Insensitive Target Research Articles

LPS–induced activation of phospholipase A2 phospholipase C and protein kinase C of murine macrophage–like cell lines (J774 and P388D1)

A murine macrophage–like cell line, J774, acquired, in response to LPS, an ability to kill tumor necrosis factor (TNF)–insensitive target P815 mastocytoma cells, whereas another cell line, P388D1, did not. LPS–triggered signaling mechanisms between the two cell lines were compared with an aim to inquire about the possible nature of the above-mentioned difference. The results showed that two cell lines respond to LPS–treatment by parallel activation of both phospholipases C and A2 (PLC and PLA2) to approximately the same extent. The maximum response of both enzymes of J774 cells was noted within 10 min of the treatment, whereas that of P388D1 cells required more than 20 min. The other properties of LPS-responsive enzymes studied were similar between two cell lines, including Ca2+ augmentation of enzyme activation, participation of guanine nucleotide binding (G) proteins in the initial activation processes, and inhibition of enzyme activation by the prior treatment of cells with cholera or pertussis toxins etc. Moreover, LPS–triggered activation of PLC and PLA2 was found to be followed by the increase of PKC activities in both cell lines. In spite of these similarities, J774 cells possessed both basic and acidic forms of PKC activities, while P 388D1 cells owned only PKC of basic form. Nevertheless, the question why J774 cells, but not P388D1 cells, can acquire the tumoricidal activity, aganist P815 cells following LPS–treatment remains to be answered.

Occurrence of Pyrethroid Resistance in Pear Psylla (Homoptera: Psyllidae) Populations from Southern Ontario

Adult pear psylla, Cacopsylla pyricola Foerster, from the Niagara peninsula of Ontario were tested for susceptibility to pyrethroid insecticides from 1980 to 1989. A Potter spray tower was used. Results of tests at IS, 22, and 30°C indicated a negative temperature-toxicity relationship for permethrin and fen vale rate. Resistance to pyrethroids (first discovered in 1986 at levels of approximately 50-fold for permethrin at the LC50) has become widespread in the Niagara region. Resistance extends to all types of pyrethroids but is highest to fenvalerate (approximately 140-fold). Combinations of permethrin and potential synergists piper only but oxide or DEF did not increase the toxicity to resistant populations, suggesting that resistance was due to an insensitive target site or kdr-like mechanism. In one resistant population studied over 4 yr without further selection with pyrethroids, resistance declined from 54-fold to approximately 3D-fold after 1 yr and stabilized at 20-30-fold.

Insecticide Resistance in Spotted Tentiform Leafminer (Lepidoptera: Gracillariidae): Mechanisms and Management

Resistance to organophosphorous insecticides, pyrethroids, and methomyl occurred in populations of spotted tentiform leafminer, Phyllonorycter blancardella (F.), from southern Ontario. Resistance to organophosphorous insecticides occurred in all populations from commerical orchards. Resistance appeared to be due to an insensitive target acetylcholinesterase (AChE). Addition of several types of synergists to azinphosmethyl solutions did not affect toxicity. Resistance to methomyl appeared to be partially due to enhanced metabolism by aliesterases and partially to reduced inhibition of AChE. Selection for methomyl resistance was separate from resistance to organophosphorous insecticides. Increased activity of glutathione S-transferases was not implicated in resistance to either organophosphorous insecticides or methomyl. With current pest control practices, management of resistance to organophosphorous insecticides is not feasible in Ontario apple orchards. Resistant populations are regularly subjected to selection pressure by organophosphorous insecticides applied for control of other orchard pests. Pyrethroid resistance has been managed by modifications in the timing of applications to coincide with a predominance of the most sensitive life stages. Use of this strategy over three seasons has not resulted in detectably higher levels of resistance. Resistance to methomyl was relatively rare compared with organophosphorous or pyrethroid resistance. Methomyl resistance could be managed by strategies similar to those used for pyrethroids, i.e., modification of spray timing to coincide with the presence of early larval stages, or by restricted use. Where use was restricted for 3 yr after resistance was detected, resistance to methomyl declined from 9-fold to approximately 4.5-fold.

Metabolism of primisulfuron by barnyard grass

Barnyard grass ( Echinochloa crus galli (L.) P.B.) is a grass weed species not controlled by the experimental sulfonylurea herbicide, primisulfuron (BEACON). The mechanism of resistance employed by barnyard grass was determined to be metabolism and not an insensitive target enzyme, acetohydroxy acid synthase (AHAS). Of the five weed species examined, barnyard grass was the fastest at metabolizing primisulfuron with an in vivo half-life between 1 and 2 hr. Two major classes of metabolites were produced and were found to be noninhibitory to AHAS. We have monitored the relative abundance of these metabolites by HPLC and have partially purified and characterized the metabolites. For one of the metabolite classes the initial step appears to be an hydroxylation reaction followed by glycosylation.

Studies on the induction and expression of T cell-mediated immunity. XV. Role of non-MHC papain-sensitive target structures and Lyt-2 antigens in allogeneic and xenogeneic lectin-dependent cellular cytotoxicity (LDCC).

The present study examines the role of nonclass I MHC target membrane structures involved in lysis by cytotoxic lymphocytes in the lectin-dependent cellular cytotoxicity (LDCC) system. Cytotoxic cells of rodents and humans have been shown to nonspecifically lyse target cells of different origins and species. The mechanism by which such cytotoxicity takes place is not known, although several hypotheses have been put forth. This study examines the role of class I MHC expression on target cells and Lyt-2 antigens on the cytotoxic cells in both allogeneic and xenogeneic LDCC systems. Human peripheral blood lymphocytes, and murine-allosensitized cytotoxic T lymphocytes (CTL) are used as effector cells, and class I MHC-negative Daudi and class I MHC-positive Raji human target cells are used. The studies reveal that in LDCC, human lymphocytes lyse HLA-negative Daudi targets to the same extent as HLA-positive Raji targets. This was shown by the 51Cr-release assay as well as the single cell assay. Furthermore, allosensitized murine CTL lyse Daudi and Raji in LDCC, demonstrating that lysis of class I negative targets is achieved by xenogeneic CTL. Target cells treated with papain were found to be resistant to lysis in LDCC. However, incubation of papain-treated targets for 1 to 2 hr resulted in recovery of cytotoxicity. In the single cell assay, the papain treatment of targets slightly reduces the frequency of binders but significantly reduces the frequency of killers, revealing that the papain-sensitive structures are essential during the lethal hit stage of lysis. Monoclonal anti-Lyt-2 antibody inhibits lysis of both Daudi and Raji targets by murine CTL. The lack of discrimination between mouse and human targets by murine CTL suggests that the Lyt-2 molecule may have a different role than merely interacting with the target cell. In the LDCC system, we propose that class I MHC molecules are not essential for lysis. Several papain-sensitive and -insensitive target cell structures may participate in the cytolytic mechanism, and we propose that both the lectin and Lyt-2 molecules are involved in the perturbation of cell membrane conformation essential for lysis.

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO 4 for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the 51Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.

Correlation of glycosphingolipids and sialic acid in YAC-1 lymphoma variants with their sensitivity to natural killer-cell-mediated lysis.

Sialoglycoconjugates and glycosphingolipids were quantitated in a series of variants derived from the YAC-1 lymphoma, known to be highly sensitive to natural killer (NK)-cell-mediated lysis. The variants, which had widely diverging sensitivities to NK cells, were obtained by a number of methods, including selection in the presence of NK cells, antibody to H-2, or antibody to the murine leukemia-virus-induced antigen, and by fusion of sensitive cells with an NK-resistant cell line, A9HT. The sensitivities of these cells to NK-cell-mediated lysis did not correlate with their sensitivities to anti-H-2a cytotoxic T cells. While no correlation could be made between the NK-sensitivity of these variants and their total cellular sialic acid, a statistically significant inverse correlation was observed between the levels of percentage neuraminidase releasable surface sialic acid of total labelled sialyl components and sensitivity to NK cells. This correlation with cell surface sialic acid was observed with either endogenous or lymphocytic choriomeningitis virus-induced activated NK cells as effectors. Neuraminidase treatment of insensitive target cells caused a moderate increase in sensitivity but failed to render the resistant targets as sensitive as YAC-1. Analysis of glycosphingolipids among the variants revealed a strong positive correlation between the total cell neutral glycolipid with chromatographic migration of asialo-GM2 and sensitivity to endogenous or activated NK-cell-mediated lysis. Significant correlations were not found with any other neutral glycolipids. However, ganglioside homologues with chromatographic mobility of GM1, GD1a, GD1b, And GT also showed a positive correlation with both endogenous and activated NK-cell-mediated lysis. The ratio of asialo-GM2 to GM2 had a highly significant positive correlation with sensitivity. These correlative results suggest that asialo-GM2 and certain gangliosides could be involved in binding or lytic events in NK cell:target cell interactions, and that high levels of sialic acid and sialylation on the surface may inhibit and/or modify such interactions. Further studies with these YAC variants should be useful for examining the biochemical bases of target cell-effector cell interactions in the NK-system.

Cholinesterase Autoanalysis: a Rapid Method for Biochemical Studies on Susceptible and Resistant Insects

An automated enzyme assay, which combines ease and speed of operation with high accuracy and reproducibility, was used to investigate activities and inhibition properties of acetylcholinesterases from susceptible and resistant strains of Spodoptera littoralis Boisduval and Musca domestica L . The resistant races of both species were found to contain modified target enzymes characterized by increases in Km and Vmax, and by a decrease in sensitivity against organophosphorus and, to a lesser extent, carbamate insecticides. The different biochemical properties of acetylcholine sterases from susceptible and resistant individuals provide an opportunity for monitoring insect populations before, under, or after selection pressure for frequencies of genes associated with an insensitive target enzyme.