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At least 937 records · Page 52Linked to original sources
PubMed2004

Laminar organization of the mouse dentate gyrus: insights from BETA2/Neuro D mutant mice.

The dentate gyrus of rodents is characterized by a highly laminar organization: above a compact granule cell layer, commissural/associational (C/A) fibers terminate on proximal granule cell dendrites and entorhinal fibers terminate on distal granule cell dendrites in a nonoverlapping manner. To gain insights into mechanisms that underlie the formation of this laminar structure, we studied mice deficient for BETA2/NeuroD, a basic helix-loop-helix transcription factor essential for granule cell differentiation. Anterograde tracing was used to label C/A and entorhinal fibers and combined with confocal double immunofluorescence for calbindin, calretinin, parvalbumin, and reelin to visualize putative target cells. The dentate gyrus of mutant mice contained only few granule cells, which formed a cap-like structure adjacent to area CA3. Despite the severe hypoplasia of the dentate gyrus, the remaining BETA2/NeuroD-deficient granule cells expressed mature markers, extended dendrites into the molecular layer, and extended mossy fibers into area CA3. Entorhinal and C/A fibers terminated in a nonoverlapping manner in the dendritic field overlying the rudiment. Entorhinal fibers terminated in the outermost portion of the dentate gyrus where they surrounded reelin-positive Cajal-Retzius cells, and C/A fibers terminated above and within the dentate rudiment. The laminar termination of C/A fibers was closest to normal in zones of the rudiment in which granule cells were densely packed. These data indicate that granule cells are able to differentiate in the absence of BETA2/NeuroD and suggest that the signals underlying the laminar anatomy of the dentate gyrus are present in the absence of most target cells.

Animals↗
PubMed2005

Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous system.

The peptide transporter PEPT2 mediates transmembrane uptake of small peptides. So far, its expression has not been evidenced in the gastrointestinal tract. We have investigated peptide transport activity in the neuromuscular layers of the gastrointestinal tract by using the fluorescent tracer-dipeptide beta-Ala-Lys-Nepsilon-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Whole-mount preparations from mouse, rat, and guinea pig stomach and small and large intestine were incubated with Ala-Lys-AMCA in the presence or absence of the uptake-inhibitors L-histidine, D-phenylalanyl-L-alanine (D-Phe-Ala), glycyl-L-sarcosine (Gly-Sar), glycyl-L-glutamine (Gly-Gln), benzylpenicillin, and cefadroxil. Fluorescence microscopy revealed that Ala-Lys-AMCA specifically accumulated in both ganglionic layers of the enteric nervous system (ENS) in all regions and species studied. This could be inhibited by Gly-Sar, D-Phe-Ala, Gly-Gln, and cefadroxil, but not by free histidine and benzylpenicillin, indicating uptake via PEPT2. Accordingly, dipeptide uptake was completely abolished in PEPT2-deficient mice. Reverse transcriptase-polymerase chain reaction analysis detected a PEPT2-specific transcript in extracts from the ganglionic ENS layers of mouse small and large intestine, further proving that enteric dipeptide transport activity is specifically mediated via PEPT2. The cellular site of dipeptide uptake was immunohistochemically localized to enteric glial cells and tissue-resident macrophages. In addition, dipeptide uptake occurred in a neurochemically defined subset of neurons in the guinea pig ENS. Our results constitute the first functional evidence for dipeptide transport activity in the ENS. PEPT2-mediated dipeptide transport in enteric glia could contribute to the clearance of neuropeptides in the ENS. In addition, the fluorophore-coupled dipeptide uptake via PEPT2 is a novel vital marker for glial cells in the ENS.

Animals↗
PubMed1991

Characterization of a maize cDNA that complements an enolase-deficient mutant of Escherichia coli.

A cDNA encoding maize enolase (2-phospho-D-glycerate hydrolase) was purified by functional genetic complementation using an enolase deficient mutant of Escherichia coli, DF261. This cDNA, pZM245, was characterized by restriction mapping and DNA sequence analysis. The cDNA contained an open reading frame encoding a protein of 446 amino acids with a high degree of similarity to enolase sequences from other organisms (72% identity to yeast enolase and 82% identity to human enolase). The pZM245 contains a correctly positioned consensus prokaryotic translation initiation sequence. The specific activity of enolase in maize increases to about twice its initial level after 48 hours of anaerobiosis. Northern-blot analysis showed a five-fold anaerobic induction in enolase mRNA, while heat shock or cold shock increased enolase mRNA levels only slightly. Southern-blot analysis of maize genomic DNA indicated that there is one copy of the pZM245 hybridizing sequence per haploid genome in maize.

Amino Acid Sequence↗
PubMed1987

The structure and expression of neuron-specific enolase gene.

Neuron-specific (gamma gamma) enolase (NSE) is an isoenzyme form of glycolytic enzyme, enolase. We isolated genomic clones for NSE and clarified NSE gene structures. The NSE-gene spanned about 9 kb and consisted of twelve exons and eleven introns. Multiple transcriptional start points were identified by a combination of S1 nuclease mapping and primer extension analysis. In the 5'-flanking region we found a TATA-like sequence TCTATAGGC which was only partially homologous to the consensus sequence, but we did not find a CAAT box. The sequence in the immediate 5'-flanking region was of a relatively high G + C content and contained GC-box-like clusters that did not correspond to the typical GC box. In addition, we found seven classes of the repeated sequences. In the introns 1, 5 and 10 there were tandem repeats (GT)33, (GT)21 and (GT)24, respectively. The 3' end contains a single polyadenylation site and an identifier sequence 2 kb downstream from the poly(A)-addition site. The in vitro cell-free transcription of the truncated genomic DNA fragment using HeLa cell extract showed that the transcription start points have been correctly identified and the putative promoter sequences appear to be functional.

Amino Acid Sequence↗
PubMed2004

Dexamethasone inhibits proliferation of adult hippocampal neurogenesis in vivo and in vitro.

Activation of glucocorticoid receptor (GR) induces a reduction of adult hippocampal neurogenesis found in dentate gyrus (DG). However, the nature of specific effects by glucocorticoid in hippocampal neurogenesis is not known. In this report, we show differential effects of dexamethasone (DEX), a glucocorticoid receptor agonist, on proliferation and functional differentiation of adult hippocampal progenitor cells in DG. Two-month-old adult rats received daily injections of DEX for 9 days and were sacrificed 12 h and 28 days after the ninth injection. Proliferation assays showed that DEX inhibited proliferation of neural progenitor cells and the inhibitory effects of DEX was not detected 28 days after recovery. Functional differentiation studies using B-cell lymphoma protein-2 (Bcl-2), brain-derived neurotrophic factor (BDNF), p-ERK, and neuronal nuclear protein (NeuN) antibodies revealed that the expressions of Bcl-2 and BDNF were not significantly different between control and DEX-treated rats. In contrast, however, the activation of extracellular signal-regulated kinase (ERK) was downregulated 12 h, but not 28 days, after the DEX treatment. When adult hippocampal progenitor cell cultures were treated with subchronic DEX, proliferation of the progenitor cells was suppressed. Taken these in vitro and in vivo results together, it is concluded that glucocorticoid receptor activation blocks only proliferation, but not differentiation, in hippocampal neurogenesis.

Animals↗
PubMed2005

Seizures induce proliferation and dispersion of doublecortin-positive hippocampal progenitor cells.

One neuropathological hallmark of temporal lobe epilepsy is granule cell dispersion, a widening of the hippocampal granule cell layer (GCL) with abnormally positioned excitatory neurons. The finding that seizure activity also induces adult hippocampal neurogenesis was taken largely as indicative of a regenerative attempt, not as part of the pathology. The aim of our study was to characterize a potential relationship between granule cell dispersion and seizure-induced neurogenesis. Kainic acid (KA)-induced seizures in mice led to increased cell proliferation and new neurons persisted for months after the seizures. We show that the proliferative stimulus did not affect nestin-expressing early precursor cells that primarily respond to physiologic mitogenic stimuli, but stimulated the division of late type-3 progenitor cells, which express doublecortin (DCX), a protein associated with cell migration. This delayed proliferation presumably interfered with migration, leading to a significant dispersion of DCX-positive progenitors and early postmitotic neurons within the dentate gyrus granule cell layer. We propose that initial seizures induce ectopic precursor cell proliferation resulting in the dispersion of immature neurons within the adult granule cell layer. Thus, seizure-generated neurons might contribute to the disease process of epilepsy.

Analysis of Variance↗
PubMed2006

Recognition of enolase in the Escherichia coli RNA degradosome.

In Escherichia coli, the glycolytic enzyme enolase is a component of the RNA degradosome, which is an RNase E mediated assembly involved in RNA processing and transcript turnover. The recruitment of enolase by the RNA degradosome has been implicated in the turnover of certain transcripts, and it is mediated by a small segment of roughly a dozen residues that lie within a natively unstructured sub-domain of RNase E. Here, we present the crystal structure of enolase in complex with its recognition site from RNase E at 1.6A resolution. A single molecule of the RNase E peptide binds asymmetrically in a conserved cleft at the interface of the enolase dimer. The recognition site is well conserved in RNase E homologues in a subfamily of the gamma-proteobacteria, including enzymes from pathogens such as Yersinia pestis, Vibrio cholera and Salmonella sp. We suggest that enolase is recruited into putative RNA degradosome machinery in these bacilli, where it plays common regulatory functions.

Amino Acid Sequence↗
PubMed2006

Nestin expression in Müller glial cells in postnatal rat retina and its upregulation following optic nerve transection.

This study examined the nestin immunoexpression and its specific cellular localization in the developing retina of rats and investigated its putative changes in an altered environment. At postnatal day 0, nestin immunoexpression was detected in radially oriented cells considered to be neural progenitors that were glutamine synthetase (GS) negative. With age, it was localized in differentiating and differentiated GS positive Müller glial cells. Nestin expression was down-regulated as maturation proceeded, so that by 12 weeks, it was almost completely diminished as confirmed also by real time-polymerase chain reaction analysis. Nestin expression along with that of glial fibrillary acidic protein (GFAP) was induced and upregulated in mature Müller glial cells following optic nerve transection. It is suggested that both nestin and GFAP may be useful biomarkers in retinal injuries. In view of their cytoskeletal nature, the marked expression of nestin and GFAP may provide a structural support for the framework of retina which would be disrupted as a result of loss of neurons in optic nerve lesion. It may also be neuronal protective taking into consideration the close spatial and functional links between Müller glial cells and the axotomized ganglion cells.

Age Factors↗
PubMed2003

Losses of NG2 and NeuN immunoreactivity but not astrocytic markers during early reperfusion following severe focal cerebral ischemia.

The ability of glia to recover essential functions following a period of focal cerebral ischemia is likely to be one important factor influencing the severity of tissue damage that subsequently develops. In this study, we have compared changes in immunoreactivity of markers specific for astrocytes, NG2-positive glia and neurons in tissue subregions during early reperfusion following 3 h of middle cerebral artery occlusion to provide insights into possible differential susceptibility of these cell populations. Under the conditions used, infarction ultimately encompasses most of the perfusion territory of the occluded artery. Nonetheless, alterations in immunoreactivity during the first 3 h of recirculation were restricted to brain regions that had been subjected to severe ischemia. In the striatum, cellular immunoreactivity for NG2 and neuronal markers, NeuN and microtubule-associated protein 2, was greatly reduced by 1 h of reperfusion and declined further at 3 h. NG2 labeling of blood vessels in the striatum appeared post-ischemically, mimicking expression of this protein during development. Less severe changes were seen in the neuronal markers in overlying cerebral cortex. In contrast to the losses of other cellular proteins, immunoreactivity for the astrocytic marker, glial fibrillary acidic protein, was preserved in all tissue that had been subjected to severe ischemia and labeling of another astrocytic protein, glutamine synthetase, was increased by 3 h of reperfusion. These findings provide the first evidence of marked sensitivity of NG2-immunoreactivity to severe ischemia and suggest a greater initial resistance of astrocytes compared with neurons and NG2-positive glia to ischemia-reperfusion damage.

Animals↗
PubMed1998

Identification and characterization of autoantibodies against catalase and alpha-enolase in patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology. Recent studies have shown that genetic factors and both cellular and humoral immunological abnormalities are important in the pathogenesis of PSC. The most prominent autoantibodies in PSC are anti-neutrophil cytoplasmic antibodies (ANCA). The autoepitopes of ANCA in PSC are not well defined. The aim of this study was to identify corresponding ANCA autoantigens in patients with PSC. A biochemical approach with enrichment and partial purification of soluble neutrophil proteins, detection of autoantibodies by Western blot and partial amino acid sequencing were used. Two new autoantigen/autoantibody systems in patients with PSC were detected: catalase and alpha-enolase. The presence of catalase autoantibodies in 9/15 (60%) and alpha-enolase autoantibodies in 4/15 (27%) was confirmed by ELISA and Western blot. Furthermore, we showed immunoreactions of PSC sera with human biliary epithelial cells, showed the reduction of fluorescence in anti-catalase absorption experiments and observed partial co-localization of anti-catalase antibodies and PSC sera in double-staining experiments on biliary epithelial cells. The anti-catalase antibody-positive PSC patients had a more severe course of disease with a significantly higher alkaline phosphatase compared with the anti-catalase-negative PSC patients (P < 0.06). All ulcerative colitis control sera were anti-catalase antibody-negative. The identified antigens catalase and alpha-enolase can partly explain the ANCA fluorescence on ethanol-fixed and formaldehyde-fixed granulocytes in patients with PSC. Catalase is an important anti-oxidant enzyme and prevents cell damage from highly reactive oxygen-derived free radicals. Catalase autoantibodies might play a pathogenic role in patients with PSC. Our findings support the hypothesis that oxidative stress is one of the pathogenic mechanisms in patients with PSC.

Amino Acid Sequence↗
PubMed1999

[Immunohistochemical characterisation of surgically excised choroidal neovascularisation in age-related macular degeneration].

AIM: The objective of the study was to gather further information about the pathogenesis of choroidal neovascularisations (CNV), which is still not clearly understood, and to establish criteria for making decisions on a appropriate therapy. Immunohistochemical characteristation should allow a more comprehensive evaluation of cellular components of the membranes and their functional role. PATIENTS AND METHODS: In 29 patients (16 women, 13 men) with age-related macular degeneration ranging in age from 46 to 91 years (mean age, 76.4 years), CNV were excised by pars-plana vitrectomy. Sections were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) and examined by light microscopy. For the immunohistochemical characterisation of the surgical specimens the following anti-genetic determinants were used: glial fibrillary acid protein (GFAP) for glial cells, synaptophysin for neuronal cells, neuron-specific enolase (NSE) for neuronal and neuroectodermal cells, CD 31 for endothelial cells and pancytokeratin (KL1) for cells of the retinal pigment epithelium (RPE). Cells undergoing apoptosis were labeled with the TUNEL technique. RESULTS: 22 (76%) surgical specimens showed TUNEL positive cells in the connective tissue, vascular endothelium and retinal pigment epithelium. Positive immunostaining of neuronal antigenetic determinants was found for glial fibrillary acid protein in 22 patients (76%), for synaptophysin in 28 patients (97%) and for neuron-specific enolase in 21 patients (72%) CNV. The epithelial marker KL1 was positive in 28 patients (97%) and the endothelial marker CD 31 in 20 patients (69%). CONCLUSION: The immunohistochemical analyses of CNV showed that in the majority of cases during the excision of choroidal neovascularizations in addition to scar tissue and connective tissue also parts of the native retinal pigment epithelium and of the neurosensory retina are removed which is only partly visible with standard staining techniques. These findings suggest that the mostly not satisfying postoperative results are partly due to the damage of neuronal cells and a partial loss of the retinal pigment epithelium. Apoptosis as a regulating mechanism in choroidal neovascularization. The variable appearence of apoptosis suggests that it is possibly related to the degree of activity of CNV.

Aged↗
PubMed2005

The effect of enzyme treatment on the in vitro fermentation of lucerne incubated with equine faecal inocula.

A series of experiments was conducted to determine the effects of a fibrolytic enzyme preparation (enzyme 1; E1) on the in vitro fermentation of lucerne incubated with equine faecal inocula. In experiment 1, high-temperature-dried (HT) lucerne was treated with five levels of E1 (0 to 2.4 ml/g DM) and incubated at 50 degrees C for 20 h. Samples then received a simulated foregut digestion (SFD) treatment before DM and NSP analysis. In experiment 2, HT lucerne was treated with the same enzyme levels used in experiment 1. Samples were then split into two groups; plus or minus an SFD treatment before in vitro fermentation using an equine faecal inoculum. In experiment 3, fresh and wilted lucerne were treated with the same levels of E1 as experiments 1 and 2, incubated at 50 degrees C for 20 h, then fermented in vitro. For experiment 4, fresh and wilted lucerne were treated with low levels (0 to 0.008 ml/g DM) of E1 before fermentation. E1 significantly (P<0.05) enhanced DM and NSP losses from HT lucerne following SFD treatment compared with the control. High levels of E1 significantly (P<0.05) enhanced the rate, but not extent, of fermentation of HT, wilted and fresh lucerne; however, low levels of E1 were ineffective. At higher application levels, E1 appears to have considerable potential to enhance the nutritive value of lucerne for horses. Information on the fermentation kinetics of the substrates was valuable; all end-point measurements showed no effect of enzyme treatment.

Animal Feed↗
PubMed2006

Distribution of AAV2-hAADC-transduced cells after 3 years in Parkinsonian monkeys.

The present report describes for the first time, the stability of recombinant adeno-associated virus serotype 2 (AAV2) human aromatic L-amino acid decarboxylase (hAADC) gene transfer after 3-year survival time in a non-human primate model of Parkinson's disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys were treated with six injections of 30 microl/site of AAV2-hAADC at a concentration of 2 x 10(12) vg/ml into the caudate and putamen. Stereological analysis revealed a 46.6% increase in the total number of AAV2-hAADC-transduced cells in the striatum between 8 weeks and 3 years after gene transfer survival time. In the 8-week animals, the distribution of the AADC+ cells was dispersed and heterogeneous, whereas in the 3-year animals it was widespread and homogenous. Confocal analysis demonstrated that approximately 85% of the AADC+ cells were neuronal nuclei immunoreactive.

Animals↗
PubMed1999

Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

Adenoviridae↗
PubMed2000

Cell surface-associated enolase in Actinobacillus actinomycetemcomitans.

Cell surface-associated materials of Actinobacillus actinomycetemcomitans were extracted by a short incubation of the cell suspension in a Tris-buffered saline in the presence and absence of a restriction enzyme, EcoRI. The supernatants (which we termed EcoRI extract and surface extract, respectively) contained a number of extracellularly released proteins. Of these proteins, four major proteins were identified by N-terminal sequencing to be the 34 and 39 kDa outer membrane proteins, the GroEL-like protein, and a 47 kDa protein homologous to Haemophilus influenzae enolase. Enolase activity was found in the extracts and its relative amount of activity in the EcoRI extract from a culture of the mid-exponential growth phase was estimated as 5.7% of total enzyme activity. In contrast, the relative amount of activity of another cytosolic enzyme, lactate dehydrogenase, was extremely low in the extracts and also in the culture supernatant. These results suggest the external localization of enolase in this bacterium.

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis.

The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, phosphoglycerate mutase (PGM), and enolase, respectively, have been cloned and sequenced. These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for phosphoglycerate kinase) followed by tpi, pgm, and eno. The triose phosphate isomerase and enolase from B. subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic. However, B. subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diphosphoglycerate (DPG) for activity. Instead, B. subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B. subtilis PGM for Mn2+. The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme. Inactivation of pgm plus eno in B. subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate. However, these mutants were asporogenous with or without malate supplementation.

Amino Acid Sequence↗
PubMed2006

Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system.

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered carboxy-peptidase responsible for the formation of vasodilatory peptides such as angiotensin-(1-7). We hypothesized that ACE2 is part of the brain renin-angiotensin system, and its expression is regulated by the other elements of this system. ACE2 immunostaining was performed in transgenic mouse brain sections from neuron-specific enolase-AT(1A) (overexpressing AT(1A) receptors), R(+)A(+) (overexpressing angiotensinogen and renin), and control (nontransgenic littermates) mice. Results show that ACE2 staining is widely distributed throughout the brain. Using cell-type-specific antibodies, we observed that ACE2 staining is present in the cytoplasm of neuronal cell bodies but not in glial cells. In the subfornical organ, an area lacking the blood-brain barrier and sensitive to blood-borne angiotensin II, ACE2 was significantly increased in transgenic mice. Interestingly, ACE2 mRNA and protein expression were inversely correlated in the nucleus of tractus solitarius/dorsal motor nucleus of the vagus and the ventrolateral medulla, when comparing transgenic to nontransgenic mice. These results suggest that ACE2 is localized to the cytoplasm of neuronal cells in the brain and that ACE2 levels appear highly regulated by other components of the renin-angiotensin system, confirming its involvement in this system. Moreover, ACE2 expression in brain structures involved in the control of cardiovascular function suggests that the carboxypeptidase may have a role in the central regulation of blood pressure and diseases involving the autonomic nervous system, such as hypertension.

Angiotensin-Converting Enzyme 2↗