Protective up-regulation of CK2 by mutant huntingtin in cells co-expressing NMDA receptors

l-Homocysteic acid (HCA), an endogenous excitatory amino acid in the mammalian CNS, potently activates N-methyl-d-aspartate (NMDA) receptors in hippocampal neurons. However, the responses to HCA in Purkinje cells, which lack functional NMDA receptors, have been largely unexplored: HCA may activate conventional non-NMDA receptors by its mixed agonistic action on both NMDA and non-NMDA receptors, or it may activate a novel non-NMDA receptor that has high affinity for HCA. To test these possibilities, we compared the responses to HCA in cultured Purkinje cells with those in hippocampal neurons by using the whole cell patch-clamp technique. To clearly isolate HCA responses mediated by non-NMDA receptors, we complemented pharmacological methods by using neurons from mutant mice (NR−/−) that lack functional NMDA receptors. A moderate dose of HCA (100 μM) induced substantial responses in Purkinje cells. These responses were blocked by non-NMDA receptor antagonists but were insensitive to NMDA receptor antagonists. HCA also activated responses mediated by non-NMDA receptors in both wild-type and NR1−/− hippocampal neurons. HCA responses in Purkinje cells had a pharmacological profile (EC50 and Hill coefficient) very similar to that of non-NMDA receptor components of HCA responses in hippocampal neurons. Moreover, the amplitude of the non-NMDA receptor component of HCA responses was directly correlated with that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)– and kainate-induced responses in both types of neurons. Finally, in both types of neurons, HCA currents mediated by non-NMDA receptors were potently blocked by the AMPA receptor antagonist GYKI52466. These findings indicate that HCA-activated AMPA receptors in Purkinje cells are similar to those in hippocampal neurons and that there is no distinct HCA-preferring receptor in Purkinje cells. We also found that in hippocampal neurons, the EC50s of HCA for non-NMDA receptors and for NMDA receptors were more similar than originally reported; this finding indicates that HCA is similar to other mixed agonists, such as glutamate. HCA responses may appear to be selective at NMDA receptors in cells that express these receptors, such as hippocampal neurons; in cells that express few functional NMDA receptors, such as Purkinje cells, HCA may appear to be selective at non-NMDA receptors.

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Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063792 ◽

1969 ◽

Vol 27 ◽

pp. 376-377

Author(s):

A. M. Watrach

Keyword(s):

Tissue Culture ◽

Small Proportion ◽

Chicken Embryo ◽

Culture Cells ◽

Tissue Culture Cells ◽

Morphologic Characteristics ◽

Infectious Laryngotracheitis ◽

Laryngotracheitis Virus ◽

In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

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Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066796 ◽

1971 ◽

Vol 29 ◽

pp. 548-549

Author(s):

Awtar Krishan ◽

Dora Hsu

Keyword(s):

Granular Material ◽

Rna Synthesis ◽

Culture Medium ◽

Actinomycin D ◽

Metabolic Inhibitors ◽

Protein And Rna Synthesis ◽

Apparent Reduction ◽

Plant Alkaloids ◽

In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

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Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162545 ◽

1996 ◽

Vol 54 ◽

pp. 16-17

Author(s):

J. R. Hully ◽

K. R. Luehrsen ◽

K. Aoyagi ◽

C. Shoemaker ◽

R. Abramson

Keyword(s):

Nucleic Acids ◽

Viral Infections ◽

Anatomical Location ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Cellular Source ◽

Gene Transcripts ◽

Initial Description ◽

In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166257 ◽

1996 ◽

Vol 54 ◽

pp. 758-759

Author(s):

D. W. Fairbain ◽

M.D. Standing ◽

K.L. O'Neill

Keyword(s):

Heat Shock ◽

Cell Lines ◽

Chromatin Condensation ◽

Membrane Integrity ◽

Resistant Cell ◽

Membrane Blebbing ◽

Late Loss ◽

Induced Apoptosis ◽

Dying Cells ◽

In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

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