Interaction of C-phycocyanin with lipid monolayers under nitrogen and in the presence of air

The surface interaction of C-phycocyanin with lipids was studied using the monolayer technique. The surface activity of the protein was found to be higher at the lipid-water interface than at the nitrogen-water interface, particularly at high surface pressures of the lipid monolayer. The maximum initial surface pressures beyond which phycocyanin could not penetrate the dipalmitoylphosphatidylcholine and monogalactosyldiglycerol monolayers were 27 and 30 mN m-1, respectively. Below these values the protein demonstrated preferential interaction with the monogalactosyldiglycerol monolayer. The surface properties of the unfolded protein at pH 2.5 at the lipid-water interface were compared with those of the protein at pH 7.0. Higher affinity of the three-dimensional structure of the protein to lipid monolayers was observed, in particular by high subphase protein concentration. When the lipid films were subjected to oxidation stress by exposure to air, the surface properties of C-phycocyanin and dipalmitoylphosphatidylcholine were not greatly affected but the surface activity of monogalactosyldiacylglycerol was reduced dramatically by autoxidation. The oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-phycocyanin molecules at the lipid-water interface.     

Effect of calcium on kinetic and structural aspects of dilution-induced micellar to lamellar phase transformation in phosphatidylcholine-cholate mixtures

Previously, we have shown [Almog, S., Kushnir, T., Nir, S., & Lichtenberg, D. (1986) Biochemistry 25, 2597-2605] that the distribution of cholate between phosphatidylcholine (PC) vesicles and aqueous media apparently obeys a single distribution coefficient, K. In PC-cholate mixed micellar systems, the monomer concentration does not rise much above the cholate's critical micelle concentration (cmc). Consequently, for vesicular systems, the cholate:PC molar ratio in the mixed aggregates (Re) is given by Re = [cholate]/([PC] + 1/K) whereas for mixed micellar systems Re = ([cholate] - cmc)/[PC]. Dilution of mixed micellar systems results in a decrease of Re, due to an increase in the fraction of monomeric PC. If the decrease in Re is to values lower than 0.3, micellar to lamellar transformation occurs. This process involves a sequence of three steps, namely, micellar equilibration followed by vesiculation and subsequent vesicle size growth via a lipid transfer mechanism. The ultimate size of the resultant vesicles is an increasing function of Re. This work is devoted to the effect of calcium on the dilution-induced vesicle formation. Its major findings and conclusions are as follows: (i) Calcium reduces the cmc of the detergent and raises its distribution coefficient between PC vesicles and the aqueous medium. Thus, for any given cholate and PC concentrations, calcium causes an increase of Re. (ii) The rate of all the steps which ultimately lead to an apparent equilibrium vesicle size distribution increases dramatically with increasing calcium concentration. Thus, equilibration is attained in seconds to minutes rather than many hours required in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Precipitation of calcium palmitate from bile salt-containing dispersions

Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.     

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.     

Epidermal keratin 5 expression and distribution is under dermal influence

Human skin melanin pigmentation is regulated by systemic and local factors. According to the type of melanin produced by melanocytes, the transfer and degradation of melanosomes differ, thus accounting for most variations between ethnicities. We made the surprising observation that in a drastically changed environment, white and black phenotypes are reversible since Caucasian skin grafted onto nude mice can become black with all black phenotypic characteristics. Black xenografts differed essentially from other grafts by the levels of epidermal FGF‐2 and keratin 5. In vitro analysis confirmed that FGF‐2 directly regulates keratin 5. Interestingly, this phenomenon may be involved in human pathology. Keratin 5 mutations in Dowling–Degos Disease (DDD) have already been associated with the pheomelanosome–eumelanosome transition. In a DDD patient, keratin 5 was expressed in the basal and spinous layers, as observed in black xenografts. Furthermore, in a common age‐related hyperpigmentation disorder like senile lentigo (SL), keratin 5 distribution is also altered. In conclusion, modulation of keratin 5 expression and distribution either due to mutations or factors may account for the development of pigmentary disorders.     

Cole Disease Results from Mutations in ENPP1

The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.     

Comparison of progesterone concentration determination by 12 non-isotopic immunoassays and gas chromatography/mass spectrometry in 99 human serum samples

A single serum progesterone determination may be highly predictive for early pregnancy and in vitro fertilisation and embryo-transfer outcomes. We therefore compared 12 direct non-isotopic progesterone immunoassays with gas-chromatography/mass spectrometry (GC/MS). For each assay, data from the analysis of 99 individual sera were compared with data obtained by GC/MS, using regression and bias plot analyses and the ratio method. We observed a larger difference in concentration between high and low values and a broader distribution of results for immunoassays than for GC/MS. All immunoassays displayed bias in the calibration process and a lack of specificity and/or sensitivity, to various degrees. We tried to identify the parameters of the assay procedure that might contribute to these discrepancies. None of the criteria investigated (antibodies, control and preparation of calibrators, blocking agents and choice of tracer) had a significant effect when studied alone.     

Functional restitution of cardiac control in heart transplant patients

Cardiovascular control is fundamentally altered after heart transplantation (HT) because of surgical denervation of the heart. The main goal of this work was the noninvasive characterization of cardiac rate control mechanisms after HT and the understanding of their nature. We obtained 25 recordings from 13 male HT patients [age = 28-68 yr, time after transplant (TAT) = 0.5-62.5 mo]. The control group included 14 healthy men (age = 28-59 yr). Electrocardiogram, continuous blood pressure (BP), and respiration were recorded for 45 min in the supine position and then during active change of posture (CP) to standing. The signals were analyzed in the time domain [mean and variance of heart rate (HR) and rise time of HR in response to CP] and the frequency domain [low and high frequency (LF and HF)]. Our principal finding was the consistent pattern of evolution of the HR response to standing: from no response, via a slow response (>40 s, TAT > 6 wk), to a fast increase (<20 s, TAT > 24 mo). HR response correlated with TAT (P < 0.001). LF correlated with HR response to CP (P < 0.0001); HF and HR did not. An important finding was the presence of very-high-frequency peaks in the power spectrum of HR and BP fluctuations. Extensive arrhythmias tended to appear at the TAT that corresponds to the transition from slow to fast HR response to CP. Our results indicate a biphasic evolution in cardiac control mechanisms from lack of control to a first-order control loop followed by partial sympathetic reinnervation and, finally, the direct effect of the old sinoatrial node on the pacemaker cell of the new sinoatrial node. There was no indication of vagal reinnervation.     

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk)

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.