Dynamic changes in endoglin expression in the developing mouse heart.

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied. Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart. ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme. In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.

Dexamethasone treatment of pregnant F0 mice leads to parent of origin-specific changes in placental function of the F2 generation.

Dexamethasone treatment of F0 pregnant rodents alters F1 placental function and adult cardiometabolic phenotype. The adult phenotype is transmitted to the F2 generation without further intervention, but whether F2 placental function is altered by F0 dexamethasone treatment remains unknown. In the present study, F0 mice were untreated or received dexamethasone (0.2µgg(-1)day(-1), s.c.) over Days 11-15 or 14-18 of pregnancy (term Day 21). Depending on the period of F0 dexamethasone treatment, F1 offspring were lighter at birth or grew more slowly until weaning (P<0.05). Glucose tolerance (1gkg(-1), i.p.) of adult F1 males was abnormal. Mating F1 males exposed prenatally to dexamethasone with untreated females had no effect on F2 placental function on Day 19 of pregnancy. In contrast, when F1 females were mated with untreated males, F2 placental clearance of the amino acid analogue (14)C-methylaminoisobutyric acid was increased by 75% on Day 19 specifically in dams prenatally exposed to dexamethasone on Days 14-18 (P<0.05). Maternal plasma corticosterone was also increased, but F2 placental Slc38a4 expression was decreased in these dams (P<0.05). F0 dexamethasone treatment had no effect on F2 fetal or placental weights, regardless of lineage. Therefore, the effects of F0 dexamethasone exposure are transmitted intergenerationally to the F2 placenta via the maternal, but not paternal, line.

Wastewater treatment modelling in practice: a collaborative discussion of the state of the art.

Three consulting teams conducted independent modelling projects for three different wastewater treatment plants ranging in size from approximately 113,800 m(3)/d (30 mgd) to 530,000 m(3)/d (140 mgd), in different parts of the world (USA and Finland). The plants have different treatment objectives ranging from nitrification and partial denitrification (nitrate plus nitrite <8.7 mg/L) to enhanced nutrient removal (total nitrogen <3 mg/L, total phosphorus <0.3 mg/L). Commonly-used models were applied in the case studies, including ASM3 (using the GPS-X simulator), New General (using GPS-X), Dold (using BioWin), and a variation of the Dold model methanol degradation capabilities (NGmeth within GPS-X). The authors compare and contrast the modelling approaches taken, including calibration and validation approaches, sensitivity analyses, and the application of results to full-scale studies, designs and operations. Despite several differences between the approaches, there are many similarities which are discussed in light of the IWA draft uniform protocol for activated sludge modelling. The authors also discuss current modelling limitations and offer suggestions to improve the state of the art.

The effect of nitric oxide on the growth of Plasmodium falciparum, P. chabaudi and P. berghei in vitro.

Protective immune mechanisms to the asexual erythrocytic stages of the malaria parasite Plasmodium chabaudi AS strain include antibody-independent mechanisms. Nitric oxide (NO) is produced during the infection and indirect evidence suggests that it can contribute to the antiparasitic mechanisms. We examined the effect of an NO producer, S-nitroso-acetyl-penicillamine (SNAP), on the growth and survival in vitro of P. chabaudi AS, P. berghei and P. falciparum. Growth of the parasites was monitored by the uptake of tritiated hypoxanthine and, in the case of P. falciparum, by morphological examination in stained blood smears. DL-penicillamine and sodium nitrite, as controls, had no inhibitory activity at the concentrations used. The results showed that at SNAP concentrations of approximately 182 microM and above NO was cytotoxic to P. falciparum but, at lower concentrations, there was a cytostatic effect and some parasites resumed growth and division after NO production had ceased. Rings were less susceptible to NO effects than later stages in the asexual cycle. The antimalarial activity of NO from SNAP also extended to the rodent parasites but, under the experimental conditions used, they were less sensitive than the human species. In the cultures of P. chabaudi, increasing the numbers of noninfected erythrocytes present did not diminish the antimalarial activity of SNAP, suggesting that here at least haemoglobin was not scavenging NO significantly.

Modifying influence of enalaprilat on mesangial cell DNA synthesis induced by hydrogen peroxide.

This study examined the effect of the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, on mesangial cell (MC) DNA synthesis induced by H2O2, IL-6 and PDGF. MC were incubated with enalaprilat (2.5-100 mumol/l) alone and together with combinations of H2O2 (3 daily pulses of 10(-6) mol/l), IL-6 (5 ng/ml) and PDGF (10 ng/ml). DNA synthesis was assessed after 72 h using [3H]thymidine (3H-TdR) incorporation. Enalaprilat alone had no effect on MC DNA synthesis. Stimulation of MC by H2O2, PDGF and IL-6 alone resulted in increases in 3H-TdR of 4936.6 +/- 1147.5, 5640.5 +/- 1537.6 and 4413.5 +/- 998.4 cpm, respectively (P < 0.05 above control). Only 2.5 mumol/l enalaprilat effected a significant reduction in IL-6 and PDGF-induced DNA synthesis. Incubation of MC with H2O2 + PDGF or H2O2 + IL-6 resulted in increases of 3H-TdR of 6471.9 +/- 1785.1 and 5507.2 +/- 1270 cpm, respectively (P < 0.05 above control). Addition of enalaprilat with either H2O2 + PDGF or H2O2 + IL-6 effected significant reductions in DNA synthesis over the range 2.5-100 mumol/l. These data demonstrate that ACE inhibitors modulate MC DNA synthesis induced by reactive oxygen species.

Germ-layer surface tensions and "tissue affinities" in Rana pipiens gastrulae: quantitative measurements.

The morphogenetic properties causing germ-layer spreading and stratification in amphibian gastrulation were called "tissue affinities" by Holtfreter. The differential adhesion hypothesis (DAH) attributes such liquid-like tissue rearrangements to forces generated by intercellular adhesions within and between the migrating cell populations. This theory predicts that, among the primary germ layers, the cohesiveness of deep ectoderm should be the greatest, that of deep mesoderm should be intermediate, and that of deep endoderm should be the least. Also, the cohesiveness of differentiating neural ectoderm should increase after induction, causing it to internalize and segregate from epidermis. The DAH also explains why the cohesiveness of "liquid" tissues, whose cells are free to rearrange, should be measurable as tissue surface tensions. Using a specially designed tissue surface tensiometer, we demonstrate that (i) aggregates of Rana pipiens deep germ layers do possess liquid-like surface tensions, (ii) their surface tension values lie in precisely the sequence necessary to account for germ-layer stratification in vitro and in vivo, and (iii) the surface tension of deep ectoderm just underlain by the archenteron roof is twice that of not-yet-underlain deep ectoderm. These measurements provide direct, quantitative evidence that the "tissue affinities" governing germ-layer flow during early stages of vertebrate morphogenesis are reflected in tissue surface tensions.

Mesangial cell DNA synthesis induced by hydrogen peroxide, interleukin-6, and platelet-derived growth factor: effects of indomethacin and dazmegrel.

This study indirectly examined the role of prostanoids (PG) in mediating rat mesangial cell (MC) DNA synthesis induced by hydrogen peroxide (H2O2), interleukin 6 (IL-6), and platelet-derived growth factor (PDGF). MC were exposed to three daily pulses of 10(-6) mol/l H2O2 alone or in combination with IL-6 (5 ng/ml) or PDGF (10 ng/ml). In order to examine (indirectly) the role of PG in mediating changes in MC DNA synthesis, indomethacin (1.5 x 10(-5) mol/l) or the thromboxane A2 synthetase inhibitor Dazmegrel (10(-5) mol/l) was added to the medium and DNA synthesis assessed after 72 h using 3H-thymidine incorporation (3H-TdR). Stimulation of MC by H2O2 alone resulted in an increase in 3H-TdR of 34.7 +/- 5.5% (p < 0.01). H2O2 enhanced the mitogenic effects of IL-6 and PDGF, 3H-TdR increasing by 52 +/- 12.1% (p < 0.01) and 100 +/- 21% (p < 0.001), respectively. Indomethacin suppressed the DNA synthesis induced by H2O2 alone, 3H-TdR decreasing by 33 +/- 12% (p < 0.05). Indomethacin also reduced the mitogenic response to H2O2 plus IL-6 and H2O2 plus PDGF by 91 +/- 17 and 97 +/- 12%, respectively (p < 0.05). Dazmegrel reduced 3H-TdR when MC were exposed to H2O2 alone by 31.8 +/- 16% (p < 0.05) and when combined with IL-6 or PDGF by 80 +/- 26 and 120 +/- 13%, respectively (p < 0.05). These data suggest that the pathways through which H2O2-induced growth of MC is mediated appear, at least in part, to involve PG, particularly thromboxane A2.

Interactions of hydrogen peroxide with interleukin-6 and platelet-derived growth factor in determining mesangial cell growth: effect of repeated oxidant stress.

1. This study examined the influence of H2O2, interleukin-6 and platelet-derived growth factor on the proliferation of rat mesangial cells. Mesangial cells were exposed to either a single pulse or three daily pulses of H2O2 (10(-8)-10(-4) mol/l), alone or in combination with interleukin-6 (5 ng/ml) and/or platelet-derived growth factor (10 ng/ml). Proliferation was assessed after 24 h and 72 h of incubation using [3H]thymidine incorporation and cell counts. 2. Although one pulse of H2O2 had no significant effect on mesangial cell proliferation, three daily pulses of 10(-6) mol/l H2O2 resulted in a significant increase in [3H]thymidine incorporation of 31 (52.6, 10.3)% (median and 75th-25th interquartile range) (P < 0.001). Both interleukin-6 and platelet-derived growth factor were also mitogenic to mesangial cells, [3H]thymidine incorporation increasing by 19 (36.7, -6.7)% (P < 0.05) and 53.5 (107, 21.9)% (P < 0.001), respectively. The mitogenic effect of interleukin-6 was enhanced by 10(-6) mol/l H2O2 [49.9 (77.7, 12.3)%] (P < 0.01), whereas the addition of 10(-6) mol/l H2O2 to platelet-derived growth factor resulted in a summated increase in [3H]thymidine incorporation of 82.7 (113, 57.4)% (P < 0.001). Incubation with all three substances simultaneously resulted in down-regulation of growth compared with H2O2 plus platelet-derived growth factor by 55.4 (77.7, 10.3)% (P < 0.05). 3. These findings suggest that reactive oxygen species may play a major role in determining the mesangial cell proliferation that occurs in certain forms of glomerulonephritis, acting either alone or in combination with other growth factors.

Definitive estimate of rate of hemoglobin switching: measurement of percent hemoglobin F in neonatal reticulocytes.

The transition from fetal to adult erythropoiesis starts at 32-36 wk postconception. The rate of this switchover has been controversial. Studies of globin chain synthesis by reticulocytes in vitro have indicated gradual switching with a time for 50% reduction in fetal Hb synthesis ("half-time") of more than 6 wk. This may not accurately reflect fetal/adult Hb synthetic balance in vivo. By contrast, histochemical studies and also indirect mathematical analysis of postnatal changes in circulating fetal Hb and adult Hb may imply abrupt patterns of switching with half-times of less than 1 wk. We have resolved this discrepancy by direct measurement of the changing proportions of fetal and adult Hb in reticulocytes prepared by flourescence activated cell sorting from 10 full-term cord and 45 preterm postnatal blood samples. This method overcomes problems both of extrapolation from in vitro measurements and of mathematical analysis. We find a gradual transition from fetal Hb to adult Hb synthesis. The half-time is approximately 16-18 wk. Values of fetal Hb in reticulocytes were on average higher than predicted from in vitro synthesis studies. We find considerable individual variation. Infants differ in their switching behaviour, many showing prolonged dependence on fetal Hb.

Determination of red-cell mass in assessment and management of anaemia in babies needing blood transfusion.

A new method for the rapid determination of red-blood-cell mass (RCM) in infants needing blood transfusion is described. RCM is estimated from the fall in the baby's fetal haemoglobin level resulting from transfusion of a known mass of adult-haemoglobin-containing red cells. In severe blood loss and refractory anaemias in preterm infants, in addition to the red-cell deficit, the plasma volume is low, leading to a falsely high haematocrit of about 0.30, which conceals a deficiency of 50-70% in circulating red cells. Red-cell transfusion in such infants based on haematocrit often fails to restore RCM to normal, leading to repeated transfusions. The frequency of transfusions may be reduced by giving enough red cells fully to correct the deficiency based on RCM estimation.

The ability of fibrinogens, FI and FII, to support ADP-induced platelet aggregation.

Fibrinogen plays an integral part in ADP-induced platelet aggregation. Controversy exists in regard to the role of the carboxy termini of fibrinogen A alpha chains in this reaction. We have attempted to clarify this problem in view of the availability of a highly purified FII fibrinogen fraction. Kabi fibrinogen or its purified fractions FI, FII and FIII-IV-V were added to washed platelets in the presence of Tyrode-HEPES buffer pH 7.4. Aggregation was initiated by the addition of calcium and ADP. These fibrinogen fractions equally promoted ADP-induced platelet aggregation. The major difference among these fractions is in their A alpha chains. The FI fraction contains intact A alpha chains while FII and FIII-IV-V fractions have one and two partially degraded A alpha chains at the carboxy terminal portion respectively. We conclude that the carboxy terminal portion of the A alpha chain does not play an important role in promoting ADP-induced platelet aggregation.

Isolation of size homogeneous preparations of high molecular weight and low molecular weight fibrinogens.

Molecular sizes of fibrinogen (F) similar to FI, higher molecular weight form, and FII, lower molecular weight form, have been found by Lipinska and colleagues. A procedure has been developed to isolate for the first time each of the FI and FII forms of fibrinogen which are free of each other and of high molecular weight fibrin-fibrinogen complexes. This process involved removing the complexes by A-5 m chromatography. This chromatography also reduced a protein contaminant (X) and removed plasminogen. (NH4)2SO4 subfractionation at pH 5.9 was then done. A subfraction (16-18%) containing 90% FI and another (22-25% or 25-28%) containing 96% FII were obtained. Reprecipitation of the first 16-18% subfraction yielded a subfraction containing 97% FI. sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis of FII revealed that it contains one intact a alpha chain per (b beta, gamma)2. Clot opacity studies on FII suggested that the carboxyl terminal portion of the alpha chain of fibrin plays an important role in the lateral associations in fibrin polymerization. Also, the pattern of (NH4)2SO4 precipitation of the endogenous fibrin-fibrinogen complexes was studied. This revealed that the complexes precipitated mostly in the least soluble subfractions, but small amounts could be found in all subfractions. Examination of the complexes by SDS-polyacrylamide gel electrophoresis showed that most of the complexes could be dissociated to FI and FII. However, there were complexes which remained and these were found to be covalently cross-linked forms probably produced by factor XIII.

Liquid-tissue behavior and differential cohesiveness during chick limb budding.

Emerging chick limb-buds at first grow only in length, not width. The growth parameters of limb mesoderm--cell shapes, distributions, division patterns and cleavage orientations--are incompatible with representations of this tissue as an elongating solid composed of proliferating but immobile cells. We observe that samples of both early limb mesoderm and also surrounding flank mesoderm round up like liquid droplets in organ culture. Therefore, liquid-like tissue rearrangments, including cell shuffling movements and neighbor exchanges, may occur in limb and flank mesoderm during in vivo limb budding. If so, differences in limb-flank surface tension properties would have to be present to keep these two fluid cell populations segregated into distinct tissues and properly positioned underneath limb and flank ectoderm. Previous studies have shown that tissue surface tensions are reflected in the spreading behavior of fused pairs of cell aggregates. To determine whether or not they possess differing surface tension properties, we pair excised pieces of early leg-bud, wing-bud or intervening flank mesoderm with pieces of 5 3/4-day heart or liver in hanging drop cultures. For more rapid determinations of relative liquid-tissue cohesiveness than can be obtained in conventional, long-term experiments, aggregate pairs are fixed shortly after fusion. Since partial-envelopment configurations depend upon relative aggregate sizes as well as their tissue surface tensions, new procedures are used to deduce relative aggregate cohesiveness from cross-sections of these briefly fused aggregate pairs. The envelopment tendencies of aggregates fixed 6--9 h after fusion are similar to those fixed 15--19 h after fusion: heart tends to surround leg; heart and wing surround each other with similar frequencies, but flank tends to surround heart. Also, liver tends to surround leg and wing, but flank tends to surround liver. When the effects of relative aggregate size are taken into account, these non-random, tissue-specific patterns of aggregate envelopment indicate that the relative cohesiveness of these tissues falls into the sequence: leg greater than heart congruent to wing greater than liver greater than flank. The in vitro behavior of early limb-bud and neighboring flank mesoderm in these studies suggests that they are not simply mechanically identical portions of a single liquid tissue. We have previously proposed that early limb-bud mesoderm may act like a non-dispersing, cohesive liquid droplet which is embedded within a less cohesive fluid layer of flank tissue (and which is molded distally into paddle-shaped conformations by solid-like limb ectoderm and/or subjacent extracellular matrix). This proposal is not only compatible with the growth parameters of limb-bud mesoderm in vivo, but is also consistent with our observation that flank mesoderm surrounds tissues which surround limb mesoderm in these aggregate-fusion experiments. Our model suggests that differences in the surface tension properties of limb vs...

Embryonic tissues as elasticoviscous liquids. I. Rapid and slow shape changes in centrifuged cell aggregates.

Certain embryonic tissue masses and cell aggregates behave like deformable solids during brief experimental manipulations but like viscous liquids in long-term organ cultures. To investigate these seemingly paradoxical physical properties, we have mechanically deformed cell aggregates derived from several embryonic chick organs by centrifuging them against solid substrata. Aggregate shapes during brief centrifugation were observed directly in a microscope-centrifuge. In addition, techniques were devised for fixing cell aggregates during prolonged centrifugation. Evidence presented here shows that these fixative-injection procedures accurately preserve the prefixation shapes of living centrifuged aggregates. According to a simple viscous-liquid model for cell aggregates, cohering cells slide past one another when external forces and/or tissue surface tensions cause gradual rearrangements in aggregate conformations. In earlier experiments, 2 types of behaviour predicted from this model were confirmed for several embryonic chick tissues subjected to prolonged centrifugation. First, initially flat aggregates rounded up against the centrifugal force to adopt the same shapes that initially round aggregates reached by flattening. Second, the relative roundness of centrifuged aggregates of different tissues at shape equilibrium correlated with the relative positions that these tissues assumed when they were combined in aggregate-spreading and cell-sorting experiments. By contrast, the brief centrifugation experiments described here provide some support for a simple elastic-solid model in which aggregate shape changes are accompanied by cell deformations rather than cell redistributions. In particular, since cell migration tends to occur quite slowly, the very rapid aggregate flattening observed during the first few minutes of centrifugation presumably requires cell stretching. Moreover, since they do also round up very rapidly following brief centrifugation, these aggregates exhibit considerable elasticity that presumably reflects the swift relaxation of cell stretching as the centrifugal force is removed. Athough both elastic-solid and viscous-liquid properties can be recognized in cell aggregates, we note that, in the prolonged centrifugation experiments described here, rapid initial aggregate flattening is followed by much more gradual, continued flattening. Similarly, after prolonged centrifugation, rapid partial aggregate rounding-up is also followed by much more gradual, continued rounding-up during subsequent culture at Ig. Such rapid-then-slow shape changes contradict both simple elastic-solid and simple viscous-liquid models for cell aggregates. These bimodal shape changes are instead consistent with both compound-viscoelastic-solid and elasticoviscous-liquid models for cell aggregates, although only the latter can also account for long-term liquid-like aggregate behaviour...

Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.