Effect of the growth-promoting alpha-globulin (GPAG) on in vitro incorporation of exogenous DNA into mammalian cells

Summary The uptake of exogenous³H-DNA by mammalian cells is increased in the presence of a specific serum protein complex — growth-promoting alpha-globulin (GPAG).³H-DNA is retained in the cell nucleus in a quantity 3 times higher than for control cultures without GPAG even after 47h of additional cultivation in the medium without³H-DNA.     

The influence ofBordetella pertussis on the kinetics of antibody production to sheep red blood cells in NMRI mice

Zusammenfassung Die simultane i.p. Injektion von 4 × 10⁸ Schaferythrozyten und 2 × 10⁹ Zellen vonB. pertussis führt im Vergleich zur alleinigen Injektion des Erythrozytenantigens bei NMRI-Mäusen zu einer gesteigerten und verlängerten Proliferation M- und G/A-Antikörper produzierender Milzzellen.

---

---

This work was supported by research grant No. Fi 115/1 of the Deutsche Forschungsgemeinschaft.     

Denaturation map of the ribosomal DNA ofLytechinus variegatus sperm

Summary Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6±0.2 m, corresponding to a mol.wt of 7.2±0.4×10⁶. Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47±0.11 m (mol.wt 4.9±0.22×10⁶) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16±0.09 m (mol.wt 2.3±0.18×10⁶) is presumed to contain non-transcribed spacer sequences.Acknowledgments. This paper is dedicated to the memory of Dr D. P. Costello whose insprration made this work possible. I gratefully acknowledge the valuable advice of Drs D.W. Stafford and M.A. Bleyman.     

The uptake of iodinated protein by spleen cells of guinea-pigs in vitro

Zusammenfassung 6×10^–10 g bis 10^–4 g J 125 Rinderserum-Albumin (BSA) bzw. 1,0×10^–7 bis 1,0×10^–6 g J 125 Rinderserum--Globulin (BGG) werden 3 × 10⁷ Milzzellen normaler Meerschweinchen in vitro angeboten. Unabhängig von der Höhe des Angebotes beträgt die Menge des aufgenommenen BSA 1%, die des aufgenommenen BGG 10% des Angebotes. Durch 4C Inkubations-temperatur wird die Aufnahme z.T. behindert. Die Aufnahme findet nach wenigen Sekunden Inkubationszeit statt und ist nach etwa 2–3 min abgeschlossen.

---

---

The work was supported by a grant of the Stiftung Volkswagenwerk and of the Deutsche Forschungsgemeinschaft.     

The effects of sodium and amiloride on the motility of the caudal epididymal spermatozoa of the rat

Summary The forward motility of the rat caudal epididymal spermatozoa has been studied in different Na⁺ concentrations. When spermatozoa were suspended in a completely Na⁺-free solution, the forward motility suffered a progressive fall and after 3 h was completely suppressed. This effect was fully reversible on resuspending the spermatozoa in a solution containing Na⁺. Amiloride caused a fall in motility and the effect was similar to that of Na⁺ removal. The inhibition by amiloride of the motility was concentration dependent and the dose response curve showed an IC₅₀-value of about 5×10^–5 M. The role of Na⁺ influx in the maintenance of sperm motility was discussed.This work was supported by the World Health Organization.The technical assistance of Mr C.M. Li and the gift of amiloride from Merck, Sharp and Dohme are gratefully acknowledged.     

Cartsian diver studies on respiration of rat bone marrow cells in the presence of erythropoietin and anti-erythropoientin

Résumé L'étude de la respiration des cellules de la moelle des os du rat fut faite avec un plongeur Cartésien. Ces cellules, avec ou sans glucose, repirent à raison de 0,73±0,15 et 0,97±0,17×10^–6 l O₂/cellule/h. Comparée aux mesures de contrôle, la respiration des cellules médullaires de rats pléthoriques est plus fabile en présence de ESF (0,45 contre 0,99×10^–6 l, O₂/cellule/h). Cette inhibition fut supprimée par la présence d'anti-ESF (0,98 contre 0,99×10^–6 l O₂/cellule/h).

---

---

This research was supported by funds from a Damon Runyon Cancer Research Training Grant and the US Public Helath Seervice. The senior author was a Damon Runyon Post-Doctoral Traince.     

Persistance of maternally derived3H-estradiol in fetal and neonatal rats

Summary Injection of³H-estradiol into pregnant rats resulted in fetal blood radioactivity 5 times higher than in maternal blood. Significant amounts of³H-estradiol were found in fetal blood 24 h later and in the offspring 5 days after birth.Supported by NIH Grant HD 08844.     

Androgen receptor in the thumb pads ofRana esculenta

Riassunto E' stato messo in evidenza un recettore per gli androgeni nel sopranatante a 105.000×g della callosità del pollice diRana esculenta. Si é visto che competono fortemente col testosterone-H³ il testosterone, il diidrotestosterone, l'11-chetotestosterone e l'acetato di ciproterone; mentre non competono l'estradiolo-17, il cortisolo ed il ciproterone libero. La costante di associazione varia tra 1,25 e 3,26×10⁹ M ed il numero di siti varia tra 1,15 e 3,01×10^–10 M.     

Effect of enkephalins in the presence of the antibiotic bacitracin in the longitudinal muscle strip preparation from guinea-pig ileum

Summary The antibiotic bacitracin (5×10^–5–4×10^–4 M) increases the inhibition of the contractile response caused by both enkephalin release and direct application of Met-enkephalin 5×10^–7 M in the longitudinal muscle strip preparation from guinea-pig ileum. This effect is attributed to an inhibition of enkephalin degrading peptidases by bacitracin.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

A histochemical demonstration of the Na++K+-ATPase activity in the thyroid and the effect of cyclic adenosine monophosphate (c-AMP)

Summary With a suitable modification of the Farquhar and Palade technique the Na⁺+K⁺-ATPase activity in guineapig thyroid is demonstrated. The addition of c-AMP (5×10^–6 M or 1.5×10^–5 M) to the incubation media produced an apparent intensification of the Na⁺+K⁺-ATPase activity in the thyroid.This work was supported by a grant from ZMNU of Serbia.     

K252a: A new blocker of the cell-cycle at G1 phase in a human hepatoma cell line

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [³H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and -fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.     

A study of DNA synthesis in sea urchin hybrids by the incorporation of H3-thymidine

Zusammenfassung (1) Es wird die DNS-Synthese der letalen SeeigelbastardeParacentrotus ×Arbacia (PA) undParacentrotus ×Sphaerechinus (PS) mit H³-Thymidin untersucht. (2) Dem verschiedenen, der Letalität vorausgehenden Entwicklungstpus (PA ohne, PS mit Chromosomenelimination) entspricht ein verschiedener Verlauf der Hemmung der DNS-Synthese. Parallel dazu wird die Vermehrung in der Anzahl der Kerne verglichen.     

Effect of strychnine on the electroretinogram of the isolated rabbit retina

Zusammenfassung Strychnin in einer Konzentration von 3×10^–6 bis 3,5×10^–5 M/l verursacht eine vorübergehende Vergrösserung derb-Welle des ERG der isolierten Kaninchennetzhaut, in einer Konzentration von 7×10^–5 bis 7×10^–4 M/l verkleinert es dieb-Welle. Aus den Ergebnissen wird geschlossen, dass die Substanz zumindest teilweise schon auf die Schicht des zweiten Neurons wirkt.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Induction of the in vitro p-hydroxylation of14C-amphetamine stereoisomers in phenobarbital-treated rats

Summary The rate of p-hydroxylation of¹⁴C-(-)-amphetamine by liver microsomes was higher than that of (+)-isomer in phenobarbital-treated male rats. The apparent K_m values for (-)- and (+)-amphetamine hydroxylation were 4.54×10^–5 M and 2.27×10^–5 M respectively, in both treated and control animals.     

Effects of retinoic acid on embryonic development of mice in culture

Summary The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first viscreral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of³H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0×10^–7 M alltrans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.     

Differential sensitivity of the two phases of ear artery contraction to intimal and adventitial norepinephrine

Summary The biphasic contraction of the rabbit ear artery to norepinephrine (NE) was investigated in the normal (adventitial stimulation) and the everted (intimal stimulation) segment of ear artery. The 2nd phase response showed an intimal ED₅₀ of 8.2×10^–8 M which was significantly (p<0.05) lower than the adventitial ED₅₀ of 42.6×10^–8 M. This difference was abolished by inhibition of neuronal and extraneuronal uptake for NE. The 1st phase response also showed an ED₅₀ for the intimal stimulation (6.9×10^–8 M) which was significantly (p<0.05) lower than adventitial (65.5×10^–8 M). This difference was reduced but not abolished by NE uptake inhibition. This suggsets that some feature of the adrenergic neuroeffector apparatus is asymmetrically arranged to favor fast responses to blood borne NE.Supported by American Heart Association-Greater Los Angeles Affiliate Grant No. 602. We wish to thank Dr John Bevan and Dr Alasdair MacLean for helpful advice.     

In vitro effects of methionine-enkephalin,somatostatin and insulin on cultured gonadal cells of the snailHelix aspersa

Isolated snail gonadal cells were cultured in the presence of synthetic neuropeptides in order to determine the subsequent effect of these substances on gonadal synthetic activities. Gonadal cells were incubated for 24 h in concentrations of methionine-enkephalin, somatostatin and insulin ranging from 10^–4 M to 10^–9 M, in medium 199 supplemented with 6% Ultroser G. Synthesis of DNA and protein by the cultured cells were simultaneously estimated by measuring incorporation of³H thymidine and³⁵S methionine. The rate of labelled precursor incorporation was measured using the liquid scintillation technique. All substances tested exerted a dose-dependent effect. The synthetic activity of the cultured cells was highest when the concentration of the peptides added to the medium approximated the physiological levels. Methionine-enkephalin, somatostatin and insulin at 2×10^–8 M significantly increased³H thymidine incorporation, by 62%, 69% and 69% respectively, and protein synthesis by 42%, 57% and 57%, respectively. In the case of juvenile gonadal cultured cells, a similar increase in³H and³⁵S incorporation was registered for a 10^–7 M peptide concentration. Both lower and higher peptide concentrations inhibited³H thymidine and³⁵S methionine incorporation. Pharmacological studies suggest the existence of methionine-enkephalin and somatostatin-like receptors on snail gonadal cells. These results indicate that our gonadal cell culture model provides a useful tool for the study of the neuroendocrinological control of the activity of snail gonadal cells.     

Growth ofNostoc muscorum mutants in the presence of diuron (DCMU) and L-methionine-DL-sulfoximine

Summary Diuron (DCMU) is inhibitory to the photoautotrophic and photoheterotrophic growth of the N₂-fixing blue-green algaNostoc muscorum at concentrations of 1.0×10^–5 M and 2.0×10^–5 M, respectively. A mutant of this organism resistant to 5.0×10^–5 M DCMU under its photoheterotrophic growth conditions, with the ability to utilize DCMU as a carbon and nitrogen source for growth, and complete inability to grow photoautotrophically has been isolated. With the apparent defect in its photosynthetic ability, it is suggested that theDCMU ^r mutant lacks the step inhibited by 1.0×10^–5 M DCMU, and metabolizes DCMU by an existing enzyme system in the absence of such inhibition. That this enzyme may be glutamine synthetase (GS) is explained with the help of a L-methionine-DL-sulfoximine (MSO)-resistant mutant ofN. muscorum which is able to grow faster with 2.0×10^–5 DCMU and is known to contain an altered GS.Thanks are due to the Council of Scientific and Industrial Research, CSIR Complex, Govt. of India, New Delhi-110012, for appointing the author to the Scientists' Pool for undertaking researches on the physiological and genetic controls of nitrogen metabolism in blue-green algae, a part of which is presented in this literature.