Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.     

Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle.

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.     

A comparison of chloride, bromide, and sucrose dilution volumes in neonatal pigs

Use of 36Cl, 82Br, and [3H]sucrose to estimate extracellular water volume was evaluated in 14 piglets (7-14 days old). 36Cl and 82Br were distributed in approximately the same volume, but a period of 5-6 hr after injection was required to reach equilibrium in the neonatal pig. Dilution volumes calculated before equilibration (2-5 hr) for 36Cl (326 +/- 11 ml/kg) and 82Br (328 +/- 13 ml/kg) were different from equilibration (6-8 hr) phase volumes (356 +/- 13 ml/kg and 355 +/- 13 ml/kg, respectively; P less than 0.001). A 3-hr sample estimated the same volume distribution calculated by extrapolation of the 6- to 8-hr period because of the relationship between the two slopes of the plasma clearance curves. After the 82Br and 36Cl had achieved equilibration, each was distributed in a volume equivalent to total body chloride space (362 +/- 29 ml/kg) measured by neutron activation; no statistical differences were found (P = 0.6). The early equilibration phase measured a 10% smaller, faster exchangeable fraction of total body Cl. Sucrose dilution volume (332 +/- 19 ml/kg) required multiple plasma samples for extrapolation and measured a dilution volume 7% smaller (P less than 0.05) than total body chloride space.     

Human axillary secretions influence women''s menstrual cycles: The role of donor extract of females

Menstrual synchrony in human females has previously been demonstrated among women attending a predominantly female university as well as among women attending coeducational universities. In each of these studies, women who spent the most time together were most likely to show the menstrual synchrony. In this experiment, the possibility that substances in axillary secretions might mediate this effect was tested using a prospective, double-blind research design and a combined axillary extract from a group of female donors. Female subjects who reported themselves to have normal (29.5 +/- 3 day) cycles were exposed to the axillary extracts or blank/ethanol for 10 to 13 weeks. Recipients of the axillary extracts showed a significant reduction in "days' difference in menses onset" relative to the donor cycle, no change was evident for recipients of blank/ethanol. These results demonstrate that constituents from the axillary region of donor females can shift the time of menstrual onset of another group to conform with the donors' cycle and that this effect can occur even in the absence of social contact.     

Extracellular volume estimation from ratios of bromide to chloride in urine or saliva.

Use of either urine or saliva samples to estimate extracellular water volume was investigated in 10 men using nonradioactive bromide (Br) and in seven newborn piglets using radioactive Br (82Br) and chloride (36Cl). The relation to Br to Cl concentrations in urine enabled an estimation of Br dilution volume from human urine (267 +/- 42 ml/kg, mean +/- SD) that was not significantly different (P = 1.0) from the Br dilution volume calculated from plasma Br concentration (268 +/- 20 ml/kg). Although the Br dilution volume estimated from saliva was not different from that of plasma, the error in the estimates of Br dilution volume from saliva was too large (mean difference, -36 +/- 64 ml/kg) to make its use practical. The data from piglets showed good agreement between 82Br and 36Cl dilution volumes calculated from 4-hr plasma samples (356 +/- 14 ml/kg and 347 +2- 12 ml/kg; P greater than 0.1) and between 82Br dilution volumes calculated from urine 82Br:36Cl and plasma 82Br (360 +/- 31 ml/kg and 356 +/- 14 ml/kg; P greater than 0.1). Extracellular water volume can be estimated in both adult and young animals using the Br dilution volume calculated from urine samples. It requires (i) two urine collections: one before and one 4 to 8 hr after administration of Br; (ii) a measurement or estimate of plasma Cl concentration; and (iii) a correction factor that describes the relationship of the ratio of Br to Cl in urine to that ratio in plasma.     

Comparative trial of treatment with Prioderm lotion and Kwellada shampoo in children with head lice.

The safety and efficacy of treatment with a new pediculicide lotion, Prioderm (0.5% malathion in isopropanol), were compared with those of treatment with Kwellada (1% lindane) shampoo in a randomized trial of children with head lice. The children''s scalps were examined for live lice immediately, 7 days and 4 to 6 weeks following treatment. To determine the in-vitro ovicidal effect of treatment, samples of hair with nits were removed before and immediately following treatment; the subsequent rates of hatching were compared. No live lice were present immediately following either treatment. At 7 days after treatment 2 of the 29 children treated with Prioderm lotion and 4 of the 33 children treated with Kwellada shampoo were infested with live lice, whereas at 4 to 6 weeks after treatment 5 and 3 children respectively were infested. The initial effectiveness of treatment was 93% in the children treated with Prioderm lotion and 88% in those treated with Kwellada shampoo; however, a large difference in efficacy could have been missed owing to the small number of children in the study. Both preparations demonstrated in-vitro ovicidal activity, but neither totally abolished post-treatment hatching. No side effects were reported from either preparation. Because ovicidal activity was incomplete two treatments with the same pediculicides should be given about 7 days apart.     

Effect of solution non-ideality on erythrocyte volume regulation

A non-ideal, hydrated, non-dilute pseudo-binary salt-protein-water solution model of the erythrocyte intracellular solution is presented to describe the osmotic behavior of human erythrocytes. Existing experimental activity data for salts and proteins in aqueous solutions are used to formulate van Laar type expressions for the solvent and solute activity coefficients. Reasonable estimates can therefore be made of the non-ideality of the erythrocyte intracellular solution over a wide range of osmolalities. Solution non-ideality is shown to affect significantly the degree of solute polarization within the erythrocyte intracellular solution during freezing. However, the non-ideality has very little effect upon the amount of water retained within erythrocytes cooled at sub-zero temperatures.     

Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase.

We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.     

Effects of cyclic GMP on the secondary structure of cyclic GMP dependent protein kinase and analysis of the enzyme's amino-terminal domain by far-ultraviolet circular dichroism

Far-UV circular dichroism spectra of bovine lung cyclic GMP dependent protein kinase (G-kinase) show that the enzyme contains alpha-helical and beta-pleated sheet elements. Binding of cyclic GMP changes the spectra in a way consistent with the induction of beta-sheet from random coil. Examination of the amino-terminal sequence of G-kinase indicates the presence of a strongly alpha-helical segment with several features in common with the leucine zipper motif. We propose that this sequence may be the important part of the dimerization domain of the enzyme. A synthetic peptide corresponding to amino acids 1-39 of G-kinase has a strongly alpha-helical CD spectrum, supporting the predicted secondary structure of this amino-terminal sequence. In contrast to the native enzyme, a structure reduced in alpha-helix was found when a constitutively active form of G-kinase, which lacks amino acids 1-77, was studied.