Studies of PPLO infection. IV. The neurotoxicity of intact mycoplasmas, and their production of toxin in vivo and in vitro.

Concentrated suspensions of washed Mycoplasma neurolyticum produce rolling disease in mice and rats, with neurological manifestations and pathological lesions similar to those seen with the exotoxin of this organism. Pretreatment of animals with tetracycline protects completely against the toxic effects of washed suspensions of mycoplasmas, while tetracycline affords no protection against the exotoxin. Freeze-thawing disruption of mycoplasma suspensions eliminates their neurotoxicity, while the same treatment does not affect exotoxin. The toxicity of intact organisms is not affected by exposure to the sedimentable component of brain, nor to ganglioside. These observations are interpreted to indicate that the neurotoxicity of living mycoplasmas must be due to their production of toxin after they have been injected into the animal. Resting mycoplasmas, suspended in Ringer's solution in dialysis sacs submerged in PPLO broth) produce considerable amounts of toxin within 15 min of incubation at 37 degrees C. Toxin is also produced, although in somewhat less amount, by washed organisms suspended in phosphate buffer containing glucose. The formation of toxin is prevented by the presence of puromycin, but not by the aminonucleoside analogue of puromycin, indicating that active protein synthesis is involved in the elaboration of toxin. The similarities between the neurotoxicity of the intact organisms of M. neurolyticum and Mycoplasma gallisepticum are discussed.

Studies of PPLO infection. II. The neurotoxin of Mycoplasma neurolyticum.

Rolling disease has been produced and studied in rats and mice, using the exotoxin of the A strain of Mycoplasma neurolyticum. The primary lesion of the brain consists of spongiform degeneration, associated with vesicle formation in the cortex and underlying white matter of the cerebral hemispheres, and in the molecular layer of the cerebellum. The brains of animals surviving 2 days or longer show extensive necrotizing lesions resembling ischemic necrosis, in both cerebral hemispheres. The brains of rats and mice with rolling disease become deeply stained by intraperitoneally injected trypan blue, indicating early disruption of the blood brain barrier. The toxin appears to be a thermolabile protein with a molecular weight exceeding 200,000. It is only active when injected by vein, and causes no disease when injected intracerebrally, intraperitoneally or subcutaneously, suggesting the existence of specific receptors within the vascular bed of the central nervous system. Protection is afforded by rabbit antibody against the toxin, but only when antibody is injected within less than 3 min after intravenous injection of toxin, indicating rapid fixation to receptors in the brain. The toxin is inactivated by incubation for 10 min at 37 degrees C with suspensions of the sedimentable component of normal brain. The inactivating factor in brain sediment is very thermostable, not affected by trypsin, and eliminated by treatment with periodate. Similar inactivation of toxin is demonstrable with water-soluble gangliosides of brain. A theoretical concept to explain the action of the toxin is proposed.

Suggested mechanism for the selective excretion of glucosylated albumin. The effects of diabetes mellitus and aging on this process and the origins of diabetic microalbuminuria.

In previous studies in the Sprague-Dawley rat, Williams and coworkers reported the phenomenon of selective urinary excretion of glucosylated albumin (editing, i.e., the percent glucosylation of urinary albumin is more than that of plasma albumin) by the mammalian kidney. Ghiggeri and coworkers subsequently found that the extent of editing is reduced in human diabetics. Moreover, the reduction in editing in diabetes correlates inversely with levels of microalbuminuria. We also find reduction in the extent of editing in diabetic humans. We find a striking inverse correlation not only with the magnitude of microalbuminuria but also with the extent of plasma albumin glucosylation. In contrast, we found little correlation between the reduction in editing and the duration of diabetes in human subjects. Stz induced diabetes in the Sprague-Dawley rat is associated with a striking and rapid reduction in editing which develops virtually with the same kinetics exhibited by the appearance of hyperglycemia. This loss of editing is rapidly reversed by daily administration of insulin but not by aldose reductase inhibitors. Mannitol infusion in anesthetized Wistar rats resulted in an increase in urine volume, GFR, and microalbuminuria, and was also accompanied by a marked reduction in editing. This reduction was rapidly reversed by a cessation of mannitol infusion. We propose here that glucosylated albumin (in contrast to unmodified albumin) is not reabsorbed by the proximal tubule, and thus, is preferentially excreted in the urine. We postulate that the increase in GFR which emerges as a consequence of increased plasma osmolality in diabetes mellitus delivers more albumin to the proximal tubule than can be reabsorbed. This results in a dilution of excreted glucosylated albumin molecules by excreted unmodified albumin, which appears as the early microscopic albuminuria of diabetes. Paradoxically, the fall in apparent editing is accompanied by an absolute increase in the total quantity of glucosylated albumin excreted. In contrast, we found that editing of glucosylated albumin by the normal kidney is found to gradually decline as a function of age without the appearance of microalbuminuria. This suggests that a different mechanism operates to produce the loss of editing seen with aging in man, and as clearly (but in a shorter absolute time intervals) in the Fischer-344 rat.

Digitonin effects on photoreceptor adenylate cyclase.

Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.

Cyclic adenosine monophosphate: function in photoreceptors.

Inactivation of adenylate cyclase in outer segments of retinal photoreceptor cells is proportional to the bleaching of rhodopsin. Membranes of the outer segments also contain a particulate, light-insensitive phosphodiesterase of high specific activity. In electrophysiological experiments, application of cyclic adenosine monophosphate along with a methylxanthine mimics the effects of illumination on the photoreceptor cell of the compound eye of Limulus.

Guanosine triphosphate activation of brain adenylate cyclase: enhancement by long-term antidepressant treatment.

Activation of adenylate cyclase by a stable guanosine 5'-triphosphate analog was augmented in brain membrane preparations from rats treated on a long-term basis with tricyclic antidepressants or electroconvulsive shock. These treatments may facilitate cyclase activation by promoting the interaction of the regulatory and catalytic subunits of the enzyme. This finding suggests a possible mechanism for the changes in sensitivity to various neurotransmitters seen after antidepressant administration.

Agaridoxin: a fungal catecholamine which acts as an alpha 1 agonist of mammalian hypothalamic adenylate cyclase.

Agaridoxin, a catecholamine isolated from mushrooms, and 4 synthetic analogues cause activation of adenylate cyclase in the presence of guanylyl imidodiphosphate (Gpp(NH)p) in membrane particles prepared from rat hypothalamus. These compounds also activate adenylate cyclase preparations from rat kidney, liver and cerebral cortex. In the presence of tyrosinase, these compounds are readily oxidized to quinones which lack agonist activity. Studies with selective adrenergic blockers suggest that agaridoxin acts at an alpha 1-type receptor. Agaridoxin-mediated adenylate cyclase stimulation is most effectively antagonized by WB-4101 and phenoxybenzamine, while propranolol and yohimbine are without inhibitory effect. Agaridoxin and the alpha 1 agonist methoxamine inhibited the binding of [3H]WB-4101 in rat hypothalamic and cerebral cortical membranes. The values of Ki for both compounds are lower than that of norepinephrine. The agaridoxin analogue, 4-aminocatechol hydrochloride, is a more effective and potent adenylate cyclase activator than agaridoxin or methoxamine.

Multiple opsin mRNA species in bovine retina.

Two retina specific cDNAs have been isolated by differential colony hybridization to retina and brain, and one of them, pCR-394, was identified as an opsin cDNA. By Northern hybridization experiment, the opsin cDNA hybridized to two species of bovine mRNA, one approximately 18 S (1800 bp) and the other 22 S (2600 bp). Using pCR-394 as a probe two opsin clones, R-5 (about 1200 bp) and LR-8 (about 2500 bp), were isolated from a cDNA library which was prepared by the method of Okayama-Berg. Each had a different length of 3'-untranslated DNA. The nucleotide sequences of R-5 and LR-8, as well as Northern and Southern hybridization experiments suggest that at least two species of opsin mRNA are expressed from a single gene. When the effects of illumination were examined by Northern hybridization and translation assays, the ratio of the two opsin mRNA species was changed between light- and dark-adapted eyes.