Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by insulin-like growth factors and insulin.

1. The binding of 125I-labelled insulin-like growth factor 1 (125I-IGF-1) to Swiss mouse 3T3 fibroblasts was time- and concentration-dependent. Unlabelled IGF-1 had a slightly higher potency than multiplication-stimulating activity (MSA) in inhibiting the binding of 125I-IGF-1, and insulin gave a parallel inhibition curve at 300-1000-fold lower potency. Chemical cross-linking of bound 125I-IGF-1 to its receptors, followed by polyacrylamide-gel electrophoresis under reducing conditions, revealed a major band of Mr 130,000, the labelling of which was inhibited by IGF-1 or high concentrations of insulin. 2. The binding of 125I-IGF-1 was not affected by either co-incubation or preincubation of the cells with a range of heterologous growth factors and mitogens. However, IGF-1 and MSA each induced down-regulation of 125I-IGF-1 binding sites. 3. The maximal stimulations of DNA synthesis induced by IGF-1, MSA and insulin, in the presence of a synergizing mitogen, were similar. The dose-response curve for insulin was not parallel to those for IGF-1 and MSA; in particular, low concentrations of insulin induced a greater stimulation than expected on the basis of its potency in the inhibition or down-regulation of 125I-IGF-1 binding. 4. The preincubation of 125I-IGF-1 with Swiss 3T3 cells at 37 degrees C decreased its ability to bind to a second batch of cells. This inactivation did not occur when the preincubation was performed at 4 degrees C or in the presence of cycloheximide. Chemical cross-linking revealed that the cells released an IGF-binding protein, giving a complex of Mr about 48,000. 5. It is concluded that type I IGF receptors mediate the stimulation of Swiss 3T3 cells by insulin-like mitogens, but that insulin probably stimulates the cells through insulin receptors. The cells can modulate the amount of ligand binding, both by down-regulation of the receptors and by the secretion of an IGF-binding protein.     

Dexamethasone influences the lipid fluidity, lipid composition and glycosphingolipid glycosyltransferase activities of rat proximal-small-intestinal Golgi membranes.

Experiments were performed to examine the effects of subcutaneous administration of the synthetic glucocorticoid dexamethasone (100 micrograms/day per 100 g body wt.) on the lipid fluidity, lipid composition and glycosphingolipid glycosyltransferase activities of rat proximal-small-intestinal Golgi membranes. After 4 days of treatment, Golgi membranes and liposomes prepared from treated rats were found to possess a greater fluidity than their control (diluent or 0.9% NaCl) counterpart, as assessed by steady-state fluorescence-polarization techniques using three different fluorophores. Moreover, analysis of the effects of temperature on the anisotropy values of 1,6-diphenylhexa-1,3,5-triene, using Arrhenius plots, demonstrated that the mean break-point temperatures of treated preparations were 4-5 degrees C lower than those of control preparations. Changes in the fatty acyl saturation index and double-bond index of treated membranes, secondary to alterations in stearic acid, linoleic acid and arachidonic acid, at least in part, appeared to be responsible for the differences in fluidity noted between treated and control Golgi membranes. Concomitant with these fluidity and lipid-compositional alterations, treated membranes possessed higher specific activities of UDP-galactosyl-lactosylceramide galactosyltransferase and CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase than their control counterparts. Experiments utilizing benzyl alcohol, a known fluidizer, furthermore suggested that the fluidity alteration induced by dexamethasone may be responsible for the increased activity of the former, but not the latter, glycosphingolipid glycosyltransferase.     

Endogenous lipid pneumonia in opossums from Louisiana

Endogenous lipid pneumonia was present in 19 of 27 opossums (Didelphis virginiana) trapped in the vicinity of Baton Rouge, Louisiana. The severity of lesions varied from small pleural and subpleural aggregates of foamy macrophages with minimal disruption of pulmonary architecture to large nodular accumulations of foam cells with cholesterol clefts and localized emphysema. The cause of the lipid pneumonia may be related to pulmonary nematode parasite infections, which were evident in 13 of the affected animals.     

Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines

The central nucleus of the amygdala (Ce) mediates cardiovascular and autonomic changes associated with defense or fear responses. At least 16 different neuropeptides have been identified within nerve terminals within the Ce. The role that these peptides play in the Ce regulation of cardiovascular and autonomic function has been assessed. Neuropeptides were microinjected into the region of the Ce and mean arterial pressure (MAP), heart rate (HR) and plasma catecholamine concentrations were measured. Five of the 16 peptides caused changes of MAP and HR. Thyrotropin releasing factor (TRF) and calcitonin gene-related peptide (CGRP) induced increases of MAP and HR. Angiotensin-II (A-II) and somatostatin-28 (SS-28) injection produced increases of MAP and decreases of HR. Bombesin (Bom) injections into the Ce induced an increase of MAP but did not alter HR. Corticotropin releasing factor (CRF), TRF and CGRP were the only peptides found to increase plasma catecholamine concentrations. These results support the conclusion that the Ce contains several peptides that could be involved in the regulation of cardiovascular and autonomic nervous system function. A role of the amygdala in mediating the observed effects of CRF, TRF, CGRP, A-II, SS-28, and Bom is suggested by these studies.     

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.     

Importance of changing photoperiod and melatonin secretory pattern in determining the length of the breeding season in the Suffolk ewe

Three groups of ovariectomized Suffolk ewes bearing s.c. Silastic implants of oestradiol were subjected to a 90-day priming treatment of an inhibitory long photoperiod (16 h light/day; 16L:8D). On Day 0 of the experiment, they were moved to stimulatory photoperiods. One control group was transferred to 12L:12D and a second control group was transferred to 8L:16D; both groups remained in those photoperiods to determine the timing of reproductive induction and refractoriness. The experimental group was transferred to 12L:12D on Day 0 and then to 8L:16D on Day 55 to determine whether the further reduction in daylength could delay the development of refractoriness. Reproductive neuroendocrine condition was monitored by serum concentrations of LH and FSH. Both gonadotrophins remained elevated for a longer period of time in the experimental group receiving the second reduction in daylength than in either control group, indicating that the second photoperiodic drop delayed the onset of photorefractoriness. Measurement of 24-h patterns of circulating melatonin suggests that the prolonged stimulation of reproductive neuroendocrine activity in the experimental group resulted from a lengthening of the nocturnal melatonin rise. These findings indicate that refractoriness to an inductive photoperiod can be temporarily overcome by exposure to a shorter daylength, and that the change in duration of the nocturnal increase in melatonin secretion is important in photoperiodic signalling. Thus, in natural conditions, the decreasing autumnal daylength, and the resulting expansion of the nocturnal elevation in melatonin secretion, may be utilized to produce a breeding season of normal duration.     

Analgesia induced by N-acetylserotonin in the central nervous system

The relationship between N-acetylserotonin (NAS) in the central nervous system (CNS) and responses to pain was investigated. Using the rat tail-flick model, we initially replicated the work of others showing that intraventricular (IVC) injection of a dipeptide structurally similar to both NAS and serotonin was capable of inducing analgesia in the rat. We then showed that IVC-NAS, but not serotonin elicited analgesia in much the same manner as the dipeptide. This effect proved to be very specific as it required the presence of both an acetyl group on the terminal side chain amine as well as a hydroxyl group on the C-5 position of the indole ring. Substitution of the C-5 hydroxyl by a methoxyl group (melatonin) abolished the analgesic effect. Similarly, removing the N-acetyl substitution (serotonin) also eliminated the analgesia. IVC injection of highly specific antiserum to NAS induced hyperalgesia. Furthermore, an interaction was found between NAS and opiate systems. We demonstrated that while naloxone, the opiate antagonist, has no hyperalgesic properties of itself, it did counteract the analgesia induced by NAS. Similarly, NAS antiserum reversed the analgesia induced by the opiate morphine. This work provides evidence that NAS is an endogenously active substance within the CNS pain network.     

DNA adduct formation by alachlor metabolites

The extent of DNA adduct formation by alachlor [ArN(CH2OCH3)C(O)CH2Cl wherein Ar is 2,6-diethylphenyl] and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. [14C-phenyl]Alachlor is compared to its two metabolic cleavage products, [14C-phenyl]2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) [ArNHC(O)CH2Cl] and [14C-phenyl]2,6-diethylaniline (DEA) (ArNH2), and to [14C-methoxy]alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CDEPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from [14C-methoxy]- than from [14C-phenyl]alachlor. This 4-fold preferential DNA labeling from the 14C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with [14C-phenyl]alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with [14C-methoxy]alachlor. Metabolic studies indicate that ArN(CH2OCH2OH)C(O)CH2Cl is an intermediate in forming CDEPA and presumably formaldehyde in the mouse liver microsomal mixed-function oxidase system and in yielding the O-glucuronide of ArN(CH2OH)C(O)CH2Cl in the urine of alachlor-treated mice. These findings point to the N-CH2OCH2OH metabolite or formaldehyde as a reactive intermediate in forming a DNA-adduct and as a candidate proximate carcinogen.