Biomedical subjects

Nigel G F Cooper

Publications and source records attributed to Nigel G F Cooper.

6 recordsLinked to original sources

PubMed2005

The role of CaMKII in BDNF-mediated neuroprotection of retinal ganglion cells (RGC-5).

The purpose of the study is to determine if expression or secretion of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells (RGC-5) is mediated by NFkappaB or Ca2+/calmodulin-dependent protein kinase II (CaMKII). RGC-5 cells were exposed to 1 mM glutamate for various periods of time, in the presence or absence of prospective regulatory molecules. BDNF mRNA and protein expression were assessed with the aid of real-time PCR and immunoblots, respectively, and BDNF secretion was determined by ELISA. The NFkappaB inhibitor (TLCK and PTD-p65), or a specific CaMKII inhibitor (m-AIP), was used to study association of NFkappaB or CaMKII with BDNF expression/secretion in RGC-5 cells. Glutamate stimulated a transient increase in BDNF mRNA and protein in RGC-5 cells, and also stimulated an early release of BDNF into the culture media. Neutralizing the BDNF or blocking the TrkB receptor enhanced the glutamate-induced cytotoxicity. NFkappaB nuclear translocation was revealed in response to glutamate treatment. Application of TLCK or PTD-p65 inhibited the glutamate-induced BDNF expression and secretion. Inhibition of CaMKII by m-AIP did not affect expression but significantly enhanced the release of BDNF from glutamate challenged cells. Our data suggest that glutamate treatment may stimulate expression of BDNF in RGC-5 cells through NFkappaB activation. A novel mechanism for neuroprotection is proposed for the CaMKII inhibitor, AIP, which appears to protect RGC-5 cells from cytotoxicity by enhancing the release of BDNF from glutamate challenged cells.

Animals↗

PubMed2005

Retinal ganglion cell death and neuroprotection: Involvement of the CaMKIIalpha gene.

The purpose of this study is to determine if calcium/calmodulin-dependent protein kinase-II (CaMKII) plays a role in neuronal cell death and if inhibition of this kinase affords some neuroprotection in the RGC-5 retinal ganglion cell line. The RGC-5 cells were treated with glutamate at various concentrations for increasing increments of time. Cytotoxicity was assayed by measuring the lactate dehydrogenase (LDH) leakage from non-viable cells and TUNEL assays. The involvement of caspase-3, Bcl-2 and caspase-8 in glutamate-induced cytotoxicity was determined by immunoblots and/or real time RT-PCR. In addition, the autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of CaMKII, was used to determine the involvement of CaMKII in glutamate-induced RGC-5 cell death. Application of increasing concentrations of glutamate to RGC-5 cells caused a dose-dependent increase in the level of cell death after 24 h. There was a glutamate-stimulated increase in the expression of caspase-8 and caspase-3 and a corresponding decrease in Bcl-2. The active fragment of caspase-3 increased in glutamate-treated cells. An early transient increase in the expression of CaMKIIalpha(B) gene and a corresponding CaMKIIalpha nuclear translocation was found in glutamate-treated cells. Treatment with AIP blocked the activation of caspase-3 and protected RGC from glutamate-mediated cell death but did not alter the glutamate-enhanced expression levels of caspase-8 or caspase-3. This report shows the likely involvement of a transcript of the CaMKIIalpha gene in the cytotoxicity response of RGC-5 cells similar to previous reports in the neural retina. AIP is shown to be a neuroprotectant for RGC-5 cells as was reported for the neural retina.

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PubMed2005

MPrime: efficient large scale multiple primer and oligonucleotide design for customized gene microarrays.

BACKGROUND: Enhancements in sequencing technology have recently yielded assemblies of large genomes including rat, mouse, human, fruit fly, and zebrafish. The availability of large-scale genomic and genic sequence data coupled with advances in microarray technology have made it possible to study the expression of large numbers of sequence products under several different conditions in days where traditional molecular biology techniques might have taken months, or even years. Therefore, to efficiently study a number of gene products associated with a disease, pathway, or other biological process, it is necessary to be able to design primer pairs or oligonucleotides en masse rather than using a time consuming and laborious gene-by-gene method. RESULTS: We have developed an integrated system, MPrime, in order to efficiently calculate primer pairs or specific oligonucleotides for multiple genic regions based on a keyword, gene name, accession number, or sequence fasta format within the rat, mouse, human, fruit fly, and zebrafish genomes. A set of products created for mouse housekeeping genes from MPrime-designed primer pairs has been validated using both PCR-amplification and DNA sequencing. CONCLUSION: These results indicate MPrime accurately incorporates standard PCR primer design characteristics to produce high scoring primer pairs for genes of interest. In addition, sequence similarity for a set of oligonucleotides constructed for the same set of genes indicates high specificity in oligo design.

Animals↗

PubMed2005

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.

Adult↗

PubMed2002

Enhanced expression of TNF-R1 protein in NMDA-mediated cell death in the retina.

Multiple apoptosis-related genes are expressed in the retina after exposure to N-methyl-D-aspartic acid (NMDA). For example, the mRNAs for TNF-R1, FasL, and Nur77 are up-regulated between 2.8 and 7-fold [Mol. Brain Res. 91 (2001) 34-42]. The purpose of the present study is to examine prospective changes in protein expression for these genes and to determine their cellular localizations subsequent to NMDA stimulation. Following anesthesia, a single intravitreal injection of 4 mM NMDA was administered into the right eye of anesthetized rats. The left eye was injected with phosphate-buffered saline. Retinae were harvested at 2 and 24 h postinjection. Western-blot and immunocytochemical techniques were used to detect changes in protein expression levels, and to localize their distributions within the retina. Analyses of Western blots demonstrated a significant increase in TNF-R1 (100 and 80%) compared to the sham-controls at 2 and 24 h postinjection with NMDA. Immunolabeling of TNF-R1 was observed in the inner nuclear layer (INL) at 2 h postinjection with NMDA. TNF-R1 was also clearly evident in cells within the INL and ganglion cell layers (GCL) at 24 h post-injection with NMDA. In contrast to these changes in TNF-R1 there were no significant changes in the levels or distributions of FasL or Nur77 in NMDA-stimulated animals at either 2 or 24 h when compared to the sham-controls. These results implicate the TNF-R1 signal transduction pathway in NMDA-induced cell death in the INL and GCL of the retina.

Animals↗

PubMed2002

Developmentally regulated expression of CaMKII and iGluRs in the rat retina.

Calcium/calmodulin-dependent protein kinase II (CaMKII) and the ionotropic glutamate receptors (iGluRs) have been shown to be pivotal in the maturation of synapses during development of the central nervous system. The purpose of the current study was to assay the expression profiles of these molecules during the development of the rat retina. The mRNA levels of CaMKII were determined by the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. The protein levels of CaMKII were assayed in slot blots. The CaMKII enzyme activity was also measured. In addition, the protein levels of iGluRs in a retinal membrane-enriched fraction were evaluated in Western blots. The results show that the levels of CaMKII (mRNA, protein, and activity) and distinct subunits of iGluR proteins increased during the first 2 weeks after birth. The highest level of CaMKII was reached during the second postnatal week, coincident with the peak of synaptogenesis in the inner plexiform layer of the rat retina. The expressions of NMDAR-NR1 and -NR2A were relatively low in the first postnatal week but rose quickly thereafter. However, NMDAR-NR2B was relatively high at postnatal day 5 (P5) and increased steadily during the postnatal period. Thus, the subunit compositional profile of the retinal NMDARs was altered during retinal maturation. The developmental pattern of AMPAR-GluR1 was similar to that of NMDAR-NR2B, with high expression at P5, and modest increases thereafter. The patterns of CaMKII and NR1/NR2A were better correlated than were CaMKII and NR2B, or CaMKII and GluR1. The temporal differences in subunit expression of these synaptically relevant molecules suggest that they play distinct roles during the development of the retina.

Animals↗