Formation and antiproliferative effect of prostaglandin E(3) from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E(3) (PGE(3)) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE(3.) The extracellular ratio of PGE(3) to PGE(2) increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE(3), respectively, than that produced by COX-1. Exposure of A549 cells to 1 microM PGE(3) inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE(3), however, had no effect. When A549 cells were exposed to EPA (25 microM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 microM). This effect appears to be associated with a 50% reduction of PGE(3) formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE(2), an increase in the level of PGE(3), and PGE(3)-mediated inhibition of tumor cell proliferation.     

Parallel increases in sister-chromatid exchanges at base level and with UV treatment in human opiate users

The SCE base level frequency and SCE levels induced by far-UV (254 nm) treatment of cells in early G1 and early S phases of the cell cycle were significantly higher in leukocytes from heroin addicts as compared to controls. The increased SCE levels in addicts was greatest at base level and smallest after UV irradiation of cells in S phase. These results corroborate and extend our previous findings of increased chromosome damage and reduced DNA-repair synthesis in heroin users. Since opiates do not directly damage DNA, the elevated cytogenetic effects associated with opiate use probably arise from secondary promotional effects related to opiate-mediated alterations in leukocyte metabolism.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

Daily hypoxia increases basal monocyte HSP72 expression in healthy human subjects

Heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. In vitro, research demonstrates HSP72 induction in response to hypoxia. Recently, in vivo, an acute hypoxic exposure (75 min at 2,980 m) was sufficient to induce significant increases in monocyte expressed HSP72 (mHSP72) and a marker of oxidative stress in healthy human subjects. The purpose of the current study was to identify the impact of 10 consecutive days of hypoxic exposures (75 min at 2,980 m) on mHSP72 and erythropoietin (EPO) expression, markers of oxidative stress, and maximal oxygen consumption in graded incremental aerobic exercise. Eight male subjects were exposed to daily normobaric hypoxic exposures for 75 min at 2,980 m for 10 consecutive days, commencing and ceasing at 0930 and 1045, respectively. This stressor was sufficient to induce significant increases in mHSP72, which was significantly elevated from day 2 of the hypoxic exposures until 48 h post-final exposure. Notably, this increase had an initial rapid (30% day on day compared to baseline) and final slow phase (16% day on day compared to baseline) of expression. The authors postulate that 7-day hypoxic exposure in this manner would be sufficient to induce near maximum hypoxia-mediated basal mHSP72 expression. Elevated levels of mHSP72 are associated with acquired thermotolerance and provide cross tolerance to non-related stressors in vivo, the protocol used here may provide a useful tool for elevating mHSP72 in vivo. Aside from these major findings, significant transient daily elevations were seen in a marker of oxidative stress, alongside sustained increases in EPO expression. However, no physiologically significant changes were seen in maximal oxygen consumption or time to exhaustion.     

Controlled destabilization of a liposomal drug delivery system enhances mitoxantrone antitumor activity.

Programmable fusogenic vesicles (PFVs) are lipid-based drug-delivery systems that exhibit time-dependent destabilization. The rate at which this destabilization occurs is determined by the exchange rate of a bilayer-stabilizing component, polyethylene glycol-phosphatidylethanolamine (PEG-PE) from the vesicle surface. This exchange rate is controlled, in turn, by the acyl chain composition of the PEG-PE. We describe in vitro and in vivo studies using PFVs as delivery vehicles for the anticancer drug mitoxantrone. We demonstrate that the PEG-PE acyl composition determined the rate at which PFVs are eliminated from plasma after intravenous administration, and the rate of mitoxantrone leakage from PFV. The nature of the PEG-PE component also determined the antitumor efficacy of mitoxantrone-loaded PFV in murine and human in murine and human xenograft tumor models. Increased circulation time and improved activity were obtained for PFV containing PEG-PE with an 18-carbon acyl chain length, as a result of slower vesicle destabilization.     

The ubiquitous nature of the progesterone receptor binding factor-1 (RBF-1) in avian tissues

The avian oviduct receptor binding factor-1 (RBF-1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF-1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF-1. The heart and pancreas had the highest and lowest RBF-1 levels, respectively; the concentration ranging by ～ 50-fold in these tissues. The 10 kDa size of the RBF-1 detected in all tissues suggests no significant tissue-specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF-1 have identical amino-terminal sequence. Using a recently isolated cDNA to RBF-1, the levels of RBF-1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue-specific differences in concentration. The presence of RBF-1 in non-progesterone responsive tissues suggests the possibility that RBF-1 may not be specifically involved in PR-DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor. © 1994 Wiley-Liss, Inc.     

Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach

Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function.     

Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli

The effect of iron concentrations in culture media on supernatant yields of campylobacter cytotonic toxin (CCT) was studied. Of the 118 Campylobacter spp. strains surveyed, 78.8% produced toxin in brucella broth or in casamino acids--yeast extract (CYE) broth. When the iron concentration of CYE was increased from 0.44 microgram/mL (7.9 microM) to 0.65 microgram/mL (11.6 microM) by the addition of ferric chloride, 94.9% of the strains were positive for toxin in a ganglioside GM1 based, enzyme-linked immunosorbent assay, using antibody to affinity-purified CCT. The addition of iron as ferrous sulfate was less effective. When four toxin-positive strains were grown in a deferrated medium of conalbumin-treated CYE with 0.04-0.08 microgram iron/mL (0.72-1.43 microM), two of the culture supernatants became negative (absorbance at 410 nm, less than 0.1 and less than 10 ng CCT/mL), and two produced about 90% less CCT but were still classified as positive (absorbance, greater than or equal to 0.1 and greater than or equal to 10 ng CCT/mL). It was therefore concluded that the production of CCT by Campylobacter spp. is influenced by iron concentration.     

Thorns as induced defenses: experimental evidence

Summary We report evidence from controlled experiments that long straight thorns deter herbivory by browsers. Cut branches of three woody species that had their thorns removed suffered significantly greater herbivory by a tethered goat than did paired intact branches. Branches on living Acacia seyal plants that had their thorns removed suffered significantly greater herbivory by a wild population of free-ranging giraffes than did intact branches on the same plants. These differences in herbivory resulted in long term losses of branch length in clipped as opposed to control branches. In addition, branches within reach of giraffes produced longer thorns and a greater density of thorns than did higher branches. These results imply that increased thorn length is an induced defense.No significance should be attached to order of authorship     

Comparison of serotonin uptake by dense bodies inside and outside human platelets

ATP-dependent proteolysis in reticulocyte extracts is stimulated by ubiquitin, a polypeptide which is covalently conjugated to proteins. It has been proposed that ATP and ubiquitin act by repressing an inhibitor of an ATP-independent protease, rather than by conjugation to substrate proteins [Speiser, S. and Etlinger, J.D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3577-3580]. We find that the inhibitor preparation used by these authors contains a positively required factor of the ATP-ubiquitin proteolytic system, which can be separated from two types of protease inhibitors by gel filtration chromatography. The following observations indicate that the "inhibitors" are endogenous protease substrates which compete with the labeled substrate: (a) inhibition is competitive with exogenous substrate; (b) inhibition is abolished by a preincubation of "inhibitor" with protease prior to the addition of labeled substrate. These findings are not consistent with the notion that the inhibitors play a regulatory role in the ATP-ubiquitin proteolytic pathway.