a couple of new publications......
Endocrinology 2000 Feb;141(2):498-504
Selective impairment of corticotropin-releasing factor1 (CRF1)
receptor-mediated function using CRF coupled to saporin.
Maciejewski-Lenoir D, Heinrichs SC, Liu XJ, Ling N, Tucker A, Xie Q, Lappi DA, Grigoriadis DE
Neurocrine Biosciences, Inc., San Diego, California 92121, USA. dmaciejewski@neurocrine.com
[Medline record in process]
CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and
physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a
variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human
CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF
receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent
and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on
CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by
coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective
nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors,
CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the
same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with
CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of
CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.
J Biol Chem 2000 Jan 7;275(1):580-4
The copper transport protein atox1 promotes neuronal survival.
Kelner GS, Lee M, Clark ME, Maciejewski D, McGrath D, Rabizadeh S, Lyons T, Bredesen D, Jenner P, Maki
RA
Department of Molecular Biology, Neurocrine Biosciences, San Diego, California 92121, USA.
[Medline record in process]
Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast
lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with
the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In
this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1
expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are
significantly protected against this stress. This level of protection is comparable with the level of protection seen with
copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal
cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding,
do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular
pathways used for protection against oxidative stress.
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