Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present study further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n = 16). PAF infusion (1-9 nmol/min) produced triphasic changes in CBF (n = 7): an initial brief phase of coronary dilation (14 +/- 2% above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B2 levels, while 6-keto-PGF1 alpha and leukotriene C4-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A2 mediated mechanism.     

Indication of selective growth of human endometrial epithelial cells on extracellular matrix

Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix (ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping, closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture dishes. This work was supported by PHS grant no. CA 30289 to J.V.     

On the Relation between Effector Concentration and the Rate of Induced Enzyme Synthesis

The Jacob and Monod scheme for the regulation of enzyme formation leads to the following relation between the relative rate of enzyme synthesis α and cellular effector concentration E (the lower sign is for repressible systems): log (α/1 - α - α_b) = ± n log [E] + log α_b ± log K₁. This equation permits linear plotting of experimental data and the evaluation of three quantities: n, the number of effector molecules combining with a repressor molecule, K₁, the dissociation constant of this interaction and K₂/R_t, the ratio of repressor-operator dissociation constant to total repressor concentration. Measurements on the repression of alkaline phosphatase in Escherichia coli as a function of phosphate concentration are reported and fit the proposed equation with n = 1, indicating that the binding of a single phosphate to the repressor species may be sufficient to cause repression. K₁ of this interaction was found to be 0.58 ±0.11 × 10^-3 M. The available data regarding the enzymes of the lac operon in a variety of E. coli strains, and several other enzymes are analyzed. It is confirmed that the lac repressor interacts with 2 isopropyl thiogalactoside (IPTG) molecules to relieve repression with a K₁ = 50 ±20 × 10^-12 M². In some strains, separate binding constants for the first and second IPTG molecules can be evaluated.     

Properties of a Maize Glutathione S-Transferase That Conjugates Coumaric Acid and Other Phenylpropanoids

A glutathione S-transferase (GST) enzyme from corn (Zea mays L. Pioneer hybrid 3906) that is active with p-coumaric acid and other unsaturated phenylpropanoids was purified approximately 97-fold and characterized. The native enzyme appeared to be a monomer with a molecular mass of approximately 30 kD and an apparent isoelectric point at pH 5.2. The enzyme had a pH optimum between 7.5 and 8.0 and apparent Km values of 4.4 and 1.9 mM for reduced glutathione (GSH) and p-coumaric acid, respectively. In addition to p-coumaric acid, the enzyme was also active with o-coumaric acid, m-coumaric acid, trans-cinnamic acid, ferulic acid, and coniferyl alcohol. In addition to GSH, the enzyme could also utilize cysteine as a sulfhydryl source. The enzyme activity measured when GSH and trans-cinnamic acid were used as substrates was enhanced 2.6- and 5.2-fold by the addition of 50 [mu]M p-coumaric acid and 7-hydroxycoumarin, respectively. 1H- and 13C-nuclear magnetic resonance spectroscopic analysis of the conjugate revealed that the enzyme catalyzed the addition of GSH to the olefinic double bond of p-coumaric acid. Based on the high activity and the substrate specificity of this enzyme, it is possible that this enzyme may be involved in the in vivo conjugation of a number of unsaturated phenylpropanoids.     

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.     

The effect of (2''-5'') and (3''-5'') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution.

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.     

Efficient Flp-Int HK022 dual RMCE in mammalian cells

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.     

Multiple quantitative trait locus analysis of bovine chromosome 6 in the Israeli Holstein population by a daughter design

Nine Israeli Holstein sire families with 2978 daughters were analyzed for quantitative trait loci effects on chromosome 6 for five milk production traits by a daughter design. All animals were genotyped for 2 markers. The three families with significant effects were genotyped for up to 10 additional markers spanning positions 0-122 cM of BTA6. Two sires were segregating for a locus affecting protein and fat percentage near position 55 cM with an estimated substitution effect of 0.18% protein, which is equivalent to one phenotypic standard deviation. This locus was localized to a confidence interval of 4 cM. One of these sires was also heterozygous for a locus affecting milk, fat, and protein production near the centromere. The hypothesis of two segregating loci was verified by multiple regression analysis. A third sire was heterozygous for a locus affecting milk and protein percentage near the telomeric end of the chromosome. Possible candidates for the major quantitative gene near position 55 cM were determined by comparative mapping. IBSP and SSP1 were used as anchors for the orthologous region on human chromosome 4. Twelve genes were detected within a 2-Mbp sequence. None of these genes have been previously associated with lactogenesis.