Phenytoin-induced DNA synthesis and inositol 1,4,5-triphosphate formation in L-929 fibroblasts

Summary Culture of L-929 fibroblasts in the presence of phenytoin (2.5–5.0 g/ml) increased DNA synthesis, as indicated by increased [³H]thymidine uptake, while a higher dose (20 g/ml) inhibited DNA synthesis. In like manner, a low dose of phenytoin (5.0 g/ml) was effective in increasing inositol 1,4,5-trisphosphate formation while a higher dose (10 g/ml) tended to inhibit this activity. These data suggest that the formation of inositol phosphate second messengers may play a role in phenytoin-induced fibroblast proliferation and connective tissue growth.     

Tannic acid inhibits insulin-stimulated lipogenesis in rat adipose tissue and insulin receptor function in vitro

Tannins occur naturally in relatively abundant amounts in fruits, herbal medicines and common beverages. Thus an understanding of how these polyphenols affect peptide hormone action is of importance. We report here that tannic acid (a hydrolysable tannin) inhibits insulin-stimulated lipogenesis in rat adipose tissue in vitro, with an IC₅₀ estimated to be about 350 M. However, its monomer, gallic acid, did not show a similar inhibitory effect at concentrations up to 1 mM. The inhibition by tannic acid was less evident with higher concentrations of bovine serum albumin in the incubation buffer. This was attributed to the formation of a tannin-protein complex between bovine serum albumin and tannic acid. In a binding assay, it was observed that the specific binding of insulin to its receptor was not inhibited by tannic acid in the concentration range 0–200 M. However, insulin-stimulated autophosphorylation of the insulin receptor, and receptor-associated tyrosine kinase phosphorylation of RR-SRC peptide, were inhibited by tannic acid at concentrations as low as 25 M. Our data do not support the current speculation that tannins affect the activity of peptide hormones by binding to them. Therefore, our finding opens up a new perspective in the understanding of the mode of action of tannins on such hormones.     

Central nicotinic receptor blockade inhibits emotionally conditioned pressor responses in rats

A conditioned stimulus previously paired with electric footshock produced an increase in blood pressure in conscious, freely moving rats. The conditioned pressor response was reproducible. Intracerebroventricular injection of the nicotinic receptor antagonists hexamethonium (1–10 g) or pentolinium (10 g) but not the muscarinic receptor antagonist methylatropine (3 g) produced an inhibition of the conditioned pressor response, whereas intraarterial injection of hexamethonium (10 g) did not affect the response. Intraventricular injection of the cholinesterase inhibitor physostigmine (3–10 g) produced an enhancement of the conditioned pressor response. These results are consistent with the possibility that central nicotinic receptors play a role in the expression of the emotionally conditioned pressor response in rats.     

Platelet aggregation is inhibited by phycolectins

Lectins from four marine algal species were examined for interaction with human platelets. The lectin designated hypnin A, from the red algaHypnea japonica, inhibited adenosine diphosphate (ADP)-or collagen-induced human platelet aggregation in a dose-dependent manner. Complete inhibition was observed at concentrations of 100 and 5 g/ml of the lectin with, ADP (2 M) and collagen (0.2 g/ml)-induced platelet aggregation, respectively. At the inhibitory concentration of 0.5 to 100 g/ml, the lectin did not induce aggregation of resting platelets. Lectins from the other three algal species also inhibited ADP-induced human platelet aggregation. These results indicate that the algal lectins are a new group of inhibitors and may be useful to study glycoconjugates on platelet membranes and to design novel platelet aggregation inhibitors.     

Giant pigment granules in dermal melanocytes of rat scrotal skin

Summary Giant melanosomes, ellipsoidal (1.5 m×1.3 m) or spherica (1.1 m–1.4 m in diameter) exist in the scrotal skin of the black pelted Long Evans rat. They are longer and wider than normal stage IV melanosomes (0.7 ×0.4 m) found in theses dermal melanocytes.The authors acknowledge with gratitude, the technical assistance of Evelyn Ann Flynn. This work was supported by grant AM 20669-16 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, USPHS.     

A sensitive protein assay method using micro-titer plates

Summary A commercially available protein-assay, based on the reaction between bicinchoninic acid (4,4-dicarboxy-2,2-biquinoline) and copper was adapted for use in microtiter plates. The assay is sensitive between 10 m/ml and several mg/ml, and it uses only 10 l of sample material. The results obtained with the method were in accordance with other protein determination methods. The assay was shown to be reproducible, reliable and insensitive to non-ionic detergents.Acknowledgments. The authors are grateful to Miss Barbara Moser who skillfully performed a number of these experiments. This research was supported by the Swiss National Science Foundation, Grant No 3.300–0.82, to U.B.     

Depletion of hypothalamic norepinephrine reduces the fever induced by polyriboinosinic acid: polyribocytidylic acid (Poly I:Poly C) in rats

Summary Administration of either Poly I:Poly C (0.05–0.50 g) or norepinephrine (2–8 g) into the anterior hypothalamic area produced a dose-related fever in rats. The fever induced by Poly I:Poly C was attenuated after selective depletion of norepinephrine in the hypothalamus. However, selective depletion of hypothalamic norepinephrine did not affect the fever induced by intrahypothalamic norepinephrine. The data indicate that Poly I:Poly C may act to induce fever through the endogenous release of norepinephrine from the rat's hypothalamus.This work was supported by grants from the National Science Council (Taipei, Republic of China).     

Gamma-aminobutyric acid uptake by rat kidney brush-border membrane vesicles

Summary Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for -aminobutyric acid (GABA), a high affinity system (Km=10.9 M) and a low affinity system (Km=1203 M). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, -alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMW uptake systems were clearly different from the GABA transport systems present in brain tissue.     

Repercussions de l'hyperthyroïdie sur le contenu total en ADN du cervelet de rat âgé de 6 et 35 jours. Effets comparés de la LT3 et de la DLT4

Summary The total cerebellar proteins RNA and DNA contents from DLT4- and LT3-treated rats was studied at 6 and 35 days of age. The effect of injections of 5 g/j of DLT4 is comparable to that of 25 g of LT3 at birth, followed by 0.5 g every 2 days. On the other hand, injection of 0.5 g of LT3 every 2 days does not induce any significant modification of the total DNA contents in the cerebellum.     

Uptake of D-glucose anomers by rat retina

Summary Uptake ofd-glucose anomers by isolated rat retina was studied. After 3 min incubation at 37°C in the presence of or anomer (750 g/ml), a significantly greater uptake (1.32 mg/g wet tissue) of -anomer was observed compared with that of -d-glucose (1.11 mg/g wet tissue). This result and other data suggest that the carrier ford-glucose transport in the retina prefers the -anomer stereospecifically.Acknowledgment. The authors are grateful to Mr S. Suzuki for his technical assistance.     

Effect of dipyridamole on glomerular mesangial cell ecto-5′-nucleotidase expression

Although dipyridamole has been extensively studied as an anti-aggregating agent, its mechanism of action has not been elucidated. Cultured mesangial cells were treated with dipyridamole 1–100 M from 6–72 h. Ecto-5-nucleotidase activity approximately doubled (from 115±11 to 226±14 nmol/min/mg) after treatment with 100 M dipyridamole for 72 h. This effect was concentration- and time-dependent. Cycloheximide, an inhibitor of protein synthesis, did not alter basal 5-nucleotidase activity. However, it prevented stimulation by 5 M dipyridamole. Adenosine availability at the receptor sites was increased by dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), which inhibit adenosine uptake into the cell. Addition of dipyridamole or NBTI to the adenosine-treated mesagial cells produced an additive increase in ecto-5-nucleotidase activity. Dipyridamole, through its effect on extracellular adenosine and ecto-5-nucleotidase, may have an influence upon regulation of the glomerular microcirculation.     

Artificial spawning effected in the fresh water teleost,Cyprinus carpio by clomiphene citrate

Summary Triweekly i.m. injections of clomiphene citrate (group 1, 25 g/0.5 ml and group II, 50 g/1.0 ml) were administered for a period of 3 months during the preparatory period to female fresh water teleosts exhibiting ovarian recrudescence, while a control group received 0.5 ml of physiological saline throughout the period of experimentation i.e., from February through April. 50 g clomiphene citrate treatment brought about a steady increase in ovarian size, and oocytes began to enlarge and mature and finally ovulation took place in April. This is 4 months ahead of their normal occurrence.The authors wish to express their gratitude to Richardson Merrel Co. for supplying the sample of clomiphene citrate, and Dr K.N. Udupa, Institute of Medical Sciences, Banaras Hindu University, Varanasi for facilities.     

Antidiuretic effects of oxytocin in the Brattleboro rat

Summary The antidiuretic activity of oxytocin (OT) was measured in Brattleboro rats with congenital diabetes insipidus. A dose dependent antidiuretic response was found in animals receiving chronic infusions of 0.1 g/h, 1.0 g/h, and 5 g/h of OT. OT infused at the rate of 5 g/h over a 7-day period completely reversed the symptoms of diabetes insipidus. The results support the concept that OT serves as a weak agonist of vasopressin at the level of the kidney and at pharmacological levels exhibits antidiuretic activity.     

Different effects of D-glucose anomers for respiration of bacterial germinated spores

Summary Effects of - or -D-glucose on the respiration of germinated spores (only germinated spores not including swollen spores and elongated spores) ofBacillus subtilis andB. megaterium were studied. In our conditions, net amount of oxygen consumed by 10¹⁰ germinated spores ofB. subtilis per min after addition of - or -D-glucose was 1.6 g or 6.6 g (/=4.13), while that byB. megaterium was 4.5 g or 6.8 g (/=1.51), respectively. However, the net amounts of oxygen consumed by 10¹⁰ vegetative cells per min after addition of - or -D-glucose were identical, forB. subtilis in both cases 443.0 g and forB. megaterium in both cases 604.4 g.     

Physicochemical studies of indocyanine green (ICG): absorbance/concentration relationship,pH tolerance and assay precision in various solvents

Summary Indocyanine green (ICG) obeyed the Beer-Lambert law within the concentration range 1.25 g/ml–10.0g/ml in distilled water, methanol, dimethylformamide (DMF), 1.2-propanediol and aqueous buffers (pH 9.0), but only up to 7.5 g/ml in human bile and 0.5% human albumin, and only to 5.0 g/ml in human duodenal fluid. ICG was rapidly (<1 h) decomposed to a colorless derivative at pH<5 and >11, but remained relatively stable for 48 h at pH 8–10. ICG is an indicator and a weak acid with a pKa of 3.27. In bile stabilized with 25% methanol, the precision of the method (CV) is 5% and the accuracy is 106–127%.     

Failure of somatostatin to influence experimental tumor cell growth in vivo and in vitro

Summary The influence of somatostatin on tumor cell growth was studied in vivo in mice (sarcoma 180 ascites tumor and Lewis lung tumor) and in vitro on nontransformed and polyoma-transformed cell lines. 4 or 20 g/100 g of cyclic somatostatin and 4 g/100 g of linear protamin Zn-bound somatostatin were injected s.c. twice daily in the in vivo study. Cyclic somatostatin (1, 4 or 10 g/ml) was added twice daily to the cell cultures. Somatostatin administration influenced neither the survival of animals nor the growth rate of cultured cell lines. *** DIRECT SUPPORT *** A2025117 00011     

Dopamine and cerebral cortical blood flow in the rat

Summary Dopamine topically applied to the cerebral cortex (1–20 g/ml) or administered i.v. (0.5–64 g/kg/min) has no effects on cerebral cortical blood flow in the rat.     

ATP-dependent Ca++-Extrusion from human red cells

Zusammenfassung In einer Lösung mit 1,39M/ml Ca⁺⁺ waren rekonstituierte menschliche Erythrozyten im Stande, ihren ursprünglichen Ca-Gehalt von 2,39M/ml Zellen in 60 min bei 37 °C auf 0,46M/ml Zellen zu senken, sofern sie Mg-ATPat enthielten. Dieser aktive Transport war durch g-Strophanthin nicht hemmbar, konnte aber mit 5 · 10^–4 M Salyrgan unterdrückt werden.     

Inhibition and stimulation by 5-bromodeoxyuridine of erythropoiesis by chick blood island cells

Summary Erythropoiesis in liquid cultures of cell populations resolved from chick blastodiscs at the primitive streak and head-fold stages was totally inhibited by 5–8 g/ml of 5-bromodeoxyuridine. However, concentrations of 0.2 /ml of the nucleoside enhanced the number of erythroid cells formed.This work was supported by grants from the Medical Research Council and National Research Council of Canada. We thank MissesK. Beall andN. N. McGrath for technical assistance. S. D. W. is an Associate of the Medical Research Council.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages ofPlasmodium falciparum and some tumor cell lines

Summary In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages ofP. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 g/ml respectively which have no effect on the growth ofP. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain ofCandida remained below 2 g/ml. These experiments suggest that 5-FC (at 50 g/ml) alone or in combination with KTZ (at 1 g/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.