Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-β2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.     

Secretion of a suppressor cell inducing factor by an interleukin-3 dependent cell line with natural cytotoxic activity

This report describes the morphology, surface markers, growth requirements, and functional activity of the M1-A5 cell line, which was established by the limiting dilution of spleen cells from a mouse bearing a large methylcholanthrene-induced fibrosarcoma. The M1-A5 cells share many of the morphological features of large granular lymphocytes and, in addition, express asialo GM1 and Ly-5 surface markers which are commonly found on natural killer cells (NK) cells. There is no expression of T-cell differentiation antigens, surface immunoglobulin, or the granulocyte/macrophage marker, MAC-1. M1-A5 cells are dependent on exogenous growth factor(s) for survival and will proliferate if cultured in interleukin 3 (IL-3), but not in interleukin 1 (IL-1), interleukin 2 (IL-2), or granulocyte/macrophage colony stimulating factor (GM-CSF). In addition, the M1-A5 cells do not absorb IL-2. Despite their morphology and surface characteristics, the M1-A5 cells do not lyse NK targets such as YAC-1 and RLM1 in 4- or 18-hr cytotoxic assays but do lyse the natural cytotoxic (NC) susceptible target, WEHI-164, and to a very small extent, the M-1 fibrosarcoma cells, in an 18-hr assay. Thus they exhibit NC-like cytotoxic activity. In addition, the M1-A5 cells secrete a small molecular weight factor which activates suppressor cells capable of inhibiting antibody synthesis by cocultured syngeneic spleen cells.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Effects of progesterone pretreatment on fertility of gilts mated at an induced pubertal estrus

The effects of progesterone (100 mg/d, im) on pubertal fertility were examined in 247 gilts over 3 experiments. In the first experiment, 128 gilts were exposed to progesterone for 0, 2, 4 or 8 d before receiving PMSG (750 IU) 1 d later. The number of large (>4mm) follicles or corpora lutea (CL) were determined on the day of PMSG injection, Day 0 (onset of estrus), Day 1 or Day 10 (n=8). In the second experiment, embryonic survival was observed in 68 gilts after induction of estrus with PG600 (400 IU PMSG, 200 IU hCG). Vehicle or progesterone was previously administered for 2 d to these gilts, and they were allowed 1, 2, or 3 d between the last progesterone injection and PG600. In Experiment 3, a field trial was conducted in which 51 gilts received vehicle or progesterone for 2 d, followed by a 3-d interval before injection of PG600 to induce estrus. The gilts were allowed to farrow. Treatment with progesterone 1 d before PMSG increased (P<0.05) the number and size of preovulatory follicles and increased (P<0.05) the number of corpora lutea. However, the percentage of gilts pregnant by Day 10, the number of embryos recovered per gilt and embryonic survival were reduced (P<0.05) with progesterone pretreatment. Utilizing a smaller dose of PMSG (750 vs 400 IU) with PG600 negated the effects of progesterone pretreatment on ovulation rate. When the interval between progesterone treatment and PG600 was lengthened to 3 d embryonic survival to Day 30 improved but was similar to that of the vehicle/PG600 treated gilts. Fertility, as defined as conception rate and litter size, was similar between gilts exposed to vehicle or progesterone. These results indicate that pretreatment with progesterone up to the day before PMSG might improve follicular development and ovulation rate at the pubertal estrus with a dose of 750 IU of PMSG but not with the 400 IU (PG600). Reducing the dose of PMSG to 400 IU and allowing for 3 d between progesterone and gonadotropin treatment reduced the incidence of uterine infections but resulted in a fertility rate similar to that of gilts receiving PG600 alone.     

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.     

Susceptibilities of Algae and Legionella pneumophila to Cooling Tower Biocides

Nine algal strains and nine Legionella pneumophila strains were tested in laboratory culture for their susceptibility to inhibition by a variety of commercially available microbiocides. The responses ranged from ineffective to effective at 1/100 the manufacturers' recommended pulse doses. Tests were also performed to determine whether the action of the microbiocide was bacteriostatic or bacteriocidal.     

Treating hop plants with (Z)-jasmone increases colonization by Phorodon humuli (Hemiptera: Aphididae) spring migrants

Hop plants were sprayed with (Z)-jasmone, at a rate of 50 g ha(-1), during the spring migration of the damson-hop aphid Phorodon humuli (Schrank) in 2002 and 2003. Numbers of P. humuli spring migrants colonizing hop plants, Humulus lupulus L., 2-6 and 7-11 days after applying this treatment were assessed in both years. During the first five-day period, significantly more spring migrants were found on hop plants treated with (Z)-jasmone, in comparison with control plants, in 2002. By contrast, no significant difference was evident in the second five-day period. Although the migration in 2003 was much lighter than in 2002, greater numbers of migrants were again removed from treated plants. Indeed, more spring migrants were removed from plants sprayed with (Z)-jasmone in this year during both five-day periods (11 and 44%, respectively) compared with the 23% greater numbers removed in the first five-day period in 2002. Therefore, unlike some other species of aphid, where numbers were consistently lower on plots sprayed with (Z)-jasmone, spraying the secondary host of P. humuli with this compound appears to increase colonization by spring migrants.     

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.