Lipid composition of human serum lipoproteins

1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.     

Glucokinase gene mutations: structural and genotype-phenotype analyses in MODY children from South Italy

Background

Maturity onset diabetes of the young type 2 (or GCK MODY) is a genetic form of diabetes mellitus provoked by mutations in the glucokinase gene (GCK).

Methodology/Principal Findings

We screened the GCK gene by direct sequencing in 30 patients from South Italy with suspected MODY. The mutation-induced structural alterations in the protein were analyzed by molecular modeling. The patients'' biochemical, clinical and anamnestic data were obtained. Mutations were detected in 16/30 patients (53%); 9 of the 12 mutations identified were novel (p.Glu70Asp, p.Phe123Leu, p.Asp132Asn, p.His137Asp, p.Gly162Asp, p.Thr168Ala, p.Arg392Ser, p.Glu290X, p.Gln106_Met107delinsLeu) and are in regions involved in structural rearrangements required for catalysis. The prevalence of mutation sites was higher in the small domain (7/12: ∼59%) than in the large (4/12: 33%) domain or in the connection (1/12: 8%) region of the protein. Mild diabetic phenotypes were detected in almost all patients [mean (SD) OGTT = 7.8 mMol/L (1.8)] and mean triglyceride levels were lower in mutated than in unmutated GCK patients (p = 0.04).

Conclusions

The prevalence of GCK MODY is high in southern Italy, and the GCK small domain is a hot spot for MODY mutations. Both the severity of the GCK mutation and the genetic background seem to play a relevant role in the GCK MODY phenotype. Indeed, a partial genotype-phenotype correlation was identified in related patients (3 pairs of siblings) but not in two unrelated children bearing the same mutation. Thus, the molecular approach allows the physician to confirm the diagnosis and to predict severity of the mutation.     

Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

Multicopper oxidases can act on a broad spectrum of phenolic and non‐phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid), 2,6‐dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where k_cat and K_m values at 60°C were 8.1 (± 0.8) s^?1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half‐life of over 10 h at 80°C and over 7 h at 90°C. Site‐directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10‐fold increase in activity compared with the wild‐type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta‐position of aromatic substrates.     

A Novel, Drug-based, Cellular Assay for the Activity of Neurotoxic Mutants of the Prion Protein

In prion diseases, the infectious isoform of the prion protein (PrP^Sc) may subvert a normal, physiological activity of the cellular isoform (PrP^C). A deletion mutant of the prion protein (Δ105–125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP^C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Δ105–125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Δ105–125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Δ105–125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.     

Alteration in mitochondrial Ca<Superscript>2+</Superscript> uptake disrupts insulin signaling in hypertrophic cardiomyocytes

Background

Cardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca²⁺ release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood.

Results

In the present study we investigated insulin-dependent mitochondrial Ca²⁺ signaling in normal and norepinephrine or insulin like growth factor-1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca²⁺-fluorescent probes we showed that insulin increases mitochondrial Ca²⁺ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca²⁺ uniporter, as well as by siRNA-dependent mitochondrial Ca²⁺ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor-1-stimulated cells. This resulted in a reduction in mitochondrial Ca²⁺ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca²⁺ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.

Conclusions

Mitochondrial Ca²⁺ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.

     

Airborne fungal spores in the coastal plain of Israel: A preliminary survey

Airborne spores were monitored during the years 1993–1995 in three cities along the coastal plain of Israel: Ramat Gan, Tel Aviv (Ramat Aviv) and Haifa. Seasonal fluctuations in the concentration of airborne spores were recorded. The following genera of fungi were identified:Alternaria, Cladosporium, Coprinus, Curvularia, Drechslera. Diplococcum, Epicoccum, Fusarium, Leptosphaeria, Pithomyces, Puccinia, Sphacelotheca, Stemphylium andUstilago. Unidentified spores were very rare and in negligible numbers. The dominant airborne fungal spores wereCladosporium andAlternaria. The monthly variations in airborne spores, observed among the three cities, seem to be rather minor. The recorded levels of airborne spores were below the concentrations that are accepted as threshold levels for provocation of clinical responses.