A new missense mutation (Cys297→Phe) of the low density lipoprotein receptor in Italian patients with familial hypercholesterolemia (FHTrieste)

During a survey of the mutations of the low density lipoprotein receptor (LDL-R) gene in Italian patients with familial hypercholesterolemia (FH), we identified a novel point mutation, that creates a new EcoRI site at the 5 end of exon 7, in a heterozygous FH subject (FH-100). The sequence of a cDNA fragment encompassing exon 7 showed the presence of a GT transversion in codon 297; this created a new EcoRI site and produced a missense mutation, leading to a Cys₂₉₇Phe substitution in repeat A of the epidermal growth factor (EGF) precursor homology domain of LDL-R. Since the substitution of Cys₂₉₇ disrupts the intracellular transport of the LDL-R protein, as previously demonstrated by site-directed mutagenesis, we suggest that this mutation is the cause of FH in the FH-100 proband. We screened the DNA of 303 Italian FH patients by amplification of exon 7 from genomic DNA followed by digestion with EcoRI or by Southern blotting. Two individuals (FH-64 and FH-127) were found to be carriers of the Cys₂₉₇Phe mutation. Restriction fragment length polymorphism analysis demonstrated that, in two kindreds (FH-64 and FH-100), the haplotype in linkage with the Cys₂₉₇Phe mutation was the same, suggesting the presence of a common ancestor. The Cys₂₉₇Phe mutation has been designated FH_Trieste after the name of the city in Northern Italy from which probands FH-100 and FH-127 originate.     

Morphogenetic potential and in vitro micropropagation of endangered plant species Leucojum aestivum L. and Lilium rhodopaeum Delip.

Summary The present work deals with the in vitro methods for rapid propagation, and morphogenetic potential of the rare and endangered bulb species Leucojum aestivum L., Amaryllidaceae, and Lilium rhodopaeum Delip., Liliaceae. The morphogenetic potential of different plant organs (bulb, stem, leaves and ovaries) was studied. Leaves of Leucojum aestivum L. and basal parts of the bulb in Lilium rhodopaeum Delip. possess the highest regeneration activity. Murashige and Skoog (MS) medium + 1 mg/l 6-benzylaminopurine (BAP) + 1 mg/l kinetin and Linsmaier and Skoog (LS) medium + 0.5 mg/l 1-naphthaleneacetic acid (NAA) + 0.1 mg/l kinetin were favourable for direct organogenisis from these explants. A stimulating effect of alow gamma-irradiation dose (5 Gy) upon the quantity and growth intensity of the bulbs formed by the explants in in vitro conditions is observed.     

Concerning the mechanism of increased thermogenesis in rats treated with dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) treatment of rats decreases gain of body weight without affecting food intake; simultaneously, the activities of liver malic enzyme and cytosolic glycerol-3-P dehydrogenase are increased. In the present study experiments were conducted to test the possibility that DHEA enhances thermogenesis and decreases metabolic efficiency via trans-hydrogenation of cytosolic NADPH into mitochondrial FADH₂ with a consequent loss of energy as heat. The following results provide evidence which supports the proposed hypothesis: (a) the activities of cytosolic enzymes involved in NADPH production (malic enzyme, cytosolic isocitrate dehydrogenase, and aconitase) are increased after DHEA treatment; (b) cytosolic glycerol-3-P dehydrogenase may use both NAD⁺ and NADP⁺ as coenzymes; (c) activities of both cytosolic and mitochondrial forms of glycerol-3-P dehydrogenase are increased by DHEA treatment; (d) cytosol obtained from DHEA-treated rats synthesizes more glycerol-3-P during incubation with fructose-1,6-P₂ (used as source of dihydroxyacetone phosphate) and NADP⁺; the addition of citratein vitro further increases this difference; (e) mitochondria prepared from DHEA-treated rats more rapidly consume glycerol-3-P added exogenously or formed endogenously in the cytosol in the presence of fructose-1,6-P₂ and NADP⁺.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

Osmotic water permeability of cell membranes from Mesembryanthemum crystallinum leaves: effects of age and salinity

The osmotic water permeability ( P _os) of cell membranes isolated from leaves of 40-, 50- and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P _os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P _os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 m M NaCl caused a decline in the P _os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P _os of the leaf-cell membranes. These variations in the P _os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.     

Analysis of the effect of periodate oxidation and phenol extraction on the antitumour properties of C. parvum

Summary The antitumour activity of C. parvum and its relationship to spleen weight increase have been analysed in a tumour model using syngeneic M₄ fibrosarcoma cells. It was found that treatment of C. parvum with low concentrations of periodate (2 mM) produced a minor decrease in the ability to abrogate tumour growth, following intratumour injection, although the capacity to increase spleen weight after IP injection was abolished. Higher concentrations of periodate (20 mM) produced complete inactivation of the bacteria. A phenol extract of C. parvum was weakly active in the intratumour test but amounts up to 1 mg injected IP were unable to increase the spleen weight. The data support the hypothesis that a surface carbohydrate is responsible for the increase in spleen weight. It is suggested that the antitumour activity may reside in a molecule that also reacts with periodate but at a slower rate, perhaps because of a more complex structure or a different location in the cell, for instance in the cytoplasm. The phenol extract was soluble in chloroform and at least two major components were glycolipids, so it is conceivable that a molecule of this type, which could be degraded by periodate, might be responsible for the antitumour activity of C. parvum. The precise biochemical nature of the active component is, as yet, undetermined.     

Perthamide C Inhibits eNOS and iNOS Expression and Has Immunomodulating Activity In Vivo

Here we have characterized perthamide C, a cyclopeptide from a Solomon Lithistid sponge Theonella swinhoei, which displays an anti-inflammatory/immunomodulatory activity. The study has been performed using the carragenan-induced mouse paw edema that displays an early (0–6 h) and a late phase (24–96 h). Perthamide C significantly inhibits neutrophils infiltration in tissue both in the early and late phases. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) in the early phase while cyclooxygenase-1 and 2 (COX-1, COX-2), and inducible NOS (iNOS) expression were unaffected. In the late phase perthamide C reduced expression of both NOS isoforms without affecting COXs expression. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation. We found that perthamide C inhibited the proliferation of peripheral lymphocytes, and that this effect was secondary to its metabolic activation in vivo. Indeed, in vitro perthamide C did not inhibit proliferation as opposite to its metabolite perthamide H.In conclusion, perthamide C selectively interferes with NO generation triggered by either eNOS or iNOS without affecting either COX-1 or COX-2. This in turn leads to modulation of the inflammatory response through a reduction of vascular permeability, neutrophil infiltration as well as lymphocyte proliferation.