Nora Frontali

Peptides in Invertebrate Nervous Systems

Throughout the Metazoa, nervous and endocrine systems are involved in the coordination of the activities of various organs and tissues. However, in the less developed invertebrate phyla, conventional epithelial endocrine glands appear to be absent (Highnam and Hill, 1969), and organismic integration is primarily under the influence of neurosecretory cells. Since the pioneering studies of the Scharrers (Scharrer and Scharrer, 1937), a vast number of investigations into invertebrate neuroendocrine systems have been made. Several reviews on this subject have recently been published (Gersch, 1975; Golding, 1974; Goldsworthy and Mordue, 1974; Truman and Riddiford, 1974a).

Effects of black widow spider venom on acetylcholine release from rat cerebral cortex slices in vitro

Abstract Addition of a whole extract of Latrodectus mactans tredecimguttatus venom glands to rat cerebral cortex slices incubated in an eserinized low-potassium saline solution elicits over the following hour a more than 2-fold increase in the amount of acetylcholine (ACh) released into the medium, and a corresponding decrease in the ACh content of the tissue. The total amount of ACh synthesized in 1 hr is not significantly altered; however, if the tissue is incubated in a high-potassium medium, the resulting enhanced ACh synthesis is markedly reduced. Protein concentrations of venom ranging around 15 μg/ml in the incubation medium are sufficient to elicit a full response. Neurotransmitter depletion of cholinergic nerve terminals may contribute to explain the intoxication symptoms in man and in other vertebrates.

Experimental Neurotoxicity and Urinary Metabolites of the C5-C7 Aliphatic Hydrocarbons Used as Glue Solvents in Shoe Manufacture

Rats were intermittently exposed (9 to 10 h/d, 5 to 6 d/week) to controlled concentrations of single analytical grad solvents in ambient air. After periods ranging from 7 to 30 weeks the animals were perfused with glutaraldehyde and samples of nerves were processed for light microscopy of sections and of teased fibers. Animals treated with n-hexane at 5000 ppm (14 weeks) or 2500 ppm (30 weeks) developed the typical giant axonal degeneration already described in rats treated continuously with 400 to 600 ppm of the same solvent for 7 weeks or more. No such alterations were found in rats subjected to the following intermittent respiratory treatments: n-hexane 500 ppm (30 weeks) or 1500 ppm (14 weeks), cyclohexane 1500 or 2500 (30 weeks), n-pentane 3000 ppm (30 weeks), n-heptane 1500 ppm (30 weeks), 2-methylpentane 1500 ppm (14 weeks), and 3-methylpentane 1500 ppm (14 weeks). The following metabolites were found in the urine of rats according to treatment (in parenthesis): 2-methyl-2-pentanol (2-methylpentane); 3-methyl-2-pentanol and 3-methyl-3-pentanol (3-methylpentane), 2-hexanol, 3-hexanol, gamma-valerolactone, 2,5-dimethylfuran, and 2,5-hexanedione (n-hexane). 2-Hexanol was found to be the main urinary metabolite of n-hexane, while 2,5-hexanedione was present only in a lesser proportion. This feature of rat metabolism suggests that in this species 2,5-hexanedione reaches an effective level at its site of action during intermittent respiratory treatment with n-hexane with difficulty and explains the high concentrations necessary to cause polyneuropathy in rats subjected to this treatment.

Urinary excretion of n-hexane metabolites

Exposure to n-hexane, a component of many industrial solvent mixtures, is known to cause polyneuropathy in man. The concentration of metabolites in urine following exposure may be useful in biological monitoring. In a comparative study experimental animals (rat, rabbit and monkey) were subjected to single inhalatory treatments of 6, 12 and 24 h with 5,000 ppm of pure n-hexane. At the end of the treatments and at intervals thereafter, urine, and in rats also blood, were collected and analyzed for n-hexane and its metabolites. While the urine of rats contained 2-hexanol, 3-hexanol, methyl n-butyl ketone, 2,5-dimethylfuran, γ-valerolactone and 2,5-hexanedione, rabbit and monkey urine were found to contain only 2-hexanol, 3-hexanol, methyl n-butyl ketone and 2,5-hexanedione. Within 72 h of the end of exposure, the principal metabolite was 2,5-dimethylfuran in rats and 2-hexanol in rabbits and monkeys. In all three species the excretion rates of methyl n-butyl ketone, 3-hexanol and 2-hexanol peaked several hours earlier than 2,5-hexanedione (and γ-valerolactone and 2,5-dimethylfuran in rats). In all species 2,5-hexanedione was still detectable in urine 60 h following exposure. n-Hexane metabolites in rat blood were 2-hexanol, methyl-n-butyl ketone, 2,5-dimethylfuran and 2,5-hexanedione. The first two, as well as n-hexane itself, were found in maximum concentration immediately after termination of exposure, while 2,5-dimethylfuran and 2,5-hexanedione, with the longer exposure times, peaked some hours later. The data from urine collected at the end of exposure were compared with those obtained in a parallel study in humans occupationally exposed to a mixture of hexane isomers. Humans chronically exposed to 10–140 ppm n-hexane had 2,5-hexanedione concentrations in urine ranging from 0.4 to 21.7 mg/l, i.e., in the same proportion as rats exposed once for 6 or 12 h to 5,000 ppm.

Action of Latrodectus mactans tredecimguttatus venom and fractions on cells cultivated in vitro.

The crude venom of the spider Latrodectus mactans tredecimguttatus and three of its protein fractions obtained electrophoretically (LV1, LV2 and LV3) were tested on cells cultivated in vitro, and on vertebrates and invertebrates in vivo. The crude venom exerts a cytotoxic action on KB and Amnion cells. Of the three protein fractions tested, only LV1 shows cytotoxic activity: the effect on the cells is the same as with crude venom, and the activity in reference to the protein content is ten times higher. This fraction is toxic to houseflies, and if injected intravenously, to mice. The other two fractions are devoid of cytotoxic activity. LV2 is toxic to houseflies but not to mice and guinea pigs, and LV3 injected into mice and guinea pigs produces the characteristic symptoms of Latrodectus bite in mammals. The crude venom and LV1 lack proteolytic activity. The cytotoxic activity is not associated with proteolytic activity, nor with the main toxic activity towards mammals. Neutralization experiments were performed using an antiserum prepared against Latrodectus crude venom. The cytopathic effect is neutralized by antiserum. The immunological test doses for crude venom were established in vivo and in tissue culture. Tissue cultures provide a more sensitive system of assay than the whole animal for fraction LV1 and its antibodies. Moreover tissue cultures behave as selective systems when employing crude preparations of venom, as cells react only to one component.

Heterogeneous visceral nerve changes in acrylamide intoxication

SummaryA variety of visceral nerves were studied by intermediate filament immunocytochemistry in rats intoxicated with acrylamide. In such animals, oesophageal and diaphragmatic motor end-plates were invaded and deformed by neurofilament protein-like material, while afferent fibres of diaphragmatic neuromuscular spindles and myelinated sensory fibres of the iris showed striking terminal accumulation of similar material. Conversely, the rich population of thin afferent fibres of the iris showed no obvious abnormality, while pre-terminal changes were seen along the extrinsic nerve fibres supplying the cornea and myenteric ganglia. Multiple lesions were demonstrated in gut nerves of acrylamide-treated rats, while scattered “enteric glial cells” showed abnormally coarse morphology and a striking increase in glial fibrillary acidic protein immunoreactivity. A distinct, delicately varicose appearence was revealed by neurofilament protein-immunostaining in bladder nerve fibres of normal rats, which was changed to one of coarse dilations by acrylamide. In conclusion, apparently selective changes were found along different types of axons, in dicating marked heterogeneity in cytoskeletal organisation among visceral nerves. Taken together with the proposed inhibition by acrylamide of neurofilament proteins degradation, the above findings may suggest a non-uniform distribution of neurofilament degradation sites along distal regions of different axons.

Experimental ββ′-iminodipropionitrile (IDPN) neuropathy: neurofilament profile of sensory, motor and autonomic nerves as seen by immunocytochemistry on whole-mount preparations

IDPN-induced changes in a variety of sensory, motor and autonomic nerves were studied by whole-mount immunocytochemistry. A full range of proximo-distal accumulations of neurofilament-like material was found, from paranuclear round bodies in perikarya to distal and preterminal axonal dilations. Conversely, both terminal areas and nodal-paranodal regions of myelinated axons showed striking, sharply localized loss of neurofilament-immunostaining. The latter change, when transport of neurofilaments is halted by IDPN, may indicate their local processing and/or differential transport at nodal-paranodal regions.

Effects of 2,5-hexanedione on the ovary and fertility. An experimental study in mice

Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 11.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.

2,5-Hexanedione modifies skeletal proteins of the red blood cells and increases the binding of hemoglobin to the membrane

The effects of 2,5-hexanedione (2,5 HD) on skeletal proteins of red blood cells (RBCs) were investigated both in vitro (human RBCs) and in vivo in male Sprague-Dawley rats which had been treated with the drug for several days. We found that 2,5 HD induced the following major changes in the electrophoretic pattern of the skeletal proteins: (i) the appearance of high-molecular weight bands, (ii) a dose-dependent decrease in spectrin Bands 1 and 2, and (iii) a dose-dependent increase in the amount of hemoglobin (Hb) associated with the membrane. Membranoskeletons, prepared from resealed ghosts which had been previously treated with 2,5 HD, were able to bind an increased amount of Hb from untreated RBCs, thus suggesting a drug-induced modification of the membrane. Extraction of spectrin and actin from ghosts did not remove the membrane-bound Hb and, furthermore, Hb bound to 2,5 HD-treated membranes mainly bearing Band 3 and free of peripheral proteins. These data suggested a 2,5 HD-induced modification of an intrinsic membrane protein, probably Band 3. This hypothesis was consistent with the observation that 2,5 HD also induced a modification of Band 3 aminogroups, as evidenced by a dose-dependent decrease in the binding of eosin probes. Furthermore, RBCs treated in vitro with 2,5 HD bound an increased amount of autologous immunoglobulins (IgG). As reported by Kay and Low et al. the binding of autologous IgG is a phenomenon associated with the aging process of RBCs and may involve a modification of Band 3. Our data show that RBCs treated with 2,5 HD acquired various characteristics of senescent cells such as spectrin cross-linking, Hb-membrane binding and increased IgG binding, and suggest that 2,5 HD treatment might affect RBC survival.