Toxicity versus avoidance response of golden shiner, Notemigonus crysoleucas, to five metals

Avoidance thresholds and 96-h LC50 values were determined for golden shiners, Noiemigonus crysoleucas , for five individual elements: chromium, Cr; copper, Cu; cadmium, Cd; arsenic, As; selenium, Se. The avoidance concentrations were 73, 26 and 28 μgl^-1 for Cr-VI, Cu and As-III, respectively. Cadmium and Se were not avoided at experimental concentrations up to 68 and 3489 μg1^-1, respectively. Acute flow-through 96-h LC50 values were Cr-VI 55·0, Cu 84·6, and As-III 12·5 mg 1^-1, which were more than two orders of magnitude above avoidance concentrations. The acute flow-through 96-h LC50 values for Cd and Se were 2·8 and 11·2 mg 1^-1, respectively. These concentrations are 31 and 2·2 times the highest concentration employed in the avoidance tests, neither of which were avoided by the test organisms. Thus, simple toxicity tests do not identify the environmental hazard of some elements, and the most toxic elements may not elicit a behavioural response. When used in concert with tests of organism function, more realistic indicators of environmental hazard or safety may be determined.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

The Actions of Calmodulin Antagonists W-7 and TFP and of Calcium on the Gating Kinetics of the Calcium-activated Large Conductance Potassium Channel of the Chara Protoplasmic Drop: A Substate-sensitive Analysis

The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca²⁺-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte plants. The large-conductance (170 pS), voltage- and Ca²⁺-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the Maxi-K channel from animal cells. This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes of action of- and different bindings sites for these calmodulin antagonists. Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten curve. W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent. In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the closed state at −50 mV but is distinguishable from it at voltages more negative than −100 mV. This substate may represent times that trifluoperazine is bound to the channel. Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect of Ca²⁻. This is good kinetic evidence against the view that the channel is activated by Ca²⁺-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca²⁻-calmodulin less available. Received: 26 August 1996/Revised: 7 October 1996     

Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.     

Immunogenicity, macrophage sensitivity, and therapeutic response to C. parvum of fibrosarcomas induced in C. parvum-treated and untreated mice

Summary No correlation has been observed between the therapeutic response of isogeneic transplants of methylcholanthrene-induced murine fibrosarcomas to Corynebacterium parvum (CP) and the sensitivity of the tumour cells to the cytotoxic action of CP-activated macrophages (Mø) in vitro. Neither of these parameters appeared to be influenced by repeated administration of CP during induction of the primary tumour or by whether the carcinogen was administered in solution or as crystals on a Millipore disc. On the other hand, tumours which developed in CP-treated mice after injection of a small dose (0.1 mg) of methylcholanthrene (MC) were more immunogenic than those which developed after the same dose of MC in untreated mice, as judged by the capacity of an injection of an irradiated tumour cell suspension to protect isogeneic mice against subsequent challenge with viable cells. Further work is needed to explain these findings.     

Effects of gamma-irradiation on chromosomes of cultured lymphocytes from rheumatoid arthritis patients and their family members

Increased rates of spontaneous or induced chromosome breakage are seen in many types of diseases, including the 'collagen-type' autoimmune diseases. Using a 60Co gamma cell, we irradiated lymphocyte cultures from three related rheumatoid arthritis patients, their immediate family members, and two unrelated rheumatoid arthritis patients. Although these individuals had not shown abnormally high levels of spontaneous chromosome breakage, they did show an abnormal sensitivity to irradiation, which was manifested in several ways. Two of the probands showed induced breakage rates that were twice as high as those seen in controls. In addition, the reduction of mitotic index, due either to increased cell death or to induction of a G2 lag period, was higher in the arthritis group (including non-symptomatic family members) than in the control group. Finally, we observed a high frequency of an unusual type of cell in the arthritis group. These unusual cells resembled c-anaphases seen with extended colcemid treatment, and may indicate that the mitotic apparatus in cells from this group is particularly sensitive to ionizing radiation.     

Effects of testosterone on messenger ribonucleic acid and protein synthesis in rat seminal vesicle

In a previous report [Higgins et al. (1976) Biochem. J. 158, 271–282] we described the effects of alterations in androgen status on the synthesis of two basic secretory proteins of the rat seminal vesicle. In the present paper we examine the effects of testosterone on the activity of mRNA in the seminal vesicle. Total cellular poly(A)-rich RNA was isolated and translated in a cell-free system prepared from wheat germ. Translation products were separated on denaturing polyacrylamide gels and the protein bands corresponding to the two basic secretory proteins were identified immunologically. Incorporation of radioactive methionine into these bands was taken as a measure of the individual mRNA activities. Total mRNA activity was estimated by radioactivity in total acid-precipitable material. The results show that 1 to 2 weeks after castration the activities of mRNA molecules for the basic secretory proteins were decreased 10–20-fold on a tissue basis. Testosterone given in vivo rapidly and substantially restores mRNA activity to normal. Since these changes correlate closely with variations in the rates of synthesis of the secretory proteins in whole cells it suggests that androgenic steroids control protein synthesis chiefly via mRNA availability. In this respect their action resembles those of other steroid hormones acting in other systems. However, these effects of testosterone on the mRNA molecules for the major secretory proteins could not be distinguished from those on total mRNA. Thus the proportion of the total mRNA population accounted for by the two specific mRNA molecules showed less than a 2-fold variation with androgen status. Similarly the two secretory proteins always accounted for 25–33% of general protein synthesis. This is in sharp contrast with the markedly differential effects of other steroid hormones controlling synthesis of major proteins in other well-studied systems. We interpret our results as indicating that testosterone regulates the mRNA population of the seminal vesicle as a whole.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.