Stimulation of Murine Interferon by a Substituted Pyrimidine

2-Amino-5-bromo-6-methyl-4-pyrimidinol (U-25,166) induced high levels of circulating interferon in mice when administered either parenterally or orally. Peak titers of interferon were found in the serum between 6 and 12 h after inoculation of the drug. Lower but significant levels of interferon were found in rat serum after administration of U-25,166 by either the intraperitoneal or oral route, and good levels of circulating interferon were observed in cats after oral treatment. Repeated intraperitoneal doses (50 mg/kg) of U-25,166 protected mice against intranasal encephalomyocarditis virus challenge. The minimal effective acute oral dose for antiviral activity was approximately 250 mg/kg. This was also the minimal dose that produced detectable levels of interferon. Maximum tolerated doses in mice were four to six times the minimal effective doses. A single oral treatment was protective in mice against challenge virus inoculated 24 h later. The compound protected mice from challenge with high levels of encephalomyocarditis virus, up to 20,000 mean lethal doses. Antiviral activity in mice was retained when certain minor substitutions were made in the U-25,166 structure.     

Adherence of L1210 murine leukemia cells to sephacryl- aminopropylcobalamin beads treated with transcobalamin-II

Sephacryl beads containing an immobilized aminopropylcobalamin- transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.     

Consistent, high-level ethanol consumption in pig-tailed macaques via a multiple-session, limited-intake, oral self-dosing procedure

Background:  Alcohol abuse is a major public health burden that can lead to many adverse health effects such as impaired hepatic, gastrointestinal, central nervous system and immune system function. Preclinical animal models of alcohol abuse allow for experimental control over variables often difficult to control in human clinical studies (e.g., ethanol exposure before or during the study, history of other drug use, access to medical care, nutritional status, etc). Nonhuman primate models in particular provide increased genetic, anatomic and physiologic similarity to humans, relative to rodent models. A small percentage of macaques will spontaneously consume large quantities of ethanol; however, most nonhuman primate models of "voluntary" ethanol intake produce relatively low daily ethanol intake in the majority of monkeys.
Methods:  To facilitate study of chronic exposure to high levels of ethanol intake, a macaque model has been developed that induces consistent, daily high-level ethanol consumption. This multiple-session procedure employed 4 drinking sessions per day, with sessions occurring once every 6 hours.
Results:  The group average alcohol consumption was 4.6 g/kg/d (SEM 0.4), roughly twice the group average consumption of previous reports. Ethanol drinking sessions produced group mean blood ethanol levels of 95 mg/dl after 60 minutes, and fine motor control was impaired up to 90 minutes after a drinking session.
Conclusion:  This model of multiple-session, limited access, oral ethanol self-dosing produced consistent, high-level ethanol consumption with each session qualifying as a "binge" drinking session using the definition of "binge" provided by the NIAAA (>80 mg/dl/session). This model of ethanol drinking in macaques will be of great utility in the study of immunological, physiological and behavioral effects of ethanol in nonhuman primates.     

Activation of the CPP32 protease in apoptosis induced by 1-beta-D- arabinofuranosylcytosine and other DNA-damaging agents

The response of human myeloid leukemia cells to treatment with 1-beta- arabinofuranosylcytosine (ara-C) includes the induction of apoptosis. Ara-C induced apoptosis is associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. However, the signals involved in this response are unknown. The present studies show that ara-C treatment of U-937 cells is associated with induction of a protease activity that cleaves the tetrapeptides Ac-DEVD- pNA and Ac-DMOD-pNA found at the cleavage sites of PARP and PKC delta, respectively. The ara-C-induced protease activity was sensitive to overexpression of the anti-apoptotic protein Bcl-xL and the baculovirus protein p35. By contrast, overexpression of the cowpox virus protein CrmA blocked apoptosis induced by engagement of the Fas receptor but not that induced by ara-C. CrmA overexpression also had no detectable effect on ara-C-induced cleavage of PKC delta. The results further show that ara-C induces activation of the CPP32 protease by a CrmA- insensitive and p35-sensitive mechanism. Similar results were obtained with cisplatinum, etoposide, and camptothecin. These findings indicate that ara-C and other DNA-damaging agents activate a CrmA-insensitive apoptotic pathway involving CPP32 and that these signals differ from those associated with apoptosis induced by the Fas receptor.     

Identification and characterization of a low-affinity granulocyte- macrophage colony-stimulating factor receptor on primary and cultured human melanoma cells

Hematopoietic growth factor receptors are present on cells of normal nonhematopoietic tissues such as endothelium and placenta. We previously demonstrated functional human granulocyte-macrophage colony- stimulating factor (GM-CSF) receptors on small cell carcinoma of the lung cell lines, and others have reported that certain solid tumor cell lines respond to GM-CSF in clonogenic assays. In the current study, we examine human melanoma cell lines and fresh specimens of melanoma to determine whether they have functional GM-CSF receptors. Scatchard analyses of 125I-GM-CSF equilibrium binding to melanoma cell lines showed a mean of 542 +/- 67 sites per cell with a kd of 0.72 +/- 0.14 nmol/L. Cross-linking studies in the melanoma cell line, M14, showed a major GM-CSF receptor species of 84,000 daltons. Under the conditions tested, the M14 cells did not have a proliferative response to GM-CSF in vitro, nor was any induction of primary response genes detected by Northern analysis in response to GM-CSF. Studies to determine internal translocation of the receptor-ligand complex indicated less than 10% of the 125I-GM-CSF internalized was specifically bound to receptors. Primary melanoma cells from five surgical specimens had GM-CSF receptors; Scatchard analysis was performed on one sample, showing 555 sites/cell with a kd of 0.23 nmol/L. These results indicate that human tumor cells may express a low-affinity GM-CSF receptor protein that localizes to the cell surface and binds ligand, but lacks functional components or accessory factors needed to transduce a signal.     

A weight-of-evidence review of colorectal cancer in pesticide applicators: the agricultural health study and other epidemiologic studies

Objective

To systematically evaluate epidemiologic studies on pesticides and colon cancer and rectal cancer in agricultural pesticide applicator populations using a transparent “weight-of-evidence” (WOE) methodological approach.

Methods

Twenty-nine (29) publications from the Agricultural Health Study (AHS) and 13 additional epidemiologic studies were identified that reported data for pesticide applicators and/or specific pesticide compounds and colorectal, colon, or rectal cancer. The AHS evaluated pesticide applicators as well as dose–response associations for specific pesticide compounds, whereas the large majority of non-AHS evaluated applicators but did not analyze specific compounds or dose–response trends. This WOE assessment of 153 different pesticide–outcome pairs emphasized several key evidentiary features: existence of statistically significant relative risks, magnitude of observed associations, results from the most reliable exposure assessments, and evidence of convincing dose–response relationships (i.e., those monotonically increasing, with statistically significant trend tests).

Results

Occupation as a pesticide applicator or pesticide application as a farming-related function was not associated with increasing the risk of colon or rectal cancer. Deficits of colon or rectal cancer were observed across most studies of pesticide applicators. After applying the WOE methodology to the epidemiologic studies of specific pesticide compounds and colon or rectal cancer, a number of pesticide–outcome pairs were identified and evaluated further based on positive statistical associations. Of these, only two—aldicarb and colon cancer and imazethapyr and proximal colon cancer—appears to warrant further discussion regarding a possible causal relationship, although the epidemiologic data are limited. For the remainder, a lack of a clear dose–response trend, inconsistencies in associations between exposure metrics and comparison groups, imprecise associations, variable participation rates for analyses of specific compounds, and the reliance upon data from one study (the AHS) limit interpretation regarding risk.

Conclusion

The available epidemiologic evidence does not support a causal relationship between occupation as a pesticide applicator or specific pesticide exposures and colon or rectal cancer.     

The inhibitory effects of exogenous arachidonic acid on rabbit platelet aggregation and the release reaction

Although arachidonic acid causes rabbit platelet aggregation and the release of granule contents in suspensions of washed platelets when used in concentrations of approximately 50-300 microM, higher concentrations (500 microM) cause neither aggregation nor release. Suspensions of platelets from rabbits wee exposed to arachidonic acid (250 microM) for 15 min, allowed to recover in the presence of PGE1 for 30 min, washed, and resuspended; in some experiments, the platelets were treated with aspirin before being exposed to arachidonic acid. Aggregation of platelets pretreated with arachidonic acid was inhibited in response to ADP; this effect was greater with the non-aspirin- treated platelets and persisted for at least 4 hr after resuspension. The association of 125I-fibrinogen with the platelets as a result of ADP stimulation was also inhibited. Aggregation and release of granule contents in response to collagen and low concentrations of thrombin was inhibited, but the inhibition could be overcome by higher concentrations. Thrombin induced further release of granule contents from platelets exposed to arachidonic acid without pretreatment with aspirin. Platelets that had been exposed to arachidonic acid, either with or without pretreatment with aspirin, did not aggregate or undergo further release upon stimulation with arachidonic acid after they were washed and resuspended. Inhibition of the lipoxygenase pathway with eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) did not affect the inhibition caused by arachidonic acid, so it is unlikely that a product of this pathway is responsible for the inhibition. Mixing experiments indicated that the pretreated platelets did not form a thromboxane-A2-like activity, and that they were unresponsive to aggregation and release induced by products formed from arachidonic acid. Experiments with 3H-arachidonic acid showed that after 45 min of incubation with platelets, only 1.1% of the 3H-arachidonic acid remained as free arachidonic acid in the platelets. Although cyclic-AMP was slightly increased 1 min after the addition of arachidonic acid, the cyclic-AMP concentration was the same as that of control platelets after the platelets were washed and resuspended, indicating that increased cyclic-AMP is not likely to be responsible for the persistent inhibitory effect. Thus, the inhibitory effect of pretreatment with arachidonic acid is a general effect on responses to a variety of aggregating agents that act through different mechanisms, and the inhibition is not related to thromboxane-A2 formation. The possibility of membrane perturbation resulting in the unavailability of receptors may explain the persistent inhibitory effect, but the responsible reactions have not been identified.