Classification of mutants on the basis of their “in vitro” altered properties and the associated clinical phenotypes.

a<p>++: highly conserved; + conserved in vertebrates; − not conserved.</p>b<p>according to Palmai et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032065#pone.0032065-Palmai1" target="_blank">[9]</a>.</p>c<p>catalytic efficiency toward 3-PG or MgATP: +++ <1%; ++ <10%; + <25%; − comparable to wild-type.</p>d<p>heat stability (T₅₀): +++ nearly 10°C lowered; ++ nearly 3–7°C lowered; + nearly 2°C lowered; − comparable to wild-type.</p><p>A: anemia (+/−: compensated hemolytic anemia with occasional hemolytic crises); M: muscular disorders after physical exercises; N: neurological disorders.</p

Expression of recombinant PGK1 enzymes.

a<p>Obtained dividing the total PGK activity of the free cell extract by the specific activity of the purified enzyme. Data are referred to 1 L of cell culture.</p>b<p>Determined at 5 mM 3PG and 5 mM Mg-ATP.</p

Ribbon representation of the human PGK1.

<p>Three-dimensional structure of open (A) and closed (B) human PGK1. The figures were generated from the atomic coordinates of Protein Data Bank, entry 2XE7 and 2WZC, using the Swiss-Pdb viewer (<a href="http://expasy.org/spdbv/" target="_blank">http://expasy.org/spdbv/</a>). The black spheres indicate the Cα atoms of the amino acid residues subjected to mutagenesis. The arrows point to the substrates binding sites.</p

Thermal stability parameters of recombinant PGK1 enzymes.

<p>Results are means (SE) for 3 determinations from at least 2 different protein preparations.</p

Steady state kinetics of PGK1 enzymes.

<p>Steady state kinetics of PGK1 wild-type and variants as a function of Mg-ATP at fixed 5 mM 3-PG (panel A) and as a function of 3-PG at fixed 5 mM MgATP (panel B). All experiments were performed at 37°C as reported in the "<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032065#s2" target="_blank">Material and Methods</a>" section. •, wild-type; ▪, p.I47N; ▴, p.L89P; ▾, p.G158V; ⧫ p.D164V; , p.K191del; ○, p.R206P; □, p.E252A; ▵, p.I253T; ▿, p.V266M; ⋄, p.D268N; , p.D285V; , p.D315N; , p.C316R; , p.S320N; , p.A354P; , p.T378P.</p

Thermal stability of PGK1 enzymes.

<p>Thermal stability of the PGK1 wild-type and variants at 45°C (panel A) and at 37°C (panel B). Each enzyme was incubated in buffer A and aliquots were collected at intervals for measuring residual activity. Plot of the residual activities at 10 minutes versus temperatures (panel C). Each enzyme was subjected to heat inactivation in a range of temperature from 25°C to 60°C. After 10 minutes of incubation at a given temperature, the enzyme sample was chilled and the residual activity measured. Residual activity was expressed as percentage of initial activity. •, wild-type; ▪, p.I47N; ▴, p.L89P; ▾, p.G158V; ⧫ p.D164V; , p.K191del; ○, p.R206P; □, p.E252A; ▵, p.I253T; ▿, p.V266M; ⋄, p.D268N; , p.D285V; , p.D315N; , p.C316R; , p.S320N; , p.A354P; , p.T378P.</p

Main interactions of the PGK1 amino acids involved in the mutations.

a<p>Atomic coordinates of Protein Data Bank entry 2XE7;</p>b<p>atomic coordinates of Protein Data Bank entry 2WZC;</p>c<p>calculated with the CCP4 Suite, residues are considered solvent accessible when accessible surface area is >5 Ų.</p

Image_1_Use of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) in the Diagnosis of RBC Membrane Disorders, Enzyme Defects, and Congenital Dyserythropoietic Anemias: A Monocentric Study on 202 Patients.TIF

<p>Chronic hemolytic anemias are a group of heterogeneous diseases mainly due to abnormalities of red cell (RBC) membrane and metabolism. The more common RBC membrane disorders, classified on the basis of blood smear morphology, are hereditary spherocytosis (HS), elliptocytosis, and hereditary stomatocytoses (HSt). Among RBC enzymopathies, the most frequent is pyruvate kinase (PK) deficiency, followed by glucose-6-phosphate isomerase, pyrimidine 5′ nucleotidase P5′N, and other rare enzymes defects. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a variety of laboratory tests. The ektacytometer laser-assisted optical rotational cell analyser (LoRRca MaxSis), able to assess the RBC deformability in osmotic gradient conditions (Osmoscan analysis), is a useful diagnostic tool for RBC membrane disorders and in particular for the identification of hereditary stomatocytosis. Few data are so far available in other hemolytic anemias. We evaluated the diagnostic power of LoRRca MaxSis in a large series of 140 patients affected by RBC membrane disorders, 37 by enzymopathies, and 16 by congenital diserythropoietic anemia type II. Moreover, nine patients with paroxysmal nocturnal hemoglobinuria (PNH) were also investigated. All the hereditary spherocytoses, regardless the biochemical defect, showed altered Osmoscan curves, with a decreased Elongation Index (EI) max and right shifted Omin; hereditary elliptocytosis (HE) displayed a trapezoidal curve and decreased EImax. Dehydrated hereditary stomatocytosis (DHSt) caused by PIEZO1 mutations was characterized by left-shifted curve, whereas KCNN4 mutations were associated with a normal curve. Congenital diserythropoietic anemia type II and RBC enzymopathies had Osmoscan curve within the normal range except for glucosephosphate isomerase (GPI) deficient cases who displayed an enlarged curve associated with significantly increased Ohyper, offering a new diagnostic tool for this rare enzyme defect. The Osmoscan analysis performed by LoRRca MaxSis represents a useful and feasible first step screening test for specialized centers involved in the diagnosis of hemolytic anemias. However, the results should be interpreted by caution because different factors (i.e., splenectomy or coexistent diseases) may interfere with the analysis; additional tests or molecular investigations are therefore needed to confirm the diagnosis.</p

Online_supplement_777441 – Supplemental material for Effects of sodium citrate, citric acid and lactic acid on human blood coagulation

<p>Supplemental material, Online_supplement_777441 for Effects of sodium citrate, citric acid and lactic acid on human blood coagulation by Vittorio Scaravilli, Luca Di Girolamo, Eleonora Scotti, Mattia Busana, Osvaldo Biancolilli, Patrizia Leonardi, Andrea Carlin, Caterina Lonati, Mauro Panigada, Antonio Pesenti and Alberto Zanella in Perfusion</p