On acetyl-CoA as a gauge of cellular metabolic state.

Many activities within a cell must be intimately coordinated with its metabolic state. Understanding these connections to metabolism is critical to deciphering the regulation of a variety of cellular processes. However, despite intensive research in these areas, the precise mechanisms by which a cell monitors its metabolic state remain controversial and poorly understood. Herein, we discuss the thesis that intracellular levels of the metabolite acetyl-CoA represent a critical gauge of cellular metabolic state, which is cleverly monitored by the cell through dynamic acetylation modifications to direct a variety of outputs connected to energy metabolism, cell growth, and survival.

The yeast metabolic cycle: insights into the life of a eukaryotic cell.

The budding yeast Saccharomyces cerevisiae undergoes robust oscillations in oxygen consumption during continuous growth under nutrient-limited conditions. Comprehensive microarray studies reveal that more than half of the yeast genome is expressed periodically as a function of these respiratory oscillations, thereby specifying an extensively orchestrated program responsible for regulating numerous cellular outputs. Here, we summarize the logic of the yeast metabolic cycle (YMC) and highlight additional cellular processes that are predicted to be compartmentalized in time. Certain principles of temporal orchestration as seen during the YMC might be conserved across other biological cycles.

Mechanisms of chondrocyte adhesion to cartilage: role of beta1-integrins, CD44, and annexin V.

The initial adhesion of transplanted chondrocytes to surrounding host cartilage may be important in the repair of articular defects. Adhesion may position cells to secrete molecules that fill the defect and integrate repair tissue with host tissue. While chondrocytes are known to become increasingly adherent to cartilage with time, the molecular basis for this is unknown. The objective of this study was to investigate the role of beta1-integrin, CD44, and annexin V receptors in chondrocyte adhesion to cartilage. Chondrocytes were cultured in high density monolayer, released with trypsin, and allowed to recover in suspension for 2 h at 37 degrees C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed beta1-integrins, CD44, and annexin V. In a rapid screening assay to assess chondrocyte adhesion to cartilage, cell detachment decreased from 79% at 10 min following transplantation to 10% at 320 min. Treatment of cells with a monoclonal antibody to block beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel-plate shear flow adhesion assay showed that blocking beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls at each level of applied shear (0-70 Pa). In both assays, treatment of cells with reagents that block CD44 (hyaluronan oligosaccharides or monoclonal Ab IM7) or annexin V (polyclonal Ab #8958) had no detectable effect on adhesion. With cartilage treated with chondroitinase ABC, blocking beta1-integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, beta1-integrins appear to mediate chondrocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical implications by allowing cell manipulations or matrix treatments to enhance chondrocyte adhesion and retention at a defect site.

Biochemical basis of oxidative protein folding in the endoplasmic reticulum.

The endoplasmic reticulum (ER) supports disulfide bond formation by a poorly understood mechanism requiring protein disulfide isomerase (PDI) and ERO1. In yeast, Ero1p-mediated oxidative folding was shown to depend on cellular flavin adenine dinucleotide (FAD) levels but not on ubiquinone or heme, and Ero1p was shown to be a FAD-binding protein. We reconstituted efficient oxidative folding in vitro using FAD, PDI, and Ero1p. Disulfide formation proceeded by direct delivery of oxidizing equivalents from Ero1p to folding substrates via PDI. This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfide bonds.

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Protein footprinting at cysteines: probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II.

Cysteine-substitution mutants of yeast DNA topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9. For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein. For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent. The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA topoisomerase II.