Difference between revisions of "Serine Racemase"
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There are two approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, and by characterization across development. | There are two approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, and by characterization across development. | ||
| − | ==Pharmacological | + | |
| + | ==Pharmacological manipulations== | ||
<b>MK-801: </b> Hasimoto et al. PMID 17109841 used qRT-PCR and HPLC to detect changes in SR and DAAO in adult rat brain after acute or chronic injections of MK-801, the non-competitive NR antagonist. Changes in mRNA were only seen at 400 ug/kg and above, Very large changes (5x - 10x at 1 h, still elevated 2-3x at 4 h) were seen in all brain regions examined (striatum hippocampys, cortex, diencephalon, midbrain, pons/medulla and cerebellum (although absolute levels were lower in more caudal brain regions.) DAAO mRNA levels were decreased by MK-801 at 1 h, but increased at 4 h (and absolute levels of DAAO mRNA were high in more caudal brain regions.) DAAO showed a more dose-dependent response. Chronic MK-801 (400 ug/kg for 14 days) also increased SR mRNA (2x in forebrain) but did not affect DAAO expression. There is little discussion of the possible mechanisms, aside from pointing out that MK-801 can induce c-fos and there is an AP-1 element in the SR promoter, and a CRE element in the DAAO promoter. | <b>MK-801: </b> Hasimoto et al. PMID 17109841 used qRT-PCR and HPLC to detect changes in SR and DAAO in adult rat brain after acute or chronic injections of MK-801, the non-competitive NR antagonist. Changes in mRNA were only seen at 400 ug/kg and above, Very large changes (5x - 10x at 1 h, still elevated 2-3x at 4 h) were seen in all brain regions examined (striatum hippocampys, cortex, diencephalon, midbrain, pons/medulla and cerebellum (although absolute levels were lower in more caudal brain regions.) DAAO mRNA levels were decreased by MK-801 at 1 h, but increased at 4 h (and absolute levels of DAAO mRNA were high in more caudal brain regions.) DAAO showed a more dose-dependent response. Chronic MK-801 (400 ug/kg for 14 days) also increased SR mRNA (2x in forebrain) but did not affect DAAO expression. There is little discussion of the possible mechanisms, aside from pointing out that MK-801 can induce c-fos and there is an AP-1 element in the SR promoter, and a CRE element in the DAAO promoter. | ||
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| + | ==Developmental changes== | ||
| + | |||
| + | <b>Aging</b> Mothet et al. PMID 16842499 found a decrease in d-serine and serine racemase expression in the aged hippocampus. (Don't have access to the original article) | ||
| + | |||
| + | <b>Postnatal</b> Puyal et al PMID 16739185 found low levels of d-serine (by chemiluminescent assay) at PO, then a surge of d-serine at P7 that declined until a very low level at P45. Western blot analysis of SR and DAAO showed that SR declined from birth to P45, while DAAO increased from P14 and later. Some nice colocalization by fluroescent double-labeling showed that d-serine was mostly in glial cells at P0-P21, but after P28 the d-serine was mostly in neurons. No discussion of what regulates SR or DAAO expression. | ||
=Probe for in situ hybridization= | =Probe for in situ hybridization= | ||
Revision as of 14:03, 20 February 2007
Regulation of SR gene expression
There are two approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, and by characterization across development.
Pharmacological manipulations
MK-801: Hasimoto et al. PMID 17109841 used qRT-PCR and HPLC to detect changes in SR and DAAO in adult rat brain after acute or chronic injections of MK-801, the non-competitive NR antagonist. Changes in mRNA were only seen at 400 ug/kg and above, Very large changes (5x - 10x at 1 h, still elevated 2-3x at 4 h) were seen in all brain regions examined (striatum hippocampys, cortex, diencephalon, midbrain, pons/medulla and cerebellum (although absolute levels were lower in more caudal brain regions.) DAAO mRNA levels were decreased by MK-801 at 1 h, but increased at 4 h (and absolute levels of DAAO mRNA were high in more caudal brain regions.) DAAO showed a more dose-dependent response. Chronic MK-801 (400 ug/kg for 14 days) also increased SR mRNA (2x in forebrain) but did not affect DAAO expression. There is little discussion of the possible mechanisms, aside from pointing out that MK-801 can induce c-fos and there is an AP-1 element in the SR promoter, and a CRE element in the DAAO promoter.
Developmental changes
Aging Mothet et al. PMID 16842499 found a decrease in d-serine and serine racemase expression in the aged hippocampus. (Don't have access to the original article)
Postnatal Puyal et al PMID 16739185 found low levels of d-serine (by chemiluminescent assay) at PO, then a surge of d-serine at P7 that declined until a very low level at P45. Western blot analysis of SR and DAAO showed that SR declined from birth to P45, while DAAO increased from P14 and later. Some nice colocalization by fluroescent double-labeling showed that d-serine was mostly in glial cells at P0-P21, but after P28 the d-serine was mostly in neurons. No discussion of what regulates SR or DAAO expression.
Probe for in situ hybridization
Primer sequences taken from Yoshikawa et al., PMID 16973158
Rat serine racemase. Genbank Ascension # NM_198757
PCR product Length: 295 bases (+22 bases for T7 promoter]
Forward Primer 664 to 683
- ATT GCA AGA AAC TGG CCA TC
Reverse Primer [with T7 promoter] 958 to 939
- [CTT AAT ACG ACT CAC TAT AGG G] TC AGC AGC GTA CAC CTT CAC
Amplification conditions (from Yoshikawa et al, PMID 15337321):
- 10-min predenaturation at 95 °C
- 15-s denaturation at 95 °C.
- 20-s at 58 °C for serine racemase
- followed by a 20-s extension at 72 °C.