483
edits
Changes
Jump to navigation
Jump to search
Line 1:
Line 1: + + − + + + + + + + + + + + + + Line 12:
Line 26: + + + + + + + + + + + + Line 32:
Line 58: + + + + + + + + + + + + + + + + + + + + + + + + + +
Fix IUPAC link (QMUL was incorrectly redirecting old URL).
Serine Racemase is the enzyme that interconverts between the two enantiomers of serine, D-serine and L-serine. (the nomenclature for chiral amino acids is well explained on the [https://www.qmul.ac.uk/sbcs/iupac/AminoAcid/AA3t5.html IUPAC site].
=Regulation of SR gene expression=
=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.
There are three approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, by characterization across development, and by pathological conditions.
==Pharmacological manipulations==
==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.
<b>MK-801: </b> Yoshikawa et al. PMID 15337321 using qt-PCR showed an increase in SR mRNA at 2-6h after systemic administration of MK-801 (0.4 mg/kg). Very nice description of qRT-PCR controls and calibration.
<b>Ketamine</b> Takeyama et al. PMID 16716293 using qRT-PCR showed an increase in SR mRNA and DAAO at 4 h (but not 2 h or 6h) after systemic ketamine (50 mg/kg) A dose response curve at the 4 h time point showed a dose dependent increse in SR and DAAO at 50 and 100 mg/kg
<b>Morphine</b> Yoshikawa et al. PMID 16256980 using qRT-PCR found a small increase(25-50%) in SR mRNA and DAAO at 4 h (but not 2 h or 6 h) after systemic morphine ((20 mg/kg, i.p.) A dose response curve at the 4 h time point showed no change after 10 mg/kg, but a dose dependent increase in sR and DAAO at 20 (2x) and 40 mg/kg (5x). Again, discussion focussed on morphine inducing AP-1 and CREB transcription factors.
<b>Beta-Amyloid</b> Wu et al. PMID 15285800
<b>inflammatory stimuli</b> Wu and Barger PMID 15681805
==Developmental changes==
==Developmental changes==
<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.
<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.
<b>Postnatal</b> Wang and Zhu PMID 14531937
==Pathological conditions==
<b>ischemia</b> Wang and Zhu PMID 15066209
<b>schizophrenia</b>
<b>alzheimers</b> Wu et al. PMID 15285800
=Probe for in situ hybridization=
=Probe for in situ hybridization=
:20-s at 58 °C for serine racemase
:20-s at 58 °C for serine racemase
:followed by a 20-s extension at 72 °C.
:followed by a 20-s extension at 72 °C.
=Probes for real time PCR=
Primer sequences taken from Yoshikawa et al. 2004, PMID 15337321 (did not use BioRad system)
Rat [actually mouse] serine racemase. Genbank Ascension # NM013761
Forward (bases 784-803)
:CCT GCA GTG ATA GCT GGA CA
Backward (bases 1052-1033)
:AAG CCA ATG CTG GAT TTG AC
Primer sequences taken from Yoshikawa et al. 2006, PMID 16973158 (did not use BioRad system)
Rat serine racemase mRNA (accession number NM198757)
forward primer 664 - 683
:ATT GCA AGA AAC TGG CCA TC
reverse primer, 958-939
:TCA GCA GCG TAC ACC TTC AC
[[Category:Taste_Aversion]]