Mice with targeted disruption of spermidine/spermine N1-acetyltransferase gene maintain nearly normal tissue polyamine homeostasis but show signs of insulin resistance upon aging

The N¹-acetylation of spermidine or spermine by spermidine/spermine N¹-acetyltransferase (SSAT) is the ratecontrolling enzymatic step in the polyamine catabolism. We have now generated SSAT knockout (SSAT-KO) mice, which confirmed our earlier results with SSAT deficient embryonic stem (ES) cells showing only slightly affected polyamine homeostasis, mainly manifested as an elevated molar ratio of spermidine to spermine in most tissues indicating the indispensability of SSAT for the spermidine backconversion. Contrary to SSAT deficient ES cells, polyamine pools in SSAT-KO mice remained almost unchanged in response to N¹, N¹¹-diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polymine pools in the wild-type animals and ES cells. Furthermore, SSATKO mice were more sensitive to the toxicity exerted by DENSPM in comparison with wild-type mice. The latter finding indicates that inducible SSAT plays an essential role in vivo in DENSPM treatmentevoked polyamine depletion, but a controversial role in toxicity of DENSPM. Surprisingly, liver polyamine pools were depleted similarly in wild type and SSAT-KO mice in response to carbon tetrachloride treatment. Further characterization of SSAT knockout mice revealed insulin resistance at old age which supported the role of polyamine catabolism in glucose metabolism detected earlier with our SSAT overexpressing mice displaying enhanced basal metabolic rate, high insulin sensitivity and improved glucose tolerance. Therefore SSAT knockout mice might serve as a novel mouse model for type 2 diabetes.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

Quantification of point mutations associated with Leber hereditary optic neuroretinopathy by solid-phase minisequencing

About two-thirds of patients with Leber hereditary optic neuroretinopathy (LHON) harbor mutations in mitochondrial DNA at positions 11778 (ND4) or 3460 (ND1). Thus, the clinical diagnosis of LHON can often be confirmed with mutation analysis. Detection of pathogenic mutations and quantification of heteroplasmy has mainly relied on PCR and restriction site analysis and densitometric scanning. We applied the recently developed solid-phase minisequencing method, based on primerguided nucleotide incorporation, to the simultaneous detection and quantitation of the ND4/11778 and ND1/3460 mutations. The method was highly sensitive, heteroplasmy as low as 1.5% being easily detected. Rapid, reproducible, and accurate results prove solid-phase minisequencing to be the method of choice for quantitative analysis of LHON mutations.     

RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.     

Synthesis and Antiviral (RNA) Evaluation of Nucleoside Analogs of Tiazofurin Modified at the Carboxamide Moiety

Abstract

The carboxamide functionality of tiazofurin 1a has been modified to produce the following analogs: carboximidates 5a,b, carboxamidines 6, 10, tetrahydropyrimidine 7, N-glycine 8 and N-glutamine 9. These structural modifications abolished the in vitro antiviral (RNA) activity exhibited by tiazofurin against the flaviviruses (yellow fever and Japanese encephalitis viruses), bunyavirus (Punta Toro virus) and togavirus (Venezuelan equine encephalomyelitis virus). Only carboximidates 5a,b retained marginal activity against bunyaviruses.     

HDL Subspecies in Young Adult Twins: Heritability and Impact of Overweight

The association between abdominal obesity and atherogenic lipid profile emerges from complex interactions of genes and environment. We aimed to explore the heritability and effects of overweight on serum lipid profile (high‐density lipoprotein‐cholesterol (HDL‐C), HDL mean particle size, percentages of HDL_{2b, 2a, 3a, 3b, and 3c}, low‐density lipoprotein‐cholesterol (LDL‐C), LDL peak particle size and triglycerides (TGs)) in healthy, young adults. HDL‐C, LDL‐C, and TG were measured in 52 monozygotic (MZ) and 89 dizygotic (DZ) twin pairs, aged 23–32 years, chosen to represent a wide range of BMIs (17.6–42.9 kg/m²). Of them, 24 MZ and 26 DZ pairs were chosen at random for measurements of HDL mean and LDL peak particle sizes and percentages of HDL subspecies. The heritabilities of the lipid parameters adjusted for BMI were HDL‐C 73%, HDL mean particle size 56%, HDL subspecies 46–63%, LDL‐C 79%, LDL peak particle size 49%, and TG 64%. Genetic and environmental correlations between BMI and HDL‐C, LDL‐C, and TG were modest (0.3–0.4). Abdominal overweight (waist circumference ≥94 cm for males and ≥80 cm for females) associated with decreased HDL‐C, increased LDL‐C, and TG concentrations, smaller HDL mean particle size, lower HDL_2b, and higher HDL_3c percentages in both genders. Within MZ twins, controlling for genetic influences, within‐pair differences in HDL_3c percentage were associated with those in waist (r = 0.46, P = 0.032) and BMI (r = 0.51, P = 0.013). In conclusion, serum lipid parameters, including LDL peak and HDL mean particle sizes and HDL subspecies distribution are under strong genetic control. Overweight associated with significant lipid profile changes, particularly, small HDL_3c increased in overweight independent of genetic influences.