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PubMed2006

Nipped-A, the Tra1/TRRAP subunit of the Drosophila SAGA and Tip60 complexes, has multiple roles in Notch signaling during wing development.

The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. Here we show, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, mastermind, during wing development. Nipped-A and mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes. We find that, like Nipped-A, the Ada2b component of SAGA and the domino subunit of Tip60 are also required for mastermind function during wing development. Based on these results, we propose that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Human peripheral blood eosinophils express stem cell factor.

Stem cell factor (SCF) or c-Kit ligand is a cytokine associated with the differentiation, survival, and activation of mast cells. Eosinophils have pleiotropic functions in several diseases and, together with mast cells, are key cells in allergy. Mast cell-eosinophil interactions can take place during the late and chronic phases of allergy. It was, therefore, investigated whether eosinophils can produce SCF and consequently influence mast cells. Human peripheral blood eosinophils variably expressed mRNA for the soluble and uncleaved forms of SCF (reverse transcription-polymerase chain reaction) and produced the 18.5-kd protein backbone of SCF (Western blot analysis). After overnight incubation in medium, eosinophils also produced SCF of higher molecular weight (42-45 kd) that might represent its glycosylated forms. Eosinophils expressed cytoplasmic SCF that colocalized with major basic protein (confocal laser microscopy). Freshly isolated eosinophils contained 8.9 +/- 1.7 pg SCF/10(6) (mean +/- SEM; enzyme-linked immunosorbent assay). Although overnight incubation of the eosinophils in either culture medium or in phorbol 12-myristate 13-acetate-calcium ionophore did not cause the secretion of SCF, the addition of chymase induced SCF release. In summary, it was demonstrated that human peripheral blood eosinophils are a source of SCF. These results may contribute to a better understanding of the interactions between eosinophils and mast cells in allergic inflammation.

Blood Proteins↗
PubMed2005

Hydrogen peroxide alters membrane and cytoskeleton properties and increases intercellular connections in astrocytes.

Excess hydrogen peroxide (H2O2) is produced in the pathogenesis of brain injuries and neurodegenerative diseases. H2O2 may damage cells through direct oxidation of lipids, proteins and DNA or it can act as a signaling molecule to trigger intracellular pathways leading to cell death. In this study, H2O2 caused plasma membranes of primary astrocytes to become more gel-like, while artificial membranes of vesicles composed of rat brain lipid extract became more liquid crystalline-like. Besides the effects on membrane phase properties, H2O2 promoted actin polymerization, induced the formation of cell-to-cell tunneling nanotube (TNT)-like connections among astrocytes and increased the colocalization of myosin Va with F-actin. Myosin Va was also observed in the H2O2-induced F-actin-enriched TNT-like connections. Western blot analysis suggests that H2O2 triggered the phosphorylation of the p38 mitogen-activated protein kinase (MAPK), and that SB203580, a specific inhibitor of p38 MAPK, suppressed the changes in membrane phase properties and cytoskeleton resulting from H2O2 treatment. These results suggest that H2O2 alters astrocyte membranes and the cytoskeleton through activation of the p38 MAPK pathway.

Actins↗
PubMed2000

Formation of glycine receptor clusters and their accumulation at synapses.

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor alpha subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an alpha mutant subunit bearing the gephyrin-binding motif of the beta subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR beta subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.

Animals↗
PubMed1999

Ovarian steroid action on tryptophan hydroxylase protein and serotonin compared to localization of ovarian steroid receptors in midbrain of guinea pigs.

The effect of estrogen (E) and progesterone (P) on the protein expression of the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase (TPH), and the level of serotonin in the hypothalamic terminal field was examined in guinea pigs. In addition, we questioned whether serotonin neurons of guinea pigs contain ovarian steroid receptors (estrogen receptoralpha[ERalpha], estrogen receptor beta[ERbeta], progestin receptors [PRs]) that could directly mediate the actions of E or P. Western blot and densitometric analysis for TPH were used on raphe extracts from untreated-ovariectomized (OVX), OVX-E-treated (28 d), and OVX-E+P-treated (14 d E+14 d E+P) guinea pigs. The medial basal hypothalami from the same animals were extracted and subjected to high-performance liquid chromatography analysis for serotonin, dopamine, 5-hydroxyindole acetic acid, and homovanillic acid. The brains from other animals treated in an identical manner were perfusion fixed and examined for the colocalization of ERalpha plus serotonin and PR plus serotonin with double immunohistochemistry or for expression of ERbeta mRNA with in situ hybridization. E and E+P treatment significantly increased TPH protein levels compared to the untreated control group (p < 0.05), but TPH levels were similar in the E and E+P-treated groups. By contrast, serotonin (nanogram/milligram of protein) in the hypothalamus was significantly increased by E+P treatment, but not by E alone. Neither ERalpha nor PR proteins were detected within serotonin neurons of the guinea pig raphe nucleus. However, ERbeta mRNA was expressed in the dorsal raphe. In summary, E alone increased TPH protein expression and the addition of P had no further effect, whereas E+P increased hypothalamic serotonin and E alone had no effect. The localization of ERbeta, but not ERalpha or PR, in the dorsal raphe nucleus suggests that E acting via ERbeta within serotonin neurons increases expression of TPH, but that P acting via other neurons and transsynaptic stimulation may effect changes in TPH enzymatic activity, which in turn, would lead to an increase in serotonin synthesis.

Animals↗
PubMed2026

FANCI promotes esophageal squamous cell carcinoma progression and cell cycle regulation and interacts with FANCD2.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor clinical outcomes, and reliable molecular biomarkers and therapeutic targets remain limited. Fanconi anemia group I protein (FANCI) is a core component of the Fanconi anemia (FA) pathway, but its expression pattern, clinical significance, and functional role in ESCC have not been comprehensively defined. This study aimed to investigate FANCI expression and prognostic value in ESCC, assess its effects on malignant cellular phenotypes and tumor growth, and explore its potential mechanistic relationship with Fanconi anemia group D2 protein (FANCD2) and cell-cycle regulation. METHODS: Multi-cohort analyses were performed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, together with ESCC single-cell RNA sequencing (RNA-seq) data. FANCI functions were assessed by bidirectional gain- and loss-of-function experiments in vitro (proliferation, colony formation, migration, invasion, apoptosis, and cell-cycle assays) and by xenograft models in vivo. Mechanistic studies included protein-protein interaction (PPI) analyses, co-immunoprecipitation (Co-IP), and immunofluorescence (IF) colocalization. RESULTS: FANCI was consistently upregulated in ESCC across bulk transcriptomic datasets and was further supported by quantitative polymerase chain reaction (qPCR), Western blotting, and immunohistochemistry (IHC). FANCI discriminated ESCC from normal tissues in TCGA-ESCC and was independently validated in GSE53624 [area under the curve (AUC) =0.940 and 0.975, respectively]. FANCI was associated with poorer overall survival (OS) and shorter disease-free interval (DFI), and these findings were validated in an independent GEO cohort. Functionally, FANCI promoted ESCC cell proliferation, migration, and invasion, while inhibiting apoptosis; FANCI knockdown suppressed tumor growth in vivo and induced G2/M cell-cycle arrest. Mechanistically, FANCI physically interacted with FANCD2, colocalized with FANCD2 in the nucleus, and was associated with altered FANCD2 protein abundance, consistent with cell-cycle and DNA repair-related programs. Single-cell analysis indicated that FANCI was enriched in epithelial cells and associated with higher activity of malignant functional programs. In TCGA-ESCC, FANCI-high tumors showed distinct mutation profiles, a trend toward increased tumor mutation burden (TMB), and altered immune-associated signatures. CONCLUSIONS: FANCI is upregulated in ESCC and is associated with diagnostic and prognostic value. It promotes malignant phenotypes and tumor growth, potentially through a FANCI-FANCD2-linked cell-cycle/DNA repair program, supporting FANCI as a candidate biomarker and therapeutic target in ESCC.

Esophageal squamous cell carcinoma (ESCC)↗
PubMed1990

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.

Animals↗
PubMed2003

New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific binding sites.

Nuclear presence of galectins suggests a role of these endogenous lectins in the regulation of transcription, pre-mRNA splicing and transport processes. Therefore, detection and localization of nuclear binding sites for galectins by a new methodological step, has potential to further functional analysis. In the first step of our model study we monitored the nuclear expression of galectins-1 and -3 in cultured stromal cells of human bone marrow and human/porcine keratinocytes. To enable detection and localization of galectin-specific binding sites, we used purified galectins biotinylated without loss of activity as cytochemical tool. The degree of labeling of the probes was determined by adapting two-dimensional gel electrophoresis and calculating pI changes in response to stepwise chemical modification of basic and acidic side chains by the biotinylation reagents. Binding studies revealed positivity for galectin-1, whereas galectins-3, -5, and -7 were not reactive with nuclear sites under identical conditions in bone marrow stromal cells and keratinocytes prepared from hair follicle enriched for stem cells. Inhibition by lactose indicated an involvement of the carbohydrate recognition domain in nuclear binding of galectin-1. Colocalization of the galectin-1-dependent signal with the SC35 splicing factor and sensitivity toward RNase treatment argued in favor of galectin binding in nuclear speckles, albeit only for a small fraction of the cells. Epidermal cells positive for galectin-1-binding sites expressed DeltaNp63 known as a potential marker of stem cells. Based on cytokeratin expression cells with nuclear binding of labeled galectin-1 were basal and not suprabasal cells. Regarding proliferation, no relationship to the expression of a proliferation marker, Ki-67, was observed. The nucleolar signal colocalized with fibrillarin and nucleophosmin/B23 as representatives of nucleolar proteins in both types of studied cells. In conclusion, the application of labeled galectins to localize accessible binding sites adds a new aspect to the functional analysis of these lectins in the nucleus.

Animals↗
PubMed2001

Colocalization of GABA and glycine in the ventral nucleus of the lateral lemniscus in rat: an in situ hybridization and semiquantitative immunocytochemical study.

We have studied by in situ hybridization for GAD65 mRNA in thick sections and by semiquantitative postembedding immunocytochemistry in consecutive semithin sections, the expression of gamma-aminobutyric acid (GABA) and glycine in cell bodies and axosomatic puncta of the rat ventral nucleus of the lateral lemniscus (VNLL), a prominent monaural brainstem auditory structure. The in situ hybridization and the densitometric analysis of the immunostaining suggest that the rat VNLL contains two main populations of neurons. Approximately one-third of neurons are unstained with either technique and are presumably excitatory; their cell bodies are enveloped by a large number of glycine-immunoreactive puncta. Most if not all of the remaining two-thirds colocalize GABA and glycine and are assumed to be inhibitory. These two populations show a complementary distribution within the VNLL, with inhibitory neurons located mainly ventrally and excitatory neurons dorsally. In scatterplots of gray values measured from cell bodies, the double-labeled cells appear to form a single cluster in terms of their staining intensities for the two transmitter candidates. However, this cluster may have to be further subdivided because cells with extreme GABA/glycine ratios differ from those with average ratios with respect to location or size. The VNLL seems unique among auditory structures by its large number of neurons that colocalize GABA and glycine. Although the functional significance of this colocalization remains unknown, our results suggest that the VNLL exerts convergent excitatory and inhibitory influences over the inferior colliculus, which may underlie the timing processing in the auditory midbrain.

Animals↗
PubMed2001

Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Puralpha in NIH3T3 cells.

Levels of Puralpha, a conserved, sequence-specific single-stranded DNA and RNA binding protein, fluctuate during the cell cycle, declining at the onset of S-phase and peaking at mitosis. In early G1 Puralpha is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Puralpha into NIH3T3 cells arrests the cell cycle at either G1/S or G2/M checkpoints with distinct morphological consequences. Here we ask whether expression of Puralpha can affect colony formation and anchorage-independent growth in ras-transformed NIH3T3 cells. Two to five-fold elevated levels of Puralpha in stably-transfected cell lines retard entry into and progression through S phase in both ras-transformed and non-transformed cells. Puralpha significantly inhibits colony formation by ras-transformed cells but not by non-transformed cells. In addition, cells transfected to express Puralpha formed only about 1/5 the number of large colonies in soft agar as control-transfected cells, demonstrating a marked inhibition of anchorage-independent growth by Puralpha. Biochemical analysis of nuclear and cytoplasmic Puralpha proteins and confocal microscopic analysis of Puralpha location indicate that access of Puralpha to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Puralpha domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215-231. In the nucleus Puralpha colocalizes with CDK2 and cyclin A. Puralpha and cyclin D1, however, do not colocalize in the nucleus. At mitosis Puralpha is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Puralpha to interact with proteins regulating cell proliferation and transformation is controlled by signals that govern its intracellular localization.

3T3 Cells↗
PubMed1998

HLA-DM is expressed on the cell surface and colocalizes with HLA-DR and invariant chain in human Langerhans cells.

The subcellular distribution patterns of molecules involved in the process of antigen loading [HLA-DR, HLA-DM, and the cytoplasmic and luminal parts of the invariant chain (Ii, CD74)] were investigated in Langerhans cells (LC), both qualitatively and quantitatively. The analysis was performed by immunofluorescence labelling of acetone-fixed vertical cryostat sections from normal human skin specimens and subsequent examination using confocal laser scanning microscopy (CSLM). The intensity-modulated multiple-wavelength scanning (IMS) technique was used to enhance channel separation when scanning dual-labelled specimens. The mean (n = 9) relative epidermal volumes of reactivity were: HLA-DR 8%+/-3%, HLA-DM 6%+/-2%, luminal Ii 6%+/-2%, and cytoplasmic Ii 4%+/-1%. The difference between HLA-DR and the other epitopes was significant at the P<0.001 level. All molecule combinations, except the combination of HLA-DM and luminal Ii (which was not studied), were to various extents colocalized. Experiments performed on unfixed epidermal sheets showed that HLA-DM is present on the cell surface of LC, suggesting that HLA-DM may interact with HLA-DR on the surface to induce peptide loading.

Adult↗
PubMed1998

Enhanced regional expression of glutathione S-transferase P1-1 with colocalized AP-1 and CYP 1A2 induction in chlorobenzene-induced porphyria.

A distinct, nonfocal expression pattern was observed for glutathione S-transferase P1-1 (rGSTP1-1) in rats exposed to either hexachloro-(HCB) or pentachlorobenzene (PeCB). The nonfocal expression was localized to the centrilobular region with the most intense staining nearest the central vein. A Western blot analysis revealed a 5- and 15-fold induction of rGSTP1-1 with PeCB and HCB treatment on an equal molar basis, respectively. Evaluation of porphyrin fluorescence also revealed a centrilobular accumulation with average porphyrin measurements of 0.319, 0.590, and 0.206 micrograms/g tissue for PeCB, HCB, and corn-oil controls, respectively. Due to the role of Activator Protein-1 (AP-1) in rGSTP1-1 expression and CYP 1A2 in the pathogenesis of porphyria cutanea tarda, immunohistochemical localization of c-jun, c-fos, and CYP 1A2 was also performed. Increased expression and colocalization within the liver lobule was observed for c-jun, c-fos, CYP 1A2, rGSTP1-1, and areas of porphyrin accumulation. These observations are consistent with studies that have associated the induction of GST-P with jun- and fos-related gene products.

Animals↗
PubMed2006

Histochemical and electron microscopic analysis of spiculogenesis in the demosponge Suberites domuncula.

The skeleton of demosponges is built of spicules consisting of biosilica. Using the primmorph system from Suberites domuncula, we demonstrate that silicatein, the biosilica-synthesizing enzyme, and silicase, the catabolic enzyme, are colocalized at the surface of growing spicules as well as in the axial filament located in the axial canal. It is assumed that these two enzymes are responsible for the deposition of biosilica. In search of additional potential structural molecules that might guide the mineralization process during spiculogenesis to species-specific spicules, electron microscopic studies with antibodies against galectin and silicatein were performed. These studies showed that silicatein forms a complex with galectin; the strings/bundles of this complex are intimately associated with the surface of the spicules and arranged concentrically around them. Collagen fibers are near the silactein/galectin complexes. The strings/bundles formed from silicatein/galectin display a lower degree of orientation than the collagen fibers arranged in a highly ordered pattern around the spicules. These data indicate that species-specific formation of spicules involves a network of (diffusible) regulatory factor(s) controlling enzymatic silica deposition; this mineralization process proceeds on a galectin/collagen organic matrix.

Amino Acid Sequence↗
PubMed2000

Movement-related desynchronization of the cerebral cortex studied with spatially filtered magnetoencephalography.

Event-related desynchronization (ERD) within the alpha and beta bands on unilateral index finger extension and hand grasping was investigated on six normal volunteers with magnetoencephalography (MEG). A novel spatial filtering technique for imaging cortical source power, synthetic aperture magnetometry (SAM), was employed for the tomographic demonstration of ERD. SAM source image results were transformed into statistical parametric images. On the same hand grasping task, a functional MRI (fMRI) study was conducted on two subjects and compared with the ERD result. When the MEG data were analyzed with the fast Fourier transformation, power attenuation within the alpha and beta bands was evident on the contralateral sensorimotor area just prior to movement onset. The tomographic distribution of ERD was clearly obtained with SAM statistical imaging analysis. The equivalent current dipole (ECD) for the signal-averaged motor field was localized to the hemisphere contralateral to the hand movement, roughly at the center of the region displaying beta-band ERD. The signal increase on fMRI roughly colocalized with the ERD on the contralateral sensorimotor area. In conclusion, with the novel spatial filtering technique for the brain magnetic field, SAM, cortical regions contributing to ERD on finger movement were successfully demonstrated in a tomographic manner. The relative colocalization of the contralateral SAM ERD with ECD as well as the fMRI activation suggests that SAM is a practically useful technique to extract event-related signals from brain noise.

Adult↗
PubMed1996

Subunits of soluble guanylyl cyclase in rat and guinea-pig sensory ganglia.

Soluble guanylyl cyclase is a heterodimeric (alpha, beta) enzyme generating the second messenger, cGMP, upon activation by the gaseous messenger, nitric oxide. The occurrence and distribution of alpha 1-, alpha 2-, beta 1- and beta 2-subunits were investigated in trigeminal and dorsal root ganglia on the mRNA and the protein level. Reverse transcription PCR analysis demonstrated mRNA coding for alpha 1-, alpha 2-, and beta 1-subunits in guinea-pig trigeminal and dorsal root ganglia. In agreement with these data, immunoreactivity to the alpha 1-subunit was found in satellite and Schwann cells, while alpha 2-subunit immunoreactivity was localized to axons of large diameter. The distribution of the beta 1-subunit could not be studied on the protein level since the antiserum was ineffective in immunohistochemistry. However, previous studies and the RT-PCR data argue in favour of alpha 1/beta 1-and alpha 2/beta 1-heterodimerization and colocalization. In both species, beta 2-subunit immunoreactivity was confined to neuronal perikarya, primarily of large diameter. Although these results were obtained with two different antibodies directed against different epitopes, the corresponding mRNA could not be detected by RT-PCR analysis. The reason for this discrepancy remains unclear, at present, but could be explained by a variant beta 2- or highly homologous as yet unidentified beta-subunit. This study demonstrates the presence of soluble guanylyl cyclase in sensory ganglia with a differential, cell type-specific distribution of the individual subunits.

Amino Acid Sequence↗
PubMed2004

Global heterochromatic colocalization of transposable elements with minisatellites in the compact genome of the pufferfish Tetraodon nigroviridis.

Because of its unusual high degree of compaction and paucity of repetitive sequences, the genome of the smooth pufferfish Tetraodon nigroviridis is the subject of a well-advanced sequencing project. An astonishing diversity of transposable elements not found in the human and the mouse has been observed in the genome of T. nigroviridis. Due to the difficulty of assembling repeat-rich regions, the whole genome shotgun sequencing approach will probably fail to reveal the general organisation of this compact vertebrate genome. Therefore, in order to gain new insights into the global distribution pattern of repeated DNA in the genome of T. nigroviridis, we have reconstructed partial/complete repetitive sequences from data generated by the genome project and performed double-colour fluorescent in situ hybridization (FISH) analysis for representatives of three major categories of repeated sequences including two minisatellites (ms100 and ms104), two DNA transposons (Tol2 and Buffy1) and two non-long terminal repeat (LTR) retrotransposons (Rex3 and Babar). We show that DNA transposons and retroelements very frequently colocalize with minisatellites and mostly accumulate within heterochromatic regions. These results, which have not been reported so far for the fugu Takifugu rubripes, show that repeated elements are generally excluded from gene-rich regions in T. nigroviridis and underline the extreme degree of compartmentalization of this compact genome. The genome organization of the pufferfish is clearly different from that observed in humans, where repeated sequences make up an important fraction of euchromatic DNA, and is more similar to that observed in the fruit fly Drosophila melanogaster.

Amino Acid Sequence↗
PubMed2005

Role of neuropeptide Y in the regulation of gonadotropin releasing hormone system in the forebrain of Clarias batrachus (Linn.): immunocytochemistry and high performance liquid chromatography-electrospray ionization-mass spectrometric analysis.

Although the importance of neuropeptide Y (NPY) in the regulation of gonadotropin releasing hormone (GnRH) and reproduction has been highlighted in recent years, the neuroanatomical substrate within which these substances might interact has not been fully elucidated. Present work was undertaken with a view to define the anatomical-physiological correlates underlying the role exercised by NPY in the regulation of GnRH in the forebrain of the teleost Clarias batrachus. Application of double immunocytochemistry revealed close associations as well as colocalizations of the two peptides in the olfactory receptor neurons (ORNs), olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tract, fibers in the area ventralis telencephali/pars supracommissuralis and cells as well as fibers in the pituitary. NPY containing axons were found to terminate in the vicinity of GnRH cells in the pituitary with light as well as electron microscopy. Double immunoelectron microscopy demonstrated gold particles for NPY and GnRH colocalized on the membrane and in dense core of the secretory granules in the cells distributed in all components of the pituitary gland. To assess the physiological implication of these observations, NPY was injected via the intracranial route and the response of GnRH immunoreactive system was evaluated by relative quantitative morphometry as well as high performance liquid chromatography (HPLC) analysis. Two hours following NPY (20 ng/g body weight) administration, a dramatic increase was observed in the GnRH immunoreactivity in the ORNs, in the fibers of the olfactory bulb (163%) and medial olfactory tract (351%). High performance liquid chromatography-electrospray ionization-mass spectrometric analysis confirmed the immunocytochemical data. Significant rise in the salmon GnRH (sGnRH)-like peptide content was observed in the olfactory organ (194.23%), olfactory bulb (146.64%), telencephalon+preoptic area (214.10%) and the pituitary (136.72%) of the NPY-treated fish. However, GnRH in the hypothalamus was below detection limit in the control as well as NPY-treated fish. Present results suggest the involvement of NPY in the up-regulation of sGnRH containing system at different level of neuraxis extending from the olfactory epithelium to the pituitary in the forebrain of C. batrachus.

Animals↗
PubMed2005

NADPH-diaphorase positive neurons of the rat hippocampal formation: regional distribution, total number and colocalization with calcium binding proteins.

The present study aimed to asses the total number and distribution of the NADPH-diaphorase-positive non-pyramidal neurons in Ammon's horn and dentate gyrus of rat hippocampal formation. Cell bodies were counted according to the "disector" principle. The total numbers varied from 27 000 to 32 400. In all strains, approximately one third of the NADPH-diaphorase-reactive non-principal cells were found in the dentate gyrus and the remaining two thirds were within the Ammon's horn. Analysis of the dorsoventral differences revealed that approximately 70% of NADPH-diaphorase-positive cells were in the dorsal and 30% in the ventral hippocampus. Distribution of NADPH-diaphorase-reactive cells in the different layers of the dentate gyrus and Ammon's horn was similar in all strains. Double-labelling studies revealed colocalization of NADPH-diaphorase with calretinin, but none with calbindin or parvalbumin. NADPH-diaphorase-positive neurons appear to form the largest chemically identified subpopulation of the GABAergic inhibitory cell population of the hippocampal formation.

Animals↗