Product Focus: Anti-ACTIVE^® Caspase-3 pAb

CXCR4-activated astrocyte glutamate release via TNFα: amplification by microglia triggers neurotoxicity.

Glial cells, including astrocytes, have recently been shown to participate in neuronal signaling in ways that were not previously expected. Bezzi et al. have defined a chemokine-directed signaling pathway in which release of TNFα results in glutamate release from astrocytes. This pathway is initiated by binding of the stromal cell derived factor 1 to its receptor, CXCR4. Treatment of hippocampal slices with the SDF-1 alpha isoform was shown to result in rapid release of glutamate. Preincubation of the slices with a calcium chelator suggested that the neurotransmitter release was not from neurons, but via a calcium-dependent pathway distinct from neuronal glutamate release pathways. This was further demonstrated by showing that astrocytes in culture respond to treatment with SDF-1α by rapid, dose dependent glutamate release. Hippocampal slices from mice which are null for TNFα do not show the rapid glutamate release induced by SDF-1α treatment. Further investigation of the pathway revealed that activation of the CXCR4 receptor caused a cascade of events including ERK/MAPK activation, metalloproteinase-mediated TNFα release leading to prostaglandin E2 generation, and eventual rapid glutamate exocytosis from astrocyte cultures. SDF-1alpha induced TNFα and glutamate release from astrocyte cultures was significantly enhanced by the presence of activated microglia, demonstrating a potential relevance to pathological neurotoxicity. Pathological significance of this pathway was demonstrated by showing that this novel synergic interaction between astrocytes and microglia can be activated by the HIV glycoprotein gp120IIIB, leading to neuronal apoptosis. The gp120IIB protein can bind to the CXCR4 receptor and activate the same signaling pathway as the natural ligand SDF-1α. Prolonged exposure of hippocampal neuron-glia co-cultures to the gp120IIIB protein resulted in neuronal apoptosis. Apoptosis was demonstrated by both use of TUNEL staining and the Anti-ACTIVE^® Caspase-3 pAb (Cat.# G7481). Gp120IIIB-induced apoptosis was blocked by use of inhibitors that had also been shown to block the SDF-1α mediated glutamate release pathway.

Bezzi, P.^1,7, Domercq, M.^1,7, Brambilla, L.¹, Galli, R.^2,3, Schols, D.⁴, De Clercq, E.⁴, Vescovi, A.^2,3, Bagetta, G.⁵, Kollias, G.⁶, Meldolesi, J.², Volterra, A.^1,* (2001) Nat. Neurosci. 4(7), 702-710.

¹Department of Pharmacological Sciences, Center for Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti, 9, 20133 Milan, Italy; ²DIBIT, Department of Neurosciences, San Raffaele Institute and Vita-Salute University, 20132 Milan, Italy; ³Stem Cell Research Institute (SCRI), San Raffaele Hospital, 20132 Milan, Italy; ⁴Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium; ⁵Department of Pharmaco-Biology, Calabria University at Cosenza, 87036, Italy; ⁶Institute of Immunology, Biomedical Sciences Research Center 'Al. Fleming,', 166 72 Vari, Greece. ⁷The first two authors contributed equally to this work.

*To whom correspondence should be addressed. andrea.volterra@unimi.it

Product Focus: Anti-ACTIVE^® p38 pAb, Rabbit (pTGpY)

Asymmetric p38 activation in Zebrafish: Its possible role in symmetric and synchronous cleavage.

Cleavage in vertebrate embryos proceeds via a series of symmetric and synchronous cell divisions. In the Zebrafish (Danio verio), the synchronous and symmetric cleavages of embryogenesis are followed by asymmetry with respect to the dorsal-ventral (DV) axis of the embryo, characterized by the zygotic expression of several genes in the dorsal blastomeres. Although Zebrafish development has been described in detail, known about the molecular and signaling events that direct early cleavage and establish the DV and AP (anterior-posterior) axes of the animal are largely unknown.

The authors use Promega’s Anti-ACTIVE^® p38 pAb, Rabbit, (pTGpY, Cat.# V1211) to investigate the potential role of p38 kinase in the control of cleavage events in zebrafish embryos. They establish by immunoblot that Promega’s antibody specifically recognizes active p38 from zebrafish, and then use the antibody in immunostaining of zebrafish embryos. They show that while the p38 protein is evenly distributed during cleavage, it is activated asymmetrically. Suppressing this p38 activation results in abnormal development, inhibiting cytokinesis on one side of the blastodisc, and they describe a novel role for the p38 signal transduction cascade during early embryogenesis in vertebrates.

Fujii, R.¹, Yamashita, S.¹, Hibi, M.¹ and Hirano, T.¹* (2000) J. Cell Biol. 150, 1335–1346.

¹Division of Molecular Oncology, Biomedical Research Center (C-7), Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

*To whom correspondence should be addressed.

Product Focus: TNT^® T7 Quick for PCR DNA System

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilization (PISA method).

The completion, or near completion, of several genome sequencing projects has made high-throughput analysis of protein function a priority. Array technologies are well established in the area of nucleic acid analysis. However, application of these methods in the area of proteomics is hindered by the difficulties of comprehensive production of functional proteins. This is caused both by the poor expression, aggregation and degradation of many eukaryotic proteins in bacterial systems, as well as the time-consuming processes of DNA cloning and gene identification. The authors describe a method, termed the 'protein in situ array' (PISA) to overcome these problems. The PISA method generates protein arrays in one step, directly from DNA templates and uses a cell-free protein expression method combined with simultaneous in situ immobilization on a surface. In this method, genes or gene fragments are amplified by PCR or RT-PCR and used in the TNT^® T7 Quick for PCR DNA System (Cat.# L5540). For this study, the PCR primers were designed so that the proteins produced were double (His)₆-tagged proteins. The reactions were carried out on a Ni-NTA-coated surface to which the protein adheres as soon as it is synthesized. The authors demonstrate that single-chain antibody fragments (human single-chain aniprogesterone V_H/K-His fragment) and an enzyme (luciferase) can be functionally arrayed using the PISA method.

He, M.¹* and Taussig, M.J.¹ (2001) Nucleic Acids Research. 29, e73.

¹Technology Research Group, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.

*To whom correspondence should be addressed: mingyue.he@bbsrc.ac.uk

Product Focus: E_max^® ImmunoAssay Systems - RQ1 DNase

Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease.

Huntington's disease (HD) is an hereditary, degenerative brain disorder that begins in early adulthood and ultimately results in death. Huntingtin, a cytoplasmic protein that is mutated in HD patients, is believed to play a role in the selective death of striatal neurons in patients with Huntington's disease. This paper demonstrates that huntingtin regulates the production of brain-derived neurotrophic factor (BDNF). BDNF is produced by cortical neurons and is necessary for the survival of striatal neurons.

BDNF production in cultured CNS cells overexpressing wild-type or mutant huntingtin was measured by ELISA. Cells expressing wild-type huntingtin produced almost two-fold more BDNF than parental cells, while cells expressing the mutant protein produced approximately half the BDNF of parental cells. RNase protection assays and Rat BDNF promoter-reporter gene fusion experiments using the same cell clones showed that the wild-type huntingtin increased BDNF gene transcription while the mutant huntingtin down-regulated transcription. The same affects on BDNF protein and mRNA levels were observed in vivo in the cerebral cortex and hippocampus of transgenic mice overexpressing the wild-type or mutant huntingtin gene. These data suggest that therapies increasing normal huntingtin activity and BDNF levels could be used to treat Huntington's disease.

BDNF, NGF, NT-3 and NT-4 E_max^® ImmunoAssay Systems (Cat.# G7610, G7630, G7640, G7650) were used for the respective ELISAs. RQ1 RNase-Free DNase (Cat.# M6101) was used to remove DNA from the total RNA used in the RNase protection assays.

Zuccato, C.^1,2, Ciammola, A.^1,2,3, Rigamonti, D.^1,2, Leavitt, B.R.⁴, Goffredo, D.^1,2, Conti, L.^1,2, MacDonald, M.E.⁵, Friedlander, R.M.⁶, Silani, V.^2,3, Hayden, M.R.⁴, Timmusk, T.⁷, Sipione, S.^1,2, Cattaneo, E.^1,2* (2001) Science 293, 493–498.

¹Department of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133 Milano, Italy; ²Center of Excellence on Neurodegenerative Diseases, University of Milano, Italy; ³Centro "Dino Ferrari," Department of Neurological Sciences, University of Milan Medical School, IRCCS Ospedale Maggiore, Milano, Italy; ⁴Centre for Molecular Medicine and Therapeutic Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; ⁵Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA; ⁶Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; ⁷Program of Molecular Neuroscience, Institute of Biotechnology, Helsinki, Finland, and Department of Developmental Neuroscience, Biomedical Center, Uppsala University, Uppsala, Sweden.

*To whom correspondence should be addressed: elena.cattaneo@unimi.it

Product Focus: CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay - CytoTox 96^® Cytotoxicity Assay

Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons.

Primary cell cultures are often preferred for their near in vivo-like behavior under appropriate culture conditions. Primary cells can prove more difficult to maintain in culture though. In this paper, the authors describe a model system that could be used for high-throughput assays of potentially toxic compounds: primary cultures of cerebellar granule neurons grown under controlled conditions including poly-lysine-coated plates and lack of serum and steroids in the medium,  Culture density was assessed, and cell viability was assessed using the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (Cat.# G3580) as well as CytoTox 96^® Cytotoxicity Assay (Cat.# G3580). The CellTiter 96^® AQ_ueous Assay measures the reduction of MTS, a colorimetric analysis of cellullar respiration; the CytoTox 96^® Assay measures lactate dehydrogenase (LDH) release, an indicator of cell death. MTS reduction and LDH release were found to be directly proportional to the number of cerebellar granule cells initially plated (at appropriate densities). In fact, 0.5–1.0 x 10⁵ cells were identified as a suitable number of cells, per well in 96 well plates, for the simultaneous measurement of cell viability and cell death using the two Promega assay reagents. The researchers showed that tert-butyl-hydroperoxide and hydrogen peroxide induced oxidative stress. They also demonstrated, using CellTiter 96^® AQ_ueous Assay, that NMDA-receptor selective antagonist, MK-801, protected the cells against glutamate-mediated excitotoxicity.

Wong, J.K.¹, Kennedy, P.R.¹, and Belcher, S.M.¹* (2001) J. Neurosci. Meth. 110, 45-55.

¹Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Mail Slot 611, 4301 West Markham Street, Little Rock, AR 72205, USA.

*To whom correspondence should be addressed: belcherscottm@uams.edu

Product Focus: FluoroTect™ Green_Lys in vitro Translation Labeling System

Coupled transcription and translation within nuclei of mammalian cells.

It is generally believed that the vital cellular processes of transcription and translation are separated in eukaryotic cells, with transcription occurring in the nucleus and translation occurring exclusively in the cytoplasm. Moreover, it is widely believed that the nuclear membrane in fact evolved to segregate splicing and translation. However, the authors  f a recent Science article present evidence that points to possible translation in the nucleus. The evidence includes: i) the presence in nuclei of all components necessary for translation; ii) that isolated nuclei have been shown to amino acylate tRNAs and incorporate radiolabeled amino acids in protein and; iii) nonsense-mediated decay—a quality control process that checks newly made messages—occurs in the nucleus. The authors suggest that nuclear translation might be the reason that nuclear NMD is seen.

The authors established an in vitro translation system that used low temperature and suboptimal precursor concentrations so that few proteins with average concentrations of ~350 amino acids were completed and able to escape from synthetic sites. Cells were permeabilized in a physiological buffer and allowed to extend nascent polypeptides by ~15 residues in the presence of tagged precursor molecules (either [³H]lysine, biotin- or BODIPY-lys-tRNA), the remaining 19 amino acids, aminoacyl-tRNA synthetases, GTP and an energy generating system. Incorporation was determined to be sensitive to inhibitors of eukaryotic protein synthesis (cycloheximide and puromycin) but not to chloramphenicol (inhibitor of bacterial protein synthesis). Promega’s FluoroTect™ Green_Lys (Cat.# L5001) was used as the BODIPY-labeled tag for localizing translation sites in the nuclei.

Autoradiography confirmed that nuclei were responsible for about 15% of cellular [³H]lysine incorporation. Thus, results with the FluoroTect™ Green_Lys tag were essentially the same as for [³H]lysine or biotin-lysine-tRNA; the nuclear signal constituted 14% of the total fluorescence. This signal was abated using cycloheximide.

In addition the authors sought evidence for the coupling of transcription and translation in the nuclei. When the essential components for transcription (two additional NTPs) were added to the in vitro translation system used for these studies, the nucleoplasmic fluorescence increase was proportional to the concentration of the added components. Alpha-amanitin, an inhibitor of RNA polymerase II, blocked this increase in fluorescence. It was noted that nucleoplasmic incorporation of biotin- and BODIPY-lys-tRNA, as well as [³H]lysine, depended on concurrent transcription.

The results show that lysine coupled to [³H], biotin and BODIPY is incorporated into discrete sites within nuclei and some of this nuclear translation is linked to concurrent transcription.

Iborra, F.J.¹, Jackson, D.A.² and Cook, P.R.¹* (2001) Science 293, 1139-1142.

¹Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK; ²Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Post Office Box 88, Manchester, M60 1QD UK.

*To whom correspondence should be addressed. peter.cook@path.ox.ac.uk

Product Focus: Transforming Growth Factor β₁, Human, Natural

TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice.

Beta-amyloid plaques appear to form as a result of Amyloid Precursor Protein (APP) aggregation. The subsequent development of Alzheimer disease might therefore result from an over-production or a fault in clearing of these beta-amyloid plaques. The immunological response against the beta amyloid deposits is clear based on experiments carried out using beta-amyloid immunization of mice. This resulted in an increased production of microglial cells which are stimulated to phagocytose the beta-amyloid deposits.

Transforming growth factor beta1 (TGF-beta1) also plays a role in the process of beta-amyloid deposition in the brain. TGF-beta1 production in mice expressing the human APP (hAPP) correlates with the deposition of beta-amyloid protein in blood vessels in the brain. Using TGF-beta1/hAPP transgenic mice, the authors have shown that the overall effect of TGF-beta1 in the brain is to reduce the amount of beta-amyloid plaques in the brain tissue and to increase the amount of amyloid plaque deposition in the brain vasculature and that this is dependent on activated microglial cells.

The authors used Promega’s TGFβ₁, Human, Natural (Cat.# G1241) to test the ability of microglial cells to clear beta-amyloid plaques. Microglial BV-2 cultures exposed to synthetic amyloid peptide were cultured with TGF-beta1. Western blot analysis revealed that the amount of cell-associated amyloid peptide decreased with increasing TGF-beta1 concentration in the culture. This response was mediated by the microglial cells given that TGF-beta1 by itself (in the absence of miroglia) did not induce the clearing.

 Wyss-Coray T.¹*,  Lin C.¹, Yan F.¹, Yu G.Q.¹, Rohde M.¹, McConlogue L.¹, Masliah E.¹, Mucke L.¹ (2001) Nat. Med. 7, 527-528.

¹Gladstone Institute of Neurological Disease, University of California, San Francisco, California, USA.

 *To whom corresponance should be addressed. twysscoray@gladstone.ucsf.edu

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