the self-report Swallowing Quality of Life questionnaire (SWAL-QOL) before and 12 months after cZI DBS surgery. The postoperative data were compared to 9 controls. Median ages were 53 years (range, 40-70 years) for patients and 54 years (range, 42-72 years) for controls. No significant differences were found between the pre- or postoperative scores. The SWAL-QOL total scores did not differ significantly between PD patients and controls. The PD patients reported significantly lower scores in the burden subscale and the symptom scale. Patients with PD selected for cZI DBS showed good self-reported swallowing-specific quality of life, in many aspects equal to controls. The cZI DBS did not negatively affect swallowing-specific quality of life in this study.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

PubMed Central

2010-01-01

Background The zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians. Results In this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern. Conclusions The absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations. PMID:20849572

Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction.

PubMed

Beek, J; Nauwynck, H; Appeltant, R; Maes, D; Van Soom, A

2015-11-01

Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 10(5) spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF. Copyright © 2015 Elsevier Inc. All rights reserved.

The nucleus raphe magnus OFF-cells are involved in diffuse noxious inhibitory controls.

PubMed

Chebbi, R; Boyer, N; Monconduit, L; Artola, A; Luccarini, P; Dallel, R

2014-06-01

Diffuse noxious inhibitory controls (DNIC) are very powerful long-lasting descending inhibitory controls which are pivotal in modulating the activity of spinal and trigeminal nociceptive neurons. DNIC are subserved by a loop involving supraspinal structures such as the lateral parabrachial nucleus and the subnucleus reticularis dorsalis. Surprisingly, though, whether the nucleus raphe magnus (NRM), another supraspinal area which is long known to be important in pain modulation, is involved in DNIC is still a matter of discussion. Here, we reassessed the role of the NRM neurons in DNIC by electrophysiologically recording from wide dynamic range (WDR) neurons in the trigeminal subnucleus oralis and pharmacologically manipulating the NRM OFF- and ON-cells. In control conditions, C-fiber-evoked responses in trigeminal WDR neurons are inhibited by a conditioning noxious heat stimulation applied to the hindpaw. We show that inactivating the NRM by microinjecting the GABAA receptor agonist, muscimol, both facilitates C-fiber-evoked responses of trigeminal WDR neurons and strongly attenuates their inhibition by heat applied to the hindpaw. Interestingly, selective blockade of ON-cells by microinjecting the broad-spectrum excitatory amino acid antagonist, kynurenate, into the NRM neither affects C-fiber-evoked responses nor attenuates DNIC of trigeminal WDR neurons. These results indicate that the NRM tonically inhibits trigeminal nociceptive inputs and is involved in the neuronal network underlying DNIC. Moreover, within NRM, OFF-cells might be more specifically involved in both the tonic and phasic descending inhibitory controls of trigeminal nociception. Copyright © 2014 Elsevier Inc. All rights reserved.

AC electric field induced dipole-based on-chip 3D cell rotation.

PubMed

Benhal, Prateek; Chase, J Geoffrey; Gaynor, Paul; Oback, Björn; Wang, Wenhui

2014-08-07

The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 μm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.

Toxicological profile of ultrapure 2,2',3,4,4',5,5'-heptachlorbiphenyl (PCB 180) in adult rats.

PubMed

Viluksela, Matti; Heikkinen, Päivi; van der Ven, Leo T M; Rendel, Filip; Roos, Robert; Esteban, Javier; Korkalainen, Merja; Lensu, Sanna; Miettinen, Hanna M; Savolainen, Kari; Sankari, Satu; Lilienthal, Hellmuth; Adamsson, Annika; Toppari, Jorma; Herlin, Maria; Finnilä, Mikko; Tuukkanen, Juha; Leslie, Heather A; Hamers, Timo; Hamscher, Gerd; Al-Anati, Lauy; Stenius, Ulla; Dervola, Kine-Susann; Bogen, Inger-Lise; Fonnum, Frode; Andersson, Patrik L; Schrenk, Dieter; Halldin, Krister; Håkansson, Helen

2014-01-01

PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological

Análisis de los determinantes socioeconómicos del gasto de bolsillo en medicamentos en seis zonas geográficas de Panamá.

PubMed

Herrera-Ballesteros, Victor H; Castro, Franz; Gómez, Beatriz

2018-04-27

Caracterizar el gasto de bolsillo privado en medicamentos en función de los determinantes sociodemográficos y socioeconómicos. MATERIALES Y MéTODOS: La fuente de datos es la Encuesta de Gasto de Bolsillo en Medicamentos de 2014. Se caracterizó el gasto de bolsillo privado mediante variables explicativas sociodemográficas (SOD) y socioeconómicas (SES). Se hizo análisis factorial por componentes principales, regresión logística y lineal simple. Los Odds Ratio demuestran que la educación y la zona geográfica son determinantes fundamentales que inciden en el gasto de bolsillo. Los medicamentos son productos necesarios, en adición a que el gasto de bolsillo aumenta a un promedio del 2% por cada año de vida cronológica adicional. Existe mayor vulnerabilidad en las zonas más pauperizadas respecto del acceso a medicamentos, en especial en las indígenas e implica un mayor riesgo de gasto catastrófico a menor ingreso ante la mayor prevalencia de enfermedades crónicas. Copyright © 2018. Published by Elsevier Inc.

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

PubMed

Gleich, Alexander; Kaiser, Bastian; Schumann, Julia; Fuhrmann, Herbert

2016-06-01

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

Coniochaeta polymorpha, a new species from endotracheal aspirate of a preterm neonate, and transfer of Lecythophora species to Coniochaeta.

PubMed

Khan, Ziauddin; Gené, Josepa; Ahmad, Suhail; Cano, Josep; Al-Sweih, Noura; Joseph, Leena; Chandy, Rachel; Guarro, Josep

2013-08-01

A new species of Coniochaeta from endotracheal secretion of a preterm neonate, Coniochaeta polymorpha, is described. This anamorphic species is characterized by development of dark brown colonies after 1 week of incubation on culture medium, formation of abundant yeast-like cells and sclerotium-like structures producing discrete, brown, nearly globose phialidic conidiogenous cells and absence of chlamydospores. A combined sequence dataset of the ITS region, partial LSU rDNA, actin and β-tubulin genes sufficiently resolved the unique phylogenetic status of this species. In response to recent changes in the nomenclature for pleomorphic fungi, we transfer the Lecythophora species to Coniochaeta, and propose the following new combinations: Coniochaeta canina, Coniochaeta cateniformis, Coniochaeta decumbens, Coniochaeta fasciculata, Coniochaeta hoffmannii, Coniochaeta lignicola, Coniochaeta luteorubra, Coniochaeta luteoviridis and Coniochaeta mutabilis.

A common 'aggregation-prone' interface possibly participates in the self-assembly of human zona pellucida proteins.

PubMed

Louros, Nikolaos N; Chrysina, Evangelia D; Baltatzis, Georgios E; Patsouris, Efstratios S; Hamodrakas, Stavros J; Iconomidou, Vassiliki A

2016-03-01

Human zona pellucida (ZP) is composed of four glycoproteins, namely ZP1, ZP2, ZP3 and ZP4. ZP proteins form heterodimers, which are incorporated into filaments through a common bipartite polymerizing component, designated as the ZP domain. The latter is composed of two individually folded subdomains, named ZP-N and ZP-C. Here, we have synthesized six 'aggregation-prone' peptides, corresponding to a common interface of human ZP2, ZP3 and ZP4. Experimental results utilizing electron microscopy, X-ray diffraction, ATR FT-IR spectroscopy and polarizing microscopy indicate that these peptides self-assemble forming fibrils with distinct amyloid-like features. Finally, by performing detailed modeling and docking, we attempt to shed some light in the self-assembly mechanism of human ZP proteins. © 2016 Federation of European Biochemical Societies.

Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurachi, H.; Wakimoto, H.; Sakumoto, T.

1984-02-01

The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using /sup 125/I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodiesmore » in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women.« less

Epithelial-mesenchymal status influences how cells deposit fibrillin microfibrils.

PubMed

Baldwin, Andrew K; Cain, Stuart A; Lennon, Rachel; Godwin, Alan; Merry, Catherine L R; Kielty, Cay M

2014-01-01

Here, we show that epithelial-mesenchymal status influences how cells deposit extracellular matrix. Retinal pigmented epithelial (RPE) cells that expressed high levels of E-cadherin and had cell-cell junctions rich in zona occludens (ZO)-1, β-catenin and heparan sulfate, required syndecan-4 but not fibronectin or protein kinase C α (PKCα) to assemble extracellular matrix (fibrillin microfibrils and perlecan). In contrast, RPE cells that strongly expressed mesenchymal smooth muscle α-actin but little ZO-1 or E-cadherin, required fibronectin (like fibroblasts) and PKCα, but not syndecan-4. Integrins α5β1 and/or α8β1 and actomyosin tension were common requirements for microfibril deposition, as was heparan sulfate biosynthesis. TGFβ, which stimulates epithelial-mesenchymal transition, altered gene expression and overcame the dependency on syndecan-4 for microfibril deposition in epithelial RPE cells, whereas blocking cadherin interactions disrupted microfibril deposition. Renal podocytes had a transitional phenotype with pericellular β-catenin but little ZO-1; they required syndecan-4 and fibronectin for efficient microfibril deposition. Thus, epithelial-mesenchymal status modulates microfibril deposition.

The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3

PubMed Central

Vidal, Valerie; Sacco, Sonia; Rocha, Ana Sofia; da Silva, Fabio; Panzolini, Clara; Dumontet, Typhanie; Doan, Thi Mai Phuong; Shan, Jingdong; Rak-Raszewska, Aleksandra; Bird, Tom; Vainio, Seppo; Martinez, Antoine; Schedl, Andreas

2016-01-01

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce β-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life. PMID:27313319

Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems.

PubMed

Akshey, Yogesh S; Malakar, Dhruba; De, Arun K; Jena, Manoj K; Garg, Shweta; Dutta, Rahul; Pawar, Sachin Kumar; Mukesh, Manisha

2010-10-01

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

Comparison of oxidative stress in four Tillandsia species exposed to cadmium.

PubMed

Kováčik, Jozef; Babula, Petr; Klejdus, Bořivoj; Hedbavny, Josef

2014-07-01

This is first study comparing four morphologically variable species of the genus Tillandsia and therefore various responses to the cadmium (Cd) action were expected. In accordance, Cd accumulation increased in order Tillandsia fasciculata < Tillandsia brachycaulos < Tillandsia pruinosa < Tillandsia capillaris, reaching 29.6 and 197.4 μg g(-1) DW in first and last species after watering with 2 μM Cd(2+) solution over 30 days. Fluorescence visualization of oxidative stress confirmed increase in ROS and especially elevation in hydroperoxides though no visible symptoms appeared on the plants. At the same time, nitric oxide generation and nitroso-glutathione depletion by Cd treatment were typically observed. Fluorescence staining of Cd using two dyes (PhenGreen and Leadmium) showed that Leadmium fits better with AAS quantification. Macro- and micro-nutrients were not considerably affected except for zinc. Reduced glutathione content was the highest in control T. fasciculata while oxidized glutathione in T. capillaris. Ascorbic acid amount revealed extreme quantitative differences among species and decreased in T. fasciculata only. Free amino acids accumulation was similar among species except for T. capillaris and Cd caused both depletion and increase but without high quantitative differences. Data are explanatively discussed in the context of limited literature related to oxidative stress in epiphytic plants and with general responses of plants to cadmium/heavy metals. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Effects of porcine zona pellucida immunocontraceptives in zoo felids.

PubMed

Harrenstien, Lisa A; Munson, Linda; Chassy, Lisa M; Liu, Irwin K M; Kirkpatrick, Jay F

2004-09-01

Methods of contraception are necessary for management of zoo felids; however, the most commonly used contraceptive (melengestrol acetate implant) is associated with serious adverse reactions with long-term use. Porcine zona pellucida (pZP) vaccines are promising as contraceptives, but their safety in zoo felids has not been tested. pZP vaccine was administered to 27 female felids representing 10 species, including African lion (Panthera leo), Asian leopard (P. pardus), jaguar (P. onca), tiger (P. tigris), snow leopard (P. uncia), cougar (Felis concolor), Siberian lynx (F. lynx), Canada lynx (F. canadensis), serval (F. serval), and bobcat (F. rufus), in 15 facilities. Over 6 wk, each animal received three i.m. injections of 65 microg pZP with Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, or carbopol as the adjuvant. Behavioral signs of estrus were seen in 14 of the vaccinated felids. An unacceptably high incidence of adverse reactions was seen including injection site swelling, lameness, limb swelling, or abscessation (or all) in five felids after injection with FCA as the initial adjuvant. Adverse behavioral signs, including increased irritability and aggression, were seen in four felids. Six of the felids were assayed for antibodies against pZP during the 12 mo after vaccination; all showed antibody production. Antibody levels appeared to peak 1-4 mo after vaccination began, although elevated antibody levels persisted in two animals for > 12 mo after the first injection. All vaccinated felids were ovariohysterectomized 3-13 mo after vaccination. Folliculogenesis was present in all treated animals, and there was no histopathologic evidence of inflammatory damage to ovaries. Contraceptive efficacy was not specifically evaluated in this study; however, two of the three felids housed with an intact male became pregnant during the study, one of which gave birth to healthy cubs.

Heterogeneity of cell firing properties and opioid sensitivity in the thalamic reticular nucleus.

PubMed

Brunton, J; Charpak, S

1997-05-01

The thalamic reticular nucleus receives afferents from the dorsal thalamus, cortex and brainstem, and projects back onto most cortically projecting thalamic nuclei thus playing a key role in the synchronization of the thalamocortical network. Although this nucleus was initially thought to consist of a homogeneous population of cells using GABA as a transmitter, and sharing identical intrinsic membrane properties, some heterogeneity was subsequently reported. The morphological diversity is generally acknowledged, but only two studies have shown functional differences between two classes of cells which vary in their ability to discharge in bursts. However, the location of the non-bursting cells was not characterized with anatomical techniques. Our recent work on the action of mu-opioid agonists in the thalamus revealed a widespread K+-mediated inhibition of most, if not all, thalamic relay and diffuse projection neurons. However, in the reticular nucleus, preliminary experiments suggested that the opioid sensitivity was variable. Based on these results and on observations of a discrete localization of mu-opioid receptors in the reticular nucleus, we investigated cellular heterogeneity within the nucleus using opioid agonists as markers. Using the whole cell patch clamp technique in young rat thalamic slices, we tested the responses of 28 neurons to opioids, the intrinsic membrane properties of each cell, and their relative location within the nucleus. Two types of intrinsic membrane properties underlying distinct discharge behaviours were seen in neurobiotin-labelled cells clearly located in the reticular nucleus: type I with the typical bursting behaviour previously reported in reticularis neurons, and type II in which bursting was greatly reduced or absent. Each class of cell could be further divided into subpopulations based on their opioid sensitivity. About half of both bursting (20) and non-bursting or tonic (8) cells were strongly inhibited by the mu

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

PubMed

Johnson, Wesley; Dai, Changsheng; Liu, Jun; Wang, Xian; Luu, Devin K; Zhang, Zhuoran; Ru, Changhai; Zhou, Chao; Tan, Min; Pu, Huayan; Xie, Shaorong; Peng, Yan; Luo, Jun; Sun, Yu

2018-03-01

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Premature adrenarche: novel lessons from early onset androgen excess.

PubMed

Idkowiak, Jan; Lavery, Gareth G; Dhir, Vivek; Barrett, Timothy G; Stewart, Paul M; Krone, Nils; Arlt, Wiebke

2011-08-01

Adrenarche reflects the maturation of the adrenal zona reticularis resulting in increased secretion of the adrenal androgen precursor DHEA and its sulphate ester DHEAS. Premature adrenarche (PA) is defined by increased levels of DHEA and DHEAS before the age of 8 years in girls and 9 years in boys and the concurrent presence of signs of androgen action including adult-type body odour, oily skin and hair and pubic hair growth. PA is distinct from precocious puberty, which manifests with the development of secondary sexual characteristics including testicular growth and breast development. Idiopathic PA (IPA) has long been considered an extreme of normal variation, but emerging evidence links IPA to an increased risk of developing the metabolic syndrome (MS) and thus ultimately cardiovascular morbidity. Areas of controversy include the question whether IPA in girls is associated with a higher rate of progression to the polycystic ovary syndrome (PCOS) and whether low birth weight increases the risk of developing IPA. The recent discoveries of two novel monogenic causes of early onset androgen excess, apparent cortisone reductase deficiency and apparent DHEA sulphotransferase deficiency, support the notion that PA may represent a forerunner condition for PCOS. Future research including carefully designed longitudinal studies is required to address the apparent link between early onset androgen excess and the development of insulin resistance and the MS.

Mapping of enkephalins and adrenocorticotropic hormone in the squirrel monkey brainstem.

PubMed

Duque-Díaz, Ewing; Díaz-Cabiale, Zaida; Narváez, José Angel; Coveñas, Rafael

2017-03-01

An immunocytochemical technique has been used to study for the first time the distribution of fibers and cell bodies containing leucine-enkephalin (leu-enk), methionine-enkephalin (met-enk) or adrenocorticotropic hormone (ACTH) in the whole brainstem of the squirrel monkey Saimiri sciureus. Cell bodies containing leu-enk or met-enk were found in the superior colliculus and the formatio reticularis tegmenti mesencephali, respectively. No immunoreactive cell bodies containing ACTH were observed. Leu-enk-immunoreactive fibers were observed in 40 brainstem nuclei/tracts/regions, fibers containing met-enk were found in 38 brainstem nuclei/tracts/regions and fibers containing ACTH were found in 26 nuclei/tracts/regions. In the latter case, the density of immunoreactive fibers was always low. A high/moderate density of leu-enk- or met-enk-immunoreactive fibers were found in 18 and 16 brainstem nuclei/tracts/regions, respectively. The distribution of immunoreactive fibers containing leu-enk or met-enk was quite similar, with both leu-enk and met-enk observed in 82.5 % of the squirrel monkey brainstem nuclei/tracts/regions. This relationship is less marked for met-enk and ACTH (60.5 %) and even lower for leu-enk and ACTH (52.5 %). In 42.5 % of the nuclei/tracts/regions of the squirrel monkey brainstem (colliculus superior, substantia grisea centralis, nucleus interpeduncularis, nucleus tractus spinalis nervi trigemini, nucleus tractus solitarii, nucleus parabrachialis, formatio reticularis, substantia nigra), we observed fibers containing all three neuropeptides. The widespread distribution reported here suggests that enkephalins and ACTH can be involved in several physiological functions. The distribution of the immunoreactive fibers reported here is quite similar to that previously reported for enkephalins and ACTH in Macaca species and humans.

The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.

PubMed

Vidal, Valerie; Sacco, Sonia; Rocha, Ana Sofia; da Silva, Fabio; Panzolini, Clara; Dumontet, Typhanie; Doan, Thi Mai Phuong; Shan, Jingdong; Rak-Raszewska, Aleksandra; Bird, Tom; Vainio, Seppo; Martinez, Antoine; Schedl, Andreas

2016-06-15

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce β-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life. © 2016 Vidal et al.; Published by Cold Spring Harbor Laboratory Press.

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

PubMed

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Surface component characterization as taxonomic tools for Phytomonas spp identification.

PubMed

Abreu Filho, B A; Dias Filho, B P; Vermelho, A B; Jankevicius, S I; Jankevicius, J V; dos Santos, R L

2001-02-01

The genus Phytomonas arbitrarily includes all protozoa of the family Trypanosomatidae isolated from plants, but its differentiation is a complex task. The phase separation technique using Triton X-114 was used to analyze hydrophobic and hydrophilic surface proteins in ten strains of Phytomonas isolated from various fruits. The iodination of surface proteins by the Iodo-Gen method was also used for Phytomonas isolates from tomatoes, corn and annatto, Herpetomonas samuelpessoai and Crithidia fasciculata. The distribution of protein-bound radioactivity in acrylamide gels was determined by autoradiograms and showed the presence of protein bands of 36-68 kDa in all strains of Phytomonas: there were two major bands at 88 kDa and 94 kDa, with minor bands at 36 kDa and 142 kDa in H. samuelpessoai; and there were three bands at 74, 86 and 94 kDa, with minor bands at 23 kDa and 105 kDa in C. fasciculata. The results demonstrated that samples of plant parasites can be clearly differentiated from H. samuelpessoai and C. fasciculata. These plant parasites were also submitted to polysaccharide analysis by gas-liquid chromatography of the corresponding alditol acetate. Arabinose, galactose, glucose and mannose, were the major monosaccharides found, while fucose, rhamnose and xylose were found in smaller amounts. The results of all these methods indicated that, after extension to a wider range of trypanosomatid strains, they may be useful in Phytomonas taxonomy.

A case of refractory polyarteritis nodosa successfully treated with rituximab.

PubMed

Seri, Yu; Shoda, Hirofumi; Hanata, Norio; Nagafuchi, Yasuo; Sumitomo, Shuji; Fujio, Keishi; Yamamoto, Kazuhiko

2017-07-01

A 59-year-old man who presented with continuous fever, livedo reticularis, and left leg ischemia with multiple tibial artery stenosis and renal artery aneurysm, as demonstrated by arteriography, was diagnosed with polyarteritis nodosa (PAN) 6 years ago. Although he frequently relapsed in spite of intensive immunosuppressive therapies, the disease activity of PAN was controlled with repeated rituximab (RTX) therapies and steroid doses were tapered safely. Peripheral CD19 + B-cells disappeared soon after the 1st administration of RTX. Although CD19 + B-cells remained absent, 3.1% of CD3 + CD20 + T-cells were observed in the peripheral blood prior to the 2nd administration of RTX. Recent studies have suggested the pathogenic role of CD3 + CD20 + T-cells in autoimmune diseases in the context of RTX therapy; therefore, their roles in the pathogenesis of PAN also need to be considered.

Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores.

PubMed

Moros-Nicolás, C; Leza, A; Chevret, P; Guillén-Martínez, A; González-Brusi, L; Boué, F; Lopez-Bejar, M; Ballesta, J; Avilés, M; Izquierdo-Rico, M J

2018-01-01

The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.

Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

PubMed

Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

2010-08-01

We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

Activation of the reticulothalamic cholinergic pathway by the major metabolites of aniracetam.

PubMed

Nakamura, K; Shirane, M

1999-09-10

The aim of the study was to further investigate the effects of aniracetam, a cognition enhancer, and its metabolites on the brain cholinergic system. We measured choline acetyltransferase activity and acetylcholine release using in vivo brain microdialysis in stroke-prone spontaneously hypertensive rats (SHRSP). The enzyme activity in the pons-midbrain and hippocampus, and basal acetylcholine release in the nucleus reticularis thalami were lower in SHRSP than in age-matched Wistar Kyoto rats, indicating central cholinergic deficits in SHRSP. Repeated treatment of aniracetam (50 mg/kg p.o. x 11 for 6 days) preferentially increased the enzyme activity in the thalamus, whereas decreased it in the striatum. Among the metabolites of aniracetam, local perfusion of N-anisoyl-gamma-aminobutyric acid (GABA, 0.1 and/or 1 microM) and p-anisic acid (1 microM) into the nucleus reticularis thalami, dorsal hippocampus and prefrontal cortex of SHRSP produced a significant but delayed increase of acetylcholine release. We failed, however, to find any effect of aniracetam itself. A direct injection of N-anisoyl-GABA (1 nmol) into the pedunculopontine tegmental nucleus of SHRSP enhanced the release in the nucleus reticularis thalami. Thus, these data prove that aniracetam can facilitate central cholinergic neurotransmission via both metabolites. Based on its pharmacokinetic profile, N-anisoyl-GABA may contribute to the clinical effects of aniracetam, mainly by acting on the reticulothalamic cholinergic pathway.

New identification of the moray eel Gymnothorax minor (Temminck & Schlegel, 1846) in China (Anguilliformes, Muraenidae).

PubMed

Li, Yuan; Zhang, Liyan; Zhao, Linlin; Feng, Ji; Loh, Karhoe; Zheng, Xinqing; Lin, Longshan

2018-01-01

A new identification of Gymnothorax minor (Temminck & Schlegel, 1846) is documented based on morphological characteristics and DNA barcoding. Sixty-one individuals of G. minor were collected from the East China Sea and the South China Sea. This species was previously reported as Gymnothorax reticularis Bloch, 1795 in China because of the similarity in external shape and color. Gymnothorax minor can be easily distinguished from G. reticularis by its color pattern of 18-20 irregular dark brown vertical bars and the body having scattered small brown spots. Additionally, the teeth are uniserial on both jaws, and the vertebrae number 137-139. By combining congener sequences of the cytochrome oxidase I (COI) gene from GenBank, two groups were detected among all the COI sequences of the currently named G. minor , which further indicated that two valid species were present based on genetic distance. A divergence also occurred on the number of vertebrae between the northern and southern populations. The phylogenetic and morphological analysis strongly supports that the northern and southern populations of G. minor are two different species. Furthermore, the distribution area of the northern G. minor has expanded southward to 5°15'N in the South China Sea. More specimens of G. minor and G. reticularis are crucial in order to define their geographical distribution boundaries and provide the correct DNA barcoding.

11β-Hydoxylase Inhibitors Protect Against Seizures in Mice by Increasing Endogenous Neurosteroid Synthesis

PubMed Central

Kaminski, Rafal M.; Rogawski, Michael A.

2011-01-01

Steroid 11β-hydroxylase (CYP11B1; EC 1.14.15.4) is a mitochondrial enzyme located in the zona fasciculata of the adrenal cortex and also in the brain that mediates the conversion of 11-deoxycortisol to cortisol and 11-deoxycorticosterone (DOC) to corticosterone. Inhibitors of CYP11B1, such as metyrapone and etomidate, reduce glucocorticoid synthesis and raise levels of DOC providing greater availability for metabolic conversion to the GABAA receptor modulating neurosteroid allotetrahydrodeoxycorticosterone (THDOC). Because THDOC is a potent anticonvulsant, it is plausible that CYP11B1 inhibitors could protect against seizures. Here we demonstrate that metyrapone affords dose-dependent protection against 6-Hz seizures 30 min after injection (ED50, 191 mg/kg), but is markedly more potent at 6 h (ED50, 30 mg/kg). Similarly, etomidate is also protective at 30 min and 6 h (ED50 values, 4.5 and 1.7 mg/kg). Finasteride, an inhibitor of neurosteroid synthesis, attenuated the anticonvulsant effects of both CYP11B1 inhibitors at 6 h, but not 30 min following their injection. Plasma THDOC levels measured by liquid chromatography-mass spectrometry were markedly increased 6 h after injection of both CYP11B1 inhibitors and this increase was attenuated by finasteride pretreatment. We conclude that inhibition of CYP11B1 causes delayed seizure protection due to slow build-up of neurosteroids. Early seizure protection is independent of neurosteroids. PMID:21458468

The acute effect of ethanol on adrenal cortex in female rats--possible role of nitric oxide.

PubMed

Dikić, Dragoslava; Budeč, Mirela; Vranješ-Durić, Sanja; Koko, Vesna; Vignjević, Sanja; Mitrović, Olivera

2011-01-01

The present study was designed to investigate a possible role of endogenous nitric oxide (NO) in the adrenal response to an acute alcohol administration in female rats. To this end, N(ω)-nitro-L-arginine-methyl ester (L-NAME), a competitive inhibitor of all isoforms of NO synthase, was used. Adult female Wistar rats showing diestrus Day 1 were treated with: (a) ethanol (2 or 4 g/kg, intraperitoneally); (b) L-NAME (30 or 50 mg/kg, subcutaneously) followed by either ethanol or saline 3 h later. Untreated and saline-injected rats were used as controls. The animals were killed 30 min after last injection. Adrenal cortex was analyzed morphometrically, and plasma levels of adrenocorticotropic hormone (ACTH) and serum concentrations of corticosterone were determined. Acute ethanol treatment enhanced the levels of ACTH and corticosterone in a dose-dependent manner. Stereological analysis revealed that acute alcohol administration induced a significant increase in absolute volume of the cortex and the zona fasciculata (ZF). In addition, ethanol at a dose of 4 g/kg increased volume density and length of the capillaries in the ZF. However, other stereological parameters were unaffected by alcohol exposure. Pretreatment with both doses of L-NAME had no effect on ethanol-induced changes. Obtained findings indicate that acute ethanol treatment stimulates the activity of the adrenal cortex and that this effect is not mediated by endogenous NO in female rats under these experimental conditions.

Human sperm degradation of zona pellucida proteins contributes to fertilization.

PubMed

Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

2015-09-02

The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

Localization of vascular endothelial growth factor in the zona pellucida of developing ovarian follicles in the rat: a possible role in destiny of follicles.

PubMed

Celik-Ozenci, Ciler; Akkoyunlu, Gokhan; Kayisli, Umit Ali; Arici, Aydin; Demir, Ramazan

2003-11-01

There is increasing evidence that in many species angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), may have important roles in folliculogenesis. The aim of this study is to determine the localization of VEGF and its receptors, Flt-1 and KDR, and bFGF expression in the rat ovary and to evaluate their distributions throughout the different follicular stages. Out of 20 virginal female rats, 10 were studied during the natural ovarian cycle without any ovulation induction. The other 10 were superovulated and their ovaries were studied by western analysis and immunohistochemistry. Granulosa cells (GC) and oocytes of primordial follicles were negative for VEGF. In early primary follicles, VEGF was present in the oocyte but its immunoreactivity was weak, while newly developing zona pellucida (ZP) of primary follicles was negative for VEGF. Subsequently, with the commencement of antral spaces between GC of the secondary follicle, ZP of some secondary follicles became strongly positive for VEGF, forming a continuous ring around the oocyte. In preovulatory mature follicles granulosa and theca interna (TI) cells showed a weak immunoreactivity for VEGF. Western blot analyses have also demonstrated that VEGF, a 26-kDa protein, was present in follicles. Moreover, in ovulated cumulus-oocyte complex we observed a halo-like immunoreactivity of VEGF around the fully mature oocyte. The immunoreactivity for Flt-1 and KDR receptors in growing follicles was mostly limited to GC and TI cells. Anti-bFGF did not exhibit any immunoreactivity in ZP of follicles at any stage. Its expression was weak in GC of the follicles at different stages, whereas, it could be localized to some extent in the blood capillaries of TI of antral follicles and in blood vessels localized in the stroma. Interestingly, VEGF immunoreactivity in the ZP of some secondary follicles is very striking. Accordingly, the possibility that VEGF may be an

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

PubMed Central

Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

2013-01-01

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

Ultrastructural analysis of Phytomonas species from Euphorbia pinea reveals trans-cytoplasmic filaments 10 nm in diameter.

PubMed

Page, A M; Lagnado, J R

2000-10-01

Phytomonas sp. derived from Euphorbia pinea are digenetic plant trypanosomes that are transmitted by the squashbug Stenocephalus agilis and exist exclusively as promastigotes. The stable sub-pellicular microtubular array, the flagellar axoneme and the paraflagellar rod represent the major cytoskeletal components common to all trypanosomes. The work described in this paper examines in detail the ultrastructural morphology of the organism and highlights a number of novel structural features, and in particular, the presence of some detergent-resistant proteins which take the form of bundles of trans-cytoplasmic filaments of ca. 10 nm in diameter, seen in cells from both log- and stationary-phase cultures. The ultrastructural morphology and immunological cross-reactivity of these filaments are described, and their relationship to filamentous bundles previously reported in stationary-phase cultures of Crithidia fasciculata and to intermediate filaments of animal cells is discussed.

Beyond the Papilionoids – What can We Learn from Chamaecrista?

USDA-ARS?s Scientific Manuscript database

Expanding legume research beyond the model papilionoids is necessary if we wish to capture more of the diversity of the enormous, economically important legume family. Chamaecrista fasciculata is emerging as a non-papilionoid model, belonging to the paraphyletic subfamily Caesalpinioideae within th...

Involvement of cell proliferation in the process of follicular atresia in the guinea pig.

PubMed

Wang, Wei; Liu, Honglin; Ding, Wei; Gong, Yan; Chen, Jingwei; Hutz, Reinhold J; Mao, Dagan; Shi, Fangxiong

2010-08-01

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs. Copyright 2010 Elsevier Ltd. All rights reserved.

Orexin Gene Transfer into Zona Incerta Neurons Suppresses Muscle Paralysis in Narcoleptic Mice

PubMed Central

Liu, Meng; Konadhode, RodaRani; Begum, Suraiya; Pelluru, Dheeraj; Gerashchenko, Dmitry; Sakurai, Takeshi; Yanagisawa, Masashi; van den Pol, Anthony N.

2011-01-01

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy. PMID:21508228

Convergent synaptic inputs from the caudal fastigial nucleus and the superior colliculus onto pontine and pontomedullary reticulospinal neurons.

PubMed

Takahashi, Mayu; Sugiuchi, Yuriko; Shinoda, Yoshikazu

2014-02-01

The caudal fastigial nucleus (FN) is known to be related to the control of eye movements and projects mainly to the contralateral reticular nuclei where excitatory and inhibitory burst neurons for saccades exist [the caudal portion of the nucleus reticularis pontis caudalis (NRPc), and the rostral portion of the nucleus reticularis gigantocellularis (NRG) respectively]. However, the exact reticular neurons targeted by caudal fastigioreticular cells remain unknown. We tried to determine the target reticular neurons of the caudal FN and superior colliculus (SC) by recording intracellular potentials from neurons in the NRPc and NRG of anesthetized cats. Neurons in the rostral NRG received bilateral, monosynaptic excitation from the caudal FNs, with contralateral predominance. They also received strong monosynaptic excitation from the rostral and caudal contralateral SC, and disynaptic excitation from the rostral ipsilateral SC. These reticular neurons with caudal fastigial monosynaptic excitation were not activated antidromically from the contralateral abducens nucleus, but most of them were reticulospinal neurons (RSNs) that were activated antidromically from the cervical cord. RSNs in the caudal NRPc received very weak monosynaptic excitation from only the contralateral caudal FN, and received either monosynaptic excitation only from the contralateral caudal SC, or monosynaptic and disynaptic excitation from the contralateral caudal and ipsilateral rostral SC, respectively. These results suggest that the caudal FN helps to control also head movements via RSNs targeted by the SC, and these RSNs with SC topographic input play different functional roles in head movements.

Description and key to the fifth-instars of some Cicadas (Hemiptera: Cicadidae) associated with coffee plants in Brazil.

PubMed

Maccagnan, D H B; Martinelli, N M

2011-01-01

Fifth-instars of the cicada species Dorisiana drewseni (Stål), Dorisiana viridis (Olivier), Fidicina mannifera (Fabricius), Fidicinoides pronoe (Walker) and Carineta fasciculata (Germar) are described and illustrated. Moreover, a key to the nymphs of these species along with Quesada gigas (Olivier) is also provided.

Ant predation on an invasive herbivore: Can an extrafloral nectar-producing plant provide associational resistance to Opuntia individuals?

USDA-ARS?s Scientific Manuscript database

The legume Chamaecrista fasciculata attracts ants to its extrafloral nectar (EFN) which can lead to reduced herbivory and increased fecundity for the plant. In Florida, Opuntia stricta and O. humifusa, hosts of the invasive moth Cactoblastis cactorum, are often found growing in close association wit...

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

PubMed Central

Diestel, Uschi; Resch, Marcus; Meinhardt, Kathrin; Weiler, Sigrid; Hellmann, Tina V.; Mueller, Thomas D.; Nickel, Joachim; Eichler, Jutta; Muller, Yves A.

2013-01-01

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3. PMID:23826237

Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

PubMed Central

Viluksela, Matti; Heikkinen, Päivi; van der Ven, Leo T. M.; Rendel, Filip; Roos, Robert; Esteban, Javier; Korkalainen, Merja; Lensu, Sanna; Miettinen, Hanna M.; Savolainen, Kari; Sankari, Satu; Lilienthal, Hellmuth; Adamsson, Annika; Toppari, Jorma; Herlin, Maria; Finnilä, Mikko; Tuukkanen, Juha; Leslie, Heather A.; Hamers, Timo; Hamscher, Gerd; Al-Anati, Lauy; Stenius, Ulla; Dervola, Kine-Susann; Bogen, Inger-Lise; Fonnum, Frode; Andersson, Patrik L.; Schrenk, Dieter; Halldin, Krister; Håkansson, Helen

2014-01-01

PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological

Somato-dendritic synapses in the nucleus reticularis thalami of the rat.

PubMed

Csillik, B; Pálfi, A; Gulya, K; Mihály, A; Knyihár-Csillik, Elizabeth

2002-01-01

In the reticular nucleus of the rat thalamus, about 30% of the synapses are brought about by the perikarya of parvalbumin-immunopositive neurons, which establish somato-dendritic synapses with large dendrites of nerve cells of specific thalamic nuclei. Although the parvalbumin-immunopositive presynaptic structures bear resemblance to goblet-like or calyciform axonal endings, electron microscopic immunocytochemistry and in situ hybridization revealed that these structures are parts of the perikaryal cytoplasm studded with synaptic vesicles. In about 15% of the somato-dendritic synapses, axons are seen to be in synaptic contact with the parvalbumin-immunoreactive perikaryon. Double immunohistochemical staining revealed that the parvalbumin immunoreactive presynaptic perikarya and dendrites contained GABA. It is assumed that the peculiar somato-dendritic synaptic complexes subserve the goal of filtration of impulses arriving at the reticular nucleus from various thalamic nuclei, thus processing them for further sampling.

Somatic cell nuclear transfer in horses.

PubMed

Galli, Cesare; Lagutina, Irina; Duchi, Roberto; Colleoni, Silvia; Lazzari, Giovanna

2008-07-01

The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.

[Pseudoangiosarcomatous squamous cell carcinoma of the penis: a case report with clinicopathological and human papilloma virus analyses].

PubMed

Qi, Xiao-Ping; Lin, Guo-Bing; Zhu, Yang-Li; Wang, Jin-Quan; Dai, Xiao-Wen; Ma, Ju-Ming; Yan, Li

2009-02-01

To further understand the clinicopathological, ultrastructural and molecular features of penile pseudoangiosarcomatous squamous cell carcinoma (PASCC), and improve its diagnosis and treatment. A 47-year-old male patient with penile PASCC was reported and the relevant literature reviewed. The main clinical manifestations of the patient were a typical surface ulceration with hemorrhage and purulent secretion with a foul smell, a papillary mass about 5.0 cm x 5.0 cm x 4.0 cm for 1 year on the foreskin of the penis, and 3 enlarged bilateral inguinal lymph nodes. CT scanning showed no enlarged lymph nodes in the abdomen and pelvis, and X-ray examination revealed no abnormality in the chest. The diagnosis was established by biopsy. Partial penectomy and bilateral inguinal lymphadenectomy (T2N2M0) were performed, followed by adjuvant pelvic radiotherapy. Two months later, total penectomy was necessitated by penile flap necrosis and local recurrence. Eleven months after the first surgery, the patient died of extensive metastasis to the pelvis and lungs. Under the light microsope, the tumor was mainly composed of vessel-like lacunar reticularis spindle cells and a few local squamous cancer cells. Careful examination revealed some focal areas with evident transition from squamous nests to the more acantholytic areas extending towards the pseudoangiosarcomatous spaces. Pathogenetically, it appeared to be the variant of acantholytic squamous cell carcinoma. Immunohistochemically, most tumor cells were strongly positive for keratin (AE1/AE3) and focally positive for EMA, with the typical squamous cells focally positive for 34betaE12 and vimentin. The vessels that proliferated in the tumor were decorated by CD31, CD34 and factor VIII-related antigens, but the tumor cells were negative for HMB45, SMA, Desmin and CEA. HPV DNA (HPVpan, HPV6B/11, HPV16/18, HPV31/33) was not detected by in situ hybridization in the primary and metastatic tumors. PASCC is a specific and extremely rare

ACTH is a novel regulator of bone mass.

PubMed

Isales, Carlos M; Zaidi, Mone; Blair, Harry C

2010-03-01

Adrenocorticotropin (ACTH) is one of several peptide hormones derived from a larger molecule, proopiomelanocortin (POMC). ACTH is a classic endocrine hormone, processed and secreted from the pituitary to stimulate cortisol production from the fasciculata cells in the adrenal gland. However, ACTH is also produced by other cells, including macrophages, at many sites in the body. ACTH binds to a specific member of the melanocortin receptor family, the MC2R. MC2R is expressed in osteoblastic cells in vivo, as shown by in situ hybridization. MC2R expression is strongest at sites of active bone deposition, and thus ACTH response probably varies with osteoblastic activity or stage of osteoblast differentiation. In vitro ACTH stimulates proliferation of osteoblasts in a dose-dependent manner. ACTH at 10 nM increases collagen I mRNA in the osteoblastic cell line SaOs2, although at lower concentrations ACTH may oppose osteoblast differentiation. ACTH is thus, at high concentrations, anabolic for the osteoblast, and it is highly likely that the hormone has concentration-dependent effects on bone metabolism in vivo.

Mouse embryo attachment to substratum and interaction of trophoblast with cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glass, R.H.; Spindle, A.I.; Pedersen, R.A.

1979-06-01

Hatching, attachment, and trophoblast outgrowth of mouse embryos in vitro were examined as a model for implantation. Mouse embryos attached and grew out on glass cover slips that were partially covered with cultured mouse cells (L cells, liver cells, transformed JLS-V11 cells, and teratocarcinoma cells). Scanning electron microscopy showed that processes of these cells made contact with trophoblast, but there was no evidence of cell lysis or of phagocytosis of the cells by trophoblast. Time-lapse cinematography showed that after contact the cultured mouse cells retracted from the trophoblast, which then spread into the areas vacated by those cells. This suggestsmore » a means by which the trophoblast gains entry into the endometrium without destruction of maternal cells. Neuraminidase (100 or 250 units/ml) had no effect on attachment of mouse embryos to glass. However, attachment was inhibited by trypsin at concentrations of 0.25%, 0.025%, and 0.0025%. Treatment of early blastocysts with diazooxo-norleucine, an inhibitor of glycoprotein synthesis, decreased the number of embryos hatching from the zona pellucida; treatment at the late blastocyst stage decreased hatching to a lesser extent. Among the late blastocysts that did hatch, the number forming trophoblast outgrowths was lower than in controls. These results suggest that glycoproteins may be of importance for embryo hatching, attachment, and outgrowth.« less

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization.

PubMed

Ferrer, Marvin; Rodriguez, Hilma; Zara, Lindsay; Yu, Yang; Xu, Wei; Oko, Richard

2012-09-01

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.

The Australian endemic woodwasp genus Austrocyrta Riek (Hymenoptera: Xiphydriidae)

Treesearch

J.T. Jennings; A.D. Austin; N.M. Schiff

2009-01-01

The Australian xiphydriid woodwasp genus, previously known only from a single species, A. australiensis Riek, is revised. A second distinctive species from Carnarvon National Park in southcentral Queensland, A. fasciculata Jennings & Austin, sp. nov., is described, and a key to separate the males of the two species is provided.

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro.

PubMed

Buffone, Mariano G; Zhuang, Tiangang; Ord, Teri S; Hui, Ling; Moss, Stuart B; Gerton, George L

2008-05-02

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Mapping the potential beverage quality of coffee produced in the Zona da Mata, Minas Gerais, Brazil.

PubMed

Silva, Samuel de Assis; de Queiroz, Daniel Marçal; Ferreira, Williams Pinto Marques; Corrêa, Paulo Cesar; Rufino, José Luis Dos Santos

2016-07-01

Detailed knowledge of coffee production systems enables optimization of crop management, harvesting and post-harvest techniques. In this study, coffee quality is mapped as a function of coffee variety, altitude and terrain aspect attributes. The work was performed in the Zona da Mata, Minas Gerais, Brazil. A large range of coffee quality grades was observed for the Red Catuai variety. For the Yellow Catuai variety, no quality grades lower than 70 were observed. Regarding the terrain aspect, samples from the southeast-facing slope (SEFS) and the northwest-facing slope (NWFS) exhibited distinct behaviors. The SEFS samples had a greater range of quality grades than did the NWFS samples. The highest grade was obtained from an NWFS point. The lowest quality values and the largest range of grades were observed at lower altitudes. The extracts from the highest-altitude samples did not produce any low-quality coffee. The production site's position and altitude are the primary variables that influenced the coffee quality. The study area has micro-regions with grades ranging from 80 to 94. These areas have the potential for producing specialty coffees. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Distribution and properties of GABA(B) antagonist [3H]CGP 62349 binding in the rhesus monkey thalamus and basal ganglia and the influence of lesions in the reticular thalamic nucleus.

PubMed

Ambardekar, A V; Ilinsky, I A; Forestl, W; Bowery, N G; Kultas-Ilinsky, K

1999-01-01

GABA(B) receptors are believed to be associated with the efferents of the nucleus reticularis thalami, which is implicated in the regulation of activity in the thalamocortical-corticothalamic circuit and plays a role in absence seizures. Yet, the distribution of GABA(B) receptors in the thalamus has only been studied in the rat, and there is no comparable information in primates. The potent GABA(B) receptor antagonist [3H]CGP 62349 was used to study the distribution and binding properties of the receptor in control monkeys and those with small ibotenic acid lesions in the anterodorsal segment of the nucleus reticularis thalami. Eight-micrometer-thick cryostat sections of the fresh frozen brains were incubated in the presence of varying concentrations of the ligand. Autoradiographs were analysed using a quantitative image analysis technique, and binding parameters were calculated for select thalamic nuclei as well as basal ganglia structures present in the same sections. The overall number of GABA(B) binding sites in the monkey thalamus and basal ganglia was several-fold higher than previously reported values for the rat. In the thalamus, the receptors were distributed rather uniformly and the binding densities and affinities were high (Bmax range of 245.5-437.9 fmol/ mg of tissue, Kd range of 0.136-0.604 nM). In the basal ganglia, the number of binding sites and the affinities were lower (Bmax range of 51.1-244.2 fmol/mg of tissue; K(d) range of 0.416-1.394 nM), and the differences between nuclei were more pronounced, with striatum and substantia nigra pars compacta displaying the highest binding densities. Seven days post-lesion, a 20-30% decrease in Bmax values (P < 0.05) was found in the nuclei receiving input from the lesioned nucleus reticularis thalami sector (the mediodorsal nucleus and densicellular and magnocellular parts of the ventral anterior nucleus) without changes in affinity. No significant changes were detected in any other structures. The results

In vitro studies on normal and pathological preimplantation development. I. Events of normal mouse preimplantation development as revealed by microcinematography.

PubMed

Checiu, M; Schlechta, B; Checiu, I; Sandor, S

1990-01-01

After briefly presenting the main historical data of in vitro culture of preimplantation mouse embryos and their filming, the first own observations on normal preimplantation development made by using microcinematography are presented: development from two-cell to eight-cell embryos; compaction and cavitation. The timing and the duration of various developmental events were recorded. Own observations were compared with previous cinematographic data reported by other authors. Some processes needing further investigations are evidenced: rotation within the zona pellucida, penetration of cytoplasmic emissions through the zona, contraction and reexpansion.

Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

PubMed

Ye, P; Yu, H; Simonian, M; Hunter, N

2014-04-01

Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin

Resveratrol reverses the adverse effects of a diet-induced obese murine model on oocyte quality and zona pellucida softening.

PubMed

Jia, Zhenzhen; Feng, Zeyang; Wang, Lining; Li, Hao; Wang, Hongyu; Xu, Dingqi; Zhao, Xin; Feng, Daofu; Feng, Xizeng

2018-05-23

Reproductive dysfunction associated with obesity is increasing among women of reproductive age, including infertility and increasing risk of miscarriage. In females, reproductive disorders are linked to declining quality of oocytes. Using a model of diet-induced obesity, we have investigated the possible effects of obesity on oocyte quality, including metabolism, lipid accumulation, ROS levels, meiosis and changes in spindle structure in Metaphase II. Our study showed that obesity induced by a high fat diet can impair oocyte meiosis, destroy spindle assembly, and promote oxidative stress and abnormal mitochondrial distribution. With the addition of resveratrol, the negative impact of diet-induced obesity on the quality of oocytes was alleviated to some extent. In addition, we found that obesity causes mouse oocytes to soften, and resveratrol can restore the zona pellucida of oocytes to the same state as the control group. In conclusion, resveratrol can reverse the adverse effects of obesity on oocytes, which is beneficial for subsequent embryonic development.

Melanin-concentrating hormone and neuropeptide EI projections from the lateral hypothalamic area and zona incerta to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers.

PubMed

Bittencourt, J C; Elias, C F

1998-09-14

The projection pathways of neurons containing melanin-concentrating hormone (MCH) and neuropeptide EI (NEI), two peptides colocalized in the lateral hypothalamic area (LHA) of the rat, were mapped using the retrogradely transported fluorescent dyes, true blue (TB) and diamidino yellow (DY). TB and DY were injected into the medial septum/diagonal band complex (MS/DBC) and the thoracic level of the spinal cord (SpCd), respectively. Brains from rats receiving only one or both tracer injections were immunohistochemically stained for MCH in the spinal cord and NEI in the forebrain. In the MS/DBC, NEI-immunoreactive (-ir) fibers are concentrated in the MS and in the vertical and horizontal limbs of the DBC. In the SpCd, MCH-ir fibers are concentrated primarily in lamina X. Of the diencephalic NEI-ir neurons, 37.15% project to the MS/DBC and reside in the rostromedial zona incerta (ZIm), in the LHAt and LHAp, and in the perifornical region. Of the diencephalic MCH-ir neurons, 20.2% project to the SpCd and reside in the LHAt and LHAp. In addition, 2. 2% of the MCH-ir cells and 8.7% of the NEI-ir cells in the hypothalamus were labeled with both retrograde tracers and thus project to both the MS/DBC and SpCd. These dual projection neurons are located mainly in the LHAt and LHAp. Anterograde injections of the tracer Phaseolus vulgaris leucoagglutinin into the LHAt and ZIm corroborated our findings in the retrograde studies. Potential autonomic and behavioral roles of the NEI and MCH systems in the MS/DBC and the SpCd are discussed. Copyright 1998 Elsevier Science B. V.

Population genetic structure of Attalea vitrivir Zona (Arecaceae) in fragmented areas of southeast Brazil.

PubMed

Santos, R R M; Cavallari, M M; Pimenta, M A S; Abreu, A G; Costa, M R; Guedes, M L

2015-06-11

Attalea vitrivir Zona (synonym Orbignya oleifera) is one of the six species of Arecaceae known as "babassu". This species is used to make cosmetics, food, and detergents due to the high concentration of oil in the seeds. It is found only in fragmented areas of southern Bahia State and northern Minas Gerais State, southeast Brazil, and this fragmentation has affected both its ecological and genetic characteristics. We evaluated the genetic diversity and population genetic structure of A. vitrivir in six areas of two different regions at the extremes of its geographical range, in order to gain a better understanding of the factors that affect the distribution and partitioning of its diversity. Nine inter simple sequence repeat (ISSR) markers amplified 74 polymorphic bands, resulting in large diversity values (Shannon diversity index, 0.37-0.47; intrapopulation genetic diversity, 0.25-0.34). Analysis of molecular variance (AMOVA) revealed considerable differentiation between sampling sites (30.03%) and regions (12.08%), although most of the diversity was observed within sampling sites (69%). Further differentiation between sampling sites was noted more in the northern region than in the southern region, highlighting the genetic connectivity between the sampling sites within Rio Pandeiros Environmental Protection Area (southern region). The identification of two distinct genetic clusters (K = 2) corresponded to the northern and southern regions, and corroborated the AMOVA results. We suggest that the northern area, outside Rio Pandeiros Environmental Protection Area, must be included in future management plans for this species.

Foaling rates in feral horses treated with the immunocontraceptive porcine zona pellucida

USGS Publications Warehouse

Ransom, J.I.; Roelle, J.E.; Cade, B.S.; Coates-Markle, L.; Kane, A.J.

2011-01-01

Locally abundant feral horses (Equus caballus) can rapidly deplete available resources. Fertility control agents present promising nonlethal tools for reducing their population growth rates. We tested the effect of 2 forms of the immunocontraceptive porcine zona pellucida (PZP) on foaling rates in 3 populations of feral horses in the western United States. A liquid form requiring annual boosters was administered at Little Book Cliffs Wild Horse Range, Mesa County (CO), and Pryor Mountain Wild Horse Range, Bighorn County (WY) and Carbon County (MT), and a time-release pellet form designed to produce 2 yr of infertility was administered at McCullough Peaks Herd Management Area, Park County (WY). Average foaling rates (foals born/mare-yr) from direct observation of untreated and treated female horses (mares), 2004-2008, were 60.1% (n = 153 mare-yr) versus 6.6% (n = 91 mare-yr) at Little Book Cliffs, and 62.8% (n = 129 mare-yr) versus 17.7% (n = 79 mare-yr) at Pryor Mountain, respectively. At McCullough Peaks, mean annual foaling rates from 2006 to 2008 were 75.0% (n = 48 mare-yr) for untreated mares and 31.7% (n = 101 mare-yr) for treated mares. Controlling for age of mares and pretreatment differences in fertility, PZP reduced foaling rates in all 3 herds. The pellets used at McCullough Peaks (produced by cold evaporation) were less effective than pellets used in a previous trial and produced by heat extrusion. Immunocontraception with PZP may be a useful tool in reducing fertility rates in some western United States feral horse herds, but population growth reduction will depend on timely access to mares for inoculation and the proportion of mares that can be successfully treated. ?? 2011 The Wildlife Society.

ULTRASTRUCTURE, STEROIDOGENIC POTENTIAL, AND ENERGY METABOLISM OF THE SNELL ADRENOCORTICAL CARCINOMA 494

PubMed Central

Kimmel, G. L.; Péron, F. G.; Haksar, A.; Bedigian, E.; Robidoux, W. F.; Lin, M. T.

1974-01-01

Electron microscope studies were carried out with the adrenocortical carcinoma 494 and normal adrenal cortex tissue. The mitochondria of the tumor cells showed marked differences when compared with mitochondria from fasciculata cells of the normal adrenal cortex. These differences were primarily related to mitochondrial number and crista structure. Corticosterone production in isolated tumor cells was extremely low and neither ACTH nor dibutyryl cyclic AMP had any stimulatory effect. Normal adrenal cells showed at least a tenfold increase under identical conditions. In the presence of corticosteroid precursors the amount of corticosterone produced by the tumor cells was much less than that produced by normal cells. The results indicate a reduced capacity for 11β-hydroxylation in the tumor mitochondria and a possible reduced capacity for biosynthetic steps before the 11β-hydroxylation reaction. Glycolysis in isolated tumor cells was also lower than in normal cells. Isolated tumor mitochondria oxidized succinate normally with a good degree of coupling with phosphorylation. However, unlike normal adrenal mitochondria, the tumor mitochondria showed little or no oxygen uptake with other Krebs cycle substrates. These data suggest that the tumor mitochondria may be lacking in the flavoprotein dehydrogenases responsible for the oxidation of NADH and NADPH, although other components of the respiratory chain may be intact. PMID:4366105

Isoflurane modulates neuronal excitability of the nucleus reticularis thalami in vitro.

PubMed

Joksovic, Pavle M; Todorovic, Slobodan M

2010-06-01

The thalamus has a key function in processing sensory information, sleep, and cognition. We examined the effects of a common volatile anesthetic, isoflurane, on modulation of neuronal excitability in reticular thalamic nucleus (nRT) in intact brain slices from immature rats. In current-clamp recordings, isoflurane (300-600 micromol/L) consistently depolarized membrane potential, decreased input resistance, and inhibited both rebound burst firing and tonic spike firing modes of nRT neurons. The isoflurane-induced depolarization persisted not only in the presence of tetrodotoxin, but after replacement of Ca(2+) with Ba(2+) ions in external solution; it was abolished by partial replacement of extracellular Na(+) ions with N-methyl-D-glucamine. In voltage-clamp recordings, we found that isoflurane slowed recovery from inactivation of T-type Ca(2+) current. Thus, at clinically relevant concentrations, isoflurane inhibits neuronal excitability of nRT neurons in developing brain via multiple ion channels. Inhibition of the neuronal excitability of thalamic cells may contribute to impairment of sensory information transfer in the thalamocortical network by general anesthetics. The findings may be important for understanding cellular mechanisms of anesthesia, such as loss of consciousness and potentially damaging consequences of general anesthetics on developing mammalian brains.

Estimating Young's modulus of zona pellucida by micropipette aspiration in combination with theoretical models of ovum

PubMed Central

Khalilian, Morteza; Navidbakhsh, Mahdi; Valojerdi, Mojtaba Rezazadeh; Chizari, Mahmoud; Yazdi, Poopak Eftekhari

2010-01-01

The zona pellucida (ZP) is the spherical layer that surrounds the mammalian oocyte. The physical hardness of this layer plays a crucial role in fertilization and is largely unknown because of the lack of appropriate measuring and modelling methods. The aim of this study is to measure the biomechanical properties of the ZP of human/mouse ovum and to test the hypothesis that Young's modulus of the ZP varies with fertilization. Young's moduli of ZP are determined before and after fertilization by using the micropipette aspiration technique, coupled with theoretical models of the oocyte as an elastic incompressible half-space (half-space model), an elastic compressible bilayer (layered model) or an elastic compressible shell (shell model). Comparison of the models shows that incorporation of the layered geometry of the ovum and the compressibility of the ZP in the layered and shell models may provide a means of more accurately characterizing ZP elasticity. Evaluation of results shows that although the results of the models are different, all confirm that the hardening of ZP will increase following fertilization. As can be seen, different choices of models and experimental parameters can affect the interpretation of experimental data and lead to differing mechanical properties. PMID:19828504

Adrenal hyperandrogenism is induced by fetal androgen excess in a rhesus monkey model of polycystic ovary syndrome.

PubMed

Zhou, Rao; Bird, Ian M; Dumesic, Daniel A; Abbott, David H

2005-12-01

Adrenal androgen excess is found in approximately 25-60% of women with polycystic ovary syndrome (PCOS), but the mechanisms underlying PCOS-related adrenal androgen excess are unclear. The objective of this study was to determine whether adrenal androgen excess is manifest in a nonhuman primate model for PCOS. Six prenatally androgenized (PA) and six control female rhesus monkeys of similar age, body weight, and body mass index were studied during d 2-6 of two menstrual cycles or anovulatory 30-d periods. Predexamethasone adrenal steroid levels were assessed in the first cycle (cycle 1). In a subsequent cycle (cycle 2), occurring one to three cycles after cycle 1, adrenal steroids were determined 14.5-16.0 h after an i.m. injection of 0.5 mg/kg dexamethasone (postdexamethasone levels) and after an i.v. injection of 50 microg ACTH-(1-39). Both before and after dexamethasone, serum levels of dehydroepiandrosterone (DHEA) in PA females exceeded those in controls. After ACTH injection, PA females exhibited higher circulating levels of DHEA, androstenedione, and corticosterone but comparable levels of 17alpha-hydroxyprogesterone, cortisol, the sulfoconjugate of DHEA, and testosterone compared with controls. Enhanced basal and ACTH-stimulated adrenal androgen levels in PA female monkeys may reflect up-regulation of 17,20 lyase activity in the adrenal zona reticularis, causing adrenal androgen excess comparable with that found in PCOS women with adrenal androgen excess. These findings open the possibility that PCOS adrenal hyperandrogenism may have its origins in fetal androgen excess reprogramming of adrenocortical function.

From Input to Intake: Towards a Brain-Based Perspective of Selective Attention.

ERIC Educational Resources Information Center

Sato, Edynn; Jacobs, Bob

1992-01-01

Addresses, from a neurobiological perspective, the input-intake distinction commonly made in applied linguistics and the role of selective attention in transforming input to intake. The study places primary emphasis upon a neural structure (the nucleus reticularis thalami) that appears to be essential for selective attention. (79 references)…

BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells.

PubMed

Cheng, Xiang; Chen, Shuai; Yu, Xiaoli; Zheng, Pengsheng; Wang, Huayan

2012-07-01

The generation of oocyte-like cells (OLCs) from stem cell differentiation in vitro provides an optimal approach for studying the mechanism of oocyte development and maturation. The aim of this study was to investigate the activation of bone morphogenetic protein 15 gene (BMP15) during the differentiation of human amniotic fluid stem cells (hAFSCs) into OLCs. After 15 days of differentiation, OLCs with a diameter of 50-60 μm and zona pellucida (ZP)-like morphology were observed. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the BMP15 was activated from approximately day 10 of differentiating hAFSCs and thereafter. The reporter construct pBMP15-enhanced green fluorescent protein (EGFP) was transiently transfected into the differentiated hAFSCs and the EGFP expression driven by the BMP15 promoter was positive in the OLCs. Moreover, RT-PCR analysis showed that the oocyte-specific markers including ZP1, ZP2, ZP3, and c-kit were expressed in the differentiated hAFSCs, and the immunofluorescence assay confirmed that the ZP2 was detected in the OLCs. Quantitative RT-PCR revealed that ZP2 and ZP3 were significantly elevated in the differentiated hAFSCs. Further, in the OLCs derived from hAFSCs, the BMP15 promoter directing the EGFP reporter was colocalized with ZP2. Together, these results illustrated that the BMP15 could be used as an oogenesis marker to track hAFSCs differentiation into the OLCs.

Garlic (Allium sativum) feeding impairs Sertoli cell junctional proteins in male Wistar rat testis: microscopy study.

PubMed

Hammami, I; Nahdi, A; Atig, F; El May, A; El May, M V

2016-12-01

Sertoli cell junctions, such as adhesion junction (AJ), gap junction (GJ) and tight junction (TJ), are important for maintaining spermatogenesis. In previous studies, we showed the inhibitory effect of crude garlic (Allium sativum, As) on spermatogenesis and steroidogenesis. The aim of this work was to complete our investigation on the impact of this plant, especially on Sertoli cell junctional proteins (SCJPs). During 1 month, 24 male rats were divided into groups: group control (0% of As) and treated groups fed 5%, 10% and 15% of As. Light and electron microscopy observations were performed to localise junctional proteins: connexin-43, Zona Occluding-1 and N-cadherin (immunohistochemistry) and to describe junctions. We showed that the specific cells involved in the localisation of the SCJP were similar in both control and treated groups, but with different immunoreactivity intensity between them. The electron microscopy observation focused on TJs between Sertoli cells, constituting the blood-testis barrier, showed ultrastructural changes such as fragmentation of TJs between adjacent Sertoli cell membranes and dilatation of rough endoplasmic reticulum saccules giving an aspect of scale to these junctions. We concluded that crude garlic consumption during 1 month induces perturbations on Sertoli cell junctions. These alterations can explain apoptosis in testicular germ cells previously showed. © 2016 Blackwell Verlag GmbH.

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway

PubMed Central

Domínguez-Calderón, Alaide; Ávila-Flores, Antonia; Ponce, Arturo; López-Bayghen, Esther; Calderón-Salinas, José-Víctor; Luis Reyes, José; Chávez-Munguía, Bibiana; Segovia, José; Angulo, Carla; Ramírez, Leticia; Gallego-Gutiérrez, Helios; Alarcón, Lourdes; Martín-Tapia, Dolores; Bautista-García, Pablo; González-Mariscal, Lorenza

2016-01-01

Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size. PMID:27009203

Gender and Gonadal Status Differences in Zona Reticularis Expression in Marmoset Monkey Adrenals: Cytochrome b5 Localization With Respect To Cytochrome P450 17,20-Lyase Activity.

PubMed Central

Pattison, J Christina; Saltzman, Wendy; Abbott, David H; Hogan, Brynn K; Nguyen, Ann D; Husen, Bettina; Einspanier, Almuth; Conley, Alan J; Bird., Ian M

2007-01-01

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive. PMID:17222503

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion.

PubMed

Romero, Damian G; Zhou, Ming Yi; Yanes, Licy L; Plonczynski, Maria W; Washington, Tanganika R; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P

2007-08-01

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary.

PubMed

Mesbah, F; Kafi, M; Nili, H

2016-12-01

The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6-hr interval) in Hams-F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4-year-old and >4-year-old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2-4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4-year-old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2-6 mm follicles are cultivable. The most slaughterhouse-derived COCs retrieved from 2-6 mm follicles of non-pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24-30 hr; the optimal time for CC expansion is 24-30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM. © 2016

[Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility].

PubMed

Serres, Catherine; Auer, Jana; Petit, François; Patrat, Catherine; Jouannet, Pierre

2008-01-01

Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.

Evidence for chronic stress in captive but not free-ranging cheetahs (Acinonyx jubatus) based on adrenal morphology and function.

PubMed

Terio, Karen A; Marker, Laurie; Munson, Linda

2004-04-01

The cheetah (Acinonyx jubatus) is highly endangered because of loss of habitat in the wild and failure to thrive in captivity. Cheetahs in zoos reproduce poorly and have high prevalences of unusual diseases that cause morbidity and mortality. These diseases are rarely observed in free-ranging cheetahs but have been documented in cheetahs that have been captured and held in captive settings either temporarily or permanently. Because captivity may be stressful for this species and stress is suspected as contributing to poor health and reproduction, this study aimed to measure chronic stress by comparing baseline concentrations of fecal corticoid metabolites and adrenal gland morphology between captive and free-ranging cheetahs. Additionally, concentrations of estradiol and testosterone metabolites were quantified to determine whether concentrations of gonadal steroids correlated with corticoid concentration and to assure that corticosteroids in the free-ranging samples were not altered by environmental conditions. Concetntrations of fecal corticoids, estradiol, and testosterone were quantified by radioimmunoassay in 20 free-ranging and 20 captive cheetahs from samples collected between 1994 and 1999. Concentrations of baseline fecal corticoids were significantly higher (p = 0.005) in captive cheetahs (196.08 +/- 36.20 ng/g dry feces) than free-ranging cheetahs (71.40 +/- 14.35 ng/g dry feces). Testosterone concentrations were lower in captive male cheetahs (9.09 +/- 2.84 ng/g dry feces) than in free-ranging cheetahs (34.52 +/- 12.11 ng/g dry feces), which suggests suppression by elevated corticoids in the captive males. Evidence for similar sulppression of estradiol concentrations in females was not present. Adrenal corticomedullary ratios were determined on midsagittal sections of adrenal glands from 13 free-ranging and 13 captive cheetahs obtained between 1991 and 2002. The degree of vacuolation of cortical cells in the zona fasciculata was graded for each animal

Rhinovirus disrupts the barrier function of polarized airway epithelial cells.

PubMed

Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C; Hershenson, Marc B

2008-12-15

Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Primary human airway epithelial cells grown at air-liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (R(T)) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in R(T) without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease R(T), suggesting a requirement for viral replication. Reduced R(T) was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, IFN-gamma and IL-1beta reversed corresponding cytokine-induced reductions in R(T) but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function.

[On the problem of irreversible brain damage due to neuroleptic long-term therapy. Experimental studies on rats (author's transl)].

PubMed

Hackenberg, P; Lange, E

1975-01-01

Reports in clinical literature about persistent terminal extrapyramidal hyperkinesis in neuroleptic long-term treated patients and speculation about demential brain decomposition in such cases give rise to support irreversible psychopharmacotoxical brain damage. Histopathological and animal experimental results in this question given up to this day are not in agreement with each other. Therefore an own chlorpromazine long-term experiment in rats is reported with special regard to biometric-statistical results. Experiments were performed in 36 adult albino rats of either sex, weighing 300-400 g, fed by a standard diet, receiving food and water ad libitum. The animals were divided into 3 groups of 12 animals accidentally. Group I received 15 mg/kg/die chlorpromazine per os by means of a throat probe, group II 10 mg/kg/die, group III was left untreated (controls). The experiment was carried out for 6 months, the animals were killed by perfusion of Bouin's solution 6-8 weeks after interruption of chlorpromazine application. 8 mum paraffin sections were stained by kresylviolet. After histological examination the material was investigated statistically. In the ncl. N. XII, ncl. N. VII, ncl, orig et term. N.V., ncl. cochlearis ant., ncl. vestibularis princeps, Oliva sup., and ncl. dentatus the glia/neurons ratio was stated by counting 200 cells, and in the formation reticularis 400 cells in each animal. The calculated differences in the glia/neurons ratio between the groups were evaluated for statistical significance by the chi2-test. In the ncl. dentatus, oliva sup., ncl, cochlearis ventr., and ncl, vestibularis glia cells and neurons were counted in a plane of 1.2 mm2, in the formatio reticularis and in homologuous parts of cerebellar stratum moleculare in a plane of 2.4 mm2. The differences between the mean values of the groups were verified for statistical significance by means of the t-test. In histological examination only 50% of the animals of group I showed a

Inhibition of endothelin- and phorbol ester-stimulated tyrosine kinase activity by corticotrophin in the rat adrenal zona glomerulosa.

PubMed Central

Kapas, S; Hinson, J P

1996-01-01

1. The experiments described in this study were carried out to investigate the role of tyrosine kinase in the acute adrenal response to peptide hormone stimulation, and to determine whether the activity of this kinase may be subject to regulation by other intracellular signalling mechanisms in the adrenal zona glomerulosa. 2. Previous studies from this laboratory have shown that angiotensin II stimulates tyrosine kinase activity in the rat adrenal cortex. This study has shown, for the first time, that endothelin-1 also stimulates tyrosine kinase activity in this tissue. 3. Using the specific inhibitor of protein kinase C (PKC) activity, Ro 31-8220, we have shown that stimulation of tyrosine kinase activity, in response to endothelin-1, angiotensin II or the phorbol ester phorbol 12-myristate 13-acetate, is at least partly dependent on increased PKC activity. 4. The data presented also provide further evidence of cross-talk between signalling systems in the adrenal cortex. Corticotrophin and its intracellular second messenger, cyclic AMP, significantly attenuate the increment in tyrosine kinase activity seen in response to each of the effectors used. 5. The results of this study provide important new evidence for the regulation of protein kinases by other intracellular second messenger systems. PMID:8611168

Genetic variation in the USDA Chamaecrista Fasciculata collection

USDA-ARS?s Scientific Manuscript database

North American wild kidney bean or thicket bean (Phaseolus polystachios (L.) Britton, Sterns, & Poggenb.) is a perennial vine found in the eastern United States from Texas to Connecticut. Habitat destruction and urbanization are limiting its distribution: e.g., it was once prevalent in the Detroit R...

Genetic variation in the USDA Chamaecrista fasciculata collection

USDA-ARS?s Scientific Manuscript database

Germplasm collections serve as critical repositories of genetic variation. Characterizing genetic diversity in existing collections is necessary to maximize their utility and to guide future collecting efforts. We have used AFLP markers to characterize genetic variation in the USDA germplasm collect...

Posthurricane Survey of Experimental Dunes on Padre Island, Texas.

DTIC Science & Technology

1983-03-01

spikerush, waterhyssop ( Bacopa monnieri), green carpet weed, and frogfruit (Phyla incisa). During the 8 years from th_ summer of 1973 to 1981, the bare...14 144 265 " . Scirpus americanus 0 0 16 0 Bacopa monnieri 0 61 183 8 Baptisia leucophaea 0 0 1 36 Cassia fasciculata 0 118 9 913 Conyza canadensis 0...Scrophulariaceae_______ ______ Bacopa monnieri 5 ______ Compos itae ______ ______ Erigeron myrionactis _______ ______ _____ _________ Senecio riddellii

Two subunits of the 55 K porcine zona pellucida glycoprotein family are immunologically distinct

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subramanian, M.G.; Yurewicz, E.C.; Sacco, A.G.

1986-03-01

The 55K glycoprotein family (ZP3) of the porcine zona pellucida is comprised of two subunits of 46 K and 45 K which can be resolved by endo-..beta..-galactosidase digestion of ZP3 followed by reversed phase HPLC on Vydac C4 resin. Gel electrophoresis revealed that the 46 K component (EBDG..cap alpha..) is approx. 95% pure and the 45 K component (EBGD..beta..) is 100% pure. In the present study, these two subunits were evaluated immunologically by RIA. Under similar reaction protocols (chloramine-T iodination procedure) comparable specific activities were obtained for EBGD..cap alpha.. (33.06 +/- 7.5 ..mu..ci/..mu..gm), EBGD..beta.. (30.45 +/- 1.6) and ZP3 (26.3more » +/- 1.3). Antibody (Ab) titration studies revealed that EBGD..cap alpha.. and ..beta.. are potent immunogens and /sup 125/I-EBGD..cap alpha.. showed minimal cross reactivity to EBGD..beta..-Ab (8% bound at 1:500 dilution), whereas, /sup 125/I-EBGD..beta.. showed a greater degree of cross reactivity to EBGD..cap alpha..-Ab (23% bound at 1:500 dilution). Maximum binding for the two labeled antigens against homologous Abs (1:500) was > 60%. Dose response studies revealed that in the /sup 125/I-EBGD..cap alpha.. vs EBGD..cap alpha.. -Ab system, the 50% intercept was 3.25 +/- 0.32 ng for EBGD..cap alpha.. and 472.43 +/- 30.26 ng for EBGD..beta.. (p < 0.01), whereas, in the /sup 125/I-EBGD..beta.. vs EBGD..beta..-Ab system the 50% intercept was 3.51 +/- 0.58 for EBGD..beta.. and 166.77 +/- 49.20 for EBGD..cap alpha.. (p < 0.01). No significant differences were observed in the slopes of the dose response curves. It is concluded that the two subunits of ZP3 possess distinct immunologic characteristics as evaluated by RIA.« less

Development of the ACTH and corticosterone response to acute hypoxia in the neonatal rat

PubMed Central

Bruder, Eric D.; Taylor, Jennifer K.; Kamer, Kimberli J.; Raff, Hershel

2008-01-01

Acute episodes of severe hypoxia are among the most common stressors in neonates. An understanding of the development of the physiological response to acute hypoxia will help improve clinical interventions. The present study measured ACTH and corticosterone responses to acute, severe hypoxia (8% inspired O2 for 4 h) in neonatal rats at postnatal days (PD) 2, 5, and 8. Expression of specific hypothalamic, anterior pituitary, and adrenocortical mRNAs was assessed by real-time PCR, and expression of specific proteins in isolated adrenal mitochondria from adrenal zona fascisulata/reticularis was assessed by immunoblot analyses. Oxygen saturation, heart rate, and body temperature were also measured. Exposure to 8% O2 for as little as 1 h elicited an increase in plasma corticosterone in all age groups studied, with PD2 pups showing the greatest response (∼3 times greater than PD8 pups). Interestingly, the ACTH response to hypoxia was absent in PD2 pups, while plasma ACTH nearly tripled in PD8 pups. Analysis of adrenal mRNA expression revealed a hypoxia-induced increase in Ldlr mRNA at PD2, while both Ldlr and Star mRNA were increased at PD8. Acute hypoxia decreased arterial O2 saturation (SPo2) to ∼80% and also decreased body temperature by 5–6°C. The hypoxic thermal response may contribute to the ACTH and corticosterone response to decreases in oxygen. The present data describe a developmentally regulated, differential corticosterone response to acute hypoxia, shifting from ACTH independence in early life (PD2) to ACTH dependence less than 1 wk later (PD8). PMID:18703410

[Effect of self-microemulsifying system on cell tight junctions].

PubMed

Sha, Xian-Yi; Fang, Xiao-Ling

2006-01-01

To study the effect of negatively charged and positively charged self-microemulsifying systems (SMES) on the cellular tight junction complex was to be investigated at molecular cell level. Human intestinal epithelial Caco-2 cell model was established. Effect of formulations on the transepithelial electrical resistance (TEER) and permeability of the paracellular transport marker mannitol were measured to evaluate the cell integrity. Changes in subcellular localization of the tight junction protein zona occludens 1 (ZO-1) and cytoskeleton protein actin by immunofluorescence were also assessed after treatment of two SMESs in different dilutions. The TEER of cell monolayers was not markedly affected by negatively charged SMES in different dilutions. The positively charged SMES could significantly decrease the TEER (P < 0.05) in three dilutions. The full recovery of TEER was found after the treatment of lower dilution for 2 h, then cultured for 48 h, while the recovery of TEER was 81.3% of control in 1 : 50 dilution. Two SMESs could enhance the apparent permeability coefficient of mannitol (2.9 - 64.6 folds), which depended on the dilution times. The immunofluorescent results indicated that the distribution of ZO-1 and actin were discrete in cell membrane after the treatment of formulation. Since the positively charged microemulsion could bind to the epithelial cell membrane by electrostatic interaction, the actin of the cells undergone some kind of stress stimulated by the higher concentration of microemulsion was more markedly affected than the negatively charged SMES. Effect of formulations on ZO-1 and actin relied on the dilution. SMES is able to enhance the paracellular transport marker mannitol. The mechanism of opening of tight junctions by SMES might be the change of distribution of ZO-1 and actin.

Livedo Reticularis: When Is It a Concern?

MedlinePlus

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Outcomes from stimulation of the caudal zona incerta and pedunculopontine nucleus in patients with Parkinson's disease.

PubMed

Khan, Sadaquate; Mooney, Lucy; Plaha, Puneet; Javed, Shazia; White, Paul; Whone, Alan L; Gill, Steven S

2011-04-01

Axial symptoms including postural instability, falls and failure of gait initiation are some of the most disabling motor symptoms of Parkinson's disease (PD). We performed bilateral deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) in combination with the caudal zona incerta (cZi) in order to determine their efficacy in alleviating these symptoms. Seven patients with predominant axial symptoms in both the 'on' and 'off' medication states underwent bilateral cZi and PPN DBS. Motor outcomes were assessed using the motor component of the Unified Parkinson's Disease Rating Scale (UPDRS 3) and a composite axial subscore was derived from items 27, 28, 29 and 30 (arising from chair, posture, gait and postural stability). Quality of life was measured using the PDQ39. Comparisons were made between scores obtained at baseline and those at a mean follow-up of 12 months. In both the off and on medication states, a statistically significant improvement in the UPDRS part 3 score was achieved by stimulation of the PPN, cZi and both in combination. In the off medication state, our composite axial subscore of the UPDRS part 3 improved with stimulation of the PPN, cZi and both in combination. The composite axial subscore, in the 'on' medication state, however, only showed a statistically significant improvement when a combination of cZi and PPN stimulation was used. This study provides evidence that a combination of PPN and cZi stimulation can achieve a significant improvement in the hitherto untreatable 'on' medication axial symptoms of PD.

Evaluation of wildlife-habitat relationships data base for predicting bird community composition in central California chaparral and blue oak woodlands

USGS Publications Warehouse

Avery, M.L.; van Riper, Charles

1990-01-01

The California Wildlife-Habitat Relationships (WHR) database can be used to assist resource managers to evaluate effects of habitat manipulations on wildlife. The accuracy of predictions from WHR was evaluated using data from bird surveys conducted during winter and spring 1984 and 1985 in chamise (Adenostema fasciculata) chaparral, mixed chaparral and blue oak (Quercus douglasii) woodland. Considerable variability between habitat types was found for errors both of commission and of omission.

Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

PubMed Central

Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

2006-01-01

Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

Effect of heme oxygenase-1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro.

PubMed

Cao, Yi; Wu, Ben-Juan; Zheng, Wei-Ping; Yin, Ming-Li; Liu, Tao; Song, Hong-Li

2017-07-01

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase-1 (HO-1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO-1 recombinant adenovirus (HO-MSCs) for stable expression of HO-1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor-α (TNF-α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad-MSCs, Ad-HO + MSCs or HO-MSCs. mRNA and protein expression of Zona occludens-1 (ZO-1) and human HO-1 and the release of cytokines were measured. ZO-1 and human HO-1 in Caco2 were significantly decreased after treatment with TNF-α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO-1 was not significantly affected by Caco2 treatment with TNF-α, Ad-HO, and MSCs. In contrast, ZO-1 and human HO-1 increased significantly when the damaged Caco2 was treated with HO-MSCs. HO-MSCs showed the strongest effect on the expression of ZO-1 in colon epithelial cells. Coculture with HO-MSCs showed the most significant effects on reducing the expression of IL-2, IL-6, IFN-γ and increasing the expression of IL-10. HO-MSCs protected the intestinal epithelial barrier, in which endogenous HO-1 was involved. HO-MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti-inflammatory factors. These results suggested that HO-MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO-1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts.

PubMed

Cervera, R P; Garcia-Ximénez, F

2003-10-01

The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 micro m; II: diameter 200-299 micro m; III: diameter >/==" BORDER="0">300 micro m). Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.

Evaluation of a two-generation reproduction toxicity study adding endpoints to detect endocrine disrupting activity using vinclozolin.

PubMed

Matsuura, Ikuo; Saitoh, Tetsuji; Ashina, Michiko; Wako, Yumi; Iwata, Hiroshi; Toyota, Naoto; Ishizuka, Yoshihito; Namiki, Masato; Hoshino, Nobuhito; Tsuchitani, Minoru

2005-12-01

A two-generation reproduction toxicity study in rats adding extra endpoints to detect endocrine disrupting activity was conducted using vinclozolin by dietary administration at 0, 40, 200, and 1000 ppm, for investigation of its utility. The extra endpoints included anogenital distance (AGD), nipple development, sexual maturation (vaginal opening and preputial separation), estrous cycle, spermatogenesis, sex organ weights, and blood hormone concentrations (thyroid and sex hormones). Hepatic drug-metabolizing enzyme activities were also measured. The results revealed changes due to vinclozolin in the AGD, nipple development, sexual maturation, sex organ weights, and blood sex hormone concentrations in males of both parental animals and offspring, even at the lowest dose of 40 ppm, confirmed by results for the classical endpoints of histopathological examination at 200 ppm and mating at 1000 ppm. The effects on parental males included increased pituitary and testis weights, and decreased epididymis weights at 1000 ppm in both generations, and decreased prostate and epididymis weights at 200 and 1000 ppm and seminal vesicle weights at 1000 ppm in F1 males. Histopathological examination revealed hypertrophy of the basophilic cells in the pituitary at these two doses, and diffuse hyperplasia of the testicular interstitial cells and atrophy of the seminal vesicle mucosa at 1000 ppm in F0 and F1 males. In addition, F1 males demonstrated decrease in prostate fluid at 200 and 1000 ppm. Blood hormone analysis revealed increases in LH, FSH, testosterone, and DHT in F0 and F1 males at 1000 ppm. General toxicological effects included suppressed body weight gain in F0 and F1 females and in F1 males, and reduced food consumption in F0 and F1 females at 1000 ppm. Histopathological examination revealed centrilobular hepatocellular hypertrophy in males at 200 and 1000 ppm and in females at 1000 ppm, increased lipid droplets in the adrenal zona fasciculata and zona glomerulosa in

Hydrogen-rich Water Exerting a Protective Effect on Ovarian Reserve Function in a Mouse Model of Immune Premature Ovarian Failure Induced by Zona Pellucida 3

PubMed Central

He, Xin; Wang, Shu-Yu; Yin, Cheng-Hong; Wang, Tong; Jia, Chan-Wei; Ma, Yan-Min

2016-01-01

Background: Premature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, we explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model. Methods: To set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software. Results: Immune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P < 0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax

Caudal Zona Incerta/VOP Radiofrequency Lesioning Guided by Combined Stereotactic MRI and Microelectrode Recording for Posttraumatic Midbrain Resting-Kinetic Tremor.

PubMed

Contreras Lopez, William Omar; Azevedo, Angelo R; Cury, Rubens G; Alencar, Francisco; Neville, Iuri S; Reis, Paul R; Navarro, Jessie; Monaco, Bernardo; da Silva, Fabio E Fernandes; Teixeira, Manoel J; Fonoff, Erich T

2016-02-01

Reporting the outcome of two patients who underwent unilateral ablative stereotactic surgery to treat pharmacologic resistant posttraumatic tremor (PTT). We present two patients (31 and 47 years old) with refractory PTT severely affecting their quality of life. Under stereotactic guidance, refined by T2-weighted magnetic resonance imaging and double-channel multiunit microelectrode recording (MER), three sequential radiofrequency lesions were performed in the caudal zona incerta (cZi) up to the base of thalamus (VOP). Effects of cZi/VOP lesion were prospectively rated with a tremor rating scale. Both patients demonstrated intraoperative tremor suppression with sustained results up to 18 months follow-up, with improvement of 92% and 84%, respectively, on the tremor rating scale. Tremor improvement was associated with enhancement functionality and quality of life for the patients. The patients returned to their work after the procedure. No adverse effects were observed up to the last follow-up. Radiofrequency lesion of the cZi/VOP target was effective for posttraumatic tremor in both cases. The use of T2-weighted images and MER was found helpful in increasing the precision and safety of the procedure, because it leads the RF probe by relying on neighbor structures based on thalamus and subthalamic nucleus. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of Nicotine and Ethanol on Indices of Reward and Sensory-Motor Function in Rats: Implications for the Positive Epidemiologic Relationship Between the Use of Cigarettes and the Use of Alcohol

DTIC Science & Technology

1997-10-07

include the auditory nerve, the ventral cochlear nucleus , nuclei of the lateral lemniscus, nucleus reticularis pontis caudalis, spinal neuron, and lower...chambers. In addition, there was a significant effect of nicotine and ethanol to reduce the ratio of dopamine/DOPAC in nucleus accumbens. Because...dopaminergic activity in nucleus accumbens is known to mediate nicotine reinforcement, reductions in the ratio of dopamine/DOPAC (perhaps indicating an

Voice Tremor Outcomes of Subthalamic Nucleus and Zona Incerta Deep Brain Stimulation in Patients With Parkinson Disease.

PubMed

Karlsson, Fredrik; Malinova, Elin; Olofsson, Katarina; Blomstedt, Patric; Linder, Jan; Nordh, Erik

2018-01-17

We aimed to study the effect of deep brain stimulation (DBS) in the subthalamic nucleus (STN) and caudal zona incerta (cZi) on level of perceived voice tremor in patients with Parkinson disease (PD). This is a prospective nonrandomized design with consecutive patients. Perceived voice tremor was assessed in patients with PD having received either STN-DBS (8 patients, 5 bilateral and 3 unilateral, aged 43.1-73.6 years; median = 61.2 years) or cZi-DBS (14 bilateral patients, aged 39.0-71.9 years; median = 56.6 years) 12 months before the assessment. Sustained vowels that were produced OFF and ON stimulation (with simultaneous l-DOPA medication) were assessed perceptually in terms of voice tremor by two raters on a four-point rating scale. The assessments were repeated five times per sample and rated in a blinded and randomized procedure. Three out of the 22 patients (13%) were concluded to have voice tremor OFF stimulation. Patients with PD with STN-DBS showed mild levels of perceived voice tremor OFF stimulation and a group level improvement. Patients with moderate/severe perceived voice tremor and cZi-DBS showed marked improvements, but there was no overall group effect. Six patients with cZi-DBS showed small increases in perceived voice tremor severity. STN-DBS decreased perceived voice tremor on a group level. cZi-DBS decreased perceived voice tremor in patients with PD with moderate to severe preoperative levels of the symptom. Copyright © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa.

PubMed

Bromfield, Elizabeth G; Aitken, R John; Gibb, Zamira; Lambourne, Sarah R; Nixon, Brett

2014-02-01

While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-β-cyclodextrin, an efficient cholesterol-withdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.

Cloning and characterization of a DNA polymerase beta gene from Trypanosoma cruzi.

PubMed

Venegas, Juan A; Aslund, Lena; Solari, Aldo

2009-06-01

A gene coding for a DNA polymerase beta from the Trypanosoma cruzi Miranda clone, belonging to the TcI lineage, was cloned (Miranda Tcpol beta), using the information from eight peptides of the T. cruzi beta-like DNA polymerase purified previously. The gene encodes for a protein of 403 amino acids which is very similar to the two T. cruzi CL Brener (TcIIe lineage) sequences published, but has three different residues in highly conserved segments. At the amino acid level, the identity of TcI-pol beta with mitochondrial pol beta and pol beta-PAK from other trypanosomatids was between 68-80% and 22-30%, respectively. Miranda Tc-pol beta protein has an N-terminal sequence similar to that described in the mitochondrial Crithidia fasciculata pol beta, which suggests that the TcI-pol beta plays a role in the organelle. Northern and Western analyses showed that this T. cruzi gene is highly expressed both in proliferative and non-proliferative developmental forms. These results suggest that, in addition to replication of kDNA in proliferative cells, this enzyme may have another function in non-proliferative cells, such as DNA repair role similar to that which has extensively been described in a vast spectrum of eukaryotic cells.

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

PubMed Central

Taladriz, Soraya; Hanke, Tobias; Ramiro, María J.; García-Díaz, Miguel; Lacoba, Mario García de; Blanco, Luis; Larraga, Vicente

2001-01-01

We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. PMID:11557814

Unresolved issues in mammalian fertilization.

PubMed

Olds-Clarke, Patricia

2003-01-01

This review considers the role of the sperm in fertilization, addressing areas of misunderstanding and unfounded assumptions and taking particular advantage of the large body of data resulting from work with rodent species in vitro. Considerable attention is given to the appropriate use and interpretation of assays for capacitation, acrosomal exocytosis, hyperactivation, and sperm protein phosphorylation, as well as tests for sperm-zona and sperm-oocyte membrane interactions. The lack of general agreement on the means of sperm adhesion to and penetration of the zona pellucida is addressed, and the need for new approaches to this problem is pointed out. Some molecular advances in our understanding of specific steps in the process of fertilization are discussed in the context of intact cell-matrix and cell-cell interaction. This review should provide practical information for researchers just beginning the study of fertilization and interesting but not widely known observations to stimulate new ideas in experienced scientists.

[Sexuality and STD/AIDS prevention: social representations by rural men in a county in the Zona da Mata region in Pernambuco, Brazil].

PubMed

Alves, Maria de Fátima Paz

2003-01-01

This study analyzes the concepts displayed by rural men in the Zona da Mata region in the State of Pernambuco, Brazil, concerning their sexual practices and STD/AIDS prevention. The study adopts a qualitative methodology, having interviewed 22 men According to the interviews, their first sexual intercourse is characterized as a learning experience and is sometimes marked by violence. They make a distinction between the "woman at home" and "street women"; they acknowledge women's sexual desire and value reciprocity in sexual relations, differentiating between the kinds of sex they have with different categories of women. Seven men report homoerotic experiences during adolescence, which they ascribe to immaturity, not affecting their heterosexual identity. Condom use, perceived in a negative light, is inconstant and irregular, inversely proportional to knowing the female partner. STDs in general inspire little fear, while AIDS is associated with death; the interviewees do not see themselves at risk of acquiring HIV. Ambiguities in the men's discourse, together with a basically ineffective approach by health services and preventive campaigns, reveal a high level of exposure to the risk of contracting STDs/AIDS among the interviewees and their female or male partners.

Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

PubMed

Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

2013-10-01

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed. © 2013 Blackwell Verlag GmbH.

Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary

PubMed Central

Premer, Courtney; Lamondin, Courtney; Mitzey, Ann; Speth, Robert C.; Brownfield, Mark S.

2013-01-01

Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors. PMID:23573410

Selecting One Among the Many: A Simple Network Implementing Shifts in Selective Visual Attention.

DTIC Science & Technology

1984-01-01

Skinner, J.E.. "Gating of thalamic input to cerebrai cortex by nucleus reticularis thalami". In: Attention, voluntary contraction and event... nucleus I uHierarchical networks Cortical anatomy/physiology 20. ABSTRACT (Continue on revee side it necesary end identify by block numnber) *This study...possibility is that the saliency .-- map resides either at the level of the lateral geniculate nucleus (LGN) or in the striate , ..% cortex, V1 (see

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

PubMed

Clarke, Hugh J

2018-01-01

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic

The preclinical evaluation of immunocontraceptive vaccines based on canine zona pellucida 3 (cZP3) in a mouse model.

PubMed

Wang, Ying; Li, Yijie; Zhang, Beibei; Zhang, Fuchun

2018-05-11

Stray dogs are the reservoirs and carriers of rabies and are definitive hosts of echinococcosis. To control the overpopulation of stray dogs, zona pellucida 3 (ZP3), a primary receptor for sperm, is a potential antigen for developing contraceptive vaccines. To enhance the immune responses and contraceptive effects of canine ZP3 (cZP3), dog gonadotropin-releasing hormone (GnRH) and a T cell epitope of chicken ovalbumin (OVA) were selected to construct two fusion proteins with cZP3, ovalbumin-GnRH-ZP3 (OGZ) and ovalbumin-ZP3 (OZ), and their contraceptive effects were evaluated in mice. The synthesized DNA sequences of OGZ and OZ were cloned into plasmid pET-28a respectively. The fusion proteins OGZ and OZ were identified by SDS-PAGE and Western blot. Mice were immunized with OGZ, OZ and cZP3, and the infertility rates were monitored. Mice immunized with mouse ZP3 (mZP3) or adjuvant alone were used as positive control and negative control, respectively. cZP3- and GnRH-specific antibodies (Abs) were detected by ELISA. The bindings of the Abs to oocytes were detected by indirect immunofluorescence assay. The paraffin sections of mice ovaries were observed under microscope for analyzing pathological characteristics. SDS-PAGE and Western blot analyses showed that the two fusion proteins OGZ and OZ were correctly expressed. ELISA results showed that OGZ vaccine induced both cZP3- and GnRH-specific Abs, and OZ vaccine induced cZP3-specific Ab, which lasted for up to 168 days. The levels of follicle stimulating hormone (FSH) and estradiol (E2) in sera were significantly decreased in OGZ immunized mice. Indirect immunofluorescence results showed that Abs induced by cZP3 and mZP3 could bind to the mouse ZP and dog ZP each other. Compared with the adjuvant group, all vaccine immunized groups significantly decreased the fertility rate and mean litter size. Interestingly, the fertility rate in OGZ-immunized group is the lowest, and only 1 mouse out of 10 mice is fertile

Novel effects of edaravone on human brain microvascular endothelial cells revealed by a proteomic approach.

PubMed

Onodera, Hidetaka; Arito, Mitsumi; Sato, Toshiyuki; Ito, Hidemichi; Hashimoto, Takuo; Tanaka, Yuichiro; Kurokawa, Manae S; Okamoto, Kazuki; Suematsu, Naoya; Kato, Tomohiro

2013-10-09

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenger used for acute ischemic stroke. However, it is not known whether edaravone works only as a free radical scavenger or possess other pharmacological actions. Therefore, we elucidated the effects of edaravone on human brain microvascular endothelial cells (HBMECs) by 2 dimensional fluorescence difference gel electrophoresis (2D-DIGE). We found 38 protein spots the intensity of which was significantly altered 1.3 fold on average (p< 0.05) by the edaravone treatment and successfully identified 17 proteins of those. Four of those 17 proteins were cytoskeleton proteins or cytoskeleton-regulating proteins. Therefore, we subsequently investigated the change of size and shape of the cells, the actin network, and the tight junction of HBMEC by immunocytochemistry. As a result, most edaravone-treated HBMECs became larger and rounder compared with those that were not treated. Furthermore, edaravone-treated HBMECs formed gathering zona occludens (ZO)-1, a tight junction protein, along the junction of the cells. In addition, we found that edaravone suppressed interleukin (IL)-1β-induced secretion of monocyte chemoattractant protein-1 (MCP-1), which was reported to increase cell permeability. We found a novel function of edaravone is the promotion of tight junction formations of vascular endothelial cells partly via the down-regulation of MCP-1 secretion. These data provide fundamental and useful information in the clinical use of edaravone in patients with cerebral vascular diseases. © 2013 Elsevier B.V. All rights reserved.

The relevance of "non-criteria" clinical manifestations of antiphospholipid syndrome: 14th International Congress on Antiphospholipid Antibodies Technical Task Force Report on Antiphospholipid Syndrome Clinical Features.

PubMed

Abreu, Mirhelen M; Danowski, Adriana; Wahl, Denis G; Amigo, Mary-Carmen; Tektonidou, Maria; Pacheco, Marcelo S; Fleming, Norma; Domingues, Vinicius; Sciascia, Savino; Lyra, Julia O; Petri, Michelle; Khamashta, Munther; Levy, Roger A

2015-05-01

The purpose of this task force was to critically analyze nine non-criteria manifestations of APS to support their inclusion as APS classification criteria. The Task Force Members selected the non-criteria clinical manifestations according to their clinical relevance, that is, the patient-important outcome from clinician perspective. They included superficial vein thrombosis, thrombocytopenia, renal microangiopathy, heart valve disease, livedo reticularis, migraine, chorea, seizures and myelitis, which were reviewed by this International Task Force collaboration, in addition to the seronegative APS (SN-APS). GRADE system was used to evaluate the quality of evidence of medical literature of each selected item. This critical appraisal exercise aimed to support the debate regarding the clinical picture of APS. We found that the overall GRADE analysis was very low for migraine and seizures, low for superficial venous thrombosis, thrombocytopenia, chorea, longitudinal myelitis and the so-called seronegative APS and moderate for APS nephropathy, heart valve lesions and livedo reticularis. The next step can be a critical redefinition of an APS gold standard, for instance derived from the APS ACTION registry that will include not only current APS patients but also those with antiphospholipid antibodies not meeting current classification criteria. Copyright © 2015 Elsevier B.V. All rights reserved.

The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

PubMed Central

Takahashi, Kazumasa; Goto, Mayumi; Anzai, Mibuki; Ono, Natsuki; Shirasawa, Hiromitsu; Sato, Wataru; Miura, Hiroshi; Sato, Naoki; Sato, Akira; Kumazawa, Yukiyo; Terada, Yukihiro

2017-01-01

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality. PMID

The status of intercellular junctions in established lens epithelial cell lines.

PubMed

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that

Calpain-10 Activity Underlies Angiotensin II-Induced Aldosterone Production in an Adrenal Glomerulosa Cell Model

PubMed Central

Seremwe, Mutsa; Schnellmann, Rick G.

2015-01-01

Aldosterone is a steroid hormone important in the regulation of blood pressure. Aberrant production of aldosterone results in the development and progression of diseases including hypertension and congestive heart failure; therefore, a complete understanding of aldosterone production is important for developing more effective treatments. Angiotensin II (AngII) regulates steroidogenesis, in part through its ability to increase intracellular calcium levels. Calcium can activate calpains, proteases classified as typical or atypical based on the presence or absence of penta-EF-hands, which are involved in various cellular responses. We hypothesized that calpain, in particular calpain-10, is activated by AngII in adrenal glomerulosa cells and underlies aldosterone production. Our studies showed that pan-calpain inhibitors reduced AngII-induced aldosterone production in 2 adrenal glomerulosa cell models, primary bovine zona glomerulosa and human adrenocortical carcinoma (HAC15) cells, as well as CYP11B2 expression in the HAC15 cells. Although AngII induced calpain activation in these cells, typical calpain inhibitors had no effect on AngII-elicited aldosterone production, suggesting a lack of involvement of classical calpains in this process. However, an inhibitor of the atypical calpain, calpain-10, decreased AngII-induced aldosterone production. Consistent with this result, small interfering RNA (siRNA)-mediated knockdown of calpain-10 inhibited aldosterone production and CYP11B2 expression, whereas adenovirus-mediated overexpression of calpain-10 resulted in increased AngII-induced aldosterone production. Our results indicate that AngII-induced activation of calpain-10 in glomerulosa cells underlies aldosterone production and identify calpain-10 or its downstream pathways as potential targets for the development of drug therapies for the treatment of hypertension. PMID:25836666

Taxonomic and faunistic studies on the genus Harutaeographa (Lepidoptera, Noctuidae, Orthosiini) with description of a new species

PubMed Central

Benedek, Balázs; Saldaitis, Aidas; Rimsaite, Jolanta

2012-01-01

Abstract Description of anew Harutaeographa Yoshimoto, 1993 species, Harutaeographa shui sp. n. from China’s Sichuan province, is given. Harutaeographa yangzisherpani transformis Hreblay & Ronkay, 1999 is combined as a synonym of Harutaeographa yangzisherpani yangzisherpani Hreblay & Ronkay, 1999. Additional distributional data for Harutaeographa pallida Yoshimoto, 1993, and Harutaeographa cinerea Hreblay & Ronkay, 1998 are provided. A checklist of the genus Harutaeographa and a key to the Harutaeographa fasciculata (Hampson, 1894) species-group, based on external characters and genitalia, are presented. PMID:23378797

Cardiac involvement in antiphospholipid syndrome associated with Sneddon syndrome: a challenging diagnosis.

PubMed

Faustino, Ana; Paiva, Luís; Morgadinho, Ana; Trigo, Emília; Botelho, Ana; Costa, Marco; Leitão-Marques, António

2014-02-01

Sneddon syndrome is a rare clinical entity characterized by the association of ischemic cerebrovascular disease and livedo reticularis. The authors report a case of stroke and myocardial infarction in a 39-year-old man with Sneddon syndrome and antiphospholipid syndrome who subsequently met some criteria for systemic lupus erythematosus, highlighting the complexity of cardiovascular involvement in systemic diseases. Copyright © 2013 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Task-Evoked Pupillary Responses and Cognitive Processing.

DTIC Science & Technology

1980-02-01

Yingling, C.D., and Skinner, J.E. Gating of thalanic input to cerebral cortex by nucleus reticularis thalami. In J.E. Desmedt (Ed.), Attention, voluntary...innervated. Peripheral control of these muscles originates In the nucleus Edinger-Westphal (located within the third nerve nucleus at the level of the...thalamus is a thin neuronal shell, the reticular nucleus of the thalamus (Carpenter, 1978). Most of the axons from this shell enter the dorsal thalamus

Uncontacted Waorani in the Yasuní Biosphere Reserve: Geographical Validation of the Zona Intangible Tagaeri Taromenane (ZITT).

PubMed

Pappalardo, Salvatore Eugenio; De Marchi, Massimo; Ferrarese, Francesco

2013-01-01

The Tagaeri Taromenane People are two indigenous groups belonging to the Waorani first nation living in voluntary isolation within the Napo region of the western Amazon rainforest. To protect their territory the Ecuadorean State has declared and geographically defined, by Decrees, the Zona Intangible Tagaeri Taromenane (ZITT). This zone is located within the UNESCO Yasuní Biosphere Reserve (1989), one of the most biodiverse areas in the world. Due to several hydrocarbon reserve exploitation projects running in the area and the advancing of a large-scale deforestation front, the survival of these groups is presently at risk. The general aim was to validate the ZITT boundary using the geographical references included in the Decree 2187 (2007) by analyzing the geomorphological characteristics of the area. Remote sensing data such as Digital Elevation Models (DEM), Landsat imagery, topographic cartography of IGM-Ecuador, and fieldwork geographical data have been integrated and processed by Geographical Information System (GIS). The ZITT presents two levels of geographic inconsistencies. The first dimension is about the serious cartographical weaknesses in the perimeter delimitation related to the impossibility of linking two rivers belonging to different basins while the second deals with the perimeter line not respecting the hydrographic network. The GIS analysis results clearly show that ZITT boundary is cartographically nonsense due to the impossibility of mapping out the perimeter. Furthermore, GIS analysis of anthropological data shows presence of Tagaeri Taromenane clans outside the ZITT perimeter, within oil production areas and in nearby farmer settlements, reflecting the limits of protection policies for non-contacted indigenous territory. The delimitation of the ZITT followed a traditional pattern of geometric boundary not taking into account the nomadic characteristic of Tagaeri Taromenane: it is necessary to adopt geographical approaches to recognize the

Uncontacted Waorani in the Yasuní Biosphere Reserve: Geographical Validation of the Zona Intangible Tagaeri Taromenane (ZITT)

PubMed Central

Pappalardo, Salvatore Eugenio; De Marchi, Massimo; Ferrarese, Francesco

2013-01-01

The Tagaeri Taromenane People are two indigenous groups belonging to the Waorani first nation living in voluntary isolation within the Napo region of the western Amazon rainforest. To protect their territory the Ecuadorean State has declared and geographically defined, by Decrees, the Zona Intangible Tagaeri Taromenane (ZITT). This zone is located within the UNESCO Yasuní Biosphere Reserve (1989), one of the most biodiverse areas in the world. Due to several hydrocarbon reserve exploitation projects running in the area and the advancing of a large-scale deforestation front, the survival of these groups is presently at risk. The general aim was to validate the ZITT boundary using the geographical references included in the Decree 2187 (2007) by analyzing the geomorphological characteristics of the area. Remote sensing data such as Digital Elevation Models (DEM), Landsat imagery, topographic cartography of IGM-Ecuador, and fieldwork geographical data have been integrated and processed by Geographical Information System (GIS). The ZITT presents two levels of geographic inconsistencies. The first dimension is about the serious cartographical weaknesses in the perimeter delimitation related to the impossibility of linking two rivers belonging to different basins while the second deals with the perimeter line not respecting the hydrographic network. The GIS analysis results clearly show that ZITT boundary is cartographically nonsense due to the impossibility of mapping out the perimeter. Furthermore, GIS analysis of anthropological data shows presence of Tagaeri Taromenane clans outside the ZITT perimeter, within oil production areas and in nearby farmer settlements, reflecting the limits of protection policies for non-contacted indigenous territory. The delimitation of the ZITT followed a traditional pattern of geometric boundary not taking into account the nomadic characteristic of Tagaeri Taromenane: it is necessary to adopt geographical approaches to recognize the

Development of Monozygotic Twin Mouse Embryos from the Time of Blastomere Separation at the Two-Cell Stage to Blastocyst1

PubMed Central

Katayama, Mika; Ellersieck, Mark R.; Roberts, R. Michael

2010-01-01

The development of blastomeres separated from two-cell stage murine embryos has been compared. Blastomeres were removed from the zona pellucida (ZP) and cultured individually; the twin embryos were compared during their progression to blastocyst in terms of development rate, cell number, morphology, conformation at the four-cell stage, and CDX2 and POU5F1 (also known as OCT4) expression. In general, twin embryos, whether obtained from superovulated or normally bred dams, displayed comparable cell numbers as they advanced. They formed morulae and blastocysts more or less synchronously with each other and with control embryos, although possessing about half of the latter's cell number. Despite this apparent synchrony, the majority of twin blastocysts differed in terms of their relative complements of POU5F1+/CDX2− cells, which represent inner cell mass (ICM), and POU5F1+/CDX2+ cells, which identify trophectoderm (TE). Many, but not all, exhibited a disproportionately small ICM. By contrast, demiembryos retained within their ZP and created by randomly damaging one of the two blastomeres in two-cell stage embryos exhibited a more normal ratio of ICM to TE cells at blastocyst and significantly less variance in ICM cell number. One possible explanation is that ZP-free demiembryos only infrequently adopt the same conformation as their partners, including the favorable tetrahedral form, at the four-cell stage, suggesting that such embryos exhibit a high degree of plasticity with regard to the orientation of their first two cleavage planes and that a significant number likely deviate from paths that provide an optimal geometric progression to blastocyst. These data could explain the difficulty of creating monozygotic twins from two-cell stage embryos. PMID:20181620

Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs.

PubMed

Tanihara, Fuminori; Nakai, Michiko; Men, Nguyen Thi; Kato, Noriko; Kaneko, Hiroyuki; Noguchi, Junko; Otoi, Takeshige; Kikuchi, Kazuhiro

2014-04-01

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization. © 2014 Japanese Society of Animal Science.

Laser microbeam irradiation and renucleation of mouse eggs. Final progress report, July 1, 1979-December 31, 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, T.P.

1983-01-01

We have combined laser microbeam irradiation of mouse egg nuclei with fusion to donor cell nuclei in order to develop a new procedure for transferring nuclei into mammalian eggs. We have been using virus-treated cells injected into the perivitelline for fusion with egg cells. Binucleate cells inside the zona pellucida were often produced indicating nuclear transfer between cells had occurred. To prevent the formation of such abortive polyploidy, host nuclei were inactivated with a laser microbeam. The subsequent cleavage of the microirradiated eggs has been studied.

Ability of spermatozoa to bind to the zona pellucida during oligozoospermia induced with testosterone during a male contraceptive trial.

PubMed

Liu, D Y; Johnston, R; Baker, H W

1995-06-01

To determine the ability of spermatozoa to bind to the zona pellucida (ZP) in testosterone-induced oligozoospermia, previously fertile men participating in the World Heath Organization (WHO) male contraceptive trial in Melbourne were studied while oligozoospermic to various degrees. Semen analysis were performed according to WHO methods. One or two ejaculates from each subject were cryopreserved before commencing weekly intramuscular injections of 200 mg testosterone enanthate. The frozen spermatozoa were used as controls for ZP-binding tests of spermatozoa obtained during testosterone-induced oligozoospermia (< 10 x 10(6)/ml) in either the suppression or efficacy (n = 6) and recovery (n = 3) phases. Two other subjects in the recovery phase with normozoospermia were also tested. Human oocytes that failed to fertilized in vitro from infertile patients were used for the sperm-ZP binding test. Control (frozen) spermatozoa were labelled with fluorescein isothiocyanate and test (oligozoospermic semen) spermatozoa were labelled with tetramethylrhodamine B isothiocyanate. A mixture of equal numbers of labelled motile control and test spermatozoa were incubated with 4-6 ZP. There was a significantly (p < 0.01) lower number of spermatozoa bound per ZP in oligozoospermic samples (65 +/- 7, mean +/- SEM) than in controls (80 +/- 7). However, there were still large numbers of spermatozoa bound to the ZP for all the oligozoospermic samples. Five subjects had similar numbers of spermatozoa bound to the ZP for both control and oligozoospermic samples. Overall, the ZP-binding ratio of test and control spermatozoa averaged 0.82 (range 0.51-1.13).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

PubMed

Kitchener, Anne L; Harman, Amanda; Kay, David J; McCartney, Carmen A; Mate, Karen E; Rodger, John C

2009-01-01

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review. Copyright 2009 Wiley-Liss, Inc.

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles.

PubMed

Petersen, Claudia G; Vagnini, Laura D; Mauri, Ana L; Massaro, Fabiana C; Silva, Liliane F I; Cavagna, Mario; Baruffi, Ricardo L R; Oliveira, Joao B A; Franco, José G

2011-04-23

This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles. The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11). The current study demonstrated an absence of

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

PubMed

Hernández-Pichardo, J E; Ducolomb, Y; Romo, S; Kjelland, M E; Fierro, R; Casillas, F; Betancourt, M

2016-01-01

In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. Experiment 1, 4-20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents ( P  < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates; n  = 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively ( P  < 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) ( P  > 0.05). Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU�

Follicular growth and atresia in the mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oakberg, E. F.

1978-01-01

Follicles were classified on the basis of number of layers of follicle cells, presence and degree of development of the zona pellucida, and presence of an antrum. Formation of an antrum in follicles with less than 7-8 cell layers and/or presence of necrotic cells was considered indicative of degeneration. When classified in this manner, the data suggest that follicles and their contained oocytes are committed to either normal development or atresia by the time a third layer of granulosa cells is formed.

Chemical and Antimicrobial Profiling of Propolis from Different Regions within Libya.

PubMed

Siheri, Weam; Zhang, Tong; Ebiloma, Godwin Unekwuojo; Biddau, Marco; Woods, Nicola; Hussain, Muattaz Yassein; Clements, Carol J; Fearnley, James; Ebel, RuAngelie Edrada; Paget, Timothy; Muller, Sylke; Carter, Katharine C; Ferro, Valerie A; De Koning, Harry P; Watson, David G

2016-01-01

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.

Chemical and Antimicrobial Profiling of Propolis from Different Regions within Libya

PubMed Central

Siheri, Weam; Zhang, Tong; Ebiloma, Godwin Unekwuojo; Biddau, Marco; Woods, Nicola; Hussain, Muattaz Yassein; Clements, Carol J.; Fearnley, James; Ebel, RuAngelie Edrada; Paget, Timothy; Muller, Sylke; Carter, Katharine C.; Ferro, Valerie A.; De Koning, Harry P.; Watson, David G.

2016-01-01

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts. PMID:27195790

MicroRNA-29b regulates TGF-β1-mediated epithelial–mesenchymal transition of retinal pigment epithelial cells by targeting AKT2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Min; Li, Hui; Liu, Xiaoqiang

2016-07-15

The role of microRNA (miRNA) in proliferative vitreoretinopathy (PVR) progression has not been studied extensively, especially in retinal pigment epithelial–mesenchymal transition (EMT) which is the main reason for formation of PVR. In this study, we first investigated the miRNA expression profile in transforming growth factor beta 1 (TGF-β1) mediated EMT of ARPE-19 cells. Among the five changed miRNAs, miR-29b showed the most significant downregulation. Enhanced expression of miR-29b could reverse TGF-β1 induced EMT through targeting Akt2. Akt2 downregulation could inhibit TGF-β1-induced EMT. Furthermore, inhibition of miR-29b in ARPE-19 cells directly triggered EMT process, which characterized by the phenotypic transition andmore » the upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin and zona occludin-1 (ZO-1) with increased cell migration. Akt2-shRNA also inhibited miR-29 inhibitor-induced EMT process. These data indicate that miR-29b plays an important role in TGF-β1-mediated EMT in ARPE-19 cells by targeting Akt2. - Highlights: • MiR-29b expression is decreased in TGF-β1-induced EMT of ARPE-19 cells. • MiR-29b inhibits TGF-β1-induced EMT in ARPE-19 cells. • MiR-29b inhibitor induces EMT in ARPE-19 cells. • Akt2 is the target for miR-29b. • Downregulation of Akt2 prevents TGF-β1-induced EMT of ARPE-19 cells.« less

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

Purpose Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. Methods The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT–PCR), and localization was determined by immunofluorescence labeling. Results Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. Conclusions The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization

The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells.

PubMed

Talbot, Prue; Lin, Sabrina

2011-01-01

Numerous studies have repeatedly shown that women who smoke experience problems establishing and maintaining pregnancies, and recent work has further demonstrated that the in utero effects of smoke may not be manifested until months or even years after birth. The purpose of this review is to examine the recent literature dealing with the effects of cigarette smoke on the earliest stages of human prenatal development. Studies in this area have included the use of animal models, patients undergoing in vitro fertilization, and embryonic stem cell models. Events leading to fertilization, such as cumulus expansion, hyperactivation of sperm motility, and oocyte pick-up by the oviduct are all impaired by smoke exposure in animal models. Steps crucial to fertilization such as the acrosome reaction and sperm binding to the zona pellucida are likewise inhibited by cigarette smoke. Preimplantation embryos and stem cells that model embryos show a number of adverse responses to smoke exposure, including poor adhesion to extracellular matrices, diminished survival and proliferation, and increased apoptosis. The current literature demonstrates that the earliest stages of prenatal development are sensitive to smoke exposure and indicates that pregnant women should be advised not to smoke during this time.

Inoculation of female American black bears (Ursus americanus) with partially purified porcine zona pellucidae limits cub production.

PubMed

Lane, V M; Liu, I K M; Casey, K; vanLeeuwen, E M G; Flanagan, D R; Murata, K; Munro, C

2007-01-01

The present 2-year study investigated the feasibility of using porcine zona pellucidae (pZP) as antigen for immunocontraception in American black bears. Sows, 3-6 years of age, were administered either two doses of 250 microg pZP with Freund's adjuvant (n = 10) or adjuvant alone (n = 5), one in April and one in May, and were kept away from the boars until June. Serum samples were collected before injections and before denning (November). The presence of sows with cubs at side was observed during premature emergence from denning. First-year results indicated that anti-pZP antibody titres in vaccinated sows were 2.5-9.0-fold (range) higher compared with non-vaccinated sows and that the vaccinated sows were threefold less likely to become pregnant (P = 0.167). Control and vaccinated bears produced 1.6 and 0.2 cubs per sow, respectively (P = 0.06). The second-year study investigated the feasibility of using pZP sequestered in a controlled-release pellet and a water-soluble adjuvant (QS-21) to avoid regulatory problems associated with Freund's adjuvant. Sows in the treatment group (n = 22) were administered a single dose of an emulsion of 250 microg pZP and 150 microg QS-21 plus a pellet containing 70-90 microg pZP for delayed release as booster dose. Control sows (n = 5) received the QS-21 adjuvant in pellet alone. Serum samples were collected before inoculations (April) and before denning (November). Seven cubs were born to the five control sows, but none was born to the 22 vaccinated sows (P < 0.001). Anti-pZP antibody mean absorbance ratios in control sows remained at background levels, whereas vaccinated sows had ratios fourfold higher than controls. Two-dimensional polyacrylamide gel electrophoresis and immunohistochemical localisation confirmed immunoreactivity of sera from inoculated bears. We conclude that cub production in the American black bear can be effectively limited with either two injections of 250 microg pZP or a single inoculation of partially

Cloning of corticotropin-releasing hormone (CRH) precursor cDNA and immunohistochemical detection of CRH peptide in the brain of the Japanese eel, paying special attention to gonadotropin-releasing hormone.

PubMed

Amano, Masafumi; Mizusawa, Nanami; Okubo, Kataaki; Amiya, Noriko; Mizusawa, Kanta; Chiba, Hiroaki; Yamamoto, Naoyuki; Takahashi, Akiyoshi

2014-04-01

The stress-related corticotropin-releasing hormone (CRH) was first identified by isolation of its cDNA from the brain of the Japanese eel Anguilla japonica. CRH cDNA encodes a signal peptide, a cryptic peptide and CRH (41 amino acids). The sequence homology to mammalian CRH is high. Next, the distribution of CRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined by immunohistochemistry. CRH-ir cell bodies were detected in several brain regions, e.g., nucleus preopticus pars magnocellularis, nucleus preopticus pars gigantocellularis and formatio reticularis superius. In the brain, CRH-ir fibers were distributed not only in the hypothalamus but also in various regions. Some CRH-ir fibers projected to adrenocorticotropic hormone (ACTH) cells in the rostral pars distalis of the pituitary and also the α-melanocyte-stimulating hormone (α-MSH) cells in the pars intermedia of the pituitary. Finally, the neuroanatomical relationship between the CRH neurons and gonadotropin-releasing hormone (GnRH) neurons was examined by dual-label immunohistochemistry. CRH-ir fibers were found to be in close contact with GnRH-ir cell bodies in the hypothalamus and in the midbrain tegmentum and GnRH-ir fibers were in close contact with CRH-ir cell bodies in the nucleus preopticus pars magnocellularis. These results suggest that CRH has some physiological functions other than the stimulation of ACTH and α-MSH secretion and that reciprocal connections may exist between the CRH neurons and GnRH neurons in the brain of the Japanese eel.

Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buehler, Paul W.; Butt, Omer I.; D'Agnillo, Felice, E-mail: felice.dagnillo@fda.hhs.gov

Highlights: {yields} Toxicological implications associated with the use of NaNO{sub 2} therapy to treat systemic cell-free Hb exposure are not well-defined. {yields} Systemic Hb exposure followed by NaNO{sub 2} infusion induces acute CNS toxicities in guinea pigs. {yields} These CNS effects were not reproduced by the infusion of cell-free Hb or NaNO{sub 2} alone. {yields} NaNO{sub 2}-mediated oxidation of cell-free Hb may play a causative role in the observed CNS changes. -- Abstract: Systemic cell-free hemoglobin (Hb) released via hemolysis disrupts vascular homeostasis, in part, through the scavenging of nitric oxide (NO). Sodium nitrite (NaNO{sub 2}) therapy can attenuate themore » hypertensive effects of Hb. However, the chemical reactivity of NaNO{sub 2} with Hb may enhance heme- or iron-mediated toxicities. Here, we investigate the effect of NaNO{sub 2} on the central nervous system (CNS) in guinea pigs exposed to systemic cell-free Hb. Intravascular infusion of NaNO{sub 2}, at doses sufficient to alleviate Hb-mediated blood pressure changes, reduced the expression of occludin, but not zona occludens-1 (ZO-1) or claudin-5, in cerebral tight junctions 4 h after Hb infusion. This was accompanied by increased perivascular heme oxygenase-1 expression, neuronal iron deposition, increased astrocyte and microglial activation, and reduced expression of neuron-specific nuclear protein (NeuN). These CNS changes were not observed in animals treated with Hb or NaNO{sub 2} alone. Taken together, these findings suggest that the use of nitrite salts to treat systemic Hb exposure may promote acute CNS toxicity.« less

The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland.

PubMed

Grandclément, B; Ronsin, B; Morel, G

1997-03-01

Atrial natriuretic peptide (ANP) actions are mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANP receptor-A (or GC-A), -B (or GC-B) and -C (the so-called clearance receptor). In rat adrenal gland, the mRNA for each subtype was detected using 35S-dUTP or digoxigenin-11-dUTP specific labeled probes, and in situ hybridization at light and electron microscopic levels respectively. The three subtypes were expressed the most abundantly in the zona glomerulosa. The amount of GC-A mRNA expression, quantified using macro-autoradiography and densitometry, was higher than the amounts of GC-B mRNA and ANPR-C mRNA both in zona glomerulosa and medullary of adrenal gland. At electron microscopic level, the three subtypes of ANPR were revealed in glomerulosa cells. A noticeable signal was also present in the medullary area, especially for GC-A mRNA, in adrenaline-containing chromaffin cells. No signal was detected in noradrenaline-containing chromaffin cells. The subcellular localization of the three mRNAs is similar: in the cytoplasmic matrix and in the euchromatin of the nucleus in each cell of glomerulosa, and in the same compartments of the adrenaline-containing chromaffin cells. These data indicate that the adrenal gland is an important target tissue for ANP action both in glomerulosa cells and adrenaline-containing chromaffin cells. The mRNA expression levels were different for each ANPR subtype.

The nucleus of the optic tract (NOT) and the dorsal terminal nucleus (DTN) of opossums (Didelphis marsupialis aurita).

PubMed

Vargas, C D; Volchan, E; Nasi, J P; Bernardes, R F; Rocha-Miranda, C E

1996-01-01

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected unilaterally into the pretectocollicular region of opossums (Didelphis marsupialis aurita), primarily to investigate the existence of a commissural subcortical pathway but also to reveal afferents and efferents of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in this species. Labelled cells and terminals were observed in the contralateral NOT-DTN. Furthermore, HRP was injected bilaterally in the region of the inferior olive (IO) to verify if the distribution of labelled cells in the NOT-DTN overlapped the region of commissural labelled cells. The two subpopulations of retrogradely labelled cells coincided, being distributed within the retinal terminal field attributed to the NOT-DTN, as revealed by contralateral eye injections of HRP. The commissural cells were located slightly more ventral than the olivary cells in the optic tract. The pretectocollicular WGA-HRP injections also labelled cells and terminals bilaterally in the lateral terminal nucleus (LTN), interstitial nucleus of the superior fasciculus, posterior fibers (INSFp), ventral lateral geniculate nucleus (vLGN), and superior colliculus (SC) and ipsilaterally in the medial terminal nucleus (MTN). In addition, further caudally, labelled cells and terminals were observed bilaterally in the nuclei prepositus hypoglossi (PH) and in the medial (MVN) and lateral (LVN) vestibular nuclei. Labelled terminals were found in the ipsilateral nucleus reticularis tegmenti pontis (NRTP) and in the IO with ipsilateral predominance. This study allowed an anatomical delimitation of the NOT-DTN in this opossum species, as defined by the olivary and commissural subpopulations, as well as a hodological evaluation of this region. The existence of some common anatomical aspects with other mammalian species is discussed.

Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome.

PubMed

Meczekalski, B; Nawrot, R; Nowak, W; Czyzyk, A; Kedzia, H; Gozdzicka-Jozefiak, A

2015-07-01

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte. We analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes. The expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices. Data from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

PubMed

Coutinho da Silva, Marco A; Seidel, George E; Squires, Edward L; Graham, James K; Carnevale, Elaine M

2014-11-10

Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of β-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, β- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for β- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that β- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown. Copyright © 2014 Elsevier B.V. All rights reserved.

Subcortical neural circuits for ocular accommodation and vergence in primates.

PubMed

Gamlin, P D

1999-03-01

Our current knowledge of the neural bases of vergence and accommodation has increased significantly over the past few years. The behavior of medial rectus motoneurons during vergence, which has been reported by a number of investigators, is described. The behavior of Edinger-Westphal neurons during accommodation is also described, as are the characteristics of midbrain near-response neurons in the supraoculomotor area. Evidence that some of these near-response neurons provide the vergence input to medial rectus motoneurons and possibly the accommodation input to Edinger-Westphal neurons is reviewed. Anatomical studies have shown that the midbrain near-response region receives input from two deep cerebellar nuclei, the posterior interposed and the fastigial nucleus. Single-unit recording in the posterior interposed nucleus has revealed cells that increase their activity during the far-response, and the behavior of these neurons is reviewed. In addition, studies of a precerebellar nucleus, the nucleus reticularis tegmenti pontis, have revealed some cells that increase their activity during the near-response and others that do so during the far-response. The behavior of these neurons is reviewed. This review documents the great strides that are occurring in our understanding of the anatomy and physiology of the neural pathways controlling vergence and accommodation in the primate.

X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells.

PubMed

Ho, Bianca; Greenlaw, Keelin; Al Tuwaijri, Abeer; Moussette, Sanny; Martínez, Francisco; Giorgio, Elisa; Brusco, Alfredo; Ferrero, Giovanni Battista; Linhares, Natália D; Valadares, Eugênia R; Svartman, Marta; Kalscheuer, Vera M; Rodríguez Criado, Germán; Laprise, Catherine; Greenwood, Celia M T; Naumova, Anna K

2018-02-20

Sexual dimorphism in DNA methylation levels is a recurrent epigenetic feature in different human cell types and has been implicated in predisposition to disease, such as psychiatric and autoimmune disorders. To elucidate the genetic origins of sex-specific DNA methylation, we examined DNA methylation levels in fibroblast cell lines and blood cells from individuals with different combinations of sex chromosome complements and sex phenotypes focusing on a single autosomal region--the differentially methylated region (DMR) in the promoter of the zona pellucida binding protein 2 (ZPBP2) as a reporter. Our data show that the presence of the sex determining region Y (SRY) was associated with lower methylation levels, whereas higher X chromosome dosage in the absence of SRY led to an increase in DNA methylation levels at the ZPBP2 DMR. We mapped the X-linked modifier of DNA methylation to the long arm of chromosome X (Xq13-q21) and tested the impact of mutations in the ATRX and RLIM genes, located in this region, on methylation levels. Neither ATRX nor RLIM mutations influenced ZPBP2 methylation in female carriers. We conclude that sex-specific methylation differences at the autosomal locus result from interaction between a Y-linked factor SRY and at least one X-linked factor that acts in a dose-dependent manner.

Brainstem efferents from the interface between the nucleus medialis and the nucleus interpositus in the rat.

PubMed

Buisseret-Delmas, C; Angaut, P; Compoint, C; Diagne, M; Buisseret, P

1998-12-14

In a previous report (Buisseret-Delmas et al. [1993] Neurosci. Res. 16:195-207), the authors identified the interface between the cerebellar nuclei medialis and interpositus as the origin of the nuclear output from cortical zone X. They named this nuclear interface the interstitial cell group (icg). In this study, the authors analyzed the icg efferents to the brainstem by using the anterograde and retrograde tracer biotinylated dextran amine. The main targets of these efferents are from rostral to caudal: 1) the accessory oculomotor nuclear region, essentially, the interstitial nucleus of Cajal; 2) the caudoventral region of the red nucleus; 3) a dorsal zone of the nucleus reticularis tegmenti pontis; 4) restricted regions of the four main vestibular nuclei; and 5) three restricted areas in the inferior olive, one that is caudal in the medial accessory subnucleus and two others that are rostral and caudal in the dorsal accessory subnucleus, respectively. These data support the notion that the icg contributes to the control of gaze-orientation mechanisms, particularly those that are related to the vestibuloocular reflex.

Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death

PubMed Central

Malecki, Marek; LaVanne, Christine; Alhambra, Dominique; Dodivenaka, Chaitanya; Nagel, Sarah; Malecki, Raf

2014-01-01

Introduction The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors. Specific aim The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases). Methods The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers’ bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies’ guided DNA vectors delivered the transgenes for the human recombinant DNases’ into proliferating stem cells. Results Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases

Oxytocin in corticosterone-induced chronic stress model: Focus on adrenal gland function.

PubMed

Stanić, Dušanka; Plećaš-Solarović, Bosiljka; Mirković, Duško; Jovanović, Predrag; Dronjak, Slađana; Marković, Bojan; Đorđević, Tea; Ignjatović, Svetlana; Pešić, Vesna

2017-06-01

Chronic stress conditions can lead to considerable and extensible changes in physiological and psychological performances, and in emergence of risk for various somatic diseases. On the other hand, the neuropeptide oxytocin is reported to increase the resistance of the organism to stress and modulate activity of autonomic nervous system. Chronic corticosterone administration is used as a rat model for a state observed in terms of chronic stress exposure, when negative feedback mechanism of hypothalamus-pituitary-adrenal axis activity is disrupted. In our study, we aimed to investigate whether chronic administration of oxytocin (10 IU/400μL/day for 14days, s.c.) influenced adrenal gland morphology and activity in adult male Wistar rats during long-term corticosterone administration via drinking water (100mg/L for 21days). We examined the influence of treatments on the levels of adrenal gland hormones, corticosterone, adrenaline and noradrenaline, as well as their response to an acute stress challenge evoked by 15-min forced swimming. In addition, the expression of two main monoamine transporters, the noradrenaline transporter (NAT) and vesicular monoamine transporter 2 (VMAT2) in adrenal medulla was measured in the rats exposed to acute stress. Our results showed that oxytocin treatment prevented corticosterone-induced decrease in body weight gain, attenuated adrenal gland atrophy by increasing glandular weight, and the area of the zona fasciculate and reticularis. Chronic corticosterone intake blunted the response of all measured hormones to acute stress, whereas concomitant oxytocin treatment reversed adrenaline and noradrenaline response to acute stress. Furthermore, in adrenal medulla, oxytocin produced significant vasodilatation and stimulated expression of both catecholamine transporters detected both on mRNA and protein level. Our data suggest that oxytocin, by reducing atrophy of adrenal gland, and by increasing catecholamine storage capacity, may be

SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice.

PubMed

Zhuang, Xin-jie; Hou, Xiao-jun; Liao, Shang-Ying; Wang, Xiu-Xia; Cooke, Howard J; Zhang, Ming; Han, Chunsheng

2011-01-01

Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated. The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum. SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

PubMed

Kilani, Suha S; Cooke, Simon; Kan, Andrew K; Chapman, Michael G

2006-02-01

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.

The Maturation of Oocyte Follicular Epithelium of Podarcis s. sicula Is Promoted by d-Aspartic Acid

PubMed Central

Raucci, Franca; Di Fiore, Maria Maddalena

2010-01-01

We investigated whether the maturation of oocyte follicular epithelium of lizard is affected by d-aspartic acid (d-Asp). Our results demonstrated that d-Asp is endogenously present in the oocytes, and its distribution varies during the reproductive cycle and following intraperitoneal administration. At previtellogenesis, it is observed in the cytoplasm and nucleus of pyriform cells, in intermediate cells, in some small cells of the granulosa, in the ooplasm, and in some thecal elements. At vitellogenesis, d-Asp is localized in the proximity of the zona pellucida, in the theca, and in the ooplasm. Injected d-Asp is mainly captured by pyriform cells and ooplasm of previtellogenic oocytes, but a moderate accumulation is evident in the cytoplasm of some small granulosa cells and in the theca. d-Asp also increases the ovarian and plasmatic levels of 17β-estradiol and decreases those of testosterone. As a direct and/or indirect consequence of d-Asp, previtellogenic oocytes grow up and mature, resulting in a higher accumulation of carbohydrates in the granulosa, zona pellucida, and ooplasm, but also a reduction in the thickness of the granulosa layer and an increase of the theca stratum. Taken together, our results show that d-Asp may be related to the synchrony of reproduction, either enhancing the growth and maturation of follicular epithelium or influencing its endocrine functions. (J Histochem Cytochem 58:157–171, 2010) PMID:19826072

Feedforward inhibitory control of sensory information in higher-order thalamic nuclei.

PubMed

Lavallée, Philippe; Urbain, Nadia; Dufresne, Caroline; Bokor, Hajnalka; Acsády, László; Deschênes, Martin

2005-08-17

Sensory stimuli evoke strong responses in thalamic relay cells, which ensure a faithful relay of information to the neocortex. However, relay cells of the posterior thalamic nuclear group in rodents, despite receiving significant trigeminal input, respond poorly to vibrissa deflection. Here we show that sensory transmission in this nucleus is impeded by fast feedforward inhibition mediated by GABAergic neurons of the zona incerta. Intracellular recordings of posterior group neurons revealed that the first synaptic event after whisker deflection is a prominent inhibition. Whisker-evoked EPSPs with fast rise time and longer onset latency are unveiled only after lesioning the zona incerta. Excitation survives barrel cortex lesion, demonstrating its peripheral origin. Electron microscopic data confirm that trigeminal axons make large synaptic terminals on the proximal dendrites of posterior group cells and on the somata of incertal neurons. Thus, the connectivity of the system allows an unusual situation in which inhibition precedes ascending excitation resulting in efficient shunting of the responses. The dominance of inhibition over excitation strongly suggests that the paralemniscal pathway is not designed to relay inputs triggered by passive whisker deflection. Instead, we propose that this pathway operates through disinhibition, and that the posterior group forwards to the cerebral cortex sensory information that is contingent on motor instructions.

Atorvastatin prevents angiotensin II-induced high permeability of human arterial endothelial cell monolayers via ROCK signaling pathway.

PubMed

Yi, Ren; Xiao-Ping, Gao; Hui, Liang

2015-03-27

Intracranial aneurysm, as a common cause of cerebral hemorrhage, is often discovered when the aneurysm ruptures, causing subarachnoid hemorrhage. Unfortunately, the formation of cerebral aneurysm, which is associated with endothelial damage and macrophage migration, still cannot be prevented now. Tight junctions (TJs) open due to the disappearance of TJ proteins occludin and zona occludens-1 (ZO-1) in damaged endothelia, thus allowing macrophage migration and forming cerebral aneurysm. Therefore, cerebral aneurysm formation can be prevented by increasing TJs of the artery endothelium. Interestingly, statin, which can reduce saccular aneurysm, may prevent aneurysm formation through acting on different steps, but the underlying mechanism remains unclear. In this study, angiotensin II (Ang II) significantly increased the permeability of human arterial endothelial cell (HAEC). Moreover, the distribution of ZO-1 in cell-cell junction area and the total expression in HAECs were significantly decreased by Ang II treatment. However, the abnormal distribution and decreased expression of ZO-1 and hyperpermeability of HAECs were significantly reversed by pretreatment with atorvastatin. Furthermore, Ang II-induced phosphorylations of MYPT1, LIMK and MLC2 were significantly inhibited with atorvastatin or Rho kinase (ROCK) inhibitor (H1152) pretreatment. Knockdown of ROCK-II probably abolished Ang II-induced abnormal ZO-1 distribution and expression deficiency and hyperpermeability of HAECs. In conclusion, atorvastatin prevented Ang II-induced rupture of HAEC monolayers by suppressing the ROCK signaling pathway. Our results may explain, at least in part, some beneficial effects of statins on cardiovascular diseases such as intracranial aneurysm. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic selection for a highly functional cysteine-less membrane protein using site-saturation mutagenesis

PubMed Central

Arendt, Cassandra S.; Ri, Keirei; Yates, Phillip A.; Ullman, Buddy

2007-01-01

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiol-specific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. PMID:17481563

Urinary retention, erectile dysfunction and meningitis due to sacral herpes zoster: a case report and review of the literature.

PubMed

Erol, B; Avci, A; Eken, C; Ozgok, Y

2009-01-01

Zona zoster infection is often associated with painful erythematous vesicular eruptions of the skin or mucous membranes. Varicella zoster virus which stays latent in the sensorial root ganglia causes zona zoster infection. The most recognized feature of zona zoster is the dermatomal distribution of vesicular rashes. In the present case report, we state an unusual presentation of sacral zona zoster with urinary retention, erectile dysfunction and meningitis. Copyright 2009 S. Karger AG, Basel.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

PubMed

Tagawa, M; Matoba, S; Narita, M; Saito, N; Nagai, T; Imai, K

2008-03-15

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

Genetics Home Reference: Naegeli-Franceschetti-Jadassohn syndrome/dermatopathia pigmentosa reticularis

MedlinePlus

... that are the basis for each person's unique fingerprints. Additional features of NFJS/DPR can include a ... Mutations and Health Inheritance Pattern This condition is inherited in an autosomal dominant pattern , which means one ...

Evaluation of tight junction protein 1 encoding zona occludens 1 as a candidate gene for albuminuria in a Mexican American population.

PubMed

Lehman, D M; Leach, R J; Johnson-Pais, T; Hamlington, J; Fowler, S; Almasy, L; Duggirala, R; Stern, M P; Abboud, H E

2006-09-01

Albuminuria, a hallmark of diabetic nephropathy, has been shown to be significantly heritable in multiple studies. Therefore, the identification of genes that affect susceptibility to albuminuria may lead to novel avenues of intervention. Current evidence suggests that the podocyte and slit diaphragm play a key role in controlling the selective sieve of the glomerular filtration barrier, and podocyte-specific genes have been identified that are necessary for maintaining its integrity. We therefore investigated the role of gene variants of tight junction protein (TJP1) which encodes another slit diaphragm-associated protein zona occludens 1 as risk factors for albuminuria in the San Antonio Family Diabetes/Gallbladder Study (SAFDGS), which consists of extended Mexican-American families with a high prevalence of type 2 diabetes. Albuminuria, defined as an albumin (mg/dl) to creatinine (mg/dl) ratio (ACR) of 0.03, which is approximately equivalent to a urinary albumin excretion (UAE) >30 mg/day, was present in a total of 14.9% of participants, and 31% had type 2 diabetes. The TJP1 exons, flanking intronic sequence, and putative proximal promoter regions were investigated in this population. Twentynine polymorphisms, including 7 nonsynonymous SNPs, were identified and genotyped in all subjects of this study for association analysis. Three sets of correlated SNPs, which include 3 exonic SNPs, were nominally associated with ACR (p value range 0.007-0.049); however, the association with the discrete trait albuminuria was not significant (p value range 0.094-0.338). We conclude that these variants in TJP1 do not appear to be major determinants for albuminuria in the SAFDGS; however, they may play a minor role in its severity in this Mexican-American population. Further examination of the TJP1 gene region in this and other cohorts will be useful to determine whether ZO-1 plays a significant role in glomerular permselectivity.

Single-treatment porcine zona pellucida immunocontraception associated with reduction of a population of white-tailed deer (Odocoileus virginianus).

PubMed

Rutberg, Allen T; Naugle, Ricky E; Verret, Frank

2013-12-01

Previous reports have demonstrated gradual reductions of white-tailed deer (Odocoileus virginianus) populations through immunocontraception, with stabilization occurring after 2-4 yr of treatment, and subsequent reductions of 6-10% annually. These studies employed porcine zona pellucida (PZP) vaccines that required two initial treatments and annual retreatments. From 2005 to 2010, 258 adult and yearling female deer on Fripp Island, South Carolina, were treated with one of several PZP preparations designed to produce 2+ yr of effective contraception with a single treatment. These included several preparations of SpayVac and of native PZP-adjuvant emulsion plus PZP and QA-21 in timed-release pellets. Deer were chemically immobilized, ear-tagged, and administered initial treatments by hand in February-March. Some treated deer were boosted remotely with PZP-adjuvant emulsion 1.5 - 4.5 yr after initial treatments. Ground-based distance sampling was used to estimate deer population density at Fripp Island, a resort community, and at a relatively undeveloped neighboring control site, Hunting Island. Most vaccine preparations tested reduced fawning rates by 75% to 95% for at least 1 yr. From 2005 to 2011, deer density on Fripp Island declined by 50%, from 72 deer/km(2) to 36 deer/km(2), an average annual reduction of 11%. In contrast, population density on the Hunting Island control site fluctuated between 2005 and 2011, averaging 23 deer/km(2) (range, 19-28 deer/km(2)). Population declines on Fripp Island were associated with an increase in the proportion of treated females and with a progressive decrease in winter fawn:doe ratios, from 1.21 fawns/doe in 2005 to 0.19 fawns/doe in 2010. Winter fawn:doe ratios averaged 1.36 fawns/doe (range, 0.84 - 1.62 fawns/doe) at the Hunting Island control site. Annual survivorship averaged approximately 79% among ear-tagged females. The rate at which deer populations diminished in association with PZP treatments on Fripp Island was

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

PubMed

Wong, Chi-Wai; Lam, Kevin K W; Lee, Cheuk-Lun; Yeung, William S B; Zhao, Wei E; Ho, Pak-Chung; Ou, Jian-Ping; Chiu, Philip C N

2017-04-01

Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface

The proliferative effect of synthetic N-POMC(1-28) peptides in rat adrenal cortex: a possible role for cyclin E.

PubMed

Mendonça, Pedro O R de; Lotfi, Claudimara F P

2011-04-10

Modified synthetic N-POMC(1-28) without disulfide bridges has been shown to act as an adrenal mitogen. Cyclins and their inhibitors are the major cell cycle controls, but in the adrenal cortex the effect of ACTH and N-POMC on the expression of these proteins remains unclear. In this work, we evaluate the effect of different synthetic N-POMC peptides on the S-phase of the cell cycle. In addition, we examine the cyclin E expression in rat adrenal cortex. Rats treated with dexamethasone were injected with ACTH and/or synthetic modified N-POMC and/or synthetic N-POMC with disulfide bridges. DNA synthesis was determined by BrdU incorporation and protein expression was analyzed by immunoblotting and immunohistochemistry. The results showed that similarly to modified N-POMC without disulfide bridges, administration of synthetic N-POMC with disulfide bridges and the combination of ACTH and N-POMC promoted an increase of BrdU-positive nuclei in adrenal cortex. However, the proliferative effect of N-POMC was comparable to that of ACTH only in the zona glomerulosa. An increase in cyclin E expression was observed 6 h after N-POMC treatment in the outer fraction of the adrenal cortex, in agreement with immunohistochemical findings in the zona glomerulosa. In summary, the effect of synthetic N-POMC with disulfide bridges was similar to modified synthetic N-POMC, increasing proliferation in the adrenal cortex, confirming previous evidence that disulfide bridges are not essential to the N-POMC mitogenic effect. Moreover, cyclin E appears to be involved in the N-POMC- and ACTH-stimulated proliferation in the zona glomerulosa of the adrenal cortex. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

RGS2 is regulated by angiotensin II and functions as a negative feedback of aldosterone production in H295R human adrenocortical cells.

PubMed

Romero, Damian G; Plonczynski, Maria W; Gomez-Sanchez, Elise P; Yanes, Licy L; Gomez-Sanchez, Celso E

2006-08-01

Regulator of G protein signaling (RGS) proteins interact with Galpha-subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. Angiotensin (Ang) II interacts with its G protein-coupled receptor in zona glomerulosa adrenal cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. We studied Ang II-mediated regulation of RGS2, the role of RGS2 in steroidogenesis, and the intracellular signal events involved in H295R human adrenal cells. We report that both H295R cells and human adrenal gland express RGS2 mRNA. In H295R cells, Ang II caused a rapid and transient increase in RGS2 mRNA levels quantified by real-time RT-PCR. Ang II effects were mimicked by calcium ionophore A23187 and blocked by calcium channel blocker nifedipine. Ang II effects also were blocked by calmodulin antagonists (W-7 and calmidazolium) and calcium/calmodulin-dependent kinase antagonist KN-93. RGS2 overexpression by retroviral infection in H295R cells caused a decrease in Ang II-stimulated aldosterone secretion but did not modify cortisol secretion. In reporter assays, RGS2 decreased Ang II-mediated aldosterone synthase up-regulation. These results suggest that Ang II up-regulates RGS2 mRNA by the calcium/calmodulin-dependent kinase pathway in H295R cells. RGS2 overexpression specifically decreases aldosterone secretion through a decrease in Ang II-mediated aldosterone synthase-induced expression. In conclusion, RGS2 expression is induced by Ang II to terminate the intracellular signaling cascade generated by Ang II. RGS2 alterations in expression levels or functionality could be implicated in deregulations of Ang II signaling and abnormal aldosterone secretion by the adrenal gland.

Neuropeptide Y mRNA and peptide in the night-migratory redheaded bunting brain.

PubMed

Devraj, Singh; Kumari, Yatinesh; Rastogi, Ashutosh; Rani, Sangeeta; Kumar, Vinod

2013-11-01

This study investigated the distribution of neuropeptide Y (NPY) in the brain of the night-migratory redheaded bunting (Emberiza bruniceps). We first cloned the 275-bp NPY gene in buntings, with ≥95% homology with known sequences from other birds. The deduced peptide sequence contained all conserved 36 amino acids chain of the mature NPY peptide, but lacked 6 amino acids that form the NPY signal peptide. Using digosigenin-labeled riboprobe prepared from the cloned sequence, the brain cells that synthesize NPY were identified by in-situ hybridization. The NPY peptide containing cell bodies and terminals (fibers) were localized by immunocytochemistry. NPY mRNA and peptide were widespread throughout the bunting brain. This included predominant pallial and sub-pallial areas (cortex piriformis, cortex prepiriformis, hyperpallium apicale, hippocampus, globus pallidus) and thalamic and hypothalamic nuclei (organum vasculosum laminae terminalis, nucleus (n.) dorsolateralis anterior thalami, n. rotundus, n. infundibularis) including the median eminence and hind brain (n. pretectalis, n. opticus basalis, n. reticularis pontis caudalis pars gigantocellularis). The important structures with only NPY-immunoreactive fibers included the olfactory bulb, medial and lateral septal areas, medial preoptic nucleus, medial suprachiasmatic nucleus, paraventricular nucleus, ventromedial hypothalamic nucleus, optic tectum, and ventro-lateral geniculate nucleus. These results demonstrate that NPY is possibly involved in the regulation of several physiological functions (e.g. daily timing feeding, and reproduction) in the migratory bunting.

Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

PubMed

Schophuizen, Carolien M S; De Napoli, Ilaria E; Jansen, Jitske; Teixeira, Sandra; Wilmer, Martijn J; Hoenderop, Joost G J; Van den Heuvel, Lambert P W; Masereeuw, Rosalinde; Stamatialis, Dimitrios

2015-03-01

The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Zonal differences in the distribution and morphology of lipid droplets using 4-amino-pyrazolo-(3,4 d) pyrimidine to lower cholesterol level in the rat adrenal.

PubMed

Szabó, D; Somogyi, J; Acs, Z; Mihály, K

1980-01-01

The effect of reduced blood and adrenal cholesterol levels on adrenocortical lipid droplets have been examined by treating adult rats with 4-amino-pyrazolo-(3,4 d) pyrimidine (4-APP), a drug that inhibits hepatic secretion of lipoproteins. Lowering the blood cholesterol level and the cholesterol content of the adrenals was associated with a marked reduction in the lipid droplets and with a simultaneous increase in their electron density in the inner cortical zones. In the zona glomerulosa cells, no perceptible differences were found in the quantity and morphology of lipid droplets. These data suggest that reduced blood and adrenal cholesterol levels do not affect lipids located in the zona glomerulosa and in the inner cortical zones in the same way, probably due to differences in their intracellular lipid dynamism. Noteworthy, that in spite of the marked lipid depletion, the adrenal glands retained their responsiveness to ACTH stimulation.

GABA receptors and T-type Ca2+ channels crosstalk in thalamic networks.

PubMed

Leresche, Nathalie; Lambert, Régis C

2017-06-07

Although the thalamus presents a rather limited repertoire of GABAergic cell types compare to other CNS area, this structure is a privileged system to study how GABA impacts neuronal network excitability. Indeed both glutamatergic thalamocortical (TC) and GABAergic nucleus reticularis thalami (NRT) neurons present a high expression of T-type voltage-dependent Ca 2+ channels whose activation that shapes the output of the thalamus critically depends upon a preceding hyperpolarisation. Because of this strict dependence, a tight functional link between GABA mediated hyperpolarization and T-currents characterizes the thalamic network excitability. In this review we summarize a number of studies showing that the relationships between the various thalamic GABA A/B receptors and T-channels are complex and bidirectional. We discuss how this dynamic interaction sets the global intrathalamic network activity and its long-term plasticity and highlight how the functional relationship between GABA release and T-channel-dependent excitability is finely tuned by the T-channel activation itself. Finally, we illustrate how an impaired balance between T-channels and GABA receptors can lead to pathologically abnormal cellular and network behaviours. Copyright © 2017 Elsevier Ltd. All rights reserved.

Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.

PubMed

Woods, Stephanie E; Qi, Peimin; Rosalia, Elizabeth; Chavarria, Tony; Discua, Allan; Mkandawire, John; Fox, James G; García, Alexis

2014-01-01

The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

Fecal 20-oxo-pregnane concentrations in free-ranging African elephants (Loxodonta africana) treated with porcine zona pellucida vaccine.

PubMed

Ahlers, M J; Ganswindt, A; Münscher, S; Bertschinger, H J

2012-07-01

Because of overpopulation of African elephants in South Africa and the consequent threat to biodiversity, the need for a method of population control has become evident. In this regard, the potential use of the porcine zona pellucida (pZP) vaccine as an effective means for population control is explored. While potential effects of pZP treatment on social behavior of African elephants have been investigated, no examination of the influence of pZP vaccination on the endocrine correlates in treated females has been undertaken. In this study, ovarian activity of free-ranging, pZP-treated African elephant females was monitored noninvasively for 1 yr at Thornybush Private Nature Reserve, South Africa, by measuring fecal 5α-pregnan-3β-ol-20-on concentrations via enzyme immunoassay. A total of 719 fecal samples from 19 individuals were collected over the study period, averaging 38 samples collected per individual (minimum, maximum: 16, 52). Simultaneously, behavioral observations were made to record the occurrence of estrous behavior for comparison. Each elephant under study showed 5α-pregnan-3β-ol-20-on concentrations rising above baseline at some period during the study indicating luteal activity. Average 5α-pregnan-3β-ol-20-on concentrations were 1.61 ± 0.46 μg/g (mean ± SD). Within sampled females, 42.9% exhibited estrous cycles within the range reported for captive African elephants, 14.3% had irregular cycles, and 42.9% did not appear to be cycling. Average estrous cycle duration was 14.72 ± 0.85 wk. Estrous behavior coincided with the onset of the luteal phase and a subsequent rise in 5α-pregnan-3β-ol-20-on concentrations. Average 5α-pregnan-3β-ol-20-on levels positively correlated with rainfall. No association between average individual 5α-pregnan-3β-ol-20-on concentrations or cyclicity status with age or parity were detected. Earlier determination of efficacy was established via fecal hormone analysis with no pregnancies determined 22 mo post

The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse.

PubMed

Mugnier, Sylvie; Boittin, Stéphane; Douet, Cécile; Monget, Philippe; Magistrini, Michèle; Goudet, Ghylène

2008-11-14

In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, since the Galactosyltransferase and N-Acetylglucosamine residues have been localized on equine gametes. We masked the Galactosyltransferase and N-Acetylglucosamine residues before the co-incubation of gametes. Galactosyltransferase was masked either with an anti-Galactosyltransferase antibody or with the enzyme substrate, UDP Galactose. N-Acetylglucosamine residues were masked either with a purified Galactosyltransferase or with an anti-ZPC antibody. The number of spermatozoa bound to the zona pellucida did not decrease after the masking of Galactosyltransferase or N-Acetylglucosamine. So, these two molecules may not be necessary in the mechanism of in vitro sperm-zona pellucida interaction in the horse. The involvement of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding may have been lost during evolution in some ungulates, such as porcine and equine species.

Serotonergic innervation of mesencephalic trigeminal nucleus neurons: a light and electron microscopic study in the rat.

PubMed

Li, J; Xiong, K H; Li, Y Q; Kaneko, T; Mizuno, N

2000-06-01

Neurons of the mesencephalic trigeminal nucleus (MTN) are considered to be homologous to mechanosensitive neurons in the sensory ganglia. The sites of origin of serotonin (5HT)-immunoreactive axons on neuronal cell bodies in the MTN were studied in the rat by combining immunofluorescence histochemical techniques with retrograde tracing of Fluoro-Gold (FG) and anterograde tracing of biotin-conjugated dextran amine (BDA). The tracing studies, which were combined with multiple-labeling immunohistochemistry and confocal microscopy, indicated that 5HT-immunoreactive axon terminals on the cell bodies of MTN neurons originated from the medullary raphe nuclei, such as the nucleus raphes magmus (RMg), alpha part of the nucleus reticularis gigantocellularis (GiA) and nucleus raphes obscurus (ROb), as well as from the mesopontine raphe nuclei, such as the nucleus raphes dorsalis (DR), nucleus raphes pontis (PnR) and nucleus raphes medianus (MnR); mainly from the RMg, GiA and DR, and additionally from the ROb, PnR and MnR. The cell bodies in close apposition to 5HT-immunoreactive axon terminals were found through the whole rostrocaudal extent of the MTN. Electron microscopically a number of axon terminals that were labeled with BDA injected into the raphe nuclei were confirmed to be in asymmetric synaptic contact with the cell bodies of MTN neurons. It was also indicated that substance P existed in some of the 5HT-containing axosomatic terminals arising from the ROb, RMg and GiA. The present results indicated that proprioceptive sensory signals from the muscle spindles or periodontal ligament might be modulated at the level of the primary afferent cell bodies in the MTN by 5HT-containing axons from the mesopontine and medullary raphe nuclei including the GiA.

Substrate preferences of epiphytic bromeliads: an experimental approach

NASA Astrophysics Data System (ADS)

Zotz, Gerhard; Vollrath, Birgit

2002-05-01

Based on the known vertical distributions of three epiphyte species we tested the hypothesis that observed interspecific differences are determined at a very early ontogenetic stage. We attached 1296 first-year seedlings of the three species Guzmania monostachya, Tillandsia fasciculata, and Vriesea sanguinolenta (Bromeliaceae) to substrates differing in orientation and relative position within the crown of the host tree, Annona glabra. Surprisingly, we found no evidence for differential mortality on different substrate types for any of the three species. Hence, differences in vertical distribution cannot be explained by interspecific differences in site-specific survival at this stage. This suggests that spatial distribution patterns are determined even earlier, probably resulting from species differences in seed dispersal or during germination.

Sericin improves heart and liver mitochondrial architecture in hypercholesterolaemic rats and maintains pancreatic and adrenal cell biosynthesis.

PubMed

Ampawong, Sumate; Isarangkul, Duangnate; Aramwit, Pornanong

2017-09-15

Hypercholesterolaemia is well known to be associated with mitochondrial dysfunction, subsequently leading to multiple organ failure. Similar to other natural products, sericin is a candidate for adjunctive therapy in hyperlipidaemic conditions. However, the cholesterol-lowering mechanisms of sericin are multifactorial and controversial. Here, a high-cholesterol-fed rat model with or without sericin treatment was established using a dosage of 1000mg/kg/day for 30 days. Blood lipid profiles, oxidative stress markers (superoxide dismutase, SOD; malondialdehyde, MDA; nuclear factor erythroid 2-related factor, Nrf-2), dysmorphic mitochondria in relation to fission (dynamin-related protein-1; Drp-1) and fusion (guanosine triphosphatase mutated in dominant optic atrophy; OPA-1) markers and biosynthetic markers (aquaporin, AQP-1; tubulin-4β, Tb4B) in the pancreas and adrenal gland were evaluated. The results showed that sericin reduced blood cholesterol and increased high-density lipoprotein (HDL) by acting against oxidative stress. Hypocholesterolaemic and antioxidant conditions further preserved heart and liver mitochondrial architecture; however, this protection was not exhibited in the kidney, where a high level of renal mitophagy, indicating by LC-3 up-regulation, was presented. The steps of ultrastructural alteration of mitochondria from degenerative changes to necrosis were also demonstrated. Sericin also conserved AQP-1 and Tb4B levels in the exocrine pancreatic acinar cells and zona glomerulosa cells, which were positively correlated with serum lipase, HDL, antioxidative markers and mitochondrial integrity. The present study revealed that sericin not only has antioxidant capacity but also balances pancreatic and adrenal cell biosynthesis, especially lipase activity, which may have played an important role in improving lipid dysregulation in the hypercholesterolaemic rat model, leading to the reduction of dysmorphic mitochondria, particularly in the heart and

Oogenesis: From Oogonia to Ovulation in the Flagfish, Jordanella floridae Goode and Bean, 1879 (Teleostei: Cyprinodontidae).

PubMed

Uribe, Mari Carmen; Grier, Harry J; García-Alarcón, Adriana; Parenti, Lynne R

2016-10-01

We provide histological details of the development of oocytes in the cyprinodontid flagfish, Jordanella floridae. There are six stages of oogenesis: Oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis [PG]), secondary growth (vitellogenesis), oocyte maturation and ovulation. The ovarian lamellae are lined by a germinal epithelium composed of epithelial cells and scattered oogonia. During primary growth, the development of cortical alveoli and oil droplets, are initiated simultaneously. During secondary growth, yolk globules coalesce into a fluid mass. The full-grown oocyte contains a large globule of fluid yolk. The germinal vesicle is at the animal pole, and the cortical alveoli and oil droplets are located at the periphery. The disposition of oil droplets at the vegetal pole of the germinal vesicle during late secondary growth stage is a unique characteristic. The follicular cell layer is composed initially of a single layer of squamous cells during early PG which become columnar during early vitellogenesis. During primary and secondary growth stages, filaments develop among the follicular cells and also around the micropyle. The filaments are seen extending from the zona pellucida after ovulation. During ovulation, a space is evident between the oocyte and the zona pellucida. Asynchronous spawning activity is confirmed by the observation that, after ovulation, the ovarian lamellae contain follicles in both primary and secondary growth stages; in contrast, when the seasonal activity of oogenesis and spawning ends, after ovulation, the ovarian lamellae contain only follicles in the primary growth stage. J. Morphol. 277:1339-1354, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Exposure to high ambient temperatures alters embryology in rabbits

NASA Astrophysics Data System (ADS)

García, M. L.; Argente, M. J.

2017-09-01

High ambient temperatures are a determining factor in the deterioration of embryo quality and survival in mammals. The aim of this study was to evaluate the effect of heat stress on embryo development, embryonic size and size of the embryonic coats in rabbits. A total of 310 embryos from 33 females in thermal comfort zone and 264 embryos of 28 females in heat stress conditions were used in the experiment. The traits studied were ovulation rate, percentage of total embryos, percentage of normal embryos, embryo area, zona pellucida thickness and mucin coat thickness. Traits were measured at 24 and 48 h post-coitum (hpc); mucin coat thickness was only measured at 48 hpc. The embryos were classified as zygotes or two-cell embryos at 24 hpc, and 16-cells or early morulae at 48 hpc. The ovulation rate was one oocyte lower in heat stress conditions than in thermal comfort. Percentage of normal embryos was lower in heat stress conditions at 24 hpc (17.2%) and 48 hpc (13.2%). No differences in percentage of zygotes or two-cell embryos were found at 24 hpc. The embryo development and area was affected by heat stress at 48 hpc (10% higher percentage of 16-cells and 883 μm2 smaller, respectively). Zona pellucida was thicker under thermal stress at 24 hpc (1.2 μm) and 48 hpc (1.5 μm). No differences in mucin coat thickness were found. In conclusion, heat stress appears to alter embryology in rabbits.

[Morphometry of giant multipolar neurons of the brain stem reticular formation in rats on board the Kosmos-1667 biosatellite].

PubMed

Belichenko, P V; Leontovich, T A

1989-05-01

Giant multipolar neurons of nucleus reticularis gigantocellularis of rats which had been kept on board the biosatellite "Kosmos-1667" were morphometrically studied. There was a trend towards the increase in the cellular surface, the maximum diameter of dendritic field, the volume of the whole dendritic territory in the test group ad in the control experimental group kept on the earth. A reliable decrease in dendritic mass oriented to nucleus vestibularis and an increase in dendritic mass oriented to the midline were also found in test group, as compared to 3 control groups. Our data were discussed in the light of nervous tissue plasticity in adult mammals.

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism.

PubMed

Cowan, A E; Myles, D G; Koppel, D E

1991-03-01

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing

PubMed Central

1992-01-01

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that r

NR2C in the thalamic reticular nucleus; effects of the NR2C knockout.

PubMed

Zhang, Yuchun; Buonanno, Andres; Vertes, Robert P; Hoover, Walter B; Lisman, John E

2012-01-01

NMDAR antagonists can evoke delta frequency bursting in the nucleus reticularis of the thalamus (nRT). The mechanism of this oscillation was determined; antagonist blocks an NR2C-like conductance that has low Mg block at resting potential and thus can contribute a resting inward current in response to ambient glutamate. Block of this current hyperpolarizes the cell, deinactivating T-type Ca channels and thus triggering delta frequency bursting. The basis for assuming a NR2C-like conductance was that (1) transcripts for NR2C are abundant in the thalamus and (2) the current-voltage curve of the synaptically evoked NMDAR current has the low rectification characteristic of NR2C. In the current study, we have sought to determine whether the channels that generate the NMDAR current are NR2C-like or are actually comprised of receptors containing NR2C. We studied the current-voltage curve of synaptically evoked NMDAR current in the nRT of NR2C knockout mice. In wild-type mice, the current was weakly voltage dependent, as previously observed in rats. This weak rectification was absent in NR2C KO mice. In contrast, NR2C KO had no effect on the strongly rectifying NMDAR current in pyramidal cells of the prefrontal cortex. These results demonstrate that the low rectification normally observed in the nRT is due to NR2C.

Angio-OCT de la zona avascular foveal en ojos con oclusión venosa de la retina.

PubMed

Wons, Juliana; Pfau, Maximilian; Wirth, Magdalena A; Freiberg, Florentina J; Becker, Matthias D; Michels, Stephan

2017-07-11

Objetivo: El objetivo del estudio comprendía visualizar y cuantificar las alteraciones patológicas de la zona avascular foveal (ZAF) mediante angio-OCT en ojos con oclusión venosa de la retina (OVR) en comparación con el ojo contralateral sano. Procedimientos: La angio-OCT se llevó a cabo mediante el sistema Avanti® RTVue 100 XR (Optovue Inc., Fremont, Calif., EE. UU.). Los bordes de la capa vascular superficial (CVS) se definieron como 3 μm por debajo de la membrana limitante interna y 15 μm por debajo de la capa plexiforme interna y, para la capa vascular profunda (CVP), como 15 y 70 μm por debajo de la membrana limitante interna y de la capa plexiforme interna, respectivamente. La longitud de la ZAF horizontal, vertical y máxima de la CVS y la CVP en cada ojo se midió de forma manual. Además, se midió el ángulo entre el diámetro máximo de la ZAF y el plano papilomacular. Resultados: La angio-OCT representó los defectos dentro de la vasculatura en el área perifoveal en ojos con oclusión de rama venosa de la retina (ORVR; n = 11) y con oclusión de la vena central de la retina (OVCR; n = 8). Esto resultó en un crecimiento del diámetro máximo de la ZAF en ojos con OVR (n = 19) en comparación con el ojo contralateral (n = 19; 921 ± 213 frente a 724 ± 145 µm; p = 0,008). Además, se observó una correlación significativa entre la mejor agudeza visual corregida (MAVC) y el diámetro máximo de la ZAF en la CVP (ρ de Spearman = -0,423, p < 0,01). Por último, en los ojos con OVR, el ángulo entre el plano papilomacular y el diámetro máximo de la ZAF se dio tan solo en el 21,05% (CVS) y en el 15,79% (CVP) de los casos a 0 ± 15 ó 90 ± 15°, respectivamente. En ojos sanos, estos ángulos (que supuestamente representan una configuración de la ZAF regular) fueron más prevalentes (CVS 68,42 frente a 21,05%, p = 0,003; CVP 73,68 frente a 15,79%, p < 0,001). Conclusiones: La angio-OCT muestra alteraciones morfológicas de la ZAF en ojos con

Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

PubMed Central

Li, Ming-Wen; Kinchen, Kristy L; Vallelunga, Jadine M; Young, Diana L; Wright, Kaleb D K; Gorano, Lisa N; Wasson, Katherine; Lloyd, K C Kent

2013-01-01

In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen–thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm–zona penetration defects. PMID:23315689

Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin.

PubMed

Takeo, Toru; Nakagata, Naomi

2011-11-01

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.

[A study on the construction, expression and immunosterility of Lagurus laguru zona pellucida 3 DNA vaccine pVAX1-sig-LTB-lZP3-C3d3].

PubMed

Li, Chen-Chen; Yu, Ji-Yun; Jiang, Min; Tu, Yi-Xian; Ma, Xiao-Lin; Zhang, Fu-Chun

2011-09-01

To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway. Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level. The female C57BL/6 mice were immunized by three ways: intramuscular injection, intranasal or oral route.Antibody levels and types were detected by ELISA. ELISA results showed that recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 immunization induced specific IgG, IgA levels were significantly different comparing with control (P<0.01). Antifertility experiment showed that the experimental group reduced the average fertility significantly different compared with the control group (P<0.01). Restriction analysis, RT-PCR and Western blot showed that the recombinant plasmid constructed correctly and can be the expression of mRNA and protein levels.It resulted that the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 can induce the specific immune response efficiently and enhance the immunocontraceptive effects.

Transgenic mice reveal unexpected diversity of On-Off direction selective retinal ganglion cell subtypes and brain structures involved in motion processing

PubMed Central

Rivlin-Etzion, Michal; Zhou, Kaili; Wei, Wei; Elstrott, Justin; Nguyen, Phong L.; Barres, Ben; Huberman, Andrew D.; Feller, Marla B.

2011-01-01

On-Off direction selective retinal ganglion cells (DSGCs) encode the axis of visual motion. They respond strongly to an object moving in a preferred direction and weakly to an object moving in the opposite, ‘null’, direction. Historically, On-Off DSGCs were classified into 4 subtypes according to their directional preference (anterior, posterior, superior or inferior). Here, we compare two genetically identified populations of On-Off DSGCs: DRD4-DSGCs and TRHR-DSGCs. We find that although both populations are tuned for posterior motion, they can be distinguished by a variety of physiological and anatomical criteria. First, the directional tuning of TRHR-DSGCs is broader than that of DRD4-DSGCs. Second, whereas both populations project similarly to the dorsal lateral geniculate nucleus, they project differently to the ventral lateral geniculate nucleus and the superior colliculus. Moreover, TRHR-DSGCs, but not DRD4-DSGCs, also project to the zona incerta, a thalamic area not previously known to receive direction-tuned visual information. Our findings reveal unexpected diversity among mouse On-Off DSGC subtypes that uniquely process and convey image motion to the brain. PMID:21677160

The NHERF1 PDZ2 Domain Regulates PKA–RhoA–p38-mediated NHE1 Activation and Invasion in Breast Tumor Cells

PubMed Central

Cardone, Rosa A.; Bellizzi, Antonia; Busco, Giovanni; Weinman, Edward J.; Dell'Aquila, Maria E.; Casavola, Valeria; Azzariti, Amalia; Mangia, Anita; Paradiso, Angelo

2007-01-01

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na+/H+ exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1α expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na+/H+ exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling. PMID:17332506

Histological differences in the adrenal glands and cortisol levels of suckling dairy goat kids in enriched and non-enriched environments.

PubMed

Rosas-Trigueros, A P; Candanosa-Aranda, I E; Ducoing-Watty, A E; Gutiérrez-Molotla, J; Galindo, F; Sisto-Burt, A M

2017-12-01

Intensive production systems can negatively affect the welfare of goats. Environmental enrichment techniques can be used to reduce stress. The aim of this study was analyze the effect of environmental enrichment on the histological characteristics of adrenal glands, cortisol levels and weekly weight gain of suckling Alpine French male kids under confinement. A randomised design was used to test the effect of the treatment. The animals (n=20) were randomly assigned to two treatments (enriched and non-enriched) with two replicates each. Enrichment elements included elevated sacks of henequen, trunks, tires and coconuts. The cortisol levels were measured weekly. Male kids were euthanized when their weight reached 10kg, and immediately after euthanasia, samples of the adrenal glands were collected. The adrenal glands were weighed and measured, and histological sections were taken and fixed. Four hundred cells were measured from each animal, with two blind measurements taken from each sample. There were no significant differences between experimental groups (P>0.05) in the weigh, size, the area of cells from the glomerulosa and fasciculata areas of the adrenal glands, the cortisol levels and weekly weight gain. However, there were histological differences between the glomerular and fascicular zones of the left and right adrenal glands of the different groups (P<0.05). These findings suggest that adrenal glands of animals in non-enriched environment, contained histological changes, suggestive of increased activity. We suggest testing adrenal histology as an indicator of stress and recommend the use of environmental enrichment as a means to reduce stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lack of transmission of mouse minute virus (MMV) from in vitro-produced embryos to recipients and pups due to the presence of cumulus cells during the in vitro fertilization process.

PubMed

Mahabir, E; Bulian, D; Needham, J; Schmidt, J

2009-09-01

The risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of cumulus-enclosed oocytes (CEOs) or without cumulus cells (CDOs) in the presence of MMV-exposed (10(4) TCID(50) [mean tissue culture infective dose]/ml MMVp [prototype strain of MMV]) spermatozoa was evaluated. Also, the time after embryo transfer to detection of MMV antibody and the presence of MMV DNA in the mesenteric lymph nodes of recipients and pups were investigated. All mice were MMV free, but two seropositive recipients and four seropositive pups were found in the group with CDOs. With regard to the CEOs, two of 11 holding drops and five of 11 groups of embryos were MMV positive using PCR, while neither holding drops nor embryos carried infectious MMVp, as evidenced by the in vitro infectivity assay. From IVF with CDOs, five of 14 holding drops and four of nine groups of embryos were MMV positive, while one of 14 holding drops and no embryos carried infectious MMVp. When 10(5) cumulus cells were analyzed 5 h after exposure to 10(4) TCID(50)/ml MMVp, cells had an average titer of 10(4) TCID(50)/ml MMVp. The present data show that, in contrast to CDOs, 2-cell embryos from CEOs did not transmit infectious MMVp to the holding drops and to recipients. This observation is due to the presence of cumulus cells during the IVF process that reduce entry of MMV into the zona pellucida and absorb some of the virus. These data further confirm the efficacy of the IVF procedure in producing embryos that are free of infectious virus, leading to virus-free seronegative recipients and rederived pups.

Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen.

PubMed

Liu, De Yi; Liu, Ming Li; Garrett, Claire; Baker, H W Gordon

2007-07-01

The aim of this study was to compare the frequency of defective sperm-zona pellucida (ZP) binding (DSZPB) and defective ZP-induced acrosome reaction (DZPIAR) in subfertile men (i.e. male partners of infertile couples) with normal and abnormal semen analyses. A total of 1030 subfertile men with normal semen analysis (n=255), oligozoospermia (count<20x10(6)/ml, n=136), severe teratozoospermia (strict normal morphology

Coagulation disorders and their cutaneous presentations: Diagnostic work-up and treatment.

PubMed

Dabiri, Ganary; Damstetter, Elizabeth; Chang, Yunyoung; Baiyee Ebot, Emily; Powers, Jennifer Gloeckner; Phillips, Tania

2016-05-01

Both inherited and acquired hypercoagulable states can present with nonspecific clinical manifestations, such as petechiae, purpura, livedo reticularis, and ulcerations. A good history and physical examination are crucial to diagnoses of these conditions. Inherited conditions tend to present either in neonatal period or later in life, while acquired conditions typically occur later in life. Diagnostic studies are performed to identify the coagulation cascade deficiency or defect. Treatment primarily hinges on anticoagulation and wound care. In this article, we provide an in-depth analysis of the clinical manifestations, diagnostic considerations, and management options of patients in hypercoagulable states. Copyright © 2015 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

The evaluation of xenotransplantation of feline ovarian tissue vitrified by needle immersed vitrification technique into male immunodeficient mice.

PubMed

Demirel, Mürşide Ayşe; Acar, Duygu Baki; Ekim, Burcu; Çelikkan, Ferda Topal; Alkan, Kübra Karakaş; Salar, Seçkin; Erdemli, Esra Atabenli; Özkavukçu, Sinan; Yar, Seda Sağlam; Kanca, Halit; Baştan, Ayhan

2018-03-01

In this study, the efficiency of the "Needle Immersed Vitrification" technique was tested on cryopreserved feline ovarian tissue. For vitrification, ovarian fragments (0.5-1.5 mm 2 ) from each ovary were collected; the grafts were exposed to 7.5-15% ethylene glycol and 7.5-15% dimethyl sulfoxide at room temperature and stored in liquid nitrogen at least 1 week. Morphologic examinations, expression of genes such as B cell lymphoma 2, B-cell lymphoma-2-associated X protein, Bone morphogenetic protein 15, zone of polarizing activity, zona pellucida C protein and DNA (cytosine-5)-methyltransferase 1, ultrastructural analysis and viability tests were carried out from collected grafts. Light microscopy examinations revealed the percentage of morphologically normal primordial follicles in a fresh group which was significantly higher than the treatment groups (p < 0.001). Terminal deoxynucleotidyl transferase dUTP nick end labeling and anti-caspase-3 staining observed in oocytes, follicle cells, interstitial tissue showed higher rates of apoptosis for post-vitrification and -transplantation groups than freshly grafted ovarian tissues. Furthermore, we observed significant downregulation of zone of polarizing activity and zona pellucida C protein gene expression in vitrified ovarian tissue grafts than in the fresh grafts (p < 0.05). In conclusion, we suggest that the needle immersed vitrification method is a convenient, cheap, and feasible vitrification method for cat ovarian tissues. However, further studies need to be performed to determine more optimal vitrification solutions and equilibration times for the needle immersed vitrification method in order to adapt it for cat ovaries.

Co-occurrence of tannin and tannin-less vacuoles in sensitive plants.

PubMed

Fleurat-Lessard, Pierrette; Béré, Emile; Lallemand, Magali; Dédaldéchamp, Fabienne; Roblin, Gabriel

2016-05-01

Vacuoles of different types frequently coexist in the same plant cell, but the duality of the tannin/tannin-less vacuoles observed in Mimosa pudica L. is rare. In this plant, which is characterized by highly motile leaves, the development and original features of the double vacuolar compartment were detailed in primary pulvini from the young to the mature leaf stage. In young pulvini, the differentiation of tannin vacuoles first occurred in the epidermis and progressively spread toward the inner cortex. In motor cells of nonmotile pulvini, tannin deposits first lined the membranes of small vacuole profiles and then formed opaque clusters that joined together to form a large tannin vacuole (TV), the proportion of which in the cell was approximately 45%. At this stage, transparent vacuole profiles were rare and small, but as the parenchyma cells enlarged, these profiles coalesced to form a transparent vacuole with a convexity toward the larger-sized tannin vacuole. When leaf motility began to occur, the two vacuole types reached the same relative proportion (approximately 30%). Finally, in mature cells displaying maximum motility, the large transparent colloidal vacuole (CV) showed a relative proportion increasing to approximately 50%. At this stage, the proportion of the tannin vacuole, occurring in the vicinity of the nucleus, decreased to approximately 10%. The presence of the condensed type of tannins (proanthocyanidins) was proven by detecting their fluorescence under UV light and by specific chemical staining. This dual vacuolar profile was also observed in nonmotile parts of M. pudica (e.g., the petiole and the stem). Additional observations of leaflet pulvini showing more or less rapid movements showed that this double vacuolar structure was present in certain plants (Mimosa spegazzinii and Desmodium gyrans), but absent in others (Albizzia julibrissin, Biophytum sensitivum, and Cassia fasciculata). Taken together, these observations strongly suggest that a

Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

PubMed Central

Monticone, Silvia; Hattangady, Namita G.; Nishimoto, Koshiro; Mantero, Franco; Rubin, Beatrice; Cicala, Maria Verena; Pezzani, Raffaele; Auchus, Richard J.; Ghayee, Hans K.; Shibata, Hirotaka; Kurihara, Isao; Williams, Tracy A.; Giri, Judith G.; Bollag, Roni J.; Edwards, Michael A.; Isales, Carlos M.

2012-01-01

Context: Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). Objective: The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. Methods: A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. Results: We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2. Conclusions: KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production. PMID:22628608

Survival of sheep demi-embryos in vivo and in vitro.

PubMed

Shelton, J N; Szell, A

1988-01-01

Sheep embryos (morulae and blastocysts) were bisected either by microscalpel or by microneedle after dissolving the zona pellucida with acidified Tyrode's solution. Fourteen and 11 cryopreserved demi-embryos failed to develop when transferred to recipients or placed in culture, respectively. When fresh demi-embryos were cultured in Dulbecco's phosphate buffered saline (DPBS) plus fetal calf serum (FCS) or Whitten's medium, the survival rate was 26% compared to 68% for whole embryos (P<0.01), and there was a suggestion that the presence of a zona pellucida was beneficial to survival. When two demi-embryos each within a zona pellucida were transferred into each of 10 ewes, six of them lambed to produce a total of eight lambs, including two sets of identical twins. Of 10 ewes receiving two demi-embryos without zonae pellucidae, three lambed to produce a total of four lambs, including one set of identical twins. Of 10 ewes that each received two whole embryos, 10 lambed to produce a total of 16 lambs. There was a suggestion that the zona pellucida might enhance the survival of demi-morulae but not demi-blastocysts.

Chemotherapeutic Studies on Schistosomiasis and Clinical, Epidemiological and Immunological Studies on Malaria in Amazonas, Brazil, along the Ituxi River.

DTIC Science & Technology

1982-09-01

enfermedades infecciosas en zonas colindantes con la Carretera Transamazonica en Brasil. II. Epidemiologia de las infecciones por arbovirus. Bol. Of...F.P. 1981. Programa multidisciplinario de vigilancia de las enfermedades , infecciosas en zonas colindantes con la Carretera Transamazonica en Brasil

Identification of ZASP, a novel protein associated to Zona occludens-2.

PubMed

Lechuga, Susana; Alarcón, Lourdes; Solano, Jesús; Huerta, Miriam; Lopez-Bayghen, Esther; González-Mariscal, Lorenza

2010-11-15

With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds. Copyright © 2010 Elsevier Inc. All rights reserved.

Physiological characterization, localization and synaptic inputs of bursting and nonbursting neurons in the trigeminal principal sensory nucleus of the rat.

PubMed

Athanassiadis, T; Westberg, K-G; Olsson, K A; Kolta, A

2005-12-01

A population of neurons in the trigeminal principal sensory nucleus (NVsnpr) fire rhythmically during fictive mastication induced in the in vivo rabbit. To elucidate whether these neurons form part of the central pattern generator (CPG) for mastication, we performed intracellular recordings in brainstem slices taken from young rats. Two cell types were defined, nonbursting (63%) and bursting (37%). In response to membrane depolarization, bursting cells, which dominated in the dorsal part of the NVsnpr, fired an initial burst followed by single spikes or recurring bursts. Non-bursting neurons, scattered throughout the nucleus, fired single action potentials. Microstimulation applied to the trigeminal motor nucleus (NVmt), the reticular border zone surrounding the NVmt, the parvocellular reticular formation or the nucleus reticularis pontis caudalis (NPontc) elicited a postsynaptic potential in 81% of the neurons tested for synaptic inputs. Responses obtained were predominately excitatory and sensitive to glutamatergic antagonists DNQX and/or APV. Some inhibitory and biphasic responses were also evoked. Bicuculline methiodide or strychnine blocked the IPSPs indicating that they were mediated by GABA(A) or glycinergic receptors. About one-third of the stimulations activated both types of neurons antidromically, mostly from the masseteric motoneuron pool of NVmt and dorsal part of NPontc. In conclusion, our new findings show that some neurons in the dorsal NVsnpr display both firing properties and axonal connections which support the hypothesis that they may participate in masticatory pattern generation. Thus, the present data provide an extended basis for further studies on the organization of the masticatory CPG network.

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp; Fujiwara, Yuki; Yamaguchi, Hideki

2011-05-20

Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however,more » its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.« less

Cutaneous Polyarteritis Nodosa Presented with Digital Gangrene: A Case Report

PubMed Central

Lew, Sogu; Cho, Sung Do; Cha, Hee Jeong; Eum, Eun-A; Jung, Hyun Chul; Park, Jae Hoo

2006-01-01

Cutaneous polyarteritis nodosa (CPAN) is an uncommon form of vasculitis involving small and medium sized arteries of unknown etiology. The disease can be differentiated from polyarteritis nodosa by its limitation to the skin and lack of progression to visceral involvement. The characteristic manifestations are subcutaneous nodule, livedo reticularis, and ulceration, mostly localized on the lower extremity. Arthralgia, myalgia, peripheral neuropathy, and constitutional symptoms such as fever and malaise may also be present. We describe a 34-yr-old woman presented with severe ischemic change of the fingertip and subcutaneous nodules without systemic manifestations as an unusual initial manifestation of CPAN. Therapy with corticosteroid and alprostadil induce a moderate improvement of skin lesions. However, necrosis of the finger got worse and the finger was amputated. PMID:16614534

Possible conditioned stimulus pathway for classical eyelid conditioning in rabbits. I. Anatomical evidence for direct projections from the pontine nuclei to the cerebellar interpositus nucleus.

PubMed

Steinmetz, J E; Sengelaub, D R

1992-03-01

Wheat germ agglutinin and cholera toxin-conjugated horseradish peroxidase (HRP) were used to retrogradely and anterogradely trace connectivity between the lateral regions of the pontine nuclei and the anterior interpositus nucleus of the cerebellum in rabbits. Projections from the pontine nuclei were found to terminate in the anterior interpositus nucleus and the interpositus was found to send projections to the pontine nuclei. Projections from the nucleus reticularis tegmenti pontis, dorsal accessory inferior olive, and Larsell's lobule HVI of the cerebellum were also found to terminate in the interpositus nucleus and projections from the interpositus nucleus to the inferior olivary complex were observed. The projections from brain stem regions to the interpositus nucleus are discussed as possible pathways that are involved in classical eyelid conditioning.

GDF9 and BMP15 Expressions and Fine Structure Changes During Folliculogenesis in Polycystic Ovary Syndrome.

PubMed

Karagül, Meryem İlkay; Aktaş, Savaş; Coşkun Yılmaz, Banu; Yılmaz, Mustafa; Orekici Temel, Gülhan

2018-01-20

Polycystic ovary syndrome is the most frequently seen endocrine disorder in women of reproductive age with a prevalence of about 10%. To investigate the efficiency of growth differentiation factor 9 and bone morphogenetic protein 15 during folliculogenesis in a dehydroepiandrosterone-induced mouse Polycystic ovary syndrome model. Animal experimentation. Mice were divided into 3 groups: control, vehicle and Polycystic ovary syndrome. Polycystic ovary syndrome model mice were developed by the injection of dehydroepiandrosterone dissolved in 0.1 mL of sesame oil. Ovarian tissues were examined for growth differentiation factor 9 and bone morphogenetic protein 15 using immunofluorescent labelling and electron microscopic examinations. The immunoreactivity of growth differentiation factor 9 and bone morphogenetic protein 15 proteins decreased (p<0.05) in the Polycystic ovary syndrome group (27.73±8.43 and 24.85±7.03, respectively) compared with the control group (33.72±11.22 and 31.12±11.05, respectively) and vehicle group (33.95±10.75 and 29.99±10.72, respectively). Apoptotic changes were observed in granulosa cells, lipid vacuoles increased in Theca cells and thickening and irregularities were noted in the basal lamina of granulosa cells. An increased electron density in the zona pellucida in some of the multilaminar primary and secondary follicles in the Polycystic ovary syndrome model was also observed at the ultrastructural level. These results suggest that the decrease in the growth differentiation factor 9 and bone morphogenetic protein 15 expression initiated at the primary follicle stage effect the follicle development and zona pellucida structure and may cause subfertility or infertility in Polycystic ovary syndrome.

Gamete competence assessment by polarizing optics in assisted reproduction.

PubMed

Montag, Markus; Köster, Maria; van der Ven, Katrin; van der Ven, Hans

2011-01-01

The purpose of this study was first to give an overview of the historical development of polarization microscopy, second to describe the various applications of this technique in assisted reproduction techniques (ART) and third to discuss the potential benefit of polarization microscopy as a predictor for IVF success. The history of polarization microscopy was undertaken by performing a backward search in the scientific literature using Google and internet sites of several Societies for Microscopy and Cell Biology. Studies of polarization microscopy in ART were identified by using a systematic literature search in PubMed and Scopus. A total of 62 articles were identified by the direct search and further relevant articles were found by screening the cited literature in these articles. The topics relevant for assisted reproduction were spindle and zona imaging in combination with IVF success, meiotic cell cycle progression, pharmaceutical studies and cryopreservation. A separate topic was the use of sperm birefringence in ART. The majority of studies are observational studies and were not performed in a randomized manner and there is no direct comparison of techniques using other gamete selection markers. Despite this, most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis. Whether certain applications such as spindle or zona imaging may lead to an increase in IVF success is unclear at present. Publications on the use of polarization microscopy on sperm are still very limited.

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

PubMed Central

TAKEO, Shun; SATO, Daichi; KIMURA, Koji; MONJI, Yasunori; KUWAYAMA, Takehito; KAWAHARA-MIKI, Ryoka; IWATA, Hisataka

2013-01-01

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization. PMID:24390595

Epididymal protein CRISP1 plays different roles during the fertilization process.

PubMed

Cohen, Débora J; Maldera, Julieta A; Vasen, Gustavo; Ernesto, Juan I; Muñoz, Mariana Weigel; Battistone, María A; Cuasnicú, Patricia S

2011-01-01

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.

Parental genetic material and oxygen concentration affect hatch dynamics of mouse embryo in vitro.

PubMed

Zhan, Shaoquan; Cao, Shanbo; Du, Hongzi; Sun, Yuan; Li, Li; Ding, Chenhui; Zheng, Haiyan; Huang, Junjiu

2018-04-21

Hatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear. The effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration. Firstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O 2 concentrations. 5% O 2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O 2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O 2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O 2 , and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O 2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences. Both parental genetic material and O 2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch

Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.

PubMed

Mio, Yasuyuki; Iwata, Kyoko; Yumoto, Keitaro; Kai, Yoshiteru; Sargant, Haruka C; Mizoguchi, Chizuru; Ueda, Minako; Tsuchie, Yuka; Imajo, Akifumi; Iba, Yumiko; Nishikori, Kyoko

2012-09-01

To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography. We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization. Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s. The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.

Nervus terminalis, olfactory nerve, and optic nerve representation of luteinizing hormone-releasing hormone in primates.

PubMed

Witkin, J W

1987-01-01

The luteinizing hormone-releasing hormone (LHRH) system was examined immunocytochemically in olfactory bulbs of adult monkeys, including two New World species (squirrel monkey, Saimiri sciureus and owl monkey, Aotus trivirgatus) and one Old World species (cynomolgus macaque, Macaca fasciculata), and in the brain and nasal region of a fetal rhesus macaque Macaca mulatta. LHRH neurons and fibers were found sparsely distributed in the olfactory bulbs in all adult monkeys. There was more LHRH in the accessory olfactory bulb (which is absent in Old World monkeys). In the fetal macaque there was a rich distribution of LHRH neurons and fibers along the pathway of the nervus terminalis, anterior and ventral to the olfactory bulb, and in the nasal septum, with fibers branching into the olfactory epithelium. In addition, there were LHRH neurons and fibers in the optic nerve.

[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction].

PubMed

González-Ortega, C; Cancino-Villarreal, P; Alonzo-Torres, V E; Martínez-Robles, I; Pérez-Peña, E; Gutiérrez-Gutiérrez, A M

2016-04-01

Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live

Genetically Expressed Transneuronal Tracer Reveals Direct and Indirect Serotonergic Descending Control Circuits

PubMed Central

BRAZ, JOÃO MANUEL; BASBAUM, ALLAN I.

2016-01-01

Despite the evidence for a significant contribution of brainstem serotonergic (5HT) systems to the control of spinal cord “pain” transmission neurons, attention has turned recently to the influence of nonserotonergic neurons, including the facilitatory and inhibitory controls that originate from so-called “on” and “off” cells of the rostroventral medulla (RVM). Unclear, however, is the extent to which these latter circuits interact with or are influenced by the serotonergic cell groups. To address this question we selectively targeted expression of a transneuronal tracer, wheat germ agglutinin (WGA), in the 5HT neurons so as to study the interplay between the 5HT and non-5HT systems. In addition to confirming the direct medullary 5HT projection to the spinal cord we also observed large numbers of non-5HT neurons, in the medullary nucleus reticularis gigantocellularis and magnocellularis, that were WGA-immunoreactive, i.e., were transneuronally labeled from 5HT neurons. Fluoro-Gold injections into the spinal cord established that these reticular neurons are not only postsynaptic to the 5HT neurons of the medulla, but that most are also at the origin of descending, bulbospinal pathways. By contrast, we found no evidence that neurons of the midbrain periaqueductal gray that project to the RVM are postsynaptic to midbrain or medullary 5HT neurons. Finally, we found very few examples of WGA-immunoreactive noradrenergic neurons, which suggests that there is considerable independence of the monoaminergic bulbospinal pathways. Our results indicate that 5HT neurons influence “pain” processing at the spinal cord level both directly and indirectly via feedforward connections with multiple non-5HT descending control pathways. PMID:18273889

The pathways connecting the hippocampal formation, the thalamic reuniens nucleus and the thalamic reticular nucleus in the rat.

PubMed

Cavdar, Safiye; Onat, Filiz Y; Cakmak, Yusuf Ozgür; Yananli, Hasan R; Gülçebi, Medine; Aker, Rezzan

2008-03-01

Most dorsal thalamic nuclei send axons to specific areas of the neocortex and to specific sectors of the thalamic reticular nucleus; the neocortex then sends reciprocal connections back to the same thalamic nucleus, directly as well indirectly through a relay in the thalamic reticular nucleus. This can be regarded as a 'canonical' circuit of the sensory thalamus. For the pathways that link the thalamus and the hippocampal formation, only a few comparable connections have been described. The reuniens nucleus of the thalamus sends some of its major cortical efferents to the hippocampal formation. The present study shows that cells of the hippocampal formation as well as cells in the reuniens nucleus are retrogradely labelled following injections of horseradish peroxidase or fluoro-gold into the rostral part of the thalamic reticular nucleus in the rat. Within the hippocampal formation, labelled neurons were localized in the subiculum, predominantly on the ipsilateral side, with fewer neurons labelled contralaterally. Labelled neurons were seen in the hippocampal formation and nucleus reuniens only after injections made in the rostral thalamic reticular nucleus (1.6-1.8 mm caudal to bregma). In addition, the present study confirmed the presence of afferent connections to the rostral thalamic reticular nucleus from cortical (cingulate, orbital and infralimbic, retrosplenial and frontal), midline thalamic (paraventricular, anteromedial, centromedial and mediodorsal thalamic nuclei) and brainstem structures (substantia nigra pars reticularis, ventral tegmental area, periaqueductal grey, superior vestibular and pontine reticular nuclei). These results demonstrate a potential for the thalamo-hippocampal circuitry to influence the functional roles of the thalamic reticular nucleus, and show that thalamo-hippocampal connections resemble the circuitry that links the sensory thalamus and neocortex.

The pathways connecting the hippocampal formation, the thalamic reuniens nucleus and the thalamic reticular nucleus in the rat

PubMed Central

Çavdar, Safiye; Onat, Filiz Y; Çakmak, Yusuf Özgür; Yananli, Hasan R; Gülçebi, Medine; Aker, Rezzan

2008-01-01

Most dorsal thalamic nuclei send axons to specific areas of the neocortex and to specific sectors of the thalamic reticular nucleus; the neocortex then sends reciprocal connections back to the same thalamic nucleus, directly as well indirectly through a relay in the thalamic reticular nucleus. This can be regarded as a ‘canonical’ circuit of the sensory thalamus. For the pathways that link the thalamus and the hippocampal formation, only a few comparable connections have been described. The reuniens nucleus of the thalamus sends some of its major cortical efferents to the hippocampal formation. The present study shows that cells of the hippocampal formation as well as cells in the reuniens nucleus are retrogradely labelled following injections of horseradish peroxidase or fluoro-gold into the rostral part of the thalamic reticular nucleus in the rat. Within the hippocampal formation, labelled neurons were localized in the subiculum, predominantly on the ipsilateral side, with fewer neurons labelled contralaterally. Labelled neurons were seen in the hippocampal formation and nucleus reuniens only after injections made in the rostral thalamic reticular nucleus (1.6–1.8 mm caudal to bregma). In addition, the present study confirmed the presence of afferent connections to the rostral thalamic reticular nucleus from cortical (cingulate, orbital and infralimbic, retrosplenial and frontal), midline thalamic (paraventricular, anteromedial, centromedial and mediodorsal thalamic nuclei) and brainstem structures (substantia nigra pars reticularis, ventral tegmental area, periaqueductal grey, superior vestibular and pontine reticular nuclei). These results demonstrate a potential for the thalamo-hippocampal circuitry to influence the functional roles of the thalamic reticular nucleus, and show that thalamo-hippocampal connections resemble the circuitry that links the sensory thalamus and neocortex. PMID:18221482

A novel relay nucleus between the inferior colliculus and the optic tectum in the chicken (Gallus gallus).

PubMed

Niederleitner, Bertram; Gutierrez-Ibanez, Cristian; Krabichler, Quirin; Weigel, Stefan; Luksch, Harald

2017-02-15

Processing multimodal sensory information is vital for behaving animals in many contexts. The barn owl, an auditory specialist, is a classic model for studying multisensory integration. In the barn owl, spatial auditory information is conveyed to the optic tectum (TeO) by a direct projection from the external nucleus of the inferior colliculus (ICX). In contrast, evidence of an integration of visual and auditory information in auditory generalist avian species is completely lacking. In particular, it is not known whether in auditory generalist species the ICX projects to the TeO at all. Here we use various retrograde and anterograde tracing techniques both in vivo and in vitro, intracellular fillings of neurons in vitro, and whole-cell patch recordings to characterize the connectivity between ICX and TeO in the chicken. We found that there is a direct projection from ICX to the TeO in the chicken, although this is small and only to the deeper layers (layers 13-15) of the TeO. However, we found a relay area interposed among the IC, the TeO, and the isthmic complex that receives strong synaptic input from the ICX and projects broadly upon the intermediate and deep layers of the TeO. This area is an external portion of the formatio reticularis lateralis (FRLx). In addition to the projection to the TeO, cells in FRLx send, via collaterals, descending projections through tectopontine-tectoreticular pathways. This newly described connection from the inferior colliculus to the TeO provides a solid basis for visual-auditory integration in an auditory generalist bird. J. Comp. Neurol. 525:513-534, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Suppression of Sleep Spindle Rhythmogenesis in Mice with Deletion of CaV3.2 and CaV3.3 T-type Ca(2+) Channels.

PubMed

Pellegrini, Chiara; Lecci, Sandro; Lüthi, Anita; Astori, Simone

2016-04-01

Low-threshold voltage-gated T-type Ca(2+) channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca(2+) currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10-15 Hz), which was accompanied by an increase in the δ band (1-4 Hz). Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms. © 2016 Associated Professional Sleep Societies, LLC.

Involvement of the raphe in the respiratory effects of gigantocellular area activation.

PubMed

Richard, C A; Stremel, R W

1990-07-01

Previous reports indicate that the nucleus reticularis gigantocellularis (NGC) of the brainstem reticular formation is involved in inhibitory respiratory and cardiovascular reflexes. Stimulation of portions of the nearby bulbar raphe complex, specifically the raphe magnus (RM), have also been shown to suppress phrenic activity and to decrease blood pressure and heart rate. Since synaptic connectivity between the NGC and the RM has been demonstrated, we hypothesized that the RM may be involved in the cardiopulmonary effects of NGC stimulation. This study found that electrolytic lesions in the raphe magnus attenuated the inhibitory respiratory effects but not the cardiovascular suppression due to NGC stimulation. Lesions in the raphe magnus also lowered resting blood pressure and resting breath frequency. We conclude that the RM may mediate part of the NGC-mediated respiratory effects.

Mapa de Vulnerabilidad en Honduras

NASA Astrophysics Data System (ADS)

Sánchez, A. A.

2013-05-01

Dado que Honduras geográficamente se encuentra en una zona que esta expuesta a diferentes fenómenos sísmicos y climatológicos. Investigamos cuales son las zonas mas propensas a desastres naturales, la vulnerabilidad y el factor de resiliencia de la población de acuerdo a diferentes parámetros de estudio.

[Aldosterone response to various stimuli in hyperthyroidism: in vivo and in vitro studies].

PubMed

Kigoshi, T; Kaneko, M; Nakano, S; Azukizawa, S; Uchida, K; Morimoto, S

1993-06-20

Responses of plasma aldosterone (PA) to alpha-ACTH-(1-24) (250 micrograms, im) injection and graded angiotensin II (AII) infusions (2, 4 and 8ng/kg/min for 30 min at each dose) on a constant sodium intake (170mEq daily) were assessed in 17 patients with Basedow's disease and 13 age-matched normal subjects. Aldosterone production in response to ACTH, AII and potassium in adrenal zona glomerulosa cells from L-thyroxine-induced hyperthyroid rats (H-rats) were also examined. Basal levels of plasma renin activity (PRA) and urinary aldosterone excretion were significantly higher (p < 0.01 and p < 0.05, respectively) in the patients with Basedow's disease than in the normal subjects, whereas basal PA level was similar in the two groups. The ACTH injection induced similar increases in plasma cortisol, plasma 18-hydroxycorticosterone (18-OHB) and PA in the two groups. The graded AII infusions also produced increases in plasma 18-OHB and PA in the two groups. Responses of these two corticosteroids to AII were, however, significantly lower (p < 0.05) in the patients with Basedow's disease than in the normal subjects. In the experimental animal study, basal PRA levels and the adrenal glomerulosa cell count/adrenal were significantly higher (p < 0.05) in the H-rats than in the control rats, whereas basal PA levels were similar in the two groups. Aldosterone production in response to AII, ACTH, and potassium increased in a dose-dependent manner in the two groups. Responses of aldosterone production to AII were, however, significantly lower (p < 0.05) in the H-rats than in the control rats. These results suggest that the impaired responsiveness of adrenal zona glomerulosa cells to AII, as well as an increased metabolic clearance rate of aldosterone, may be involved in the abnormal aldosterone metabolism in hyperthyroidism.

New insights into molecular pathways associated with flatfish ovarian development and atresia revealed by transcriptional analysis

PubMed Central

Tingaud-Sequeira, Angèle; Chauvigné, François; Lozano, Juanjo; Agulleiro, María J; Asensio, Esther; Cerdà, Joan

2009-01-01

Background The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray. Results Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8), intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a). Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits), probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein C-I), chemotaxis

Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes).

PubMed

Maria, Alexandre N; Orfão, Laura H; Rizzo, Elizete; Ninhaus-Silveira, Alexandre; Viveiros, Ana T M

2015-06-01

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 μm in thickness with eight pore canals/μm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Reprogramming of single-cell derived mesenchymal stem cells into hair cell-like cells

PubMed Central

Lin, Zhaoyu; Perez, Philip; Sun, Zhenyu; Liu, Jan-Jan; Shin, June Ho; Hyrc, Krzysztof L.; Samways, Damien; Egan, Terry; Holley, Matthew C.; Bao, Jianxin

2012-01-01

Hypothesis Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. Background Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. Methods Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed by RT-PCR, and cell structures were examined by immunocytochemistry. Results Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells prior to overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. Conclusion We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells. PMID:23111404

Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

PubMed

Lee, Kun-Hsiung

2014-01-01

The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated

Regulation of aldosterone production by ion channels: From basal secretion to primary aldosteronism.

PubMed

Yang, Tingting; He, Min; Hu, Changlong

2018-03-01

Aldosterone is produced by zona glomerulosa (ZG) cells of the adrenal cortex and plays a key role in balancing water and electrolytes levels. Autonomous overproduction of aldosterone leads to primary aldosteronism (PA), which is the most common form of secondary endocrine hypertension. Recently, significant progress has been made towards understanding the genetic basis of PA, where increasing clinical evidence suggests that mutations in ion channels appear to be the major cause of aldosterone-producing adenomas. In this review, we focused on potassium and calcium channels that regulate aldosterone secretion, and their roles in the pathology of PA. Because potassium and calcium channels are differentially expressed in ZG cells in different species of mammals, the limitations of published studies are also discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

An investigation of the origin and significance of bilateral symmetry of the pronuclear zygote in the mouse.

PubMed

Gardner, R L; Davies, T J

2006-02-01

Preliminary observations revealed that advanced zygotes of the PO strain mouse are often bilaterally symmetrical, and suggested that both the plane of first cleavage and features of the blastocyst bear a consistent relationship to the zygote's bilateral plane. Spaced oil drops were injected into the zona pellucida to delineate the bilateral plane in pronuclear zygotes, and a distinct cluster of drops then placed over the second polar body. Such non-invasive marking was combined with gelation of the perivitelline space to prevent rotation of the zygotes within the zona pellucida. Nearly two-thirds of advanced pronuclear stage zygotes were bilaterally symmetrical and, regardless of whether first cleavage was meridional, it was almost invariably orthogonal to the bilateral plane. Moreover, both the axis of polarity and bilateral plane of the blastocyst bore a consistent relationship to the zygote's bilateral plane. Haploid parthenotes also exhibited bilateral symmetry, although in the absence of fertilization, first cleavage was less consistently orthogonal to the bilateral plane. Bilateral symmetry may be an intrinsic property of the oocyte that is induced by its activation and, from the reproducible way it maps on both the 2-cell conceptus and blastocyst, seems to play a role in early patterning.

Thermal effects in laser-assisted embryo hatching

NASA Astrophysics Data System (ADS)

Douglas-Hamilton, Diarmaid H.; Conia, Jerome D.

2000-08-01

Diode lasers [(lambda) equals 1480 nm] are used with in-vitro fertilization [IVF] as a promoter of embryo hatching. A focused laser beam is applied in vitro to form a channel in the zona pellucida (shell) of the pre-embryo. After transfer into the uterus, the embryo hatches: it extrudes itself through the channel and implants into the uterine wall. Laser-assisted hatching can result in improving implantation and pregnancy success rates. We present examples of zone pellucida ablation using animal models. In using the laser it is vital not to damage pre-embryo cells, e.g. by overheating. In order to define safe regimes we have derived some thermal side-effects of zona pellucida removal. The temperature profile in the beam and vicinity is predicted as function of laser pulse duration and power. In a crossed-beam experiment a HeNe laser probe detects the temperature-induced change in refractive index. We find that the diode laser beam produces superheated water approaching 200 C on the beam axis. Thermal histories during and following the laser pulse are given for regions in the neighborhood of the beam. We conclude that an optimum regime exists with pulse duration

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

PubMed

Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

2016-09-01

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

Different kinetic properties of two T-type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane

PubMed Central

Joksovic, Pavle M; Bayliss, Douglas A; Todorovic, Slobodan M

2005-01-01

Currents arising from T-type Ca2+ channels in nucleus reticularis thalami (nRT) play a critical role in generation of low-amplitude oscillatory bursting involving mutually interconnected cortical and thalamic neurones, and are implicated in the state of arousal and sleep, as well as seizures. Here we show in brain slices from young rats that two kinetically different T-type Ca2+ currents exist in nRT neurones, with a slowly inactivating current expressed only on proximal dendrites, and fast inactivating current predominantly expressed on soma. Nickel was about twofold more potent in blocking fast (IC50 64 μm) than slow current (IC50 107 μm). The halogenated volatile anaesthetic enflurane blocked both currents, but only the slowly inactivating current was affected in voltage-dependent fashion. Slow dendritic current was essential for generation of low-threshold Ca2+ spikes (LTS), and both enflurane and nickel also suppressed LTS and neuronal burst firing at concentrations that blocked isolated T currents. Differential kinetic properties of T currents expressed in cell soma and proximal dendrites of nRT neurones indicate that various subcellular compartments may exhibit different membrane properties in response to small membrane depolarizations. Furthermore, since blockade of two different T currents in nRT neurones by enflurane and other volatile anaesthetics occurs within concentrations that are relevant during clinical anaesthesia, our findings suggest that these actions could contribute to some important clinical effects of anaesthetics. PMID:15845580

[Neurologic manifestations associated with antiphospholipid antibodies. Or what remains of neurolupus?].

PubMed

Hachulla, E; Leys, D; Deleume, J F; Pruvo, J P; Devulder, B

1995-01-01

Antiphospholipid antibody is associated with a clinical syndrome of vascular thrombosis, thrombocytopenia, recurrent fetal loss and livedo reticularis, whether or not a clinical diagnosis of systemic lupus erythematosus (SLE) coexists. Central nervous system involvement in SLE is multifactorial, thrombotic events, antineuronal antibodies, hypertension, infection, side effects of drugs etc. Antiphospholipid antibodies may play a role in focal neurological manifestations in SLE. In the absence of SLE, different neurological symptoms are well associated with antiphospholipid antibodies including stroke, seizures, dementia, migraine, ocular ischemia, chorea, transverse myelopathy, cerebral phlebitis. Other association are more controversal like Guillain Barré syndrome, motor neuron disease, communicating hydrocephalus. In all patients with antiphospholipid antibodies with neurological involvement, cerebral MRI may be performed with an echocardiographic study because a possible association with Libman and Sacks endocarditis, valve dysfunction or cardiac thrombus source of cerebral ischemia.

Demonstration of circulating and tissue-fixed immune complexes in cutaneous necrotizing vasculitis.

PubMed

Imamura, S; Yanase, K

1980-01-01

Simultaneous demonstration of circulating and tissue-fixed immune complexes was attempted in 22 patients with cutaneous necrotizing vasculitis (7 anaphylactoid purpura, 9 cutaneous allergic vasculitis, 2 livedo reticularis, 1 thrombophlebitis, 2 erythema elevatum diutinum and 1 acute generalized pustular bacterid). In 16 out of the 22 patients, particularly patients with anaphylactoid purpura and cutaneous necrotizing vasculitis, there was a high Clq-binding activity. Decreased levels of C3 and C4 were seen in 2 and 3 patients, respectively. In 11 out of 16 skin lesions, the granular deposits of immunoglobulins and/or complement were demonstrated in the blood vessel walls of the dermis. IgA deposit was seen in anaphylactoid purpura, and IgM deposit in other types of vasculitis. C3 deposit was the most frequently noted. There was no definite correlation between Clq-binding activity and tissue deposits.

Thalamic Mechanisms in Language: A Reconsideration Based on Recent Findings and Concepts

PubMed Central

Crosson, Bruce

2012-01-01

Recent literature on thalamic aphasia and thalamic activity during neuroimaging is selectively reviewed followed by a consideration of recent anatomic and physiological findings regarding thalamic structure and functions. It is concluded that four related corticothalamic and/or thalamocortical mechanisms impact language processing: (1) selective engagement of task-relevant cortical areas in a heightened state of responsiveness in part through the nucleus reticularis (NR), (2) passing information from one cortical area to another through corticothalamo-cortical mechanisms, (3) sharpening the focus on task-relevant information through corticothalamo-cortical feedback mechanisms, and (4) selection of one language unit over another in the expression of a concept, accomplished in concert with basal ganglia loops. The relationship and interaction of these mechanisms is discussed and integrated with thalamic aphasia and neuroimaging data into a theory of thalamic functions in language. PMID:22831779

Eukaryotic cells and their cell bodies: Cell Theory revised.

PubMed

Baluska, Frantisek; Volkmann, Dieter; Barlow, Peter W

2004-07-01

Cell Theory, also known as cell doctrine, states that all eukaryotic organisms are composed of cells, and that cells are the smallest independent units of life. This Cell Theory has been influential in shaping the biological sciences ever since, in 1838/1839, the botanist Matthias Schleiden and the zoologist Theodore Schwann stated the principle that cells represent the elements from which all plant and animal tissues are constructed. Some 20 years later, in a famous aphorism Omnis cellula e cellula, Rudolf Virchow annunciated that all cells arise only from pre-existing cells. General acceptance of Cell Theory was finally possible only when the cellular nature of brain tissues was confirmed at the end of the 20th century. Cell Theory then rapidly turned into a more dogmatic cell doctrine, and in this form survives up to the present day. In its current version, however, the generalized Cell Theory developed for both animals and plants is unable to accommodate the supracellular nature of higher plants, which is founded upon a super-symplasm of interconnected cells into which is woven apoplasm, symplasm and super-apoplasm. Furthermore, there are numerous examples of multinucleate coenocytes and syncytia found throughout the eukaryote superkingdom posing serious problems for the current version of Cell Theory. To cope with these problems, we here review data which conform to the original proposal of Daniel Mazia that the eukaryotic cell is composed of an elemental Cell Body whose structure is smaller than the cell and which is endowed with all the basic attributes of a living entity. A complement to the Cell Body is the Cell Periphery Apparatus, which consists of the plasma membrane associated with other periphery structures. Importantly, boundary structures of the Cell Periphery Apparatus, although capable of some self-assembly, are largely produced and maintained by Cell Body activities and can be produced from it de novo. These boundary structures serve not only as

A Modular, Reconfigurable Surveillance UAV Architecture

DTIC Science & Technology

2003-09-02

Una Società Galileo Avionica A Modular, Reconfigurable Surveillance UAV Architecture METEOR, Finmeccanica Group Zona Industriale di Soleschiano Via...ES) METEOR, Finmeccanica Group Zona Industriale di Soleschiano Via Mario Stoppani 21 34077 Ronchi dei Legionari (GO) ITALY 8. PERFORMING...PMSFMS RS1Backup FMS NSU Payload Control Actuators Router Router RS2 Recovery Devices Una Società Galileo Avionica • Daylight TV Camera • IR Sensor • HR

Eukaryotic Cells and their Cell Bodies: Cell Theory Revised

PubMed Central

BALUŠKA, FRANTIŠEK; VOLKMANN, DIETER; BARLOW, PETER W.

2004-01-01

• Background Cell Theory, also known as cell doctrine, states that all eukaryotic organisms are composed of cells, and that cells are the smallest independent units of life. This Cell Theory has been influential in shaping the biological sciences ever since, in 1838/1839, the botanist Matthias Schleiden and the zoologist Theodore Schwann stated the principle that cells represent the elements from which all plant and animal tissues are constructed. Some 20 years later, in a famous aphorism Omnis cellula e cellula, Rudolf Virchow annunciated that all cells arise only from pre‐existing cells. General acceptance of Cell Theory was finally possible only when the cellular nature of brain tissues was confirmed at the end of the 20th century. Cell Theory then rapidly turned into a more dogmatic cell doctrine, and in this form survives up to the present day. In its current version, however, the generalized Cell Theory developed for both animals and plants is unable to accommodate the supracellular nature of higher plants, which is founded upon a super‐symplasm of interconnected cells into which is woven apoplasm, symplasm and super‐apoplasm. Furthermore, there are numerous examples of multinucleate coenocytes and syncytia found throughout the eukaryote superkingdom posing serious problems for the current version of Cell Theory. • Scope To cope with these problems, we here review data which conform to the original proposal of Daniel Mazia that the eukaryotic cell is composed of an elemental Cell Body whose structure is smaller than the cell and which is endowed with all the basic attributes of a living entity. A complement to the Cell Body is the Cell Periphery Apparatus, which consists of the plasma membrane associated with other periphery structures. Importantly, boundary stuctures of the Cell Periphery Apparatus, although capable of some self‐assembly, are largely produced and maintained by Cell Body activities and can be produced from it de novo. These

Ultrastructural observations of previtellogenic ovarian follicles of dove.

PubMed

Zarnescu, Otilia

2004-11-01

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

PubMed Central

Rodríguez, Lleretny; Navarrete, Felipe I.; Tovar, Heribelt; Cox, José F.

2008-01-01

Purpose In order to simplify cloning, a new method that does not require micromanipulators was used. We aimed to evaluate the developmental potential of two bovine cell lines upon cloning. Materials and methods In vitro matured bovine oocytes, were released from zona pellucida, enucleated, fused to foetal or adult somatic donor cells. The reconstructed embryos were reprogrammed, activated and cultured until blastocyst stage. No micromanipulators were used. Blastocyst rate and quality was scored. Some expanded (d7) blastocysts were transferred to recipient cattle and collected back at d17 to assess elongation. Results High developmental potential in vitro of cloned embryos to expanded (d7) blastocysts was achieved (52.6%). In one cell line, 65.7% of blastocysts was scored. Most blastocysts (87.4%) were graded as excellent. In vivo development to elongation (day-17) in temporary recipient cows also showed a high developmental potential (11/18 transferred blastocysts elongated). Conclusions Hand-made cloning is an efficient alternative for cloning in cattle. PMID:18205035

Vestibular signals in the parasolitary nucleus.

PubMed

Barmack, N H; Yakhnitsa, V

2000-06-01

Vestibular primary afferents project to secondary vestibular neurons located in the vestibular complex. Vestibular primary afferents also project to the uvula-nodulus of the cerebellum where they terminate on granule cells. In this report we describe the physiological properties of neurons in a "new" vestibular nucleus, the parasolitary nucleus (Psol). This nucleus consists of 2,300 GABAergic neurons that project onto the ipsilateral inferior olive (beta-nucleus and dorsomedial cell column) as well as the nucleus reticularis gigantocellularis. These olivary neurons are the exclusive source of vestibularly modulated climbing fiber inputs to the cerebellum. We recorded the activity of Psol neurons during natural vestibular stimulation in anesthetized rabbits. The rabbits were placed in a three-axis rate table at the center of a large sphere, permitting vestibular and optokinetic stimulation. We recorded from 74 neurons in the Psol and from 23 neurons in the regions bordering Psol. The activity of 72/74 Psol neurons and 4/23 non-Psol neurons was modulated by vestibular stimulation in either the pitch or roll planes but not the horizontal plane. Psol neurons responded in phase with ipsilateral side-down head position or velocity during sinusoidal stimulation. Approximately 80% of the recorded Psol neurons responded to static roll-tilt. The optimal response planes of evoked vestibular responses were inferred from measurement of null planes. Optimal response planes usually were aligned with the anatomical orientation of one of the two ipsilateral vertical semicircular canals. The frequency dependence of null plane measurements indicated a convergence of vestibular information from otoliths and semicircular canals. None of the recorded neurons evinced optokinetic sensitivity. These results are consistent with the view that Psol neurons provide the vestibular signals to the inferior olive that eventually reached the cerebellum in the form of modulated climbing fiber

Microheater as an alternative to lasers for in-vitro fertilization applications

NASA Astrophysics Data System (ADS)

Palanker, Daniel V.; Turovets, Igor; Glazer, Rima; Reubinoff, Benjamin E.; Hilman, Dalia; Lewis, Aaron

1999-06-01

During the last decade various lasers have been applied to drilling of the micrometer-sized holes in the zona pellucida of oocytes for in-vitro fertilization applications. In this paper we describe an alternative approach to laser instrumentation based on microfabricated device capable of precise drilling of uniform holes in the zona pellucida of oocytes. This device consists of a thin (1 micrometer) film microheater built on the tip of glass capillary with a diameter varying between a few to a few tens of micrometers. Duration of the pulse of heat produced by this microheater determines the spatial confinement of the heat wave in the surrounding liquid medium. We have demonstrated that gradual microdrilling of the zona pellucida can be accomplished using a series of pulses with duration of about 300 microseconds when the microheater was held in contact with the zona pellucida. Pulse energy applied to 20 micrometer tip was about 4 (mu) J. In vitro development and hatching of 127 micromanipulated embryos was compared to 103 non-drilled control embryos. The technique was found to be highly efficient in creating round, uniform, well defined holes with a smooth wall surface, matching the size of the heating source. The architecture of the surrounding zona pellucida was unaffected by the drilling, as demonstrated by scanning electron microscopy. Micromanipulated embryos presented no signs of thermal damage under light microscopy. The rate of blastocyst formation and hatching was similar in the micromanipulated and control groups. Following further testing in animal models, this methodology may be used as a cost- effective alternative to laser-based instrumentation in clinical applications such as assisted hatching and embryo biopsy.

Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution.

PubMed

Hredzák, R; Ostró, A; Zdilová, Viera; Maracek, I; Kacmárik, J

2006-03-01

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

PubMed Central

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg; Ettl, T.

2011-04-01

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simplemore » method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.« less

Herpesvirus infections in psittacine birds in Japan.

PubMed

Tsai, S S; Park, J H; Hirai, K; Itakura, C

1993-03-01

Herpesvirus infection was diagnosed histologically and electron microscopically in 21 out of 241 pet birds examined. The infected birds included 14 parakeets (Psittacula krameri manillensis) with respiratory infection and three parrots (Ama-zona aestiva aestiva), two cockatiels (Nymphicus hollandicus) and two rosellas (Platycercus emimius) with Pacheco's disease. The consistent lesions of respiratory herpesvirus infection were the formation of syncytial cells associated with the presence of intranuclear inclusion bodies, mainly in the lung and air sac. There was lack of an apparent cellular reaction in situ. The agent induced tubular structures containing a clear core in the nuclei of the affected cells. The present study indicated that it was a distinct entity from infectious laryngotracheitis based on tissue tropism, host reaction and morphology of the tubular structures. The striking lesions of Pacheco's disease consisted of syncytial cell formation with intranuclear inclusion bodies in various organs, especially the liver, parathyroid, ovary, bone marrow and intestine. This agent showed similar morphology to that of the respiratory herpesvirus infection, but was larger in size and had no tubular structure formation in the nuclei of affected cells.

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

PubMed Central

Wang, Hehai; Ding, Tianbing; Brown, Naoko; Yamamoto, Yasutoshi; Prince, Lawrence S.; Reese, Jeff; Paria, B. C.

2008-01-01

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells. PMID:18423437

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

PubMed Central

Ilinskaya, Anna; Dorjbal, Batsukh; Truong, Rosaline; Derse, David; Uchil, Pradeep D.; Heidecker, Gisela; Mothes, Walther

2013-01-01

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV. PMID:23308151

Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity.

PubMed

Russi, Abigail E; Walker-Caulfield, Margaret E; Guo, Yong; Lucchinetti, Claudia F; Brown, Melissa A

2016-09-01

GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Engineering Cell-Cell Signaling

PubMed Central

Milano, Daniel F.; Natividad, Robert J.; Asthagiri, Anand R.

2014-01-01

Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling based on quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilizing synthetic cells, advanced ‘chassis’ and predictive modeling to engineer the form and function of living tissues. PMID:23856592

CONSIDERATIONS OF THE HISTOLOGIC AND HISTOCHEMICAL INJURY INDUCED BY TOTAL ROENTGEN IRRADIATION IN THE FEMALE GENITAL ORGANS OF PUBERTAL AND PREPUBERTAL MICE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cecio, A.; Cerasuolo, G.

1961-07-15

Changes in uterus and ovaries were studied in mice exposed to 700 r whole-body irradiation. Endometrial glands showed an increase in mitotic figures after irradiation and somewhat decreased numbers of cells although histochemical tests for polysaccharides and ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were intensified. The results were similar in adult and immature mice. In ovary, irradiation caused degeneration of follicles with separation of granulosa cells, which was followed by hyperplasia during the reparative phase. Here also polysaccharides were increased in amounts, especially in the zona pellucida of the oocytes. Epithelium of Fallopiant tube also showed increased histochemical reactionsmore » for polysaccharides, RNA, and DNA. Most of these changes were evident within 24 hr after irradiation; they increased in intensity up to the 2nd day, then tended to decrease. (H.H.D.)« less

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens.

PubMed

Ienne, Susan; Pappas, Georgios; Benabdellah, Karim; González, Antonio; Zingales, Bianca

2012-04-01

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of γ-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas, Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. Copyright © 2012 Elsevier B.V. All rights reserved.

Mast cells enhance T cell activation: Importance of mast cell-derived TNF

NASA Astrophysics Data System (ADS)

Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

2005-05-01

Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

[The cell theory. Progress in studies on cell-cell communications].

PubMed

Brodskiĭ, V Ia

2009-01-01

Current data confirm the fundamental statement of the cell theory concerning the cell reproduction in a series of generations (omnis cellula e cellula). Cell communities or ensembles integrated by the signaling systems established in prokaryotes and protists and functioning in multicellular organisms including mammals are considered as the structural and functional unit of a multicellular organism. The cell is an elementary unit of life and basis of organism development and functioning. At the same time, the adult organism is not just a totality of cells. Multinucleated cells in some tissues, syncytial structure, and structural-functional units of organs are adaptations for optimal functioning of the multicellular organism and manifestations of cell-cell communications in development and definitive functioning. The cell theory was supplemented and developed by studies on cell-cell communications; however, these studies do not question the main generalizations of the theory.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

PubMed

Moreno-Cencerrado, Alberto; Iturri, Jagoba; Pecorari, Ilaria; D M Vivanco, Maria; Sbaizero, Orfeo; Toca-Herrera, José L

2017-01-01

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Molluscan cells in culture: primary cell cultures and cell lines

PubMed Central

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

PubMed

Feyerabend, Thorsten B; Terszowski, Grzegorz; Tietz, Annette; Blum, Carmen; Luche, Hervé; Gossler, Achim; Gale, Nicholas W; Radtke, Freddy; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2009-01-16

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

Glioma cell dispersion is driven by α5 integrin-mediated cell-matrix and cell-cell interactions.

PubMed

Blandin, Anne-Florence; Noulet, Fanny; Renner, Guillaume; Mercier, Marie-Cécile; Choulier, Laurence; Vauchelles, Romain; Ronde, Philippe; Carreiras, Franck; Etienne-Selloum, Nelly; Vereb, Gyorgy; Lelong-Rebel, Isabelle; Martin, Sophie; Dontenwill, Monique; Lehmann, Maxime

2016-07-01

Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis.

PubMed Central

Massip, A; Mulnard, J; Vanderzwalmen, P; Hanzen, C; Ectors, F

1982-01-01

The behaviour of the cow blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: continuous expansion followed by hatching; discontinuous expansion interrupted by few contractions and followed by hatching; discontinuous expansion interrupted by several rapid contractions without hatching. This demonstrated that the pulsatile activity of the blastocyst is not a necessary condition of hatching but also that only a moderate pulsatile activity is compatible with normal hatching. The time of hatching in vitro corresponded approximately with the time of zona loss in vivo (9-10 days). Rupture of the zona occurred at any point of the trophoblast layer. Hatching by herniation through a reduced opening of the zona was occasionally observed. The behavior of the embryos from a particular animal was very similar but differences were noted between embryos from different animals. Images Fig. 3 PMID:7076563

Border cell release: Cell separation without cell wall degradation?

PubMed

Mravec, Jozef

2017-07-03

Plant border cells are specialized cells derived from the root cap with roles in the biomechanics of root growth and in forming a barrier against pathogens. The mechanism of highly localized cell separation which is essential for their release to the environment is little understood. Here I present in situ analysis of Brachypodium distachyon, a model organism for grasses which possess type II primary cell walls poor in pectin content. Results suggest similarity in spatial dynamics of pectic homogalacturonan during dicot and monocot border cell release. Integration of observations from different species leads to the hypothesis that this process most likely does not involve degradation of cell wall material but rather uses unique cell wall structural and compositional means enabling both the rigidity of the root cap as well as detachability of given cells on its surface.

Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

PubMed

Shamloo, Amir

2014-09-01

This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey

2007-12-31

Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. Thesemore » cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.« less

Cell wars: regulation of cell survival and proliferation by cell competition

PubMed Central

Vivarelli, Silvia; Wagstaff, Laura; Piddini, Eugenia

2012-01-01

During cell competition fitter cells take over the tissue at the expense of viable, but less fit, cells, which are eliminated by induction of apoptosis or senescence. This probably acts as a quality-control mechanism to eliminate suboptimal cells and safeguard organ function. Several experimental conditions have been shown to trigger cell competition, including differential levels in ribosomal activity or in signalling pathway activation between cells, although it is unclear how those differences are sensed and translated into fitness levels. Many of the pathways implicated in cell competition have been previously linked with cancer, and this has led to the hypothesis that cell competition could play a role in tumour formation. Cell competition could be co-opted by cancer cells to kill surrounding normal cells and boost their own tissue colonization. However, in some cases, cell competition could have a tumour suppressor role, as cells harbouring mutations in a subset of tumour suppressor genes are killed by wild-type cells. Originally described in developing epithelia, competitive interactions have also been observed in some stem cell niches, where they play a role in regulating stem cell selection, maintenance and tissue repopulation. Thus competitive interactions could be relevant to the maintenance of tissue fitness and have a protective role against aging. PMID:22928509

Quantitative Characterization of Cell Behaviors through Cell Cycle Progression via Automated Cell Tracking

PubMed Central

Wang, Yuliang; Jeong, Younkoo; Jhiang, Sissy M.; Yu, Lianbo; Menq, Chia-Hsiang

2014-01-01

Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells. PMID:24911281

Is vitronectin the velcro that binds the gametes together?

PubMed

Fusi, F M; Bernocchi, N; Ferrari, A; Bronson, R A

1996-11-01

Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.

Aurelia aurita (Cnidaria) Oocytes' Contact Plate Structure and Development

PubMed Central

Adonin, Leonid S.; Shaposhnikova, Tatyana G.; Podgornaya, Olga

2012-01-01

One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the “contact plate”. Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high Mr bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides Mr corresponds to that of mesogleal cells, the other ones' Mr is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida. PMID:23185235

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

PubMed

Song, Wen-Yan; Peng, Zhao-Feng; Chen, Xue-Mei; Jin, Hai-Xia; Yao, Gui-Dong; Shi, Sen-Lin; Yang, Hong-Yi; Zhang, Xiang-Yang; Sun, Ying-Pu

2016-01-01

To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT. © 2016 The Author(s) Published by S. Karger AG, Basel.

Single cell RNA sequencing of stem cell-derived retinal ganglion cells.

PubMed

Daniszewski, Maciej; Senabouth, Anne; Nguyen, Quan H; Crombie, Duncan E; Lukowski, Samuel W; Kulkarni, Tejal; Sluch, Valentin M; Jabbari, Jafar S; Chamling, Xitiz; Zack, Donald J; Pébay, Alice; Powell, Joseph E; Hewitt, Alex W

2018-02-13

We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) at the single cell level. The human embryonic stem cell line BRN3B-mCherry (A81-H7), was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype.

Rate dependence of cell-to-cell variations of lithium-ion cells.

PubMed

An, Fuqiang; Chen, Lufan; Huang, Jun; Zhang, Jianbo; Li, Ping

2016-10-11

Lithium-ion cells are commonly used in a multicell configuration in power devices and electric vehicles, making the cell-to-cell variation (CtCV) a key factor to consider in system design and management. Previous studies on CtCV have two major limitations: the number of cells is usually less than one hundred, and the cells are usually commercial cells already subjected to cell-screenings. In this article, we first make a statistical analysis on the CtCV of 5473 fresh cells from an automotive battery manufacturer before the cell-screening process. Secondly, 198 cells are randomly selected from these 5473 cells and the rate dependence of the CtCV is examined, focusing on the correlations of capacity versus weight and capacity versus resistance, corresponding to thermodynamic and kinetic factors, respectively. The rate dependence of these two correlations is explained from a phenomenological model. Finally, eight cells from the 198 cells are further characterized with electrochemical impedance spectroscopy method to elucidate the kinetic origins of the CtCV.

Rate dependence of cell-to-cell variations of lithium-ion cells

PubMed Central

An, Fuqiang; Chen, Lufan; Huang, Jun; Zhang, Jianbo; Li, Ping

2016-01-01

Lithium-ion cells are commonly used in a multicell configuration in power devices and electric vehicles, making the cell-to-cell variation (CtCV) a key factor to consider in system design and management. Previous studies on CtCV have two major limitations: the number of cells is usually less than one hundred, and the cells are usually commercial cells already subjected to cell-screenings. In this article, we first make a statistical analysis on the CtCV of 5473 fresh cells from an automotive battery manufacturer before the cell-screening process. Secondly, 198 cells are randomly selected from these 5473 cells and the rate dependence of the CtCV is examined, focusing on the correlations of capacity versus weight and capacity versus resistance, corresponding to thermodynamic and kinetic factors, respectively. The rate dependence of these two correlations is explained from a phenomenological model. Finally, eight cells from the 198 cells are further characterized with electrochemical impedance spectroscopy method to elucidate the kinetic origins of the CtCV. PMID:27725767

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

PubMed Central

Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

2016-01-01

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

Suppression of Sleep Spindle Rhythmogenesis in Mice with Deletion of CaV3.2 and CaV3.3 T-type Ca2+ Channels

PubMed Central

Pellegrini, Chiara; Lecci, Sandro; Lüthi, Anita; Astori, Simone

2016-01-01

Study Objectives: Low-threshold voltage-gated T-type Ca2+ channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). Methods: We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. Results: CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca2+ currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10–15 Hz), which was accompanied by an increase in the δ band (1–4 Hz). Conclusions: Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms. Citation: Pellegrini C, Lecci S, Lüthi A, Astori S. Suppression of sleep spindle rhythmogenesis in mice with deletion of Cav3.2 and Cav3.3 T-type Ca2+ channels. SLEEP 2016;39(4):875�

9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS 2 AND 4, BASEMENT LEVEL. LOOKING SOUTHEAST. - Fairchild Air Force Base, Engine Test Cell Building, Near intersection of Arnold Street & George Avenue, Spokane, Spokane County, WA

Possible mechanisms of mammalian immunocontraception.

PubMed

Barber, M R; Fayrer-Hosken, R A

2000-03-01

Ecological and conservation programs in ecosystems around the world have experienced varied success in population management. One of the greatest problems is that human expansion has led to the shrinking of wildlife habitat and, as a result, the overpopulation of many different species has occurred. The pressures exerted by the increased number of animals has caused environmental damage. The humane and practical control of these populations has solicited the scientific community to arrive at a safe, effective, and cost-efficient means of population control. Immunocontraception using zona pellucida antigens, specifically porcine zona pellucida (pZP), has become one of the most promising population control tools in the world today, with notable successes in horses and elephants. A conundrum has risen where pZP, a single vaccine, successfully induces an immunocontraceptive effect in multiple species of mammals. This review describes the most current data pertaining to the mammalian zona pellucida and immunocontraception, and from these studies, we suggest several potential mechanisms of immunocontraception.

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.