Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.

Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 ß-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.

[Epigenetics and pseudohypoparathyroidism]. / Épigénétique et pseudohypoparathyroïdies.

Parental imprinting and the type of the genetic alteration play a determinant role in the phenotype expression of GNAS locus associated to pseudohypoparathyroidism (PHP). This imprint is tissue-specific, mainly localized in the kidney and the thyroid. Only the maternal allele is expressed at this level. An alteration in the coding sequence of the gene leads to an haplo-insufficiency and a dysmorphic phenotype (Albright's syndrome). If the alteration is on the maternal allele, there is a hormonal resistance to the PTH at the kidney level and to the TSH at the thyroid level. The phenotype is known as a PHP1a. If the alteration is on the paternal allele, there are few clinical signs with no hormonal resistance and the phenotype is known as pseudo-pseudo-hypoparathyroidism (PPHP). Methylation anomalies of GNAS locus, in particular of exon 1A, are responsible for a lack of expression of Gαs at kidney and thyroid levels only. If these anomalies concern the maternal allele (the only one expressed) with a paternal pattern, there is no haplo-insufficiency and no dysmorphic syndrome. The hormonal resistance is yet again limited to PTH and TSH. The phenotype is known as PHP1b. In the familial forms, these methylation anomalies are associated with a deletion of the syntaxine 16 gene in the maternal allele. This gene contains probably the imprinting center of the locus.

CYP24A1 Mutations in a Cohort of Hypercalcemic Patients: Evidence for a Recessive Trait.

CONTEXT: Loss-of-function mutations of CYP24A1 (which encodes the 25-OH-D3-24-hydroxylase) have recently been reported to cause hypercalcemia. OBJECTIVES: The aims of this study were: 1) to evaluate the frequency of CYP24A1 mutations in patients with medical history of hypercalcemia; 2) to show the clinical utility of a simultaneous assay of serum 25-hydroxyvitamin D3 (25-OH-D3) and 24,25-dihydroxyvitamin D3 (24,25-[OH]2D3) by liquid chromatography tandem mass spectrometry (LC-MS/MS); and 3) to investigate biochemical parameters in heterozygous gene carriers with CYP24A1 mutations. PATIENTS AND METHODS: We screened for CYP24A1 mutations in 72 patients with serum calcium levels > 2.6 mmol/L and PTH levels < 20 pg/mL and recruited 24 relatives after genetic counseling for subsequent investigations. Vitamin D metabolite concentrations were assessed in a subset of patients by LC-MS/MS and results expressed as a ratio (R) of 25-OH-D3:24,25-(OH)2D3. RESULTS: Twenty-five patients with hypercalcemia (35%) harbored CYP24A1 variations. Twenty (28%) had biallelic variations, mostly found in subjects with nephrocalcinosis or renal stones (19/20). Five patients, all neonates, were heterozygous, without renal disease. We describe 15 new variations leading to loss-of-function according to pathogenicity prediction programs, and we functionally characterized 5 of them in vitro. A dramatic increase of R, usually >80, was found in patients harboring biallelic mutations providing evidence in vivo for the loss of CYP24A1 activity. In contrast, R value remains <25 in patients without CYP24A1 mutations. Subjects carrying one mutant allele, hypercalcemic individuals, as well as gene-carrier relatives, had a detectable 24,25-(OH)2D3 level and R < 25, indicating normal 24-hydroxylase activity. CONCLUSION: CYP24A1 biallelic mutations are frequently found in patients presenting with hypercalcemia, low PTH, and renal disease. We confirm the accuracy and effectiveness of a novel blood test estimating the ratio between relevant vitamin D metabolites as a useful screening tool for CYP24A1 mutations. Haploinsufficiency is not associated with CYP24A1 deficiency.

[Positive inotropic and lusitropic effect of RP 62719, a new class III antiarrhythmia agent]. / Action inotrope et lusitrope positive du RP 62719, une nouvelle molécule antiarythmique de classe III.

Antiarrhythmic drugs, especially the Class I family, exert a negative inotropic effect on the myocardium which is particularly undesirable in patients with depressed left ventricular function. Therefore, research has been directed to the development of new, more specific molecules of the Class III family. The authors studies the mechanical effects of RP 62719 on guinea pig left ventricular papillary muscle. This new molecule is a pure Class III antiarrhythmic, known to lengthen the duration of the cardiac action potential by selectively blocking the potassium current iK1 (inward rectifier K+ current). The mechanical parameters were determined during the phases of contraction and relaxation under isotonic and isometric conditions. At 0.2 and 2 microM concentrations, RP 62719 improved cardiac contraction under both isotonic and isometric conditions with an increase of about 30% of Vmax (p < 0.001), the maximum unloaded shortening velocity delta 1 (p < 0.001), the peak isometric active force normalized per cross-sectional area [AF/S (p < 0.001)]. At these two concentrations, a positive lusitropic effect (improved relaxation) was demonstrated by an increase in negative peak of derivative per mm2-dF/s and maximum lengthening velocity VR max (p < 0.01). At higher concentrations (20 microM), the inotropic and lusitropic effects were less marked with a bell-shaped form of the dose-effect curve. This study indicates that RP 62719 has moderate but significant positive inotropic and lusitropic effects. These actions could provide significant therapeutic advantages especially in patients cardiac failure.

[Our experience with bioprostheses in French Polynesia. A follow-up study from 1975 to 1988]. / L'expérience des bioprothèses en Polynésie française. Suivi de 1975 à 1988.

French Polynesia has unique social, cultural and geographic features. The prevalence of acute rheumatic fever (ARF) of 1.2 % population justified the health authorities' classification of this endemic as "major, severe, and prioritary" in 1984. Seventy per cent of patients with ARF develop cardiac sequellae which require surgery in 25 per cent of cases. Bioprostheses were considered from 1975 as the ideal valve replacement for these young, often undisciplined patients with no facilities for haemostatic control. Reoperation for valve degeneration has been increasingly frequent since 1982 and poses an acute problem leading to this statistic study and to a reflection as to the value of continuing to use this type of valve in this population. Analysis of 178 Polynesians with one or more cardiac bioprostheses in 1988, totalling 221 valves with a mean follow-up of 55 months, shows an actuarial survival rate excluding operative mortality of 86.7 +/- 3 % at 5 years (93.8 +/- 2.5 % in patients under 25 years of age, p = 0.001). Fifty-two patients (29 %) have been reoperated with a probability of being free of reoperation at 5 years of 70.1 +/- 4.2% (85.2 +/- 3.9 % in patients over 25 years of age and 43.8 +/- 7.8 % in patients under the age of 25, p = 0.002). The authors discuss the alternative of bioprostheses in this population : mechanical valves with anti-coagulant or anti-aggregant therapy, frozen aortic homografts, mitral valvuloplasty.

[Changes in the diastolic left ventricular function in primary antiphospholipid antibody syndrome]. / Altération de la fonction ventriculaire gauche diastolique au cours du syndrome primitif des anticorps antiphospholipides.

Previous studies have shown that left ventricular (LV) diastolic function is frequently impaired in patients with systemic lupus erythematosus. We prospectively studied echo-Doppler indices of LV diastolic function in 18 patients with primary antiphospholipid syndrome (PAPS), who where compared to a group of 18 healthy controls. Heretofore undescribed LV relaxation abnormalities were found in the PAPS group: this finding suggests the existence of a causal link.

Effects of isoproterenol on myocardial relaxation rate: influence of the level of load.

The present work was undertaken to test the hypothesis that the level of the load faced by the myocardium influences the effects of isoproterenol on relaxation rate. Responses to cumulative doses of isoproterenol (from 10(-10) to 10(-6) M) were studied in rat left ventricular papillary muscle stimulated 12 beats/min at 29 degrees C in 0.5 mM extracellular calcium and preloaded at initial muscle length corresponding to apex of length-active tension curve (Lmax; group 1, n = 20) or at 95% of Lmax (group 2, n = 9). A control group (group 3, n = 8) was studied every 15 min for 75 min. We measured maximum unloaded shortening velocity (Vmax), normalized positive and negative peak force derivatives (+dF and -dF, respectively) of the fully isometric twitch, and peak lengthening velocity of the isotonic twitch with preload only (Vlmax). In group 1, Vmax and +dF increased under 10(-10) and 10(-9) M isoproterenol, respectively, and -dF increased under 10(-9) M isoproterenol (115 +/- 13 vs. 96 +/- 12 mN.s-1.mm-2, P = 0.01). Conversely, Vlmax increased under 10(-7) M isoproterenol only (2.34 +/- 0.19 vs. 1.45 +/- 0.18 Lmax/s, P < 0.001). In group 2, both -dF and Vlmax increased under 10(-7) M isoproterenol only (P = 0.015 and 0.011, respectively). In group 3, -dF and Vlmax did not vary in time. Our results suggest a load-revealed (or length-revealed) difference in the dose dependence of the various biochemical processes involved in the effects of isoproterenol during myocardial relaxation.

Positive inotropic effects of RP 62719, a new pure class III antiarrhythmic agent, on guinea pig myocardium.

The mechanical effects of RP 62719 [(-)1-[-2-(3,4-dihydro-2H-1- benzopyran-4-yl)ethyl]-4-(3,4-dimethoxyphenyl)-piperidine] were tested in vitro on guinea pig left ventricular papillary muscle. RP 62719 is a novel pure class III antiarrhythmic agent known to prolong the cardiac action potential duration by selectively blocking the inward rectifying K+ current. Mechanical parameters were determined from contraction and relaxation phases under isotonic and isometric conditions. At a concentration of 0.02 microM, RP 62719 did not produce significant effects on inotropy or lusitropy. At 0.2 and 2 microM, the drug improved contraction under both heavy and low loading conditions, as evidenced by a 30% increase in maximum unloaded shortening velocity (Vmax, P < .001), peak amplitude of shortening (delta L, P < .001), peak isometric active force normalized per cross-sectional area (AF/s, P < .001) and positive peak of the force derivative per mm2 (+dF/s, P < .001). At the same concentrations, positive lusitropic effects were evidenced by an increase in maximum lengthening velocity (maxVr) and negative peak of force derivative per mm2 (-dF/s, P < .001). At a higher concentration (20 microM), effects of RP 62719 on inotropy and lusitropy were less marked, thus accounting for the bell-shaped form of the dose-response curve. An increase in the extracellular Ca++ concentration from 2.5 to 3.75 mM improved inotropy to a similar extent (+30-50%) as did 2 microM RP 62719. However, lusitropy and mechanical coupling between contraction and relaxation were not modified in the same proportion under RP 62719 and under 3.75 mM Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of heterozygosity at the TP53 gene: independent occurrence from genetic instability events in node-negative breast cancer.

TP53 abnormalities have been reported as an early event in the process of cellular transformation of human breast cancers, and involved in mammary-tumor evolution, from in situ to invasive disease. In this study, node-negative (N-) tumors were examined for TP53 allelic loss in relation to different genetic instability events, including allelic loss at chromosome 17p13.3 and c-H-ras-1 loci, as well as alteration of the c-myc and c-erbB-2/neu oncogenes. TP53 allelic loss was analyzed to determine whether such an abnormality was the more important, among other genetic events, in the N- tumors, whether it appeared independently of these genetic events, and whether accumulation of genetic events arises in this group of breast tumors. Clinicopathological parameters were also examined. Loss of heterozygosity (LOH) at the TP53 gene appears the most frequent alteration detected (26% vs. 13%, 8%, 9% and 3% for LOH at D17S30 and c-H-ras-1 loci, and amplification of c-myc and c-erbB-2/neu respectively). There was no association between LOH at the TP53 locus and other genetic events. Among clinicopathological parameters, significant associations were observed only with estrogen-receptor-negative tumors (p = 0.05). Our results demonstrate that LOH at TP53 arises more frequently in the N- breast cancer, thus supporting earlier findings suggesting that TP53 abnormality has a role early in the pathogenesis of breast lesions. Moreover, the data indicate that accumulation of many genetic events occurs at a low level in N- breast tumors, and that TP53 abnormality occurs independently of these genetic events.

M mode and Doppler echocardiographic assessment of left ventricular diastolic function in primary antiphospholipid syndrome.

BACKGROUND: High titres of serum antiphospholipid antibodies are a possible pathogenic factor for cardiac lesions in patients with systemic lupus erythematosus. OBJECTIVE: To test the hypothesis of a causal link between high titres of antiphospholipid antibodies in the serum and myocardial involvement in patients without systemic lupus erythematosus. PATIENTS AND DESIGN: 18 patients with primary antiphospholipid syndrome (recurrent fetal loss, arterial and/or venous thrombosis, high titres of antiphospholipid antibodies, and no criteria for systemic lupus erythematosus) were prospectively studied by cross sectional, M mode, and pulsed Doppler echocardiography, and compared with 18 healthy controls. The pulsed Doppler indices of left ventricular diastolic function included isovolumic relaxation time and four mitral outflow indices: peak velocity of early flow, peak velocity of late flow, early to late peak flow velocity ratio, and rate of deceleration of early flow. Four computerised M mode indices were also measured: peak rate of left ventricular enlargement in diastole, peak rate of posterior wall thinning, peak velocity of lengthening of the posterior wall, and velocity of circumferential chamber lengthening. RESULTS: Compared with controls, patients with primary antiphospholipid syndrome had higher values for isovolumic relaxation time and peak velocity of late mitral outflow and lower values for early to late mitral peak outflow velocity ratio, rate of deceleration of early mitral outflow, peak rate of left ventricular enlargement in diastole, peak rate of posterior wall thinning, peak velocity of lengthening of the posterior wall and velocity of circumferential chamber lengthening. CONCLUSION: This abnormal pattern reflects an impairment of myocardial relaxation and filling dynamics of the left ventricle in patients with primary antiphospholipid syndrome who were free of any clinically detectable heart disease. These data suggest that high serum titres of antiphospholipid antibodies may be associated with subclinical myocardial damage.

Left ventricular diastolic function in asymptomatic and symptomatic human immunodeficiency virus carriers: an echocardiographic study.

Acquired immunodeficiency syndrome (AIDS) is a systemic illness affecting multiple organs, including the heart. Left ventricular (LV) diastolic dysfunction has been reported as the first echocardiographically detectable abnormality in several cardiovascular disorders. We tested the hypothesis that Human Immunodeficiency Virus (HIV) carriers have LV diastolic impairment when studied early in the clinical course of the infection. Doppler echocardiographic and computerized time-motion parameters of LV diastolic function were obtained in 51 HIV patients and in 25 age- and sex-matched healthy controls. The HIV population consisted of 28 totally asymptomatic subjects and 23 patients with incipient AIDS. As compared to controls, the HIV group had similar heart rate, blood pressure level, LV dimensions and fractional shortening, but increased isovolumetric relaxation time (P = 0.03), early filling duration (P < 0.001) and decreased early mitral flow peak velocity (E) (P = 0.02) and EF slope (P < 0.001). HIV patients also showed lower values for posterior wall thinning (PWT, P < 0.01) and peak lengthening velocity of the posterior wall (PVL, P < 0.05), and a trend to a decreased peak rate of LV enlargement in diastole (D+, P = 0.05). Doppler-derived parameters of diastolic function were significantly altered in the asymptomatic HIV group vs controls. The LV diastolic indices were similar in symptomatic and asymptomatic HIV patients except for PWT, which was lower in the symptomatic HIV group (P = 0.04). Since mild and focal wall motion abnormalities were detected in 11 HIV carriers (22%), comparison of LV diastolic indexes between HIV patients and controls was also performed in two subgroups; these included asymptomatic (n = 26) and symptomatic (n = 14) patients with normal contractile state. The two subgroups had abnormalities of diastolic function similar to those of the HIV group as a whole, but with somewhat lower levels of statistical significance. Our data strongly suggest that there is myocardial involvement at the early stage of HIV infection; however, its impact on the clinical course of the disease remains to be clarified.