Nature of the main contaminant in the drug primaquine diphosphate: SFC and SFC-MS methods of analysis

The drug primaquine diphosphate is used for causative treatment of malaria. Using HPLC-MS and GC-MS, this research group was previously able to show that the main contaminant of primaquine is the positional isomer quinocide [I. Brondz, D. Mantzilas, U. Klein, D. Ekeberg, E. Hvattum, M.N. Lebedeva, F.S. Mikhailitsyn, G.D. Soulimanov, J. Roe, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 800 (2004) 211-223; I. Brondz, U. Klein, D. Ekeberg, D. Mantzilas, E. Hvattum, H. Schultz, F. S. Mikhailitsyn, Asian J. Chem. 17 (2005) 1678-1688]. Primaquine and quinocide are highly toxic substances which can have a number of side effects upon use in medical treatment. A standard for quinocide is not typically commercially available. In the present work, supercritical fluid chromatography-mass spectrometry (SFC-MS) with two different columns was used to achieve a shorter analysis time for the separation between the positional isomers quinocide and primaquine in primaquine diphosphate and to elucidate additional information about differences in their MS fragmentation. Unlike using HPLC-MS, it was possible to achieve the differential fragmentation of positional isomers at branching points using the SFC-MS technique. The desired short analysis time was achieved using SFC equipped with a Discovery HS F5 column and the differential fragmentation of positional isomers during SFC-MS provides information on the differences in the structure of these substances. Using a Chiralpak AD-H chiral column, it was possible to resolve the enantiomers in primaquine and separate quinocide from those enantiomers.     

A small-molecule Psora-4 acts as a caloric restriction mimetic to promote longevity in C. elegans

GeroScience - In populations around the world, the fraction of humans aged 65 and above is increasing at an unprecedented rate. Aging is the main risk factor for the most important degenerative...     

Active p38α causes macrovesicular fatty liver in mice

One third of the western population suffers from nonalcoholic fatty liver disease (NAFLD), which may ultimately develop into hepatocellular carcinoma (HCC). The molecular event(s) that triggers the disease are not clear. Current understanding, known as the multiple hits model, suggests that NAFLD is a result of diverse events at several tissues (e.g., liver, adipose tissues, and intestine) combined with changes in metabolism and microbiome. In contrast to this prevailing concept, we report that fatty liver could be triggered by a single mutated protein expressed only in the liver. We established a transgenic system that allows temporally controlled activation of the MAP kinase p38α in a tissue-specific manner by induced expression of intrinsically active p38α allele. Here we checked the effect of exclusive activation in the liver. Unexpectedly, induction of p38α alone was sufficient to cause macrovesicular fatty liver. Animals did not become overweight, showing that fatty liver can be imposed solely by a genetic modification in liver per se and can be separated from obesity. Active p38α-induced fatty liver is associated with up-regulation of MUC13, CIDEA, PPARγ, ATF3, and c-jun mRNAs, which are up-regulated in human HCC. Shutting off expression of the p38α mutant resulted in reversal of symptoms. The findings suggest that p38α plays a direct causative role in fatty liver diseases and perhaps in other chronic inflammatory diseases. As p38α activity was induced by point mutations, it could be considered a proto-inflammatory gene (proto-inflammagene).

Inflammatory liver diseases form a serious epidemic in the Western world. Nonalcoholic fatty liver disease (NAFLD) has grown from a relatively unknown disease to the most common cause of chronic liver diseases in the world over the last two decades, with a global prevalence of 25% (1). NAFLD is characterized by steatosis of the liver, involving greater than 5% of parenchyma, with no evidence of hepatocyte injury. In nonalcoholic steatohepatitis (NASH), the liver cells become injured in a background of steatosis (2). NAFLD may further develop to NASH and, at a later stage, to liver cirrhosis that could eventually progress to liver cancer (3).Many cases of liver diseases are associated with viral infections (hepatitis B and C and HIV), alcoholism, drugs, or autoimmunity. NAFLD and NASH are commonly associated with obesity, insulin resistance (IR), and type 2 diabetes. In addition, a large number of patients develop fatty liver with no known background (3, 4).Regardless of the background conditions, the molecular basis of the triggering and the etiology of the diseases are unclear (3). As is the case for all other chronic inflammatory diseases, the current understanding of the underlying mechanism of the onset of NAFLD is that it involves some combination of genetic and environmental conditions. The latest theory is the “multiple hit” hypothesis, which suggests that a number of events, occurring in parallel in several tissues and systems, combined with environmental and genetic factors, lead to NAFLD (5–7). Such hits include IR, adipose tissue inflammation, hormones secreted from adipose tissue, nutritional factors, gut microbiota, and genetic and epigenetic factors (5, 8, 9). This explanation for the onset of NAFLD and other chronic inflammatory diseases stands in a sharp contrast to the molecular mechanism underlying the onset of cancer diseases, which are triggered by genetic alterations in proto-oncogenes and in tumor suppressor genes (10, 11). Namely, the “multiple hit” concept for the onset of NAFLD does not consider the possibility of proto-inflammatory genes that may become disease-causing genes (“inflammagenes”) following some genetic alteration. It is shown here, nonetheless, that fatty liver could be induced by a single mutated protein.The lack of deep understanding of the molecular triggers of fatty liver diseases hinders the development of accurate experimental models (12). Current models rely on treatments that indirectly lead to accumulation of lipids in the liver. These models can be broadly categorized as diet induced, genetics, or a combination of more than one intervention. Diet-induced models include mainly the use of high-fat diet (HFD) that causes obesity and in turn NAFLD-like disease in mice. In these models, the development of the disease is slow as it must be preceded by a serious increase in body weight (13). In the genetic models, lipidosis (fatty liver) is enhanced in response to HFD as the animal is engineered to develop obesity faster. The principal genetic animal models of NASH are the ob/ob (leptin-deficient) and db/db (leptin receptor–deficient) mice, the Zucker (fa/fa) rat, foz mice (deficient in the Alstrom syndrome 1 gene) and several transgenic or conditional knockout mice (12). Exposure of the animals to drugs such as the hepatotoxin CCl₄ to amplify injury and fibrosis also appears to closely resemble human NASH (12, 14). In all models, numerous biochemical pathways and systemic responses are induced in various tissues, making it difficult to extract the relevant activities that impose the liver disease. No genetic model exists in which fatty liver is induced solely by activation of a single protein by mutation or gene amplification, analogously to models of cancer diseases. By contrast, this study describes a transgenic system in which induction of a single mutated gene, exclusively in the liver, leads rapidly to the development of fatty liver, with no effect on body weight. The mutated gene encodes the mitogen-activated protein kinase (MAPK) p38α.p38α, a member of the p38 family, which is composed of four isoforms (15), is abnormally active in chronic inflammatory diseases including NAFLD (15–18). Inhibition of p38α resulted in reversal of pathological symptoms in some experimental systems, suggesting that p38α plays a role in the maintenance of the disease and perhaps in its etiology as well (17, 18). Finally, mice knocked out for various MAPK phosphatases are prone to the development of fatty liver. Although these phosphatases affect all members of the MAPK family (i.e., p38s, JNKs, and ERKs) it seems that the effect on the liver is a result of overactive p38 (19–23).In other inflammatory diseases in which p38α is abnormally active, including rheumatoid arthritis, asthma, and inflammatory bowel diseases (24–26), its role in these maladies, if any, is also not clear, similarly to the case of fatty liver diseases (18). We thus sought the establishment of an experimental model that will disclose the exact function of p38α in each tissue and may also point at its relative contribution to disease etiology. Achieving this goal necessitates in vivo activation of p38α per se in a tissue-specific and temporally controlled manner. Accomplishing such a task is far from trivial as, like all MAPKs, p38α manifests no basal catalytic activity when not activated, making an overexpression approach insufficient. Natural activation of p38α is obtained, in most cases, through a complex signaling cascade that culminates in p38α’s dual phosphorylation on a TGY motif located at its activation loop, a reaction catalyzed by the MAPK kinases MKK3 and MKK6 (27, 28). In the absence of this dual phosphorylation, p38α is catalytically impotent (27). Activation of upstream components of the p38 cascade induces the activity of various components in addition to p38α, including all p38 isoforms.In this study, the challenge of activating p38α individually in a tightly controlled manner was met by combining a unique double cassette expression system in transgenic mice, with an intrinsically active mutant of p38α, p38α^D176A+F327S (29, 30). p38α^D176A+F327S was shown to be intrinsically catalytically active in vitro and in cell cultures, confirming its independence from MKK3/6 activation or any upstream induction (31–35). Importantly, apart from gaining intrinsic activity, p38α^D176A+F327S maintained the biochemical, pharmacological, and physiological properties of naturally activated p38α molecules (35).We describe a transgenic model in which p38α could be specifically activated in a reversible manner, in vivo, in any tissue of choice. This model could be applied for studying the role of p38α per se in various inflammatory diseases, and here, we apply it to reveal the role of p38α in the etiology of fatty liver. It was originally expected that activation of p38α in the liver would render the liver prone to stimuli that impose lipidosis, such as HFD, or CCl₄. Unexpectedly, it was observed that expression of p38α^D176A+F327S in liver of mice unprovoked by any treatment was sufficient to cause an increase in serum and hepatic triglycerides and a histological appearance that resembles macrovesicular lipidosis. This observation proposes a perspective on the onset of NAFLD.Looking into the mechanism through which active p38α imposes fatty liver, it was assumed that p38α’s substrates would be strongly phosphorylated. Unexpectedly, the expression level and phosphorylation of the p38α’s substrate MAPKAPK2 (MK2) is down-regulated along with reduced phosphorylation of heat shock protein 27 (Hsp27) in mice expressing p38α^D176A+F327S in the liver. This suggests that constitutive activity of p38α may impose fatty liver by causing down-regulation of downstream components, perhaps via a feedback inhibition mechanism.     

Homozygous mutation in <Emphasis Type="Italic">MFSD2A</Emphasis>, encoding a lysolipid transporter for docosahexanoic acid,is associated with microcephaly and hypomyelination

The major facilitator superfamily domain-containing protein 2A (MFSD2A) is a constituent of the blood-brain barrier and functions to transport lysophosphatidylcholines (LPCs) into the central nervous system. LPCs such as that derived from docosahexanoic acid (DHA) are indispensable to neurogenesis and maintenance of neurons, yet cannot be synthesized within the brain and are dependent on MFSD2A for brain uptake. Recent studies have implicated MFSD2A mutations in lethal and non-lethal microcephaly syndromes, with the severity correlating to the residual activity of the transporter. We describe two siblings with shared parental ancestry, in whom we identified a homozygous missense mutation (c.1205C?>?A; p.Pro402His) in MFSD2A. Both affected individuals had microcephaly, hypotonia, appendicular spasticity, dystonia, strabismus, and global developmental delay. Neuroimaging revealed paucity of white matter with enlarged lateral ventricles. Plasma lysophosphatidylcholine (LPC) levels were elevated, reflecting reduced brain transport. Cell-based studies of the p.Pro402His mutant protein indicated complete loss of activity of the transporter despite the non-lethal, attenuated phenotype. The aggregate data of MFSD2A-associated genotypes and phenotypes suggest that additional factors, such as nutritional supplementation or modifying genetic factors, may modulate the severity of disease and call for consideration of treatment options for affected individuals.