Sarah+Wang

Journal Article Summary 1 This lab tried to determine if space radiation/and or microgravity has any effect on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space is gamma s 13 and NC 4 amoebae strains. Based on their previous research, amoebae of Dictyosterium discoideum grew and differentiated to fruiting bodies in space. Gamma s 13 strain is a radiation-sensitive mutant, and NC4 is the parent strain. They put the amoebae into cell culture kits and one cell culture kit was taken into space on a space shuttle (STS-84) and 3 cell culture kits were left on earth as controls. They used a video camera to record the observations 7 days after activation onboard. After the spores arrived back on earth, they allowed them to germinate and measured its mutation frequency. They found that there were hardly any abnormal fruiting bodies in the space environment. There was a delay in the germinations of spores formed in space as compared with those formed on the ground in the case of NC4. They also found a depression of spore formation in NC4 and no spore formation in gamma s 13. They speculate that space radiation and microgravity may affect some stages of development, especially cell differentiation from amoebic cells to spores.

Takahashi, A., K. Ohnishi, et al. (2001). "Differentiation of Dictyostelium discoideum vegetative cells into spores during earth orbit in space." Advances in Space Research 28(4): 549-553.

Journal Article Summary 2 Primordial germ cells (PGCs) generate germ cells to transmit the gene from generation to generation. Defects in PGC development often cause infertility. In the mouse embryo, PGCs undergo proliferation during a 5-day period. Dr. Means’ lab showed that a peptidyl-prolyl isomerase, Pin1, is involved in the regulation of mammalian PGC proliferation. They had Pin1 knockout mice and wild type mice, and they crossbred all of them. They used histology and immunohistochemistry to detect PGCs and Pin1 in various postnatal testes and ovaries. They also looked for PGC in embryos, and they did an apoptosis assay since the lack of Pin1 has been reported to cause mitotic arrest or apoptosis in budding yeast. They found that both male and female Pin1-deficient mice were infertile when mated together, and reduced fertility when mated with wild type mice. They also found that PGCs are compromised in the absence of Pin1. They also found that Pin1 is in fact expressed in PGCs through their embryonic development, and they found the PGC proliferation in the first 5 days was decreased in Pin1-deficient embryos. As for the apoptosis assay, no mitotic arrest or cell death was found in the absence of Pin1, which contradicted earlier results. They proposed that Pin1-deficient PGCs undergo insufficient cell cycle progression, which leads to a longer cell cycle, so PGCs divide less frequently during the 5 day proliferation period. This results in a severely reduced number of germ cells. What is unknown is how Pin1 regulates PGC proliferation and cell cycle progression. Also, it was recently found that Par14, another prolyl-isomerase, may be a companion for Pin1, so they would like to obtain some Par14 antibodies to study if PGCs lack Par14.

Atchison, F.W., Capel, B., Means, A.R. (2003) Pin1 regulates the timing of the mammalian germ cell proliferation. Development 130:3579-3586

4/9/09 Pin1 has been suggested as a molecular timer because Pin1-null mice had tissue and neuron defects, decrease in fertility (impairment of primordial germ cells), growth defects (resisted cell-cycle re-entry in response to growth factor), and increased time to complete nuclear division cycle. The problem is that several papers show that Pin1 acts as tumor suppressor, while several others show that low Pin1 level slows down the cell cycle and leads to a more selective growth disadvantage, which makes cells more vulnerable to oncogenesis. For example, some studies show that inhibition/depletion of Pin1 can decrease cancer susceptibility and increased Pin1 levels/activity may promote growth of tumors. Also, Pin1 is overexpressed in human mammary tumors, and without Pin1, mice failed to undergo epithelial expansion. The absence of Pin1 has also been shown to prevent expression of cyclin D1 (an oncogene) and the development of breast cancer in mice. On the other hand, Pin1 regulates MYC, cyclin E, and NCOA3 levels by degradation, and absence of Pin1 can facilitate formation of tumors in mice. These conflicting studies may be because each experiment used mice of different genetic background. For instance, studies that suggest Pin1 may promote tumorgenesis were carried out in mice of mixed genetic backgrounds, while studies that implicate Pin1 as a tumor suppressor were carried out in an isogenic background. To clarify the data, identical experiments are needed to be conducted on two lines of genetically altered mice. Right now, no small-molecule inhibitor of Pin1 has been developed, and overexpression of Pin1 and inhibition of Pin1 can both cause unwanted side effects. Therefore, more research is needed on its role in tumorgenesis before Pin1 can be presented as an effective target for drug development.

Yeh, E.S. and Means, A.R. (2007) Pin1, the cell cycle and cancer. Nat Rev Cancer 7:381-388

4/23/09 Journal Article Summary 4 Membrane microdomains (rafts) are thought to play critical roles in biological functions such as signal transduction, cytoskeletal organization, lipid sorting, and protein trafficking/recycling. Rafts are mainly composed of detergent-resistant membranes. Detergent-resistant membranes, DRMs, are enriched in cholesterol and sphingolipids that have saturated hydrocarbon chains while detergent-soluble membranes, DSMs, are enriched in membrane phospholipids that contain unsaturated hydrocharbon chains. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a lipid that, when present in rafts, is critical to cell functions such as cell signaling, enzyme activation, regulation of potassium channels, membrane trafficking, and regulation of the actin cytoskeleton. However, it is unfavorable for PIP2 to reside in rafts because it has unsaturated hydrocarbon chains. GAP-43, a cytoplasmic protein, contains clusters of basic amino acid residues (“basic effector domains”) that laterally sequester PIP2 in the plane of the bilayer, and it is an abundant raft-associated protein due to dual palmitoylation (convalent attachment of fatty acids) of its N-terminus. GAP-43P is a peptide that contains both the N-terminus and the effector domain. The lab conducted GAP-43 peptide binding, detergent extractions, thin layer chromatography, and confocal microscopy. They found that without the peptide, PIP2 was mainly found in DSMs (nonraft microdomains), but with acylated GAP-43P, PIP2 distribution in the bilayer shifted into rafts. Therefore, acylation changed the free energy transfer of PIP2 from DSMs to DRMs, resulting a change from a positive delta G to a negative delta G with the energy difference of -1.32 kcal/mol. The lab suggests that palmitoylated GAP-43P bound primarily to DRMs, and the effector domain of GAP-43P bound to PIP2, therefore sequestering this lipid to DRMs.

Tong, Jihong, Lam Nguyen, Adriana Vidal, Sidney A. Simon, J.H. Pate Skene, and Thomas J. McIntosh. “Role of GAP-43 in Sequestering Phosphatidylinositol 4,5-Bisphosphate to Raft Bilayers.” Biophysical Journal 94, no. 1 (January 2008): 125-133.