Variant forms of immunoreactive thyrotropin in aged rats

Immunoreactive thyrotropin, extracted by affinity chromatography from the serum of Fischer 344 male rats and fractionated by gel filtration, is more polymorphic (in terms of molecular weight) in 22- and 30-month-old rats than in younger animals (3- and 12-month-old). Changes in hormonal levels with age involve thyrotropin and thyroxine but not triiodothyronine, serum thyrotropin being significantly decreased at 30 months and thyroxine at 22 and 30 months. Low serum thyroxine levels and increased thyrotropin polymorphism may suggest an impaired pituitary-thyroid axis in aged rats.     

Myocardial structure and capillary basal laminar thickness in experimentally diabetic rats

This study was designed to investigate myocardial abnormalities in general, and the extent of capillary basal laminar thickening (CBLT) in particular, in experimentally induced diabetic rats. For this purpose, buffered streptozotocin (70 mg/kg) was administered to 34 rats; control groups consisted of 18 rats, uninjected or buffer-injected only. Animals were sacrificed at monthly intervals up to 12 months and at 15 months following induction of the diabetes. Myocardial, renal, and skeletal muscular tissues, prepared for ultrastructural examination, were subjected to quantitative procedures, in order to obtain an index of CBLT. The results indicated that abnormal CBLT was present and relatable to the length of exposure to the hyperglycemia. Six months following induction, the increments in laminar thickening in diabetic rats significantly differed from those in normal maturing rats. In the diabetic myocardium CBLT represented the single, specific, and clearly identifiable strucutural abnormality. Increased CBLT was also observed within renal glomeruli and quadriceps in diabetic rats, to a more marked extent than that seen in the myocardium. Parenchymal changes in the three tissues under study were most pronounced in the kidneys of diabetic rats. These findings, paralleling previously published observations in human diabetics, fail to reveal morphologic evidence of an intrinsic diabetic cardiomyopathy and suggest that CBLT in experimentally diabetic rats is associated with the altered metabolic state in these animals.     

Age-related changes in immune function of rats and the effect of long-term hypophysectomy.

The effect of age on the ability of rats to raise antibodies to sheep erythrocytes and on the number of IgM plaque-forming cells in the spleen was investigated. An age-related decline in both parameters was observed. Additionally, the possibility that long-term hypophysectomy coupled with minimal replacement therapy might result in a delay and/or reversal of the age-related decline in immune function was studied. It was observed that long-term hypophysectomized rats responded better to immunization with sheep erythrocytes than did their age-matched unoperated littermates. The possible relationship of this to aging is discussed.     

Tail temperature in rats after intraventricular injections of 6-hydroxydopamine or 5,7-dihydroxytryptamine

The tail vasomotor tone in rats is reduced by the intraventricular injection of 5,7-dihydroxytryptamine but not by 6-hydroxydopamine. Central serotonergic but not noradrenergic neurons participate in vasoconstriction of the tail.     

Allergens of Parietaria judaica pollen--I. Purification and characterization of a hapten and a low molecular weight allergenic peptide

A low mol. wt allergen (Pj-2) and a hapten (Pj-H3) were purified from Parietaria judaica pollen by means of long-term aqueous extraction, dialysis and gel filtrations. The yield of the Pj-2 allergen was 0.94% (w/v) of the total protein present in the aqueous extract of the pollen, while its allergenic activity was about 60% of the total dialyzable activity, as verified by skin prick tests, ELISA- and RAST-inhibition experiments. The homogeneity of this allergen was demonstrated by one single sharp peak on HPLC, one single band on PAGE-SDS and by one single arc on IEF. Its mol. wt, estimated by HPLC and amino acid composition, was 10,400. The amino acid analysis showed 73 amino acid residues, and lysine was predominant, with 20 residues. The hapten Pj-H3 was 0.2% (w/v) of the total protein found in the pollen aqueous extract. It was inactive in skin prick tests even at a protein concn of 2 mg/ml, while it was capable of inhibiting by 60% in ELISA- and RAST-inhibition experiments, suggesting an immunochemical relationship with both IgE and allergens specific to P. judaica. The homogeneity was demonstrated by one single sharp peak on HPLC and one single band on PAGE-SDS. The amino acid analysis showed 10 amino acid residues, with no specific traits, and the mol. wt determined by gel filtration and amino acid composition was 1000. An immunochemical relation between the allergen and the hapten was also suggested by the results of an ELISA-inhibition test, and by the ability of the hapten to partially inhibit the precipitin line between rabbit antibodies to whole P. judaica pollen extract and the Pj-2 allergen. The allergen and the hapten described above, purified at homogeneity and in an antigenically active state, both provide adequate material for further structural and immunological characterizations.     

Thyroxine dependency of the developing substantia nigra. Evidence from intraocular grafting experiments in rats

Small pieces of rat fetal brain stem containing immature dopaminergic substantia nigra neurons were grafted to the anterior eye chamber of thyroidectomized and control recipient rats, to elucidate the possible developmental dependence of these neuroblasts on thyroid hormones. Outgrowth of nerve fibers in sympathectomized irides was analyzed with Falck-Hillarp fluorescence histochemistry. Both total surface area of the irides innervated by the dopaminergic neurons, and the number and size of axon bundles were markedly reduced in the experimental groups. This strongly suggests a role for thyroid hormones during fiber formation by the developing dopaminergic neurons of substantia nigra.     

Development of reproductive tract lesions in male F344 rats after treatment with dimethyl methylphosphonate

Dimethyl methylphosphonate (DMMP) is an organophosphorous compound that impairs fertility in male rodents. In previous studies, male rats treated with DMMP had decreased sperm motility and count, and sired fewer litters with fewer pups per litter. The following studies examined the development of the reproductive lesions by light and electron microscopy after treatment with DMMP. Adult male F344 rats were treated po with DMMP, 1750 mg/kg, for up to 12 weeks. Tissues were perfused in situ with Karnovsky's fixative and embedded in glycol methacrylate. After 5 weeks of treatment there were occasional PAS-positive bodies in lumina of tubules in stages XII-III. These were ultrastructurally similar to cytoplasm of step 12-17 spermatids. After 7 weeks of treatment, there was an increase in the number of tubules exhibiting these bodies, as well as an increase in the number of tubules showing delayed or early spermiation, or focal exfoliation of nonnecrotic cap-phase spermatids and some spermatocytes. No multinucleated giant cells were seen. Focal loss of germ cells occurred more frequently as duration of exposure increased, and occupied 5-100% of an affected tubule. Frequently, an area of germ cell exfoliation occurred adjacent to areas of normal tubular epithelium. These lesions were not specific to any particular stages of spermatogenesis. Occasionally, elongating spermatids were without rib elements of the fibrous sheath in the tail; these were not seen in epididymal sections. Animals left to recover for 14 weeks after treatment showed approximately 80% normal tubules; affected tubules varied in their degree of recovery, but all showed the loss of normal epithelial organization, a characteristic of DMMP treatment. Epididymal epithelium was not visibly affected by treatment with DMMP. DMMP produced morphologic alterations in Sertoli cells and elongating spermatids, as well as producing functional defects in spermatozoa.     

Sodium erythorbate is not carcinogenic in F344 rats

Carcinogenicity of sodium erythorbate, a widely used antioxidant food additive, was evaluated using a total of 306 eight-week-old male and female F344/DuCrj rats. Test rats were given 1.25 or 2.5% aqueous solution as drinking water for 104 weeks. Controls were given tap water. All the rats were fed commercial pellets. None of the tumors observed was attributable to sodium erythorbate in drinking water. Neither concentration of sodium erythorbate changed the pattern of spontaneous tumor development in both sexes, except for a slight reduction in aggregate tumor incidence in the 2.5% Group females. Additionally, 2.5% solution suppressed body weight gains in both males and females. These results and prior data by others together suggest that weak mutagens may be noncarcinogenic under certain conditions.     

Cytotoxic effects of erythromycin estolate and chlorpromazine on hepatocytes isolated from rats of varying ages: A brief note

Hepatocytes isolated from young (1 month) rats were as sensitive to the cytotoxic effects of erythromycin estolate and chlorpromazine as were liver cells obtained from older (3, 10 and 24 months) rats. The hepatocytes from the 24-month-old rats released aspartate transaminase more slowly than did parenchymal cells isolated from the younger rats.     

Autoregulation of histamine release in brain by presynaptic H3-receptors

Regulation of histamine release was studied mainly on brain slices prelabeled with L-[3H]-histidine and depolarized by increased extracellular K+ concentration or veratridine in a non-superfused system. The released 3H-labeled amines, isolated by ion-exchange chromatography from a large excess of 3H-labeled precursor consisted by more than 95% of unchanged [3H]histamine. Exogenous histamine reduced the release of neosynthesized [3H]histamine via stimulation of previously characterized H3-receptors whereas it did not modify the 3H-labeled amine release from slices prelabeled with preformed [3H]histamine. The maximal inhibitory effect of exogenous histamine progressively diminished as the strength of the depolarizing stimulus or the external Ca2+ concentration were elevated. On the contrary H3-receptor antagonists like impromidine or burimamide enhanced the depolarization-induced release of [3H]histamine, an effect which was particularly marked when slices were loaded with histamine by preincubation with [3H]histidine in high concentration. These results suggest that the inhibition of [3H]histamine release by exogenous histamine acting via H3-receptor stimulation is mediated by a restricted access of Ca2+ and that its extent is influenced by the degree of autostimulation by endogenous histamine as well as, possibly, by actual internal Ca2+ concentration. In addition the decrease in external Ca2+ concentration shifted rightwards the concentration-response curve to histamine. The autoinhibitory effect of exogenous histamine was found on slices from various regions, known from lesion studies to contain terminals of extrinsic histaminergic neurons. It did not apparently involve interneurones, not being prevented in slices in which the traffic of action potentials was blocked by tetrodotoxin. It also remained unaffected in striatal slices in which the neuronal cell-bodies were selectively destroyed by prior local infusion of kainic acid. Finally exogenous histamine inhibited [3H]histamine release from depolarized synaptosomes of rat cerebral cortex, with an EC50 value similar to that found with slices and was antagonised by impromidine with an apparent Ki value similar to that displayed at H3-receptors. It is concluded that histamine modulates its own release from cerebral neurones by interacting with H3-presynaptic autoreceptors and via mechanisms similar to those previously evidenced on other aminergic systems.     

Facilitation of transmission in Ib pathways by cutaneous afferents from the contralateral foot sole in man

Changes in the quadriceps H-reflex were used to study the effect of a weak contralateral cutaneous stimulation upon transmission in Ib pathways from ankle muscles to quadriceps in man. Provided it was applied to the contralateral foot sole, such a stimulation facilitated transmission in Ib pathways to quadriceps (inhibitory from extensors as well as excitatory from flexors). The central latency of this contralateral cutaneous facilitation was 1 msec longer than that of the ipsilateral cutaneous depression of Ib pathways. These findings are discussed with regard to the requirements of bipedal gait.     

Putative dopamine-containing cells in the retina of seven species demonstrated by tyrosine hydroxylase immunocytochemistry

Immunocytochemistry with antibodies to catecholamine synthesizing enzymes has revealed cells in the retina of chick, mouse, hamster, rat, guinea-pig, piglet and marmoset which contain tyrosine hydroxylase but not dopamine beta-hydroxylase. These findings suggest that the cells in question produce dopamine but that catecholamine synthesis does not proceed further to noradrenaline. Tyrosine hydroxylase-containing amacrine cells, located in the innermost part of the inner nuclear layer, were present in all the species studied. Some species showed atypically located amacrine cells in the inner plexiform or ganglion cell layer. In the rodents, the existence of tyrosine hydroxylase-containing interplexiform cells was suggested by the presence of a few short immunoreactive ascending processes. Three different morphological types of putative dopamine-containing cells were classified according to the level of ramification.     

Induction of megamitochondria in the mouse liver by isonicotinic acid derivatives

Megamitochondria were induced in mouse hepatocytes simply by feeding the animal for 7 to 10 days with a diet containing isonicotinic acid derivatives, namely, nialamide, isoniazid, and iproniazid. Monoamine oxidase activities of megamitochondria were invariably decreased significantly. Isonicotinic acid, however, did not induce megamitochondria and had no effect on monoamine oxidase activity of mitochondria. Coupling efficiencies of megamitochondria induced by the reagents, specified above, were not affected. Cytochrome c oxidase and ATPase activities of megamitochondria were also unchanged compared to the control. Isoniazid, iproniazid, and nialamide affected monoamine oxidase activity of mitochondria not only in vivo, as described above, but also in vitro: the activity was inhibited by 50% at concentrations of 5.8 mM, 215 and 140 μM for isoniazid, iproniazid, and nialamide, respectively. Rotenone-insensitive NADH-cytochrome c reductase of megamitochondria, another marker enzyme for mitochondrial outer membrane besides monoamine oxidase, was not affected so much: the lowest activity was 88.6% of the control in the case of nialamide-induced megamitochondria. These reagents had no effect on the enzyme activity in vitro. These data seem to indicate that the reagents examined in the present study affected monoamine oxidase specifically. Mechanism of the formation of megamitochondria is discussed with respect to membrane fusion phenomenon.     

Depletion by capsaicin of substance P-immunoreactivity and acetylcholinesterase activity from nerve fibres in the guinea-pig heart

The sensitivity to capsaicin of substance P-immunoreactive nerve fibres in the hearts of guinea-pigs was examined. Capsaicin decreased considerably the substance P-immunoreactive material in nerve fibres of the parietal pericardium, atria, bicuspid and tricuspid valves. Pericardial and valvar nerve fibres localized by acetylcholinesterase (AChE) histochemistry had a distribution and density pattern similar to that of the substance P immunoreactive fibres. Capsaicin treatment also decreased the number of visible AChE-positive nerve fibres. It is known that capsaicin has a selective action on those substance P-immunoreactive fibres that are of sensory origin; thus, these results imply that substance P-immunoreactive fibres in the heart are sensory. Moreover, the results suggest that some of the nerve fibres localized in the heart by AChE histochemistry are substance P-containing sensory fibres.     

Dopamine-induced ventilatory depression in the rat,mediated by carotid nerve afferents

In pentobarbitone-anesthetized rats, i.v. injections of dopamine (DA) at 10^?8–10^?5 g/kg led to transient ventilatory depression, usually not associated with changes in systemic arterial pressure. DA-induced ventilatory depression consisted of decreases in tidal volume and respiratory frequency in 12 rats, and in bradypnea without changes in tidal volume in 5 rats. After bilateral section of the carotid nerves, ventilatory responses to DA were abolished or greatly diminished. It is concluded that DA inhibits carotid body chemosensory discharges in the rat. The possibility that chemosensory activity directly affects respiratory frequency is discussed.     

Cloning of hybridomas by a single-cell transfer technique

A method for cloning hybridomas is described which involves transfer of single cells with a capillary tubing connected to a suction apparatus. This method enables the efficient recovery of antibody-producing clones of differing abundances from the parent hybridoma cultures. The 4 cell lines submitted to this cloning procedure produced a 100% yield of positive wells upon recloning by the same method.     

Alteration in the membrane fatty acid composition of human lymphocytes and cultured transformed cells induced by interferon

The effects of interferon (IFN) treatment on the lipid composition of human peripheral blood lymphocytes or transformed cell line cells were investigated. The major phospholipid classes of lymphocytes as analyzed by 2-dimensional TLC and quantified by phosphorous content were phosphatidylcholine (PC, 43%) and phosphatidylethanolamine (PE, 28%), along with phosphatidylserine (9%) and phosphatidylinositol (8%). The membrane-impermeant reagent, trinitrobenzenesulfonate was used to covalently label cell surface PE. Fatty acid (FA) composition, determined by gas-liquid chromatography, showed a distinct pattern in each lipid class, with a predominance of 16 and 18 carbon fatty acids (FA) in PC and PE respectively. Arachidonic acid (20:4) and, to a lesser extent, docosahexanoic acid (22:6) were predominant in PE. The degree of unsaturation in each class, expressed as the ratio between unsaturated and saturated FA (U/S), was higher in PE (1.72) than in derivatized trinitrophenyl cell surface PE (TNP-PE, 0.57) or PC (0.64). Treatment with IFN resulted in an increased U/S ratio in cell surface PE (1.10) but not in other PE species (1.46). A small increase in unsaturation (0.88) was also observed in PC. Most of the increase in TNP-PE U/S was accounted for by an increase in 20:4 and a concomitant decrease in 18:0. These alterations were observed in the absence of quantitative change in the principal phospholipid classes or in the FA composition of the total lipid extract. In K562, a transformed cell line with characteristics of the erythromyeloid lineage, PE was found to be the most saturated lipid class with a predominance of 18:0. In PC, 16:0 was most abundant. Among unsaturated FA, 18:1 predominated in all lipid classes studied. Treatment with natural IFN alpha for 30 hr generally resulted in a decrease in saturated FA and an increase in unsaturated FA, which was most marked in PE. The U/S ratio in PE was highest in K562 cells during the time of maximal cell proliferation as assessed by tritiated thymidine incorporation. TNP-PE simultaneously decreased. Daudi cells, a B-lymphoblastoid cell line, demonstrated changes in FA composition of lipids with decreased saturated and monoenoic FA after IFN treatment, whereas DIF3 (a clone selected for lack of sensitivity to IFN) showed no change. These studies document changes in membrane FA composition of lymphocytes treated with IFN and correlate IFN-induced changes in transformed cell line FA with effects on proliferation. They further show the existence of a transverse molecular species asymmetry of PE in the plasma membrane of these cells which is altered after IFN treatment.     

Immunomodulation in mice by Bacillus megaterium and its dependence on culture conditions

Mice pretreated with Bacillus megaterium ATCC 33085 grown on TSA medium developed a significant increase in primary antibody response to SRBC. Conversely, pretreatment with a spore suspension harvested from nutrient Agar medium decreased this antibody response. A suspension of organisms grown on a defined, phosphorus-deficient medium (P-Medium) had no effect. Otherwise, only the spore suspension was able to enhance the contact sensitivity to dinitrofluorobenzene. Peritoneal leucocyte numbers were increased by inoculation with both TSA-cultured bacteria and the spore suspension, but not by P-Medium-cultured bacteria. Administration of both the spore suspension and P-Medium-cultured bacteria decreased the in vitro phagocytosis by peritoneal adherent cells. These immunomodulator properties are discussed in relation to characteristics of the strain tested.