Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells of different origins and strains

Pluripotent stem cell lines with similar phenotypes can be derived from both blastocysts (embryonic stem cells, ESC) and primordial germ cells (embryonic germ cells, EGC). Here, we present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Even in the differentiation-promoting conditions, these pluripotent cells showed the same general trends of gene expression changes regardless of their origin and genetic background. These data indicate that ESCs and EGCs are indistinguishable based on global gene expression patterns alone. On the other hand, a detailed comparison between a group of ESC lines and a group of EGC lines identified 20 signature genes whose average expression levels were consistently higher in ESC lines, and 84 signature genes whose average expression levels were consistently higher in EGC lines, irrespective of mouse strains. Similar analysis identified 250 signature genes whose average expression levels were consistently higher in a group of 129 cell lines, and 337 signature genes whose average expression levels were consistently higher in a group of C57BL/6 cell lines. Although none of the genes was exclusively expressed in either ESCs versus EGCs or 129 versus C57BL/6, in combination these signature genes provide a reliable separation and identification of each cell type. Differentiation-promoting conditions also revealed some minor differences between the cell lines. For example, in the presence of RA, EGCs showed a lower expression of muscle- and cardiac-related genes and a higher expression of gonad-related genes than ESCs. Taken together, the results provide a rich source of information about the similarities and differences between ESCs and EGCs as well as 129 lines and C57BL/6 lines. Such information will be crucial to our understanding of pluripotent stem cells. The results also underscore the importance of studying multiple cell lines from different strains when making comparisons based on gene expression analysis.     

Identification of a murine ICAM-1-specific peptide by subtractive phage library selection on cells

The ICAM-1 adhesion molecule is expressed selectively at low levels on endothelial cells but is strongly upregulated in dysfunctional endothelial cells associated with inflammation, cancer, and atherogenesis. Using COS-7 cells transfected with murine ICAM-1 (mICAM-1) as a target receptor, a phage display library was screened. Clones were selected by elution with a mAb specific for a functional epitope of ICAM-1 and a novel peptide sequence binding to the extracellular domain of mICAM-1 was identified that can potentially be used as a targeting vector aimed at dysfunctional endothelium. We further showed that the targeting specificity of the peptide was retained following its incorporation at the N terminal end of a large chimeric protein. Moreover, this chimeric protein containing the mICAM-1-specific sequence was found to inhibit ICAM-1-mediated intercellular adhesion during antigen presentation. Taken together, these results demonstrate the potential for improving the cell-selectivity and properties of therapeutical agents toward targeting adhesion molecules involved in cell-cell interactions.     

Serum methionine metabolites are risk factors for metastatic prostate cancer progression

Background

Clinical decision for primary treatment for prostate cancer is dictated by variables with insufficient specificity. Early detection of prostate cancer likely to develop rapid recurrence could support neo-adjuvant therapeutics and adjuvant options prior to frank biochemical recurrence. This study compared markers in serum and urine of patients with rapidly recurrent prostate cancer to recurrence-free patients after radical prostatectomy. Based on previous identification of urinary sarcosine as a metastatic marker, we tested whether methionine metabolites in urine and serum could serve as pre-surgical markers for aggressive disease.

Methodology/Principal Findings

Urine and serum samples (n = 54 and 58, respectively), collected at the time of prostatectomy were divided into subjects who developed biochemical recurrence within 2 years and those who remained recurrence-free after 5 years. Multiple methionine metabolites were measured in urine and serum by GC-MS. The role of serum metabolites and clinical variables (biopsy Gleason grade, clinical stage, serum prostate specific antigen [PSA]) on biochemical recurrence prediction were evaluated. Urinary sarcosine and cysteine levels were significantly higher (p = 0.03 and p = 0.007 respectively) in the recurrent group. However, in serum, concentrations of homocysteine (p = 0.003), cystathionine (p = 0.007) and cysteine (p<0.001) were more abundant in the recurrent population. The inclusion of serum cysteine to a model with PSA and biopsy Gleason grade improved prediction over the clinical variables alone (p<0.001).

Conclusions

Higher serum homocysteine, cystathionine, and cysteine concentrations independently predicted risk of early biochemical recurrence and aggressiveness of disease in a nested case control study. The methionine metabolites further supplemented known clinical variables to provide superior sensitivity and specificity in multivariable prediction models for rapid biochemical recurrence following prostatectomy.     

Reactions of ketones with aromatics in acid media. The effect of trifluoromethyl groups and the acidity media. A theoretical study

The reactions of acetone, 2,2,2-trifluoroacetone and hexafluoroacetone in methanesulfonic (MSA) and triflic acids (TFSA) with benzene have been studied at M06-2X/6-311+G(d,p) level using cluster-continuum model, where the carbonyl group is explicitly solvated by acid molecules. The introduction of a trifluoromethyl group into the ketone structure reduces the activation energy of the tetrahedral intermediates formation due to an increase of the electrophilicity of the carbonyl group and raises the activation and the reaction energies of the C-O bond cleavage in formed carbinol due to the destabilization of the corresponding carbocation. The introduction of the second trifluoromethyl group inhibits the hydroxyalkylation reaction due to a very strong increase of the reaction and activation energies of the C-O bond cleavage which becomes the rate determining step. The most important catalytic effect of TFSA compared to MSA is not the protonation of the ketone carbonyl, but the reduction of the activation and reaction energies of the carbinol C-O bond cleavage due to better protosolvation properties. Even for TFSA no complete proton transfer to carbonyl oxygen has been observed for free ketones. Therefore, the protonation energies of free ketones cannot be considered as a measure of ketone reactivity in the hydroxyalkylation reaction.

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Folate in demethylation: The crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate

Dimethylglycine dehydrogenase (DMGDH) is a mammalian mitochondrial enzyme which plays an important role in the utilization of methyl groups derived from choline. DMGDH is a flavin containing enzyme which catalyzes the oxidative demethylation of dimethylglycine in vitro with the formation of sarcosine (N-methylglycine), hydrogen peroxide and formaldehyde. DMGDH binds tetrahydrofolate (THF) in vivo, which serves as an acceptor of formaldehyde and in the cell the product of the reaction is 5,10-methylenetetrahydrofolate instead of formaldehyde. To gain insight into the mechanism of the reaction we solved the crystal structures of the recombinant mature and precursor forms of rat DMGDH and DMGDH–THF complexes. Both forms of DMGDH reveal similar kinetic parameters and have the same tertiary structure fold with two domains formed by N- and C-terminal halves of the protein. The active center is located in the N-terminal domain while the THF binding site is located in the C-terminal domain about 40 Å from the isoalloxazine ring of FAD. The folate binding site is connected with the enzyme active center via an intramolecular channel. This suggests the possible transfer of the intermediate imine of dimethylglycine from the active center to the bound THF where they could react producing a 5,10-methylenetetrahydrofolate. Based on the homology of the rat and human DMGDH the structural basis for the mechanism of inactivation of the human DMGDH by naturally occurring His109Arg mutation is proposed.