Mitochondrial carrier homolog 2 (MTCH2) is a regulator of apoptosis, mitochondrial dynamics, and metabolism. Loss of MTCH2 results in mitochondrial fragmentation, an increase in whole-body energy utilization, and protection against diet-induced obesity. In this study, we used temporal metabolomics on HeLa cells to show that MTCH2 deletion results in a high ATP demand, an oxidized cellular environment, and elevated utilization of lipids, amino acids, and carbohydrates, accompanied by a decrease in several metabolites. Lipidomics analysis revealed a strategic adaptive reduction in membrane lipids and an increase in storage lipids in MTCH2 knockout cells. Importantly, MTCH2 knockout cells showed an increase in mitochondrial oxidative function, which may explain the higher energy demand. Interestingly, this imbalance in energy metabolism and reductive potential triggered by MTCH2-deletion prevents NIH3T3L1 preadipocytes from differentiating into mature adipocytes, an energy consuming reductive biosynthetic process. In summary, the loss of MTCH2 leads to increased mitochondrial oxidative activity and energy demand, creating a catabolic and oxidative environment that fails to fuel the anabolic processes required for lipid accumulation and adipocyte differentiation.

Despite extensive research, our understanding of the rules according to which cis-regulatory sequences are converted into gene expression is limited. We devised a method for obtaining parallel, highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ∼10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control.

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 mu M) was similar to 80% compared to allicin treatment alone (similar to 50%). Also M-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 mu M, 30 min) the redox state for 2GSH/oxidized glutathione shifted from E-GSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 mu M, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/ 106 cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status. (C) 2008 Elsevier Inc. All rights reserved.

The possibility that mature lymphocytes play a role in the regulation of human T cell development was studied in the experimental model of fetal thymus organ cultures (FTOC), by reconstituting lymphocyte-depleted murine fetal thymus (FT) lobe with cells isolated from human umbilical cord blood (CB). Cultures were incubated with human cytokines (IL-7, FLT-3 ligand and Steel Factor), or remained untreated. When CD4+, or CD8+ CB cells, were co-cultured with FT explants, they expanded and maintained their original phenotypic markers, with no significant effect of the cytokines. Cultures of human hematopoietic stem cells (CD34+) gave rise to CD4+CD8− cells, which were mainly CD3−, with no indication of further intermediate developmental stages. However, a limited number of CD4+CD8+ (double positive [DP]) cells were detected when the CD34+ cells were co-cultured with CD4+ cells from the same CB samples. In contrast, FT with unseparated CB cells resulted in the different CD4/CD8 subsets, and their numbers increased in the presence of cytokines. The appearance of DP cells depended on the presence of either CD4+ or CD8+ cells in the cultured CB samples. Hence, DP cells were not detected when the CB was depleted of CD4+ and CD8+ cells (“depCB”) before culture, and they appeared when depCB were co-cultured with either CD4+ or CD8+ cells. In contrast, CD4+ cells inhibited the development of CD8+CD3+ cells, and this was most pronounced in the absence of the cytokines. There was no symmetrical down-regulatory effect of CD8+ cells on the development of CD4+CD3+ cells. Addition of IL-15 to the cytokine mixture led to an increased proportion of CD56+ cells in cultures of CD34+ cells. The presence of CD4+, and not CD8+ cells, interfered with this process. Our results thus imply differential effects of CD4+ and CD8+ cells on thymocytopoiesis.

Rabbit and mouse anti-Torpedo acetylcholine receptor antibodies cross-reacted partially with the highly phosphorylated protein, phosvitin. We have selected an anti-Torpedo acetylcholine receptor monoclonal antibody which binds specifically to phosvitin; this binding is inhibited by acetylcholine receptor. These findings suggest that a phosphorylated amino acid residue may be a part of the determinant on the acetylcholine receptor recognized by this monoclonal antibody.