Sanda Clejan

Blood histamine is associated with coronary artery disease, cardiac events and severity of inflammation and atherosclerosis

Background: Mast cells are prevalent in the shoulder of unstable atheromas; cardiac mast cells secrete proteases capable of activating matrix metalloproteinases. Histamine is essential in the inflammatory cascade of the unstable plaque. Ascorbate depletion has been correlated with histaminemia which has been shown to impair endothelial‐dependent vasodilation. This study evaluates whether oxidative stress as measured by isoprostanes (PGF2α) coupled with an inflammatory state characterized by histaminemia predisposes patients to acute coronary syndrome (ACS).

Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells

We detected cell‐to‐cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 μm in diameter, and from a few microns to at least 50–100 μm in length. Time‐lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1–3 μm in diameter) between adjacent cells. Immunofluorescence detected alpha‐tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anticancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids Permeability and 13C-nuclear magnetic resonance measurements

13C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of 13C-NMR relaxation enhancement measurements using cholesterol enriched with 13C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-13C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether -phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated 45Ca2+ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether -phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for 45Ca2+ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether -, and 1-ether, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. The results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether -phosphatidylcholine bilayers, the 4-13C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating‐wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex‐3 microcarrier beads to form bead aggregates with extensive three‐dimensional growth between beads and on the aggregate surface. HARV and spinner‐flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G1‐phase cells, larger bead aggregates, enhanced development of filopodia and microvilli‐like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T‐flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three‐dimensional growth.

Tri‐dimensional prostate cell cultures in simulated microgravity and induced changes in lipid second messengers and signal transduction

The high aspect rotating‐wall vessel (HARV) was designed to cultivate cells in an environment that simulate microgravity. We studied previously the effects of HARV cultivation on DU‐145 human prostate carcinoma cells. We determined that HARV cultivation produced a less aggressive, slower growing, less proliferative, more differentiated and less pliant cell than other cell cultivation methods. The result was a 3‐dimensional (3D) growth model of prostate cancer which mimics in vivo tissue growth. This work examines the signal transduction‐second messenger pathways existing temporarily in these HARV cells and correlates these features with the special properties in growth and 3D spheroid formation. We found an initial very active ceramide, a diacylglycerol increase together with increases in PI‐PLC and PLA2 a central defect in PLD (no phosphatic acid or phosphatidylethanol at any time during 15 days of HARV cultivation). There is a cross‐talk between ceramide and PI3K pathways with activation of PI3K, after 6 days of HARV growth concomitant with down‐regulation of ceramide. At this time, there is also an increase of cAMP (seen by increases in arachidonic acid). Taken together these results can explain the 3D organoidlike growth. We therefore developed a model for growth in HARV prostate cancer cells which involve temporal “switches” between second messengers, activation and cross‐talk between multiplicity of signaling pathways and a central defect in PLD pathways. Essential to the late slow growth, and 3D organotypic formation are the apoptotic, anti‐survival, anti‐proliferation and differentiation pathways in the first days of HARV, with growth of “new” different types of prostate cancer cells which set‐up for later “switch” in ceramide‐PI3K to survival and proliferation.

Prediction of respiratory distress syndrome using the Abbott FLM-II amniotic fluid assay.

BACKGROUND Most laboratories using the Abbott FLM-II assay for assessing fetal lung maturity follow the manufacturers recommendations for interpreting the surfactant to albumin ratio (S/A). Thus, values >55 mg/g are considered mature and values <40 mg/g, immature-leaving a wide range of indeterminate values. Little data is available to assist the clinician in interpreting values between 40 and 55 mg/g. The goal of this study was to determine decision levels that would more clearly identify risk for RDS based on S/A results. METHODS Respiratory distress syndrome was identified based on medical record review in 46 infants (born at six hospitals), who had S/A measurements on amniotic fluid within 72 h of delivery. An additional 257 women, who had had the S/A test requested but had non-RDS infants, were also identified for this study. The probability of RDS was calculated based on S/A values and on gestational age. Odds ratios were computed for different S/A ratios and different gestational ages. RESULTS Probability of RDS increased with decreasing S/A and decreasing gestational age. At gestational age >36 weeks, the probability of developing RDS ranged from 1% at S/A>44 mg/g to 39% at S/A</=20 mg/g. At gestational age <34, the probability of developing RDS ranged from 14% at S/A>44 mg/g to 92% at S/A</=20 mg/g. CONCLUSIONS We report a risk-based approach for the clinical interpretation of the results of Abbott FLM-II assays based on a broad range of S/A values and gestational ages.

Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.

Isolation of Tn917 insertional mutants of Bacillus subtilis that are resistant to the protonophore carbonyl cyanide m-chlorophenylhydrazone

Tn917 transposition libraries prepared from Bacillus subtilis were screened for mutants that had insertions in the chromosome resulting in resistance to the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Five such strains were characterized. Three of these were found to have distinct insertion sites that resulted in changes in fatty acid composition of the membrane lipids. The lipid changes were qualitatively similar to changes observed earlier in CCCP-resistant strains of B. subtilis that had been isolated after chemical mutagenesis. However, the extent of the changes was more modest, correlating with a lower level of protonophore-resistance. One of these mutants was disrupted in a gene homologous to the Escherichia coli rho gene, as reported earlier (Quirk et al. (1993) J. Bacteriol. 175, 647-654), one was disrupted in a new member of the two-component signalling systems, and the third was disrupted in a new gene of unknown function that apparently forms an operon with transporter genes. The other two CCCP-resistant mutants were disrupted in genes that are likely to encode membrane transporters; the disruption of these genes may have reduced the transmembrane ion leaks during growth, thus conferring modest protonophore-resistance. In one of these strains, the disrupted gene is part of an apparent operon that is a homologue of iron uptake operons from other prokaryotes.

Permeability studies of lipid vesicles from alkalophilic Bacillus firmus showing opposing effects of membrane isoprenoid and diacylglycrol fractions and suggesting a possible basis for obligate alkalophily

Previous studies of the membrane lipids of extremely alkalophilic bacilli had indicated that both facultative and obligate alkalophiles contained a substantial fraction of isoprenoid lipid as well as high concentrations of cardiolipin. Facultative alkalophiles differed from obligate strains in having a phospholipid fatty acid composition that would be expected to result in a more ordered membrane structure. Current studies of ion permeability in vesicles prepared from lipids from obligately alkalophilic Bacillus firmus RAB and its facultatively alkalophilic strain, OF4, support the suggestion that membranes of the latter strain form a tighter barrier structure, with the difference especially pronounced at near neutral pH values. The water permeability of whole cells and the reflection coefficients for acetamide in vesicles were also consistent with a tighter membrane in the facultatively alkalophilic strain than in the obligately alkalophilic strain. The permeability properties of vesicles prepared from phospholipids from these organisms were studied as a function of the addition of either homologous membrane isoprenoid or diacylglycerol. For each permeability parameter that was assayed, in lipids from both strains, the isoprenoid fraction decreased the permeability, whereas the diacylglycerol fraction increased the permeability of the vesicles to solute.

Immune responses induced by intranasal imiquimod and implications for therapeutics in rhinovirus infections

Notwithstanding the progress recently made in immunology and virology, there is yet no effective, specific treatment for the common cold. Symptomatic treatment is minimally effective. An anecdotal report of rapid clearing of the common cold of recent onset after intranasal application of imiquimod in several subjects by one of the authors, made us test the hypothesis that this treatment works through the secretion of interferon by the nasal mucosa. We decided to do an animal study in primates (Indian Macaca Mulata): 5 treatment and 3 control animals were used. Imiquimod or placebo was massaged into the nares of the animals and periodic samples of post‐nasal fluid were taken and measurements for Interferon α (IFNα) and Tumor Necrosis Factor α (TNFα) were made by ELISA methods, and kinetic studies. IFNα mRNA was also isolated and analyzed by quantitative competitive RT‐PCR. The internal standard was constructed to be complementary to and compete with oligonucleotide primers and for amplification of target sequences. One intranasal application of imiquimod rapidly (1–4 h) induced high levels of mRNA for IFNα, and minimal levels in the control animals. Rapid induction of INFα, and proportional increase of TNFα sustained for 4 and 6 h respectively were noted. No adverse reactions to treatment were found in macaques during this short period of intranasal imiquimod usage (except in one macaque with a short period of lacrimation). No animal had cytotoxic effects when examined at 6 h, 12 h, 24 h or 48 h, except one animal, which had an episode of lacrimation for 6hr post treatment. Thus both safety and efficacy of short treatment with imiquimod is proven in this animal model. Proof of principle for intranasal treatment of the common cold with imiquimod is shown. We think that this work will encourage a number of double blind clinical trials to confirm the effectiveness of the intranasal treatment of the common cold with imiquimod.