Cell-sized liposome doublets reveal active tension build-up driven by acto-myosin dynamics.

Cells modulate their shape to fulfill specific functions, mediated by the cell cortex, a thin actin shell bound to the plasma membrane. Myosin motor activity, together with actin dynamics, contributes to cortical tension. Here, we examine the individual contributions of actin polymerization and myosin activity to tension increase with a non-invasive method. Cell-sized liposome doublets are covered with either a stabilized actin cortex of preformed actin filaments, or a dynamic branched actin network polymerizing at the membrane. The addition of myosin II minifilaments in both cases triggers a change in doublet shape that is unambiguously related to a tension increase. Preformed actin filaments allow us to evaluate the effect of myosin alone while, with dynamic actin cortices, we examine the synergy of actin polymerization and myosin motors in driving shape changes. Our assay paves the way for a quantification of tension changes triggered by various actin-associated proteins in a cell-sized system.

ActA and human zyxin harbour Arp2/3-independent actin-polymerization activity.

The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.

Actin modulates shape and mechanics of tubular membranes.

The actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes for intracellular transport of material in mammalian cells, yeast, or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are formed during this process and destabilize into vesicles. While the role of actin in tubule destabilization through scission is suggested, literature also provides examples of actin-mediated stabilization of membranous structures. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatiotemporally. Depending on network cohesiveness, actin is able to entirely stabilize or locally maintain membrane tubes under pulling. On a single tube, thicker portions correlate with the presence of actin. These structures relax over several minutes and may provide enough time and curvature geometries for other proteins to act on tube stability.

Limited proteolysis of Hansenula polymorpha yeast amine oxidase: isolation of a C-terminal fragment containing both a copper and quino-cofactor.

Limited proteolysis of recombinant Hansenula polymorpha yeast amino oxidase produces a 48 kDa fragment which corresponds to the C-terminal two-thirds of the protein. The fragment contains both TOPA (2,4,5-trihydroxyphenylalanine) and copper, as well as the histidine ligands implicated in copper binding. The fragment is proposed to be the domain responsible for cofactor production in yeast amine oxidase.

Study of the left ventricular function in pregnancy-induced hypertension.

Left ventricular (LV) morphological and functional characteristics in 9 women suffering from pregnancy-induced hypertension (PIH) were studied by means of echocardiograms. In order to distinguish which changes depended on the pressure values and which were the result of pregnancy, 10 nonpregnant control women with no heart disease and 10 normal pregnant women (NP) were studied and the results of each of the groups compared. To evaluate the structure, left ventricular systodiastolic diameters and wall thickness were measured. The only statistically significant difference was in the diastolic diameters between the PIH (4.7 +/- 0.3 cm) and the control group (4.4 +/- 0.2 cm) p less than 0.01. Left ventricular mass was significantly increased (p less than 0.01) in the PIH patients (185 +/- 53.1 g) compared to the NP patients (161 +/- 29.6 g) and the control group (125 +/- 17.4 g). No statistically significant differences were found in the radius thickness ratio in the three groups. The systolic function assessed by the shortening percentage was significantly lower (p less than 0.05) in the control group (32.8 +/- 4.4%) and in the NP patients (37.8 +/- 5.2%) than in the PIH group (39 +/- 6.5%). Afterload assessed by isovolumic period stress was significantly greater (p less than 0.01) in the PIH patients (157 +/- 10.6 dyne/cm2) compared with the NP group (118.9 +/- 7.01 dyne/cm2). There were no significant differences between the first group and the control group (134.09 +/- 8.7 dyne/cm2). As evidence of the diastolic function, analysis was made, on the one hand, of diastolic isovolumic period length (DIP).(ABSTRACT TRUNCATED AT 250 WORDS)

Health status of teenage school boys in eastern Sudan.

PIP: In July, 1981 a health survey of teenage boys was conducted in a medical clinic in the eastern Sudan, run by the Lalmba Association, a non-profit aid organization based in the US. The study population was a group of 231 students ranging in age from 13-18, some of whom board at the school. Data consisted of a general health questionnaire, height and weight measurements, and a thorough physical examination including stool sample. Average height and weight were both less than Western teenagers, possibly due to a diet deficient in animal and plant protein and total calories. Hematocrit levels were comparable. Stool parasites were present in 31% of those surveyed, the most common being Giardia, Entamoeba histolytica, and Hymenolepsis nana. The most common symptomatic problem was dental caries (8%) and the most common asymptomatic problems were trachoma and anemia (15% and 9% respectively). Investigators noted an unexpected number of genitourinary abnormalities (6%). The study population was selective in that it was the well-off portion of the teenage population in this locality. The investigators suggest that health authorities in the area distribute trachoma prophylaxis (antibiotic eye ointment) to all school age children.^ieng

[Characteristics of the isovolumic relaxation period in dilated cardiomyopathy]. / Características del periódo de relajación isovolúmica en la miocardiopatía dilatada.

The duration of isovolumic relaxation (IR) was used in order to analyze several aspects of diastolic function in patients with dilated cardiomyopathy (DC). This period as measured from the aortic closure sound (A2 on phonocardiogram) to the opening of the mitral valve (on echocardiogram) was determined in 14 patients with DC and compared with findings in 10 normal volunteers (N). Heart rate (HR) and blood pressure (BP) were similar in both groups. The duration of IR was significantly increased in DC (72 ms +/- 6.3 ns) compared with N (41.6 ms +/- 2.8 ns; p less than 0.01). These data suggest that the prolongation in IR may result from alterations in ventricular relaxation.

An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha.

The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity.

Caracteristicas del periodo de relajacion isovolumica en la miocardiopatia dilatada / Characteristics of the isovolumic relaxation period in dilated cardiomyopathy

A los efectos de analizar algunso aspectos de la función diastólica en la miocardiopatía dilatada (Md) utilizamos la duración del período de relajación isovolúmico (PRI) obtenido por métodos no invasivos en un grupo de pacientes con dicha patología. Se examinaron un total de 24 pacientes, 10 de los cuales configuraron el grupo control (C) y 14 el portador de Md; la duración del PRI se midió desde el comienzo de las vibraciones rápidas del segundo ruido aórtico obtenida por fonocardiograma y hasta el momento de separación de ambas valvas de la mitral analizada por ecocardiograma. Dado que la duración del PRI no sólo depende del estado de la relajación también analizamos otros parámetros como la frecuencia cardíaca y la presión arterial. La cavidad ventricular izquierda estaba significativamente aumentada en los pacientes con Md y la función sistólica estaba severamente deprimida. La frecuencia cardíaca resultó similar en los dos grupos. La presión arterial tampoco resultó diferente. El PRI resultó prolongado significativamente en los pacientes con Md. Los datos analizados permiten concluir que la prolongación del PRI encontrada en los pacientes con Md puede estar vinculada a alteraciones en la relajación miocárdica