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PubMed2006

Effects of electromagnetic stimulation on calcified matrix production by SAOS-2 cells over a polyurethane porous scaffold.

There is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic crosslinked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells using a porous scaffold. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effect of the electromagnetic stimulation on SAOS-2 human osteoblast proliferation and calcified matrix production. Cell proliferation was twice as high; expression of decorin, osteocalcin, osteopontin, type I collagen, and type III collagen was greater (1.3, 12.2, 12.1, 10.0, and 10.5 times as great, respectively); and calcium deposition was 5 times as great as under static conditions without electromagnetic stimulation. RT-PCR analysis revealed the electromagnetically upregulated transcription specific for decorin, fibronectin, osteocalcin, osteopontin, transforming growth factor-beta, type I collagen, and type III collagen. The immunolocalization of the extracellular matrix constituents showed their colocalization in the cell-rich areas. The bioreactor and the polyurethane foam were designed to obtain cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.

Bioreactors↗

PubMed1989

Monoclonal antibody Tor 23 binds a subset of neural cells in the human cortex and displays an altered binding distribution in Alzheimer's disease.

Recent evidence suggests that alterations in molecules of the external neuronal surface may be pivotal factors in Alzheimer's disease (AD) either as primary or secondary lesions. We are studying neuronal surface components with a library of monoclonal antibodies (MAbs) made to highly purified, exclusively cholinergic nerve terminals of the Torpedo ray. The most extensively characterized of the Tor MAbs. Tor 23, binds the external membrane of some human neuronal cells in culture. Our present findings demonstrate that Tor 23, in situ, binds the apparent limiting membrane of rare neurons of the human isocortex. Tor 23 binds, in addition, internally within a subpopulation of subcortical white matter astrocytes, as identified by colocalization with glial fibrillary acidic protein. Neuronal surface binding of Tor 23 parallels our findings in other species: astrocyte staining was not observed in other species and may be unique to human. Immunoblot analysis of white matter reveals one polypeptide band with a relative mobility of 115,000 +/- 15,000 daltons. In the mid-frontal cortex from cases of AD. Tor 23 immunopositive neurons are greatly reduced in number and immunopositive astrocytes are completely absent. The reduction of the neuronal surface epitope defined by Tor 23 supports the recent hypothesis that surface molecules are altered in AD. The absence of Tor 23 positive astrocytes opens an area for specific investigation: namely, the role subcortical astrocytes may play in the pathogenesis of AD.

Aged↗

PubMed2001

Intracerebral hemorrhage-induced neuronal death.

OBJECTIVE: The mechanisms underlying neural injury in intracerebral hemorrhage (ICH) remain uncertain. The present two-part study investigated cell death in the region of ICH and its association with caspase-3 activation. METHODS: ICH was produced in adult rats by injection of 100 microl of autologous blood or saline into the right basal ganglia. The animals' brains were removed at 6 hours or at 1, 3, 7, or 14 days after hemorrhage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin in situ nick end-labeling (TUNEL) was used to detect deoxyribonucleic acid (DNA) fragmentation. TUNEL-positive cells were quantified. Caspase-3 activation was measured by Western blotting and immunohistochemistry. Double labeling was used to compare TUNEL with caspase-3 distribution and to identify the cell types affected. TUNEL-positive cells were also quantified at 6 hours, 1 day, and 3 days after injection of 5 U of thrombin into the right basal ganglion. RESULTS: At 6 hours, TUNEL-positive cells appeared in the ICH model (but not in the saline control brains) and were present for more than 2 weeks after ICH, peaking at 3 days. Western blot analysis revealed that the increase in immunoreactivity for the activated caspase-3 precedes that of DNA fragmentation, peaking at 1 day after ICH and declining thereafter. Immunohistochemistry analysis showed nucleus translocation of caspase-3 after ICH. Double-labeling studies demonstrated that both neurons and astrocytes surrounding the clot were TUNEL-positive. In addition, TUNEL and caspase-3 were colocalized in the same cells. Intracerebral thrombin injection elicited DNA fragmentation similar to that observed after the injection of blood. CONCLUSION: Double-strand breaks in genomic DNA and induction of caspase-3 were demonstrated adjacent to parenchymal hematoma in the animals' brains. These results provide evidence that cell loss after ICH is associated with activation of caspase-3.

Animals↗

PubMed2003

Expression of survivin after acute necrohemorrhagic pancreatitis in rats.

INTRODUCTION: Survivin is one of the inhibitors of the apoptosis family and has dual effects: antiapoptotic effect and regulation of the cell cycle. AIM: To show involvement of survivin in acute pancreatitis. METHODOLOGY: Acute necrohemorrhagic pancreatitis was induced in male Wistar rats by intraductal infusion of 4% sodium taurocholate. RESULTS: By northern blotting, the survivin mRNA level was significantly increased at 36 hours and peaked at 48 hours after induction of acute pancreatitis. Survivin protein was found in cytoplasm of ductal cells by immunohistochemical analysis at 48-72 hours. It was also observed in nuclei of both acinar and ductal cells as well as infiltrating cells. Apoptotic cells were observed in pancreatic acinar cells. Survivin protein partially colocalized with 5-bromo-2'-deoxyuridine in some nuclei of ductal cells. CONCLUSIONS: We showed involvement of survivin in acute pancreatitis in rats. Survivin may have some roles in the regulation of pancreatic regeneration and proliferation as well as an antiapoptotic effect after acute pancreatitis.

Acute Disease↗

PubMed2003

Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates.

Staphylococcus aureus has been shown to invade and induce the death of various cell types. Here we investigate whether the cytotoxicity of intracellular S. aureus is a general feature or rather characteristic of individual S. aureus strains. The majority of 23 randomly collected clinical S. aureus isolates were killed inside keratinocytes and fibroblasts, indicating that the uptake of S. aureus represents an important mechanism of cell-autonomous host defense. However, seven independent S. aureus isolates survived intracellularly and induced significant cytotoxicity for their host cells. Subcloning analysis revealed that the ability or inability to kill host cells is a stable, apparently genetically determined trait of a given S. aureus isolate. We show that noncytotoxic strains but not cytotoxic strains colocalize with the lysosomal marker LAMP-1, suggesting that only cytotoxic strains escape degradation by the endolysosomal pathway. In a mouse septicemic model, cytotoxic S. aureus isolates produce significantly greater lethality (96%) compared to noncytotoxic strains (41%), which corresponds to 23-, 63-, and 30,000-fold increases of bacterial loads in the liver, spleen, and kidney, respectively. Finally, cytotoxic S. aureus strains produce clinically apparent arthritis in mice at a greater frequency than compared to noncytotoxic S. aureus strains. The results of our study unravel a previously unrecognized dichotomy of cytotoxic and noncytotoxic S. aureus isolates, which may play an important role in the dissemination of, and mortality induced by, S. aureus infection.

Animals↗

PubMed2005

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly.

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.

Amino Acid Sequence↗

PubMed2005

Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: interactions with BFRF1 and with the nuclear lamina.

We have reported in the accompanying paper that the BFRF1 protein of Epstein-Barr virus (EBV) is important for efficient primary viral envelopment and egress (A. Farina, R. Feederle, S. Raffa, R. Gonnella, R. Santarelli, L. Frati, A. Angeloni, M. R. Torrisi, A. Faggioni, and H.-J. Delecluse, J. Virol. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur during the course of herpesviral replication, and (ii) while BFLF2 alone did not cause any apparent alteration, coexpression of the two proteins dramatically induced profound convolutions of the duplicated nuclear membrane. Both biochemical and morphological analysis showed association of the BFRF1-BFLF2 complex with a component of the nuclear lamina, lamin B. Taken together, these results and those of the accompanying paper (Farina et al., J. Virol. 79:3703-3712) indicate an important role of BFRF1 and BFLF2 in the early steps of EBV maturation at the nuclear membrane.

Blotting, Western↗

PubMed1991

Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

Animals↗

PubMed2004

TRAF family proteins link PKR with NF-kappa B activation.

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.

3T3 Cells↗

PubMed2005

Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction.

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.

Cell Nucleolus↗

PubMed2004

PDZ binding motif-dependent localization of K+ channel on the basolateral side in distal tubules.

Kir5.1, a nonfunctional inwardly rectifying K(+) channel by itself, can form functional channels by assembling with other proteins. We previously showed that Kir5.1 assembled with Kir4.1 and functioned as an acid-base regulator in the kidney. In this study, we examined the intrarenal distribution of Kir5.1 by RT-PCR analysis on dissected nephron segments and immunohistochemical analysis with the specific anti-Kir5.1 antibody. Strong expression of Kir5.1 was detected in distal convoluted tubules, and weak expression was also detected in thick ascending limb of Henle's loop. Colocalization of Kir5.1 with Kir4.1 indicated expression of Kir5.1/Kir4.1 heteromer in these nephron segments. In a renal epithelial cell line, Madin-Darby canine kidney cells, heteromer formation with Kir4.1 changed the localization of Kir5.1 from intracellular components to the cell surface. The COOH-terminal cytoplasmic portion that includes the PDZ binding motif of Kir4.1 was responsible for this intracellular localization. These data suggest the signals on the COOH terminus of Kir4.1, including PDZ binding motif, determine the intracellular localization of Kir5.1/Kir4.1 heteromer in distal tubules.

Animals↗

PubMed1996

Colocalization on the same synaptic vesicles of syntaxin and SNAP-25 with synaptic vesicle proteins: a re-evaluation of functional models required?

Synaptic vesicle docking and calcium dependent exocytosis are thought to require the specific interaction of proteins of the synaptic vesicle membrane (such as VAMP/synaptobrevin and synaptotagmin) and their plasma membrane-located counterparts (such as syntaxin and SNAP-25). When isolating synaptic vesicles by glycerol velocity gradient centrifugation we found cosedimentation of the presumptive presynaptic plasma membrane proteins syntaxin and SNAP-25 with synaptic vesicle membrane proteins. In order to further identify the antibody binding organelles we performed an immunoelectron microscopical analysis of synaptosomal profiles. Syntaxin and SNAP-25 were not only associated with the plasma membrane but to a large extent also with synaptic vesicle profiles. In order to answer the question whether the syntaxin and SNAP-25 containing vesicular compartment would also carry classical synaptic vesicle membrane markers we performed double labeling experiments using poly- and monoclonal antibodies. We found colocalization on the same vesicle not only of SNAP-25 and syntaxin but also of SNAP-25 with the synaptic vesicle membrane proteins SV2 and synaptotagmin and of syntaxin with the vesicular membrane protein synaptophysin. Our results demonstrate that syntaxin and SNAP-25 are colocalized with classical vesicle membrane proteins on the same vesicle and suggest that the functional models for the interaction of presynaptic proteins need to be re-evaluated.

Animals↗

PubMed2026

Programmed cell death and risk of diabetic retinopathy: a Mendelian randomization study.

BACKGROUND: Programmed cell death (PCD) plays an important role in diabetic retinopathy (DR); however, the underlying genetic mechanisms remain unclear. We used Mendelian randomization (MR) to investigate the causal relationships between PCD-related genes and DR. This study aimed to investigate the effects of PCD on the risk of DR by conducting MR analysis. METHODS: Summary statistics from gene expression quantitative trait loci (eQTL) studies (31,684 Europeans) were analyzed. Genetic instrumental variables were selected using cis-eQTL single-nucleotide polymorphisms (SNPs; P < 5 × 10- 8). Summary data-based MR (SMR) was employed to assess causal associations between PCD-related genes and DR, with three additional MR methods used for sensitivity testing. Bayesian colocalization was used to examine the shared regulatory mechanisms between PCD QTLs and DR risk loci. RESULTS: Sensitivity and colocalization analyses revealed six genes that affected DR: cathepsin H (CTSH), NAD(P)H: quinone oxidoreductase 1 (NQO1), tribbles pseudokinase 3 (TRIB3), and phosphoglycerate mutase 5 (PGAM5), which increased DR risk, and iron-responsive element binding protein 2 (IREB2) and tumor necrosis factor (TNF), which exhibited protective effects. Multivariate MR confirmed significant causal effects for CTSH, IREB2, and PGAM5 (p < 0.050). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (including 10 STRING-derived genes) revealed that 13 genes were enriched in necroptosis, apoptosis, mitophagy, and TNF signaling pathways in DR. CONCLUSIONS: This MR study supports the causal involvement of PCD in DR and identifies candidate genes (CTSH, IREB2, PGAM5, NQO1, TRIB3, and TNF) for therapeutic targeting or biomarker development in DR prevention or diagnosis.

Humans↗

PubMed1998

Immunocytochemical evidence for a diurnal rhythm of neurons showing colocalization of VIP with GRP in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), which functions as a biological clock, contains several neuropeptides such as vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and gastrin-releasing peptide (GRP). Studies from several laboratories have provided evidence for the coexistence of VIP with PHI and GRP, but reliable data about the proportions of colocalization and a possible diurnal rhythmicity are lacking. In the present study, we therefore aimed at studying these aspects. To this end, rats were killed by perfusion fixation during the middle of the day (Zeitgeber time [ZT] 7) and during the second part of the night (ZT 19). Coronal Vibratome sections through the SCN were double-immunolabeled for the presence of VIP and PHI or for VIP and GRP. Analysis of the sections was done by semi-quantitative confocal laser scanning fluorescence microscopy. It turned out that, in keeping with previous literature data, VIP and PHI always coexist at the cellular level. This was seen in all possible ratios, both during the day and at night. Part of these VIP/PHI-containing neurons (21%) and part of the GRP-containing neurons (33%) showed colocalization during the middle of the day. During the second part of the night, these percentages increased significantly to 28% and 40%, respectively. This increase in percentages was due to a significant, nocturnal increase of the number of profiles showing colocalization, in contrast to the number of profiles exclusively immunoreactive for VIP or GRP.

Animals↗

PubMed2004

Retinal colocalization and in vitro interaction of the glutamate transporter EAAT3 and the serum- and glucocorticoid-inducible kinase SGK1 [correction].

PURPOSE: The serum- and glucocorticoid-inducible kinase SGK1 regulates several epithelial channels and transporters, the related protein kinase B (PKB) regulates glucose transport. SGK1 is expressed in the brain and could thus regulate glial and/or neuronal transport processes. The present study explores whether SGK1 is expressed in the retina and whether it regulates EAAT3, a Na(+)-coupled glutamate transporter. EAAT3 is expressed in retinal ganglion cells and accomplishes the clearance of glutamate from synaptic clefts. METHODS: Immunohistochemistry was performed to test for retinal SGK1 expression. For functional analysis, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without additional injection of wild-type SGK1, constitutively active (S422D)SGK1, inactive (K127N)SGK1, and/or constitutively active (T308D,S473D)PKB. Glutamate induced current (I(GLU)) was taken as a measure for transport. RESULTS: SGK1 is indeed expressed in several retinal cells including retinal ganglion cells where it is colocalized with EAAT3. In EAAT3-expressing Xenopus oocytes, glutamate-induced current was stimulated by coexpression of wild-type SGK1, constitutively active (S422D)SGK1, and constitutively active (T308D,S473D)PKB, but not by inactive (K127N)SGK1. CONCLUSIONS: SGK1 and EAAT3 are coexpressed in retinal neurons, and SGK1 serves to stimulate EAAT3. This function is shared by protein kinase B (PKB). The experiments reveal a novel mechanism regulating EAAT3, which may be essential for the function of the retinal ganglion cells.

Amino Acid Transport System X-AG↗

PubMed2004

Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1alpha,25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway.

Macrophage cell membranes were labeled with PKH26 and subsequently incubated with latex beads to generate phagosomes surrounded by a red-fluorescent membrane suitable for flow cytometry. Following cell disruption and partial purification of phagosomes, these vesicles were readily distinguished from both cell debris and free beads released from disrupted vacuoles. Flow cytometry analysis of phagosomes stained with specific mAbs and FITC-labeled secondary antibodies showed progressive acquisition of both Rab7 and LAMP-1 consistent with movement along the endocytic pathway. Alternatively, macrophages were preloaded with the lysosomal tracer FITC-dextran before membrane labeling with PKH and incubation with latex beads. Phagosome-lysosome fusion was then quantified on the basis of the colocalization of red and green signals. Using these flow cytometry-based systems, we showed that co-internalization of beads with lysates of Mycobacterium tuberculosis, but not lysates from the nonpathogenic organism Mycobacterium smegmatis, markedly decreased phagosome acquisition of Rab7 and LAMP-1 and vesicle fusion with FITC-dextran-loaded lysosomes. Inhibition of phagolysosome fusion could be attributed, at least in part, to the mycobacterial cell wall glycolipid lipoarabinomannan, and further analysis showed complete rescue of phagosome maturation when cells were pretreated with vitamin D3 before exposure to lipoarabinomannan. Moreover, the ability of vitamin D3 to reverse the phenotype of phagosomes in the presence of the glycolipid was completely abrogated by LY-294002, suggesting that vitamin D3 promotes phagolysosome fusion via a phosphoinositide 3-kinase signaling pathway. These findings establish a robust platform technology based on labeling of phagocyte cell membranes and flow cytometry capable of supporting broad-based screens to identify microbial and other bioactive compounds that influence phagosome biology.

Antigens, CD↗

PubMed1987

Conversion of neuropeptide K to neurokinin A and vesicular colocalization of neurokinin A and substance P in neurons of the guinea pig small intestine.

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.

Animals↗

PubMed2006

Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters.

The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B(12) riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B(12) biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.

ATP-Binding Cassette Transporters↗