Announcements

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. V. Evidence that manganese inhibits a calcium-dependent step in programming for lysis

The mechanism of T-lymphocyte-mediated cytolysis consists of three successive steps: adhesion formation, programming for lysis, and killer-cell-independent lysis. Mg²⁺, but not Ca²⁺, is required for adhesion formation, whereas programming for lysis is strongly Ca²⁺ dependent. We have previously reported that the transition metal manganese can substitute for Mg²⁺ in supporting adhesion formation. In the present paper, we demonstrate that manganese inhibits programming for lysis. The inhibitory effect of Mn²⁺ on cytolysis can be reduced by increasing the concentration of Ca²⁺. Furthermore, inhibitor sequencing experiments were unable to distinguish the step blocked by Mn²⁺ from the Ca²⁺-dependent step. These results suggest that Mn²⁺ blocks a Ca²⁺-dependent step(s) in programming for lysis. Present evidence does not distinguish whether the action of Ca²⁺ in programming for lysis is via a Ca²⁺ influx (as a “second messenger?”) or whether Ca²⁺ simply serves as a cofactor at the cell exterior.     

Fluorescent/ultraviolet absorbing ester derivative formation and analysis of eicosanoids by high-pressure liquid chromatography

A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B₂ chromatographs as two species and coelutes with PGF_2α. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF_1α, 6-keto-PGE₁, 13,14-dihydro-6,15-diketo-PGF_1α). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF_1α extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF_1α quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.     

Protecting vernal pools: a model from Massachusetts,USA

Ephemeral ponded wetlands, often referred to as vernal pools in the wetland’s lexicon of the northeastern United States, are in need of protection from outright loss, catastrophic alteration, and disruption of natural processes in the surrounding landscape, because of their great importance to a characteristic wildlife fauna. The state of Massachusetts, USA provides a useful model for vernal pool protection. Vernal pools have been specifically protected under state wetlands regulations since 1987 and many of the state’s municipalities have added additional, more stringent protection under local wetland bylaws. Some vernal pools are also protected under federal wetlands protection law. The protection of vernal pools in Massachusetts is based on a certification process in which biological data are collected to demonstrate that a wetland provides vernal pool functions. Once certified, and if a pool meets jurisdictional requirements, regulatory restrictions are placed on development and other activities proposed within the pool. The regulatory protection for vernal pools in Massachusetts has resulted in the prevention of outright loss and degradation of many vernal pools across the state. However, a 2001 US Supreme Court decision, referred to as the SWANCC decision, may result in a loss of federal jurisdiction over many vernal pools, protection that is especially valuable for pools that do not meet state regulatory criteria. Of significant importance is a lack of protection in existing regulations of the upland non-breeding habitat surrounding vernal pools, which has been shown to be of critical importance to the animals that rely on the pools. Despite the shortcomings of the regulatory protection model in Massachusetts, it has been especially effective as a catalyst for environmental and wildlife awareness, public participation in the wetlands regulatory process, and as an extremely valuable tool in environmental education and outreach.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Lack of correlation between naloxone-induced changes in sexual behavior and serum LH in male rats

Four experiments were conducted to determine whether the action of opiate receptor antagonist drugs on sexual performance in male rats is mediated by the central release of luteinizing hormone releasing hormone (LHRH). First, in Experiment 1 it was demonstrated that administration of naloxone (20 mg/kg) caused a lengthening of postejaculatory intervals and an elevation of serum LH concentrations in gonadally intact male rats. In Experiment 2, manipulation of females' proceptive and receptive behaviors failed to reveal the reductions in ejaculation latencies and in the number of intromissions preceding ejaculation which have been previously reported after administration of naloxone to male rats. Again, the predominant response to treatment with naloxone was an increase in the length of the postejaculatory interval. In Experiment 3, pinching the tails of male rats every 30 sec after ejaculation partially abolished the relative refractory periods of the postejaculatory intervals; naloxone-induced increases in the lengths of these shortened postejaculatory intervals were nevertheless identical to those of control males, suggesting that naloxone acts to lengthen the absolute refractory period. Finally, in Experiment 4 naloxone was given to castrated males implanted with testosterone-filled silastic capsules ranging in length from 2 to 45 mm, which produced a wide range of basal serum LH concentrations. Naloxone caused an increase in postejaculatory intervals; however, this effect was not correlated with the degree to which naloxone stimulated serum LH, suggesting that the effects of naloxone on the postejaculatory interval are not mediated by a drug-induced release of LHRH.