MAB3872 Sigma-AldrichAnti-PPAR γ Antibody, isoform 1&2

To characterize the distribution of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the substantia nigra of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated hemiparkinsonian monkeys, in order to validate PPAR-gamma as a target for neuroprotection.Immunohistochemical analysis of PPAR-gamma expression was performed in the substantia nigra and other select brain regions of fifteen rhesus monkeys including controls (n = 3), hemiparkinsonian necropsied after 3 (n = 5) or 12 (n = 3) months after MPTP, and animals who received MPTP+5 mg/kg of the PPAR-gamma agonist pioglitazone (n = 4).PPAR-gamma expression was prominent in the subthalamic nucleus, oculomotor nucleus, ventral tegmental nucleus, and to a lesser extent, in the putamen; 3 or 12 months after MPTP, only the lesioned putamen had increased PPAR-gamma. Stereological cell quantification in normal subjects showed that approximately 50% of neurons in the substantia nigra pars compacta (SNpc) expressed PPAR-gamma. After MPTP, there was a significant loss of dopaminergic neurons in the ipsilateral SNpc and the actual numbers of tyrosine hydroxylase (TH) and PPAR-gamma cells were not significantly different at either time point. Pioglitazone dosing protected TH-positive neurons, closely matching the number of PPAR-gamma expressing cells in the ipsilateral SNpc. Nigral immunofluorescence verified colocalization of PPAR-gamma in neurons.These results demonstrate that PPAR-gamma is expressed in the SNpc and putamen of nonhuman primates and, that the dopaminergic nigral neurons expressing PPAR-gamma are more likely to survive neurotoxin challenge after ligand activation by pioglitazone, therefore providing neuroanatomical validation for the use of PPAR-gamma agonists in Parkinson's disease (PD).

Transcription factor IID (TFIID) activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit TAF4 mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs) to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of TAF4 transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.

Adipose tissue has been shown to contain adult mesenchymal stem cells that have therapeutic applications in regenerative medicine. There is evidence that the ability of adipose precursor cells to grow and differentiate varies among fat depots and changes with age. Defining these variations in cell function and molecular mechanisms of adipogenesis will facilitate the development of cell-based therapies. We compared cells harvested from 5 different subcutaneous (SC) adipose depots in 12 female patients classified into 3 age ranges (25-30, 40-45, and 55-60 years old). Capacity for differentiation of isolated adipose-derived stem cells (ASCs) with and without ciglitazone, a strong peroxisome proliferatoractivated receptors (PPAR)-gamma agonist, was assessed in vitro. ASCs were also characterized by lipolytic function, proliferation, and sensitivity to apoptosis. Additionally, PPAR-gamma-2 protein expression was determined. We observed a difference in the apoptotic susceptibility of ASCs from various SC depots, with the superficial abdominal depot (above Scarpas layer) significantly more resistant to apoptosis when compared with the 4 other depots. We have also demonstrated that a PPAR-gamma agonist aids in the induction of differentiation in cells from all depots and ages. Although sensitivity to apoptosis was linked to anatomic depot, differences in cell proliferation were related primarily to age. Stimulated free glycerol release has been shown to be highest in the arm depot. The arm depot has also consistently shown expression of PPAR-gamma-2 with and without a PPAR-gamma agonist. Younger patients have increased PPAR-gamma-2 expression in all depots, whereas the older patients have consistent elevated expression only in the arm and thigh depots. We have shown there is variability in function of ASCs that have been harvested from different SC depots. Additionally, we have shown age-related changes in function. These data will help select patients and cell harvest sites most suitable for tissue engineering therapies.

The goal of this study was to synthesize and evaluate in vivo the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist (11)C-GW7845 ((S)-2-(1-carboxy-2-{4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl}ethylamino)benzoic acid methyl ester) ((11)C-compound 1). PPARgamma is a member of a family of nuclear receptors that plays a central role in the control of lipid and glucose metabolism. Compound 1 is an analog of tyrosine (inhibitor constant, 3.7 nmol/L), which is an inhibitor of experimental mammary carcinogenesis. METHODS: Protection of the carboxylic acid moiety of compound 1 was effected by treatment with N,N-dimethylformamide di-tert-butyl acetal to provide compound 2. Hydrolysis of the carbomethoxy group of compound 2 provided the benzoic acid (compound 3) that served as an immediate precursor to radiolabeling. Compound 3 underwent treatment with (11)C-methyl iodide followed by high-performance liquid chromatography to produce a radioactive peak sample that coeluted with a standard sample of compound 1. Analysis of biodistribution was undertaken by injecting male CD-1 mice via the tail vein with 6.03 MBq (163 microCi, 2.55 microg/kg) of (11)C-compound 1. To determine the tumor uptake of the radiotracer, 6 female SCID mice bearing MCF-7 xenografts were injected via the tail vein with 10.5 MBq (283 microCi, 0.235 microg/kg) of (11)C-compound 1. RESULTS: (11)C-Compound 1 was synthesized at an 8% radiochemical yield in 29 min with an average specific radioactivity of 1,222 GBq/micromol (33,024 mCi/micromol; n = 6) at the end of synthesis. Spleen (target)-to-muscle uptake and tumor-to-muscle uptake ratios were 3.1 and 1.5, respectively, but this uptake could not be blocked with unlabeled compound 1 at 2 mg/kg. CONCLUSION: Further structural modification, perhaps to generate a less lipophilic tyrosine analog, will be necessary to enable receptor-mediated PPARgamma imaging by this class of agents.