Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.

Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al. (1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by subcloning aequorin cDNA into the pRC23 plasmid vector such that its expression is under control of the lambda PL promoter [Cormier et al. (1989) Photochem. Photobiol. 49, 509]. Purification of recombinant apoaequorin from E. coli containing this new recombinant plasmid (pAEQ1.3) was accomplished by a two-step procedure involving gel filtration and anion-exchange chromatography on Sephadex G-100 and DEAE-Sepharose, respectively. Typically, 400-500 mg of recombinant protein was obtained from 100 L of fermentation culture. The purified recombinant apoaequorin could be converted to aequorin in high yield upon incubation with synthetic coelenterate luciferin, dissolved oxygen, and a thiol reagent with a photon yield similar to the native photoprotein. Detection of recombinant aequorin in the Dynatech ML1000 Microplate luminometer was linear between 10(-18) and 10(-12) mol, and little loss of specific activity was observed when the protein was derivatized with biotin. The biotinylated derivative was stable when frozen, lyophilized, or stored at 4 degrees C. The feasibility of using biotinylated aequorin as a nonradioactive tag was established by its application in a variety of solid-phase assay formats using the high-affinity streptavidin/biotin interaction. A microtiter-based bioluminescent immunoassay (BLIA) using biotinylated aequorin and the ML1000 luminometer was developed for the detection of subnanogram amounts of a glycosphingolipid (Forsmann antigen). In addition, nanogram to subnanogram quantities of protein antigens and DNA, immobilized on Western and Southern blots, respectively, were detected on instant and X-ray films using biotinylated aequorin.     

An evaluation of Salmonella (Ames) test data in the published literature: application of statistical procedures and analysis of mutagenic potency

We searched the published literature for Salmonella test data on some 450 chemicals. Only 137 of more than 400 articles containing original data satisfied minimum criteria for a quantitative analysis [1751 experiments, comprising data on 152 chemicals (Table 1)]. Many of these papers did not report basic information about the test protocol (Table 2). We used previously described statistical procedures (Bernstein et al., 1982) to estimate the initial slopes of the dose-response curves and corresponding standard errors. We also applied tests for significance and linear goodness-of-fit. We then used the results of these analyses to examine several issues: (1) Linearity of the low dose region of the dose-response curve. We found that the overwhelming majority of curves were linear, though ability to detect non-linearity of dose-response curves in the standard plate test is only limited. 7% of all experiments to which the goodness-of-fit test was applied were curves of increasing slope, and with a few possible exceptions, these were not obviously associated with any particular mutagens, even those generally considered to produce non-linear effects such as MNNG and EMS (Table 3). (2) Performance of the statistical test for significance. Results of the statistical test for significance of the dose-response were compared with author's opinions as to positivity. In almost all cases (94%) results of the statistical test and authors opinions were the same. In the examples of conflicting opinions, the reasons were: (a) the statistical test places more weight than do most authors on the presence of a linear dose-response; (b) most authors tend to require at least a 2-fold increase over the spontaneous background for 'significance', and (c) when the number of spontaneous revertants is small (e.g., TA1537), authors tend to require a larger increase in induced revertants than when the spontaneous background is large, whereas the statistical procedure makes no such distinction. These factors result in the statistical test tending to identify more experiments as positive than do authors, provided there is a linear dose-response, and authors tending to judge more experiments as positive when the dose-response is not linear. (3) Reproducibility. Among the 1751 experiments there were 122 data-sets (a total of 333 experiments) in which the same chemical was tested by two or more different laboratories under the same protocol. 21 of the 122 data-sets had some disagreement between experiments as to whether results were positive or negative (Table 4).(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of naloxone on basal and stress-induced release of ACTH and prolactin in the male rat

The effect of i.v. injection of various doses of naloxone (NAL) on plasma adrenocorticotropin (ACTH) and prolactin (Prl) in conscious animals bearing an indwelling intrajugular catheter was assessed. The effects were evaluated in animals which were left undisturbed and in others subjected to either restraint or ether stress. The results revealed that the dose of 3 mg/kg of NAL significantly reduced basal Prl levels, whereas a dose of 6 mg/kg of NAL was required to block completely either ether or restraint stress-induced release of Prl. The behavior of ACTH contrasted with that of Prl. There was no effect whatsoever of the 3 mg/kg dose of NAL on either resting or stress-induced ACTH levels, whereas a 6 mg/kg or 12 mg/kg dose of NAL elevated resting ACTH levels and only partially attenuated the further elevation induced by stress in these animals. The results clearly indicate a NAL sensitive step in the control of resting and stress-induced Prl release but indicate that the control of resting and stress-induced release of ACTH is different in that the predominantly millimicron receptor blocker, NAL, can elevate ACTH at high doses and can only partially block the response to stress. In contrast to Prl where opioid peptide control is solely stimulatory, this control of ACTH secretion appears to have both stimulatory and inhibitory features.     

Ultrastructure and electrical activity in developing heart cells (insect)

Ultrastructural and electrophysiological studies on developing heart cells of the moth Hyalophora cecropia are reported. The myocardium is identified at 6 days postoviposition (dpo) and heartbeats are detected at 8 dpo. At 10 dpo eclosion produces a larva. Cellular ultrastructure of the heart is presented from 6, 7, and 8 dpo embryos and first-instar larvae. Electrophysiological recordings are presented to demonstrate that the developing heart is myogenic, cells generate typical cardiac-type action potentials, and some mechanism for intercellular communication is present. The structure as it relates to function is discussed.     

Compliance and side effects in sleep apnea patients treated with nasal continuous positive airway pressure.

Nasal continuous positive airway pressure (CPAP) is an effective therapy for sleep apnea. We treated 144 patients with nasal CPAP and observed them for periods of as long as 25 months. No pneumothoraces occurred in any patient. Compliance rates were between 65% (90/139) and 83% (90/108), depending on the patient population considered. Demographic factors unrelated to discontinuing using CPAP included age, sex, and the presence of a housemate. Better-educated patients were less able to tolerate the equipment. Dry throat and nose and sore eyes were the most common side effects, but only sore eyes related to the amount of pressure. Side effects were unrelated to the number of months on the treatment, and obesity was related to higher pressures. Our study provides optimistic intermediate-term follow-up observations of patients on nasal CPAP therapy for sleep apnea. Whether adverse consequences occur over longer periods of time remains to be seen.     

Studies of Impedance in Cardiac Tissue Using Sucrose Gap and Computer Techniques: I. The Influence of Sucrose and Oil as Insulating Media

Impedances of cardiac cells of an insect were determined as a function of time to test the effects of sucrose and oil as insulating media in a gap arrangement. Impedance values are shown to increase markedly with time when sucrose is used as the insulating agent. Although impedance values are steady when oil is used, it is suggested that a layer of trapped electrolyte provides a shunt pathway and seriously impairs the validity of the measurements. A quick wash with sucrose followed by oil does not alleviate the situation but leaves a layer of sucrose trapped at the tissue-medium interface into which ions diffuse. The hypotheses (a) that the diffusion of intracellular K⁺ into the sucrose would result in an increase in tissue impedance and (b) that a layer of trapped electrolyte under the oil film provides a shunt pathway are examined by computer analyses of a simple model.