Giemsa stain applied to deplasticized sections to identify pancreatic islet cells

A new application of the Giemsa stain to demonstrate endocrine cells in deplasticized sections of Epon embedded material is described. Its application to the pancreas of Rana temporaria is illustrated. The technique does not require postfixation with OsO4 and is easily performed in 30 min. It allows the easy identification of three types of endocrine cells (A, B, and D). A cells, preferentially located at the islet periphery, stain purple-blue. B cells, which occupy the interior of the islet, display a lilac color. D cells give a strong purple color; they are located both in the periphery of the islets and scattered among acinar cells. Positive identification of the cell types was made by immunocytochemistry and electron microscopy.     

Decrease in the secretion of anti-Müllerian hormone by the chick testis during development

At the end of embryonic life the chick embryonic testis possesses a low anti-Müllerian activity, as evidenced by the grafting method to female hosts. The percentage of grafted embryos presenting a Müllerian duct regression is not increased by administration of an anti-estrogenic drug (tamoxifen). This observation does not favour the hypothesis according to which the low percentage of regression could be due to a protection of Müllerian ducts by estrogens from the host ovary. It shows rather that the anti-Müllerian hormone secretion actually decreases during development.     

The blood pressure decrease-induced sympathetic discharge following atrial natriuretic factor administration may offset its natriuretic action

Conscious SHR and WKY rats were infused during 7 days with ANF (Arg 101-Tyr 126), 100 ng/hr/rat, by means of miniosmotic pumps and their basal blood pressure (BP), changes in sodium excretion and urinary catecholamines compared with those at the last day of the infusion. The SHR initial BP of 181 +/- 3 mmHg gradually declined to 137 +/- 5 mmHg. No significant change in blood pressure was observed in the ANF-infused WKY group. However, WKY rats exhibited an increased sodium excretion and urinary dopamine/norepinephrine ratio when compared to sham-infused rats. No such differences were observed in SHR. It is suggested that an ANF-induced withdrawal of the renal sympathetic tone permits the manifestation of its natriuretic action in WKY rats. When, however, a BP decrease predominates, as in SHR, this decrease results in a reflex sympathetic discharge with a renal sympathetic activity over-riding the ANF induced natriuresis seen in WKY rats. Secondary sympathetic responses to the ANF-induced BP decrease have to be thus taken into account when a dissociation between the hypotensive and natriuretic action of ANF is observed in vivo.     

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens,not achievable with untransformed protoplasts

Summary Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens shooter strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of leaf disc and stem segment cloning and co-cultivation experiments varied from 5×10^–3 to 5×10^–5. After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular shooter mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.Abbreviations B5-h Gamborg medium without hormones (Gamborg 1968) - V47 protoplast medium (Binding 1974) - D2a protoplast medium (Li et al. 1980) - MS-h Murashige and Skoog medium without hormones (Murashige and Skoog 1962) Dedicated to Professor Dr. G. Melchers in occasion of his 80th birthday     

In vitro response of leaf tissues from Lolium multiflorum — a comparison with leaf segment position,leaf age and in vivo mitotic activity

Summary Immature gramineous leaves provide a convenient system for comparing the response of cells in culture with their state of differentiation. Callusing frequency is compared with leaf segment position, leaf age and in vivo mitotic activity in Lolium multiflorum. (1) In a succession of one millimeter sections from the immature leaf base, callus was formed from the first and second sections but not the third or subsequent sections. The frequency of those explants callusing decreased with distance from the base of the leaf and with leaf age (or leaf extension growth). (2) In vivo, the proportion of cells in mitosis declined from around 10–14% at the base of young leaves to zero at 5 mm from the base and beyond. Mitotic activity also declined in leaves as they aged, and dividing cells were not observed in leaves 30 days from initiation or older. (3) A high frequency of callus formation was associated with a high mitotic index in the explant. But for corresponding mitotic indices, cells further away from the leaf base were less responsive in culture. (4) It is proposed that cells are becoming differentiated even in highly meristematically active regions of the leaf and concomitantly losing their ability to respond in culture.     

Effect of the segregation of neutral red, acridine orange and ammonium chloride by L cells (subline LSM) on lysosomal hydrolase activity

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.     

Mouse splenocyte sensitization to normal tissue antigens and natural killer activity. III. The effect of partial hepatectomy

Partial hepatectomy leads to both increasing of natural cell-mediated activity and sensibilization level (SL) of splenocytes of hepatectomized mice towards antigens of the syngeneic liver. The wave-like variability of SL was shown with sharp increase at 3, 6 and 9 days after operation. Natural killer activity was elevated on the 2nd and 10th days with a significant decrease on the 3-4th days after operation. It is assumed that the variability in the functional activity of splenocytes under study may characterized splenocytes of different populations.     

Specific antigen of granular cells of the human endometrium

Rabbits were immunized by homogenates of endometrium obtained from women during 10-12 weeks of gestation. A specific antiserum was obtained by absorption of the crude antiserum by blood cells and plasma proteins of men with different kinds of ABO and Rh antigens, till disappearance of positive reaction with men's serum protein in the Ouchterlony test. Such an adsorbed specific antiserum continued to react with the sera of pregnant women. Two antigens, numbers 1 and 2, respectively, were determined by the Ouchterlony test. Another group of rabbits was immunized by antigens detected in the precipitation test. A monospecific antidecidual antiserum (ADS 1092) was obtained against number 2 antigens. This antiserum revealed only one antigen in sera of women with gestation and did not react with sera of non-pregnant women. In the slides of endometrium of pregnant women of 10-12 weeks of gestation ADS 1092 had a strong positive reactive with the cytoplasm of one type of endometrium cells. The immunomorphological analysis by the non-direct Coons test and the PAP-test permits to identify cells with the positive reaction as granular cells. It is concluded that the granular cells may be a source of one of the serum antigens detected in women with gestation.     

Indomethacin inhibits thrombin-, but not thyroxin-stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum. Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM-10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10 uM indomethacin.