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PubMed1994

Neurotrophin-4/5 promotes survival and differentiation of rat striatal neurons developing in culture.

Cultures of dissociated striatal neurons from fetal rats were prepared, and were grown in the presence of neurotrophin-4/5 (NT-4/5) as well as the other known neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). We found that acute administration of NT-4/5 to 7-day-old cultures stimulates the hydrolysis of phosphatidylinositol, an event involved in neurotrophin signal transduction. Growth of striatal cultures in the presence of NT-4/5 resulted in increased cell survival, as indicated by elevations in cell number, protein content, and a measure of mitochondrial enzyme activity (MTT assay). NT-4/5 increased GABA uptake and staining intensity in these cultures, as indicated by GABA immunocytochemistry, indicating a trophic action on GABAergic neurons, the predominant neuron type in the striatum. To further identify responsive cell populations we analysed for calretinin, a calcium-binding protein known to colocalize with GABA in a number of neuronal cells. In cultures prepared from rats of embryonic day 15, NT-4/5 strongly increased the number of calretinin-positive cells as well as calretinin levels, as determined by Western blot analysis. When the cultures were prepared from embryonic day 18 rats, NT-4/5 very strongly increased the morphological differentiation of calretinin-positive cells, whereas the increase in cell number was less prominent. All effects produced by NT-4/5 were mimicked by BDNF with similar potency. NT-3 was less effective than NT-4/5 and BDNF, and its effects were limited to cultures prepared from embryonic day 15 rats, suggesting a role in the regulation of cell survival at early developmental stages. NGF did not affect any of the measured parameters. Our findings identify NT-4/5 as potent neurotrophic factor for striatal neurons, able to promote their survival and differentiation.

Animals↗
PubMed2007

GABA excitation in mouse hilar neuropeptide Y neurons.

Neuropeptide Y-containing interneurons in the dentate hilar area play an important role in inhibiting the activity of hippocampal circuitry. Hilar cells are often among the first lost in hippocampal epilepsy. As many types of neurons are found in the hilus, we used a new transgenic mouse expressing green fluorescent protein (GFP) in a subset of neurons that colocalized neuropeptide Y (NPY), somatostatin (SST), and GABA for whole-cell, perforated, and cell-attached recording in 240 neurons. As these neurons have not previously been identifiable in live slices, they have not been the focus of physiological analysis. Hilar NPY neurons showed modest spike frequency adaptation, a large 15.6 +/- 1.0 mV afterhyperpolarization, a mean input resistance of 335 +/- 26 M Omega, and were capable of fast-firing. Muscimol-mediated excitatory actions were found in a nominally Ca(2+)-free/high-Mg(2+) bath solution using cell-attached recording. GABA(A) receptor antagonists inhibited half the recorded neurons and blocked burst firing. Gramicidin perforated-patch recording revealed a GABA reversal potential positive to both the resting membrane potential and spike threshold. Together, these data suggest GABA is excitatory to many NPY cells. NPY and SST consistently hyperpolarized and reduced spike frequency in these neurons. No hyperpolarization of NPY on membrane potential was detected in the presence of tetrodotoxin, AP5, CNQX and bicuculline, supporting an indirect effect. Under similar conditions, SST hyperpolarized the cells, suggesting a direct postsynaptic action. Depolarizing actions of GABA and GABA-dependent burst-firing may synchronize a rapid release of GABA, NPY, and SST, leading to pre- and postsynaptic inhibition of excitatory hippocampal circuits.

Animals↗
PubMed2000

The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.

Cell Nucleus↗
PubMed2000

Bovine herpesvirus 1 U(L)3.5 interacts with bovine herpesvirus 1 alpha-transinducing factor.

The bovine herpesvirus 1 (BHV-1) U(L)3.5 gene encodes a 126-amino-acid tegument protein. Homologs of U(L)3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking U(L)3.5 is deficient in viral egress but can be complemented by BHV-1 U(L)3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). The function of BHV-1 U(L)3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 U(L)3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 alpha-transinducing factor (alphaBTIF) as a BHV-1 U(L)3. 5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, U(L)3.5 and alphaBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of U(L)3.5, but not 40 amino acids from the C terminus, abolished the U(L)3.5-alphaBTIF interaction both in vitro and in vivo. The interaction between U(L)3. 5 and alphaBTIF may be important for BHV-1 maturation and regulation of alphaBTIF transactivation activity.

Animals↗
PubMed2004

Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins.

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nsp12 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.

Animals↗
PubMed2005

Identification and localization of TRPC channels in the rat kidney.

It is well established that transient receptor potential (TRP) channels are activated following stimulation of G protein-coupled membrane receptors linked to PLC, but their differential expression in various cells of the renal nephron has not been described. In the present study, immunoprecipitations from rat kidney lysates followed by Western blot analysis using TRPC-specific, affinity-purified antibodies revealed the presence of TRPC1, -C3, and -C6. TRPC4, -C5, and -C7 were nondetectable. TRPC1 immunofluorescence was detected in glomeruli and specific tubular cells of the cortex and outer medulla. TRPC1 colocalized with aquaporin-1, a marker for proximal tubule and thin descending limb, but not with aquaporin-2, a marker for connecting tubule and collecting duct cells. TRPC3 and -C6 immunolabeling was predominantly confined to glomeruli and specific tubular cells of the cortex and both the outer and inner medulla. TRPC3 and -C6 colocalized with aquaporin-2, but not with the Na(+)/Ca(2+) exchanger or peanut lectin. Thus TRPC3 and -C6 proteins are expressed in principle cells of the collecting duct. In polarized cultures of M1 and IMCD-3 collecting duct cells, TRPC3 was localized exclusively to the apical domain, whereas TRPC6 was found in both the basolateral and apical membranes. TRPC3 and TRPC6 were also detected in primary podocyte cultures, whereas TRPC1 was exclusively expressed in mesangial cell cultures. Specific immunopositive labeling for TRPC4, -C5, or -C7 was not observed in kidney sections or cell lines. These results suggest that TRPC1, -C3, and -C6 may play a functional role in PLC-dependent signaling in specific regions of the nephron.

Animals↗
PubMed2003

BetaB1-crystallin: identification of a candidate ciliary body uveitis antigen.

PURPOSE: Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body. METHODS: Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein. RESULTS: Proteomic analysis suggested that beta B1 (betaB1)-crystallin is the primary ciliary body antigen. The presence of betaB1-crystallin in the human ciliary body was confirmed by Western blot with a betaB1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-betaB1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of betaB1-crystallin RNA in the ciliary body tissues. CONCLUSIONS: This study identified betaB1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of betaB1-crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis.

Amino Acid Sequence↗
PubMed2006

Proliferative activity through extracellular signal-regulated kinase of smooth muscle cells in vascular walls of cerebral arteriovenous malformations.

OBJECTIVE: We examined the expression and localization of phosphorylated extracellular signal-regulated kinase (pERK) and proliferation-related antigens in human cerebral arteriovenous malformations (AVMs) to clarify the role of vascular remodeling via this pathway in the development of the lesions. METHODS: Thirteen cerebral AVMs and five control specimens were analyzed using immunohistochemical methods. Specimens were obtained from the patients during the surgical procedures. Control middle cerebral artery samples were obtained during autopsies. RESULTS: We performed immunohistochemical analysis of AVMs by using an antibody specifically recognizing pERK. pERK immunoreactivity was detected in all specimens. Among the control specimens, only weak pERK immunoreactivity was detected, mainly in the intima. pERK immunoreactivity was located in nuclei of cells in the endothelial layer and media. Semiquantitative analysis for pERK immunoreactivity showed that the immunoreactivity score in the media was significantly higher for the AVM than for the control specimens. The results of double staining for pERK and proliferating cell nuclear antigen indicated that these immunoreactivities were colocalized in the same cells. Moreover, those cells in the media were immunoreactive for alpha-actin, indicating that they were smooth muscle cells. CONCLUSION: pERK was detected in smooth muscle cells of the vascular walls of AVMs. It may function in the proliferative activity of smooth muscle cells. Vascular remodeling through pERK may play an important role in the growth and maintenance of cerebral vascular malformations.

Adult↗
PubMed2006

GRIF1 binds Hrs and is a new regulator of endosomal trafficking.

Endosomal sorting of internalized cell surface receptors to the lysosomal pathway plays a crucial role in the control of cell signaling and function. Here we report the identification of GABA(A) receptor interacting factor-1 (GRIF1), a recently discovered protein of unknown function, as a new regulator of endosome-to-lysosome trafficking. Yeast two-hybrid screen and co-immunoprecipitation analysis reveal that GRIF1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), an essential component of the endosomal sorting machinery. We have mapped the binding domains of GRIF1 and Hrs that mediate their association and shown the colocalization of GRIF1 with Hrs on early endosomes. Like Hrs, both overexpression and siRNA-mediated depletion of GRIF1 inhibit the degradation of internalized epidermal growth factor receptors and block the trafficking of the receptors from early endosomes to the lysosomal pathway. Our results indicate, for the first time, a functional role for GRIF1 in the regulation of endosomal trafficking. Interestingly, overexpression of full-length GRIF1, but not the Hrs- or kinesin-interacting GRIF1 deletion mutants, causes a perinuclear clustering of early endosomes. Our findings suggest that GRIF1 may also participate in microtubule-based transport of early endosomes by acting as an adaptor linking Hrs-containing endosomes to kinesin.

Adenosine Triphosphatases↗
PubMed2002

Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes.

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of human adenosine 5'-triphosphate-binding cassette, sub-family A, transporter 2 (ABCA2).

We have isolated the full-length cDNA for human ATP-binding cassette, sub-family A, member 2 transporter (ABCA2). The ORF of this cDNA encodes a protein consisting of 2436 amino acids with apparent molecular weight of M(r) 270,000. Accordingly, ABCA2 is the largest known mammalian ABC transporter described thus far. Analysis of mRNA expression levels indicated that ABCA2 is highest in human brain and has a broad expression pattern in a panel of tumor cell lines. Using specific antibodies to ABCA2 and various organelle marker proteins, ABCA2 was found to colocalize with the lysosomal/endosomal marker LAMP1, forming discrete, punctate intracellular vesicles. In ABCA2-transfected cells, the transporter also colocalized with a fluorescently labeled steroid analogue, estramustine. The sequestration of the steroid into the lysosomal/endosomal compartment indicates a potential substrate specificity for ABCA2. Furthermore, the presence of a lipocalin signature motif in the ABCA2 sequence suggests a possible broad role for this protein in the transport of steroids, lipids, and related molecules.

ATP-Binding Cassette Transporters↗
PubMed2002

[A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines].

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.

Adenovirus E1 Proteins↗
PubMed1997

Human monocytes and neutrophils store transforming growth factor-alpha in a subpopulation of cytoplasmic granules.

Transforming growth factor-alpha (TGF-alpha) exerts several effects on target cells, such as neovascularization promotion and mitogenic signalling. Using immunoelectron microscopy, we show that monocytes and neutrophils, store TGF-alpha in cytoplasmic granules. In monocytes, TGF-alpha did not colocalize with components of peroxidase-positive granules or with albumin of secretory vesicles. Furthermore, no colocalization of TGF-alpha with components of azurophilic or specific granules or secretory vesicles was observed in neutrophils. Activated monocytes and tissue-macrophages contained much less TGF-alpha-positive granules, suggesting TGF-alpha release. Western blot analysis showed a protein of 10 kD in lysates of monocytes. TGF-alpha mRNA was detected in monocytoid cells from the bone marrow by in situ hybridization. This study shows for the first time that monocytes and neutrophils contain TGF-alpha in all stages of maturation and that TGF-alpha in monocytes is stored in a large population of peroxidase-negative granules suggesting a function for these granules. Monocytes and neutrophils are important effector cells in inflammatory reactions. The present finding that these cells contain TGF-alpha might explain complications such as fibrosis and neoplastic transformation, caused by chronic inflammation.

Bone Marrow↗
PubMed1998

Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro.

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, alpha 1 subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiation in vitro neuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by "loss of function" analysis in vitro.

Agrin↗
PubMed2001

SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells.

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins (N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK(1) cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.

Adenosine Triphosphatases↗
PubMed1999

Interactions between neuropeptide Y and gamma-aminobutyric acid in stimulation of feeding: a morphological and pharmacological analysis.

Neuropeptide Y (NPY) produced in neurons in the arcuate nucleus and brain stem and released in the paraventricular nucleus (PVN) and surrounding areas is involved in stimulation of feeding in rats. We recently reported that gamma-aminobutyric acid (GABA) is coexpressed in a subpopulation of NPY neurons in the arcuate nucleus. To determine whether GABA is colocalized in NPY terminals in the PVN, the site of NPY action, light and electron microscopic double staining for NPY and GABA using pre- and postembedding immunolabeling was performed on rat brain sections. GABA was detected in NPY-immunopositive axons and axon terminals within both the parvocellular and magnocellular divisions of the PVN. These morphological findings suggested a NPY-GABA interaction in the hypothalamic control of feeding. Therefore, the effects of muscimol (MUS), a GABA(A) receptor agonist, on NPY-induced food intake were examined in sated rats. When injected intracerebroventricularly, both NPY and MUS elicited dose-dependent feeding responses that were blocked by the administration of 1229U91 (a putative Y1 receptor antagonist) or bicuculline (a GABA(A) receptor antagonist), respectively. Coadministration of NPY and MUS intracerebroventricularly amplified the feeding response over that evoked by NPY or MUS alone. Similarly, microinjection of either NPY or MUS into the PVN stimulated food intake in a dose-related fashion, and coinjection elicited a significantly higher response than that evoked by either individual treatment. These results suggest that GABA and NPY may coact through distinct receptors and second messenger systems in the PVN to augment food intake.

Animals↗
PubMed2000

Calbindin-D28k and calretinin in the rat posterior pituitary; light and electron microscopic localization and upregulation with dehydration.

Ca(2+) binding proteins (CaBPs), calbindin-D(28k) (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca(2+) in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals.

Animals↗
PubMed2005

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere.

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Animals↗