The immunologically active site of prothymosin α is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients

Prothymosin α (proTα) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTα is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTα presenting maximum immunomodulatory activity. Calf thymus proTα was trypsinised, and the five fragments produced (spanning residues 1–14, 21–30, 31–87, 89–102 and 103–109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTα(89–102) and proTα(103–109) significantly fortified healthy donor-lymphocytes’ immune responses to levels comparable to those induced by intact proTα. These effects were more pronounced in cancer patients, where peptides proTα(89–102) and proTα(103–109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTα. ProTα(1–14), proTα(21–30) and proTα(31–87) marginally upregulated lymphocyte activation. This is the first report showing that proTα’s immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTα fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.     

Glycosylation of mouse and human immune cells: insights emerging from N-glycomics analyses

N-glycans are key players mediating cell-cell communication in the immune system, interacting with glycan-binding proteins. In the present article, we discuss key themes that are emerging from the structural analysis of complex-type N-linked glycans from human and murine immune cell lines, employing high-sensitivity MALDI (matrix-assisted laser desorption ionization)-TOF (time-of-flight) MS technology. Particular focus is given to terminal epitopes, the abundance of multiply branched N-glycans and how glycosylation can affect human health in diseases such as congenital neutropenia and glycogen storage disease.     

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.     

Assessment of locomotion in chlorine exposed mice by computer vision and neural networks

Assessment of locomotion following exposure of animals to noxious or painful stimuli can offer significant insights into underlying mechanisms of injury and the effectiveness of various treatments. We developed a novel method to track the movement of mice in two dimensions using computer vision and neural network algorithms. By using this system we demonstrated that mice exposed to chlorine (Cl(2)) gas developed impaired locomotion and increased immobility for up to 9 h postexposure. Postexposure administration of buprenorphine, a common analgesic agent, increased locomotion and decreased immobility times in Cl(2)- but not air-exposed mice, most likely by decreasing Cl(2)-induced pain. This method can be adapted to assess the effectiveness of various therapies following exposure to a variety of chemical and behavioral noxious stimuli.     

Is cancer a metabolic rebellion against host aging?: In the quest for immortality,tumor cells try to save themselves by boosting mitochondrial metabolism

Aging drives large systemic reductions in oxidative mitochondrial function, shifting the entire body metabolically toward aerobic glycolysis, a.k.a, the Warburg effect. Aging is also one of the most significant risk factors for the development of human cancers, including breast tumors. How are these two findings connected? One simplistic idea is that cancer cells rebel against the aging process by increasing their capacity for oxidative mitochondrial metabolism (OXPHOS). Then, local and systemic aerobic glycolysis in the aging host would provide energy-rich mitochondrial fuels (such as L-lactate and ketones) to directly “fuel” tumor cell growth and metastasis. This would establish a type of parasite-host relationship or “two-compartment tumor metabolism,” with glycolytic/oxidative metabolic coupling. The cancer cells (“the seeds”) would flourish in this nutrient-rich microenvironment (“the soil”), which has been fertilized by host aging. In this scenario, cancer cells are only trying to save themselves from the consequences of aging by engineering a metabolic mutiny, through the amplification of mitochondrial metabolism. We discuss the recent findings of Drs. Ron DePinho (MD Anderson) and Craig Thomspson (Sloan-Kettering) that are also consistent with this new hypothesis, linking cancer progression with metabolic aging. Using data mining and bioinformatics approaches, we also provide key evidence of a role for PGC1a/NRF1 signaling in the pathogenesis of (1) two-compartment tumor metabolism and (2) mitochondrial biogenesis in human breast cancer cells.Key words: aging, mitochondria, cancer metabolism, autophagy, mitophagy, aerobic glycolysis, oxidative phosphorylation, Metformin, drug resistance, chemoresistance, Warburg effect, metabolic compartments, parasite, PGC1a, PGC1b, NRF1, two-compartment tumor metabolism     

Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome

Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell. Because of the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell-surface glycans. As an example of the functional consequences, the inhibitors substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.