Abnormal T cell activation and cell death underlie the pathology of systemic lupus erythematosus. Although mitochondrial hyperpolarization (MHP) represents an early and reversible checkpoint of T cell activation and apoptosis, lupus T cells exhibit persistent MHP. NO has recently been recognized as a key signal of mitochondrial biogenesis and mediator of MHP in human T lymphocytes. In this study, we show that persistent MHP was associated with increased mitochondrial mass (+47.7 ± 2.8%; p = 0.00017) and increased mitochondrial (+21.8 ± 4.1%; p = 0.016) and cytoplasmic Ca2+ content in T cells from 19 systemic lupus erythematosus patients with respect to 11 control donors (+38.0 ± 6.4%; p = 0.0023). Electron microscopy revealed that lupus lymphocytes contained 8.76 ± 1.0 mitochondria, while control donors contained 3.18 ± 0.28 mitochondria per cell (p = 0.0009). Increased mitochondrial mass in T cells was associated with 2.08 ± 0.09-fold enhanced NO production by lupus monocytes (p = 0.0023). Activation of T cells through the TCR initiates a biphasic elevation in cytosolic free Ca2+ concentration, a rapid initial peak observed within minutes, and a plateau phase lasting up to 48 h. In response to CD3/CD28 costimulation, rapid Ca2+ fluxing was enhanced while the plateau phase was diminished in lupus T cells. NO-induced mitochondrial biogenesis in normal T cells enhanced the rapid phase and reduced the plateau of Ca2+ influx upon CD3/CD28 costimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Mitochondria constitute major Ca2+ stores and NO-dependent mitochondrial biogenesis may account for altered Ca2+ handling by lupus T cells.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1 -/-and Taldo1 +/-mice spontaneously developed HCC, and Taldo1 -/-mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1 -/-livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced β-catenin phosphorylation and enhanced c-Jun expression in Taldo1 -/-livers reflected adaptation to oxidative stress. Taldo1 -/-hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1 -/-mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

Fertility of spermatozoa depends on maintenance of the mitochondrial transmembrane potential (⌬ m), which is generated by the electron-transport chain and regulated by an oxidation-reduction equilibrium of reactive oxygen intermediates, pyridine nucleotides, and glutathione (GSH). Here, we report that male mice lacking transaldolase (TAL) ؊/؊ are sterile because of defective forward motility. TAL ؊/؊ spermatozoa show loss of ⌬m and mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca 2؉ stores; thus, diminished mitochondrial mass accounts for reduced Ca 2؉ fluxing, defective forward motility, and infertility. Reduced forward progression of TAL-deficient spermatozoa is associated with diminished mitochondrial reactive oxygen intermediate production and Ca 2؉ levels, intracellular acidosis, and compensatory down-regulation of carbonic anhydrase IV and overexpression of CD38 and ␥-glutamyl transferase. Microarray analyses of gene expression in the testis, caput, and cauda epididymidis of TAL ؉/؉ , TAL ؉/؊ , and TAL ؊/؊ littermates confirmed a dominant impact of TAL deficiency on late stages of sperm-cell development, affecting the electrontransport chain and GSH metabolism. Stimulation of de novo GSH synthesis by oral N-acetyl-cysteine normalized the low fertility rate of TAL ؉/؊ males without affecting the sterility of TAL ؊/؊ males. Whereas TAL ؊/؊ sperm failed to fertilize TAL ؉/؉ oocytes in vitro, sterility of TAL ؊/؊ sperm was circumvented by intracytoplasmic sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the nucleus and DNA integrity. Collectively, these data reveal an essential role of TAL in sperm-cell mitochondrial function and, thus, male fertility. F orward motility and fertility of spermatozoa depend on production of reactive oxygen intermediates (ROIs) (1) and maintenance of the mitochondrial transmembrane potential (⌬ m ) (2, 3). ⌬ m is generated by the electron-transport chain and subject to regulation by an oxidation-reduction equilibrium of ROI, pyridine nucleotides (NADH͞NAD ϩ NADPH͞NADP), and reduced glutathione (GSH) (4). In turn, NADPH, a reducing equivalent required for biosynthetic reactions and regeneration of GSH from its oxidized form, is produced by the pentose phosphate pathway (PPP) (5). The PPP was originally formulated based on metabolites and enzymes detected in yeast (6). Thus, PPP comprises two separate oxidative and nonoxidative phases. Enzymes of the oxidative phase, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase, can generate both ribose 5-phosphate (R5P) and NADPH. Although enzymes of the nonoxidative phase, transketolase (TK) and transaldolase (TAL), can convert R5P into glucose 6-phosphate (G6P) for the oxidative phase, and, thus, indirectly, these enzymes can also contribute to the generation of NADPH, the significance of the nonoxidative branch is less well established. Certain organisms (7,8) and mammalian tissu...

TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.

Melanocytes have not been described in the pituitary of mammals or in the meninges of the rat. In this paper, we report the presence of the cluster of melanocytes in the intermediate lobe of the pituitary and around the median eminence of the hypothalamus forming an 'infundibulo-hypophysial circle', and also describe the characteristics of meningeal melanocytes in Zucker rats. In the leptomeninges, numerous melanocytes were found on the ventrolateral surface of cerebral hemisphere in the area of the middle cerebral artery. Pigment granules were also observed in the surrounding tissue outside the melanocytes as well as incorporated in the cytoplasm of neural and epithelial cells. Electron microscopy revealed that melanosomes in hypophysial and meningeal melanocytes were in different (II-IV) stages of maturity. In the leptomeninges of Zucker rats, HMB-45 immunoreactivity was found in round non-melanosome-containing cells, while no HMB-45 reaction was found in the leptomeninges of the albino rat. We conclude that both obese and lean Zucker rats possess functionally active melanocytes in the meninges and the pituitary and transfer pigment granules to neighboring cells. The distributions of melanocytes in proximity to blood vessels in the leptomeninges and in the 'infundibulo-hypophysial circle' suggest an endocrine secretory function.