Biomedical subjects

J R Greene

Publications and source records attributed to J R Greene.

At least 19 recordsLinked to original sources

PubMed2001

Analyses of p53 target genes in the human genome by bioinformatic and microarray approaches.

The completion of the human genome sequence (International Human Genome Sequence Consortium (2001) Nature 409, 860-921; Venter, J. C., et al. (2001) Science 291, 1304-1351) allows for new ways to analyze global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank(TM) annotation and a computationally derived transcript map. 4,852 genes analyzed in this way contained at least one p53 consensus binding sequence. Of 13 genes randomly selected for mRNA analysis, 11 were shown to respond to p53 expression. Five promoters were analyzed by chromatin immunoprecipitation, which revealed that all were bound by p53 in vivo. We then analyzed 33,615 unique human genes on cDNA microarrays, identifying 1,501 genes that respond to p53 expression. A parameter was derived that demonstrates that in silico prediction greatly enriches for genes that are activated and repressed by p53 and assists us to suggest other signaling pathways that may be connected to p53. The methods shown here illustrate a novel approach to analysis of global gene regulatory network through the integration of human genomic sequence information and genome-wide gene expression analysis.

Computational Biology↗

PubMed2001

Structural and functional abnormalities of the hippocampal formation in rats with environmentally induced reductions in prepulse inhibition of acoustic startle.

The effects of social isolation on prepulse inhibition of acoustic startle (PPI), electrophysiology and morphology of subicular pyramidal neurons and the densities of interneuronal sub-types in the hippocampal formation were examined. Wistar rats (male weanlings) were housed socially (socials, n=8) or individually (isolates, n=7). When tested eight weeks later, PPI was lower in isolates. Rats then received terminal anaesthesia before slices of hippocampal formation were made in which the electrophysiological properties of a total of 108 subicular neurons were characterized. There were no differences in neuronal sub-types recorded in socials compared with isolates. Intrinsically burst-firing and regular spiking pyramidal neurons were examined in detail. There were no differences in resting membrane potential or input resistance in isolates compared with socials but action potential height was reduced and action potential threshold raised in isolates. A limited morphological examination of Neurobiotin-filled intrinsically burst-firing neurons did not reveal differences in cell-body area or in number of primary dendrites. Sections from the contralateral hemispheres of the same rats were stained with antibodies to calretinin, parvalbumin and the neuronal isoform of nitric oxide synthase (nNOS). In isolates, the density of calretinin positive neurons was increased in the dentate gyrus but unchanged in areas CA3, CA1 and subiculum. Parvalbumin and nNOS positive neuronal densities were unchanged. Hence in rats with environmentally induced reductions in PPI there are structural and functional abnormalities in the hippocampal formation. If the reduction in PPI stems from these abnormalities, and reduced PPI in rats is relevant to schizophrenia, then drugs that correct the reported electrophysiological changes might have antipsychotic effects.

Acoustic Stimulation↗

PubMed2001

Accumulation of calbindin in cortical pyramidal cells with ageing; a putative protective mechanism which fails in Alzheimer's disease.

There is considerable interest in the status of calbindin immunoreactive neurones in Alzheimer's disease (AD) but previous studies have produced widely differing results. Here we describe calbindin neurones in temporal neocortex from 18 severely demented patients with neuropathologically confirmed AD and 13 age and post-mortem delay matched, neurologically normal controls. Calbindin immunoreactive neurones were small and round in layers II-IV, and pyramidal in layers IIIc and V. There were significantly more calbindin positive neurones in controls than in AD (mean+/- SD, for each comparison P < 0.01): superior temporal lobe, AD = 3.32 +/- 2.24, Control (C) = 24.83 +/- 10.8; middle temporal lobe, AD = 3.6 +/- 4.94, C = 26.09 +/- 15.7; inferior temporal lobe, AD 3.69 +/- 3.6, C = 25.25 +/- 16.9. Furthermore, there was an age-related increase in immunopositive neurones in the superior (r2 = 0.37, P = 0.046) and inferior (r2 = 0.75, P = 0.01) temporal gyri in controls. In AD the number of calbindin positive neurones did not change with age. This is the first report of such an age-related increase in controls, and it suggests that this, rather than a decrease in AD, accounts for the overall difference between AD and controls. It is possible that an increase in intraneuronal calbindin protects these cells from degeneration and that failure of such a neuroprotective mechanism is a significant contributory factor in the pathogenesis of AD.

Aged↗

PubMed2001

Post-weaning social isolation of rats leads to an abnormal gait.

The purpose of this study was to further characterize the phenotype of rats that have experienced prolonged postweaning social isolation, a paradigm that produces changes relevant to neuropsychiatric disorders. At weaning, male Wistar rats from three litters were housed socially (n = 12) or in isolation (n = 13) for 10 weeks. Isolated rats could see, hear and smell other rats. A sophisticated analysis of gait revealed that the stride duration was increased in isolates (12%, P = 0.0024) as a result of increased stance duration (18%, P = 0.0005), but there was no difference in vertical reaction force or velocity. Adrenal glands were heavier in isolates (19%, P = 0.0047). There was no difference in cross-sectional area of the brain or lateral ventricles anywhere along the anterior-posterior axis. All experiments and analysis were performed blind to housing condition. This is the first study to demonstrate that socially isolated rats have an abnormal gait. Further analysis, including pharmacological manipulation, is needed in order to understand the nature of the abnormality.

Adrenal Glands↗

PubMed2001

Genetic footprinting in bacteria.

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.

Bacteriophage lambda↗

PubMed1999

Induction of hyperphosphorylated tau in living slices of rat hippocampal formation and subsequent detection using an ELISA.

Although hyperphosphorylated tau is an established feature of Alzheimer's Disease, its role in the disease process is poorly understood, partly because of lack of suitable animal models. We describe the use of living slices of rat hippocampal formation to study tau phosphorylation. Using the AT8 antibody in an ELISA, phosphorylated tau was detected in freshly frozen slices and it increased significantly in slices that were incubated in an electrophysiological recording chamber; the amount detected was greatest when the homogenisation buffer contained phosphatase and kinase inhibitors. The phosphorylated tau content of the slices increased significantly after exposure to the phosphatase 1 and 2A inhibitor okadaic acid (OA) - 1.5 microM. Electrophysiological recordings confirmed that slices were alive and that OA had no acute toxic effect. In control slices phosphorylated tau, detected immunohistochemically, was mainly in the somatodendritic compartment of neurones; in OA treated slices, there was an apparent decrease in somatodendritic AT8 staining and an increase in neuropil staining. Our system enables the induction of hyperphosphorylated tau within living slices, in an experimental environment that can be used to study the biological consequences of such a change, and may therefore help further our understanding of the significance of hyperphosphorylated tau in Alzheimer's Disease.

Alzheimer Disease↗

PubMed1999

Heparin injection into the adult rat hippocampus induces seizures in the absence of macroscopic abnormalities.

The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycans such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain. Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques.

Animals↗

PubMed1997

Morphology and distribution of electrophysiologically defined classes of pyramidal and nonpyramidal neurons in rat ventral subiculum in vitro.

Intracellular electrophysiological recordings were made from 210 ventral subicular neurons in rat brain slices. Recordings were classified as burst-firing or nonburst-firing. Eighteen burst-firing neurons were filled with Neurobiotin, and all had pyramidal morphology. Nine of these recordings were made from intrinsically burst-firing (IB) cell bodies, and nine were made from burst-firing dendrites (BD). Twelve nonburst-firing neurons were also filled with Neurobiotin. Eight were regular spiking (RS) and had pyramidal morphology, four were fast spiking (FS) and nonpyramidal. Additional electrophysiological parameters distinguished IB from BD, RS from FS, and RS from IB recordings. The distribution of IB and RS neurons was examined by using 180 recordings. Information from the first series of experiments was used to distinguish between somatic and dendritic recordings. The deep-superficial axis (alveus-hippocampal fissure) was divided into four equal rows. RS neurons accounted for 12%, 28%, 58%, and 50% of presumed somatic recordings in successively more superficial rows. The proximal-distal (CA1-perforant path) axis was divided into five equal columns. RS cells accounted for 52% of presumed somatic impalements in the central column compared with 16% in the most proximal and 10% in the most distal columns. Thus, two electrophysiological classes of pyramidal neuron were localized to particular regions of the ventral subiculum. In the light of existing knowledge of the topography of subicular inputs and outputs, our results are consistent with the hypothesis that the ratio of RS to IB pyramidal neurons will be different in different transhippocampal circuits.

Animals↗

PubMed1997

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro.

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

Animals↗

PubMed1996

Neuronal diversity in the subiculum: correlations with the effects of somatostatin on intrinsic properties and on GABA-mediated IPSPs in vitro.

1. We used intracellular current-clamp techniques to record from 33 ventral subicular neurons in slices or rat hippocampal formation. Presumed pyramidal neurons were characterized by their responses to depolarizing current pulses as either intrinsically burst firing (IB) or regular spiking (RS). Within the subiculum, IB cells were encountered most frequently in the deep cell layer, whereas RS cells were encountered most frequently in the superficial cell layer. IB cells had more depolarized resting potentials, lower input resistances, and more sag in their voltage responses to hyperpolarizing current pulses. 2. Somatostatin (5 microM) applied in the bathing medium caused a hyperpolarization and reduction in input resistance. These effects were of greater magnitude in IB cells. Somatostatin had no effect on sag in either cell type. These effects of somatostatin were unchanged in the presence of gamma-aminobutyric acid (GABA) receptor antagonists. 3. In a series of experiments conducted in RS cells only, somatostatin reduced the amplitude of the late but not the early component of evoked biphasic inhibitory postsynaptic potentials (IPSPs). 4. A second series of experiments was conducted in RS and IB cells. Somatostatin reduced the amplitude of pharmacologically isolated GABAA IPSPS in both cell types. In IB cells but not RS cells there was a correlation between this effect and the somatostatin-induced hyperpolarization. Somatostatin also reduced the amplitude of isolated GABAB IPSPS in both cell types, but more so in IB cells. 5. Somatostatin had no effect on the reversal potential of either IPSP in either cell type and no effect on the GABAA-mediated conductance in either cell type. In contrast, the GABAB-mediated conductance was reduced, especially in IB cells. 6. The effects of somatostatin on GABAA IPSPS are principally a result of membrane shunting and reductions in ionic driving force, but these mechanisms do not account for the reduction in GABAB IPSPS. 7. We suggest that the combined effects of somatostatin are likely to alter the balance between fast and slow inhibition and to do so more in IB cells than in RS cells.

Animals↗

PubMed1996

Effects of somatostatin and related peptides on the membrane potential and input resistance of rat ventral subicular neurons, in vitro.

The effects of bath-applied somatostatin (SS), and related peptides on the membrane potential and input resistance of 117 ventral subicular neurons were investigated by intracellular recording in rat brain slices. Electrophysiological properties, which included burst-firing in response to depolarizing current pulses, indicated that the neurons studied were of the pyramidal type. For the 89 cells analyzed quantitatively, membrane potential was -69.1 +/- 0.3 mV (mean +/- S.E.) and input resistance was 23.9 +/- 0.5 megohms. SS (5 microM) caused a hyperpolarization of 3.4 +/- 0.3 mV (n = 9) and reduced input resistance by 16 +/- 3.1% (n = 6). SS D-Trp8, somatostatin, octreotide, CGP 23996 and MK 678 shared these effects, but somatostatin was inactive. SS effects persisted when bathing solutions contained tetrodotoxin, reduced calcium and elevated magnesium concentrations and when both of these treatments were combined. They were unaltered by antagonists at gamma-aminobutyric acid receptors or at ionotropic glutamate receptors. The effects of MK 678, SS, SS D-Trp8 and somatostatin were concentration-dependent, and these peptides were equipotent at 500 nM and at 5 microM. For MK 678, the EC50 was 316 nM for the hyperpolarization and 90 nM for the reduction in input resistance. We conclude that SS acts directly on pyramidal neurons of the rat subiculum to cause a hyperpolarization and a decrease in input resistance. We suggest that these effects are mediated by the SSTR2 receptor subtype.

Animals↗

PubMed1996

The subiculum: a potential site of action for novel antipsychotic drugs?

In this article, recent information from neuropathological studies in humans and behavioural and electrophysiological/pharmacological studies in rats is used to examine the hypothesis that the subiculum, the major output region of the hippocampal formation, may contain sites of action for novel antipsychotic drugs. In the first section, the possible interactions between subicular neurons and the sites at which existing antipsychotic drugs act are discussed. These include the interaction implied by convergence of subicular and dopaminergic inputs to the nucleus accumbens. The second section concentrates upon subicular involvement in animal behaviours that are thought to be relevant to schizophrenia, which, in rats, include Latent inhibition and Pre-pulse inhibition of Acoustic Startle. Involvement of the subiculum in the neuropathology of schizophrenia is discussed in the third section. However, few neuropathological studies comment specifically on the subiculum. Those which suggest involvement tend to be recent and, as yet, have not all been replicated. Finally, there is discussion of the possibility that the subiculum contains chemical sites at which drugs could act specifically to produce appropriate physiological effects. Potential sites include, but are not necessarily restricted to, particular ion channels in electrophysiologically defined subclasses of subicular pyramidal neurons, the receptors for neuromodulatory peptides such as somatostatin and cholecystokinin, and the enzyme nitric oxide synthase. It is concluded that a wide range of clinical and basic neuroscience disciplines has provided evidence which, especially when viewed as a whole, is consistent with the hypothesis that the subiculum is a potential site of action for novel antipsychotic drugs.

Animals↗

PubMed1994

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10.

Morphogenesis in the yeast Saccharomyces cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.

Amino Acid Sequence↗

PubMed1993

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth.

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1- mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1- mutants. Fe(II) transporter activity and regulation is normal in gef1- mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1- deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1- cells and by mitochondrial fractions isolated from gef1- mutants was reduced 25-50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.

Amino Acid Sequence↗

PubMed1992

Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase.

Two independent pathways of transcriptional regulation that show functional homology have been identified in yeast. It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated. We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle-regulated transcription and nuclear localization similar to those seen previously for SWI5. Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation. SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not. In contrast, ACE2 is a transcriptional activator of the CTS1 gene (which encodes chitinase), whereas SWI5 is not. An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTS1 both are cell cycle regulated in the same way, and HO and CTS1 both require the SWI4 and SWI6 transcriptional activators. Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate. Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain.

Amino Acid Sequence↗

PubMed1991

Bacteriophage SPO1 middle transcripts.

Phage SPO1 middle transcripts are known to fall into two classes, m and m1l. Class m1l transcripts continue to be made late in the viral infection, while the synthesis of class m transcripts ceases soon after the onset of replication and late transcription. The experiments that are reported here deal with the regulatory nature of this diversity. The accumulation of transcripts associated with eight middle promoters was analyzed by S1 nuclease mapping. DNA sequence surrounding these middle promoters was determined or redetermined, and the stability of RNA associated with most of these promoters was also analyzed. Class m1l transcription was shown to be associated with SPO1 middle promoters that remain active at late stages of viral development, when middle promoters of class m are repressed. The consensus sequences of class m and m1l middle promoters were found to be indistinguishable and the search for sequences consensual with late promoters yielded only divergent candidates. No other consensus sequence that is specific and exclusive to either class of middle promoters was detected within a hundred base pairs upstream or downstream of these promoters. Considerable variations in the stabilities of SPO1 middle transcripts were found. Two promoters that are only 71 base pairs apart yielded transcripts that had substantially different stabilities. The 5'-flanking segment of the transcript associated with the upstream promoter apparently conferred a high degree of stability on this RNA.

Bacillus subtilis↗

PubMed1987

Subcloning.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1986

DNA binding by the bacteriophage SPO1-encoded type II DNA-binding protein, transcription factor 1. Formation of nested complexes at a selective binding site.

The interactions of the phage SPO1-encoded Type II DNA-binding protein, transcription factor 1 (TF1), with one of its preferred binding sites in SPO1 DNA have been analyzed in detail. The results suggest that TF1 recognizes a high-affinity "core" binding site and that additional protein moieties can accrue to either side of the occupied core site to form higher order complexes. Close contacts between TF1 and the core binding site as well as some of the steric requirements for recognition of the core site were determined. Comparison of the nucleotide sequences of several preferred binding sites for TF1 reveals a striking lack of precise homology but does show common features.

Bacteriophages↗