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Growth factors play a critical role in suppressing cell death during both development and in cellular homeostasis. Many growth factors activate the Ras pathway, which comprises a series of sequentially activated protein kinases, and includes MAPK (mitogen-activated protein kinase). In mammals, the mechanism by which the Ras-MAPK signaling pathway promotes cell survival has not yet been elucidated. The authors of this article investigated the mechanism by which the Ras-MAPK signaling pathway mediates BDNF (brain-derived neurotrophic factor) survival of cerebellar granule neurons. The MAPK-activated kinases Rsks catalyzed the phosphorylation of BAD, which suppressed apoptosis in neurons. Rsks also phosphorylate the transcription factor CREB (cAMP-response element binding protein), which also promoted cell survival. The findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism that phosphorylates and inactivates a component of the cell death machinery and also increases transcription of pro-survival genes. The authors utilized the Promega Anti-ACTIVE® MAPK pAb (Cat. #V8031) to demonstrate MAPK phosphorylation (activation), as well as the Anti-Beta-Galactosidase mAb (Cat.# Z3781) in immunofluorescence cotransfection studies.
Bonni, A., Brunet, A., West, A.E., Datta, S.R., Takasu, M.A. and Greenberg, M.E. (1999)
Science 286, 1358.
Division of Neuroscience, Children's Hospital, and Department of Neurobiology, Harvard
Medical School, 300 Longwood Avenue, Boston, MA 02115 USA
The p42 and p44 mitogen-activated protein kinases (MAPKs) have been implicated in both
proliferation and differentiation programs and function in cascades controlling cell fate
and cell survival in various invertebrates and mammalian cells. Although both the p42 and
p44 MAPK isoforms are highly homologous, they do exhibit 17% divergence in amino acid
sequence, which may indicate functional significance. The authors of this citation
evaluated the specific role of the p44 MAPK isoform in the whole animal through the
generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells.
The p44 MAPK-/- mice were viable, fertile and of normal size. However, in p44 MAPK-/-
mice, thymocyte maturation beyond the CD4+CD8+ stage was reduced, and proliferation in
response to activation of the T cell receptor in the presence of phorbol myristate acetate
was severely reduced even though activation of p42 MAPK was more sustained in these cells.
These results demonstrate that p44 MAPK is required for the differentiation of double- to
single-positive thymocytes and for thymocyte proliferation. The authors utilized the
Promega Anti-ACTIVE® MAPK pAb (Cat. #V8031) to monitor
the levels of inactive (unphosphorylated) and active (dually phosphorylated) p42 and p44
MAPK in both wildtype and p44 MAPK-/- mice.
Pagès, G.1, Guérin, S.2, Grall, D.1, Bonino, F.1,
Smith, A.3, Anjuere, F.2, Auberger, P.2 and Pouysségur,
J.1 (1999) Science 286, 1374.
1Institute of Signaling, Developmental Biology and Cancer Research, CNRS
UMR 6543, Centre A. Lacasagne, 33 Avenue de Valombrose, 06189 Nice, France. 2Faculte
de Medecine, INSERM U364 and CJF 96.05, Avenue de Valombrose, 06107 Nice, France; 3Centre
for Genome Research, King's Building, West Mains Road, Edinburgh, Scotland, UK.
Exit from the cell cycle during terminal differentiation requires inactivation of the
cyclin-dependent protein kinases (CDKs), which involves the action of the polypeptide CDK
inhibitors (CKIs). The mammalian CDK inhibitors exist in two distinct families, the Ink4
family (p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d),
which inhibit the cyclin D-dependent kinases, CDK4 and CDK6, and the Cip/Kip family (p21Cip1,
p27Kip1, and p57Kip1) which are potent inhibitors of the cyclin E-
or A-dependent kinase, CDK2. The timing of neuronal cell cycle exit and differentiation
are likely to be regulated by such CDK inhibitors. The expression patterns of p19Ink4d
and p27Kip1in postmitotic brain cells suggested that these proteins may play an
important role in suppressing neuronal cell growth. The authors produced crosses of
Ink4d-null mice with Kip1-null mice, which resulted in mice that exhibited bradykinesia,
proprioceptive abnormalities and seizures, and died at 18 days after birth. Subpopulations
of CNS neurons were actively proliferating in the brain, but the increased proliferation
was balanced by cell death, resulting in no gross changes in brain cytoarchitecture. The
authors concluded that p19Ink4d and p27Kip1 cooperate to maintain
differentiated neurons in a quiescent state that is potentially reversible. The DeadEnd™ Colorimetric Apoptosis Detection System (Cat.# G7130) was used
to demonstrate that apoptosis was occurring in many parts of the brain that are normally
quiescent.
Zindy, F.1, Cunningham, J.J.2, Sherr, C.J.3, Jogal, S.1,
Smeyne, R.J.2 and Roussel, M.F.1 (1999) Proc. Natl. Acad. Sci.
USA 96, 13462.
Departments of 1Tumor Cell Biology, 2Developmental Neurobiology,
and 3the Howard Hughes Medical Institute at St. Jude Children's Research
Hospital, Memphis, TN 38105 USA
The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), which regulates salt and water transport across epithelial cells. The CFTR channel is implicated in two major human diseases: cystic fibrosis and secretory diarrhea. Detailed knowledge of the mechanisms that activate or inactive this channel would greatly aid in developing strategies for the treatment of both of these diseases. The initial step in activating the CFTR channel is phosphorylation of the cytoplasmic regulatory domain (R) by protein kinase A (PKA), relieving an inhibitory effect of this domain through an undetermined mechanism. The authors of this article addressed the role of the NH2-terminal tail of CFTR in the gating process. The amino-terminal cytoplasmic tail of CFTR was found to control PKA-dependent channel gating through a physical interaction with the R domain. In mutational analysis of the amino-tail, the authors identified a cluster of acidic residues that regulate steady-state CFTR activity. CFTR activity appears to be regulated by an interdomain interaction involving the NH2-terminal tail, which may serve as a potential target for physiologic and pharmacologic molulators of channel function. The authors used Promega’s cAMP-Dependent Protein Kinase, Catalytic Subunit (Cat.# V5161), to investigate the single-channel properties of wildtype CFTR and the N-tail mutants in inside-out membrane patches excised from oocytes.
Naren, A.P.1, Cormet-Boyaka, E.1, Fu, J.1, Villain, M.1,
Blalock, J.E.1, Quick, M.W.1,2 and Kirk, K.L.1,2 (1999) Science
286, 544.
1Department of Physiology and Biophysics, 2Department of
Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama
at Birmingham, Birmingham, AL 35294 USA.
This citation was kindly suggested by author Dr. Manuela Battaglia.
One of the potential complications of autologous transplantation is the possible
contamination of the graft by residual tumour cells, which may contribute to
post-transplant tumor growth. Thus, techniques for detecting minimal residual tumor cells
in the autograft are becoming increasingly important in the field of transplantation
biology. Others have demonstrated that PCR-based methods are prone to false positive
results. The authors of this article developed a highly specific, less time-consuming and
technically simpler RT-PCR assay capable of detecting epithelial cells that would
otherwise go undetected by presently available immunocytochemical methods. They compared
the specificity and sensitivity of a ‘one tube’ RT-PCR assay with the more
widely used nested RT-PCR method for detection of cytokeratin 19-positive epithelial
cells. The one-tube RT-PCR method was highly specific in contrast to the nested method,
and could detect tumor contamination at 10-6. The authors used the Promega
Access RT-PCR System(a) (Cat.# A1250) for the
one step RT-PCR method.
Battaglia, M.1, Pedrazzoli, P.2, Palermo, B.2, Lanza, A.2,
Bertolini, F.2, Gibelli, N.2, Da Prada, G.A.2, Zambelli,
A.2, Perotti, C.3 and Robustelli della Cuna, G.2 (1998) Bone
Marrow Transplantation 22, 693.
1Blood Research Institute, The Blood Center of Southeastern Wisconsin,
Milwaukee, WI 53233 USA; 2Division of Medical Oncology and Laboratory of
Experimental Medicine, IRCCS ‘S Maugeri’ Foundation and 3Immunohaematology
and Transfusion Service, IRCCS Policlinico S Matteo, Pavia, Italy
(a)The PCR process is covered by patents issued and applicable in certain countries. Pormega does not enocurage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.
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