Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 3. Fractionation of human peripheral blood mononuclear cells (PBMCs) from lean normal donors suggested that all sub-populations of PBMCs, including T (CD3+) cells and monocytes (CD14+), probably expressed approximately the same level of p27 as unfractionated PBMCs. Panels a to d show the results of the western immunoblot analysis of p27, total 4E-BP1 and total MNK1 in unfractionated PBMCs obtained from lean normal African Americans (AAs) (n = 3) and lean normal Caucasian Americans (CAs) (n = 3). Panels a to d also include the results of the western immunoblot analysis of p27, total 4E-BP1 and total MNK1 in T (CD3+) cells (n = 3) and monocytes (CD14+) (n = 3) isolated from unfractionated PBMCs us- ing Miltenyi’s AutoMAC Simmunomagnetic cell sorting protocols (ZenBio Inc., Research Triangle Park, North Carolina, USA and Miltenyi Biotech Inc., Auburn, California, USA). The unfractionated PBMCs used to isolate T (CD3+) cells and monocytes (CD14+) were obtained from the mixtures of lean normal African and Caucasian Americans (AAs and CAs). decreased—by about 40%—relative to the unfractionated PBMCs. Surprisingly, this extent of the decrease in the expression of MNK1 in the T (CD3+) cells did not interfere with the expression of the p27 itself in the same T (CD3+) cells. In summary, the results presented above suggested that all sub-populations of PBMCs, including T (CD3+) cells and monocytes (CD14+), probably expressed the same level of p27 as unfractionated PBMCs in either lean normal African or Caucasian Americans.

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Fractionation, Western Blot, Isolation, FACS, Expressing

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 4. Regarding the results presented in Figure 3, panels a and b present the hypothetical upstream molecular signaling pathways-inclu- ding 4E-BP1 and MNK1-of the expression of p27 in human PBMCs ob- tained from lean normal donors as compared to those in (1) the livers from genetically homozygous obese diabetic rats or mice [2] and (2) hu- man breast cancer cell lines in vitro [12]-[14]. press decreased—by about 40%—relative to the unfractionated PBMCs. Surprisingly, this extent of the decrease in the expression of MNK1 in the T (CD3+) cells did not interfere with the expression of the p27 itself in the same T (CD3+) cells. In summary, the results presented above suggested that all sub-populations of PBMCs, including T (CD3+) cells and monocytes (CD14+), probably expressedthe same level of p27 as unfractionated PBMCs in either lean normal African or Caucasian Americans.

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Protein-Protein interactions, Expressing, In Vitro

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 5. Severe depression—by over 95%—of the expression of p27 in the peripheral blood mononuclear cells (PBMCs) obtained from obese type 2 diabetic African Americans (AAs) was probably caused primarily by 1) the severe depression of the MNK1 upstream molecular signaling pathways of the expression of p27 and secondarily by 2) the moderate increase in the phosphorylation of 4E-BP1 upstream molecular signaling pathways of the expression of p27. Panels a to h present the results of the western immunoblot analysis of (Panels a and b) p27 (n = 3), (Panels a and c) total 4E-BP1 (n = 3), (Panels a and d) 4E-BP1 phosphorylated at Thr 37/46 (n = 3), (Panels a and e) total MNK1 (n = 3), (Panels a and f) MNK1 phosphorylated at Thr 197/202 (n = 3), (Panels a and g) total eIF4E (n = 3), and (Panels a and h) eIF4E phosphorylated at Ser 209 (n = 3). lean normal African and Caucasian Americans (AAs and CAs) and Panel b of Figure 6 for primarily obese type 2 diabetic African Americans (AAs). Stated briefly, the severe depression of the expression of p27 in the PBMCs from obese type 2 diabetic African Americans was caused primarily by 1) the severe depression of the MNK1 upstream molecular signaling pathways and secondarily by 2) the moderate increase in the phosphoryla-

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Expressing, Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 6. Regarding the results presented in Figure 5, panels a and b present the hypothetical MNK1 and 4E-BP1 upstream mo- lecular signaling pathways of the expression of p27 in the PBMCs obtained from lean normal African and Caucasian Americans (AAs nd CAs) (Panel (a)) and primarily obese type 2 diabetic Af- rican Americans (AAs) (Panel (b)). tion of the 4E-BP1 upstream molecular signaling pathways of the expression of p27. Panels a, e and f of Figure 5 show that the expressions of total MNK1 and MNK1 phosphorylated at Thr 197/202 in the PBMCs from obese type 2 diabetic African Americans (AAs) were severely depressed-by ap- proximately 82.2% and 83.3%, respectively-relative to the lean normal African Americans (AAs). The results presented above in the previous section indicated that the depression of MNK1 by approximately 40% did not significantly decrease the expression of p27. The results presented here in the Panels a and b of Figure 5 sug- gested that the depression of total MNK1 and MNK1 phosphorylated at Thr 197/202 by over 80% was associ- ated with the severe depression of the expression of p27—by over 95%—in the PBMCs from obese type 2 dia- betic African Americans (AAs) relative to the lean normal African Americans (AAs). It could have been that the depression of the expression of MNK1 by 40% was not enough to severely depress the expression of p27. Lastly, the results presented in the Panels a, g and h of Figure 5 indicated that the expression of the total eIF4E was significantly increased but the expression of the eIF4E phosphorylated at Ser 209 was severely de- pressed in the PBMCs from obese type 2 diabetic African Americans (AAs). This observation suggested that the eIF4E unphosphorylated at Ser 209 might protect the 5’-N7-methylguanosine cap (5’-m7G cap)in the 5’-untran- slated region (5’-UTR) of the p27 mRNA, thereby severely depressing the inherent cap-independent translation initiation of p27 mRNA into p27 protein in obese type 2 diabetic African Americans [13] [15] [16].

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Protein-Protein interactions, Expressing