Immunocytochemical demonstration of calmodulin in cells secreting by exocytosis

Calmodulin is a regulator of several calcium-dependent cellular processes. It has been suggested that it plays a role in the mechanism of secretion. Employing an indirect immunoperoxidase technique at the light microscope level, this study demonstrates the presence of calmodulin in several exocytotic cells (mast cells, thyroid follicular cells, neurohypophyseal neurosecretory terminals, pancreatic beta-cells and pancreatic acinus cells) in rat and man. The positive staining reaction for calmodulin was granular and at least in the case of rat mast cells it appeared to be associated with the granule membrane.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

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.     

Smallest zinc quantities affect the histamine release from peritoneal mast cells of the rat

Summary Seven individual 0.025-mg doses of zinc administered as lactose tablets on consecutive days, significantly increase histamine release from peritoneal mast cells of the rat. Seven individual doses of 0.25 g cause a somewhat smaller, though still very pronounced increase in the release in comparison with zero control.Acknowledgments. We thank Ms T. L. Pham and Ms K. Löppen for their technical assistance.     

Zur Umwandlung von β-Pyridylcarbinol in Nicotinsäure im tierischen Organismus

Summary By way of the perfusion technique, it is shown that the liver of the rat readily converts-pyridyl-carbinol to nicotinic acid. The turnover attains a rate of 0.5M/g/min.     

Expression of the mutant gene for L-gulono-γ-lactone oxidase in scurvy-prone rats

A mutant strain of Wistar rats with L-gulono--lactone oxidase deficiency has recently been established. To investigate this deficiency by DNA and RNA blot hybridization analyses, a fragment of a previously cloned cDNA encoding rat L-gulono--lactone oxidase was used as a probe. When genomic DNA of the mutant rat was digested with several restriction enzymes, the probe hybridized to fragments of the same sizes as those produced from DNA of normal rats. Poly(A)⁺RNA from the liver of the mutant rat was found to contain an L-gulono--lactone oxidase-specific mRNA of a normal size at a comparable level to that of normal rats. An in vitro translation experiment revealed that the mRNA programmed the synthesis of an enzyme protein which had the same molecular weight as that of the translational product of the normal mRNA, although the amount synthesized was markedly reduced as compared with that synthesized with the normal mRNA. In accordance with this observation, a very low but definite degree of L-gulono--lactone oxidase activity was detected in the microsomes of the mutant rat by a newly developed, highly sensitive method.Acknowledgments. The authors thank Dr Susumu Makino, Shionogi Research Laboratories, Shionogi & Co., Ltd, Japan, for his kind donation of normal (ODS- +/+) and ODS (ODS-od/od) rats. This work was supported in part by Grant-in-Aid (59570103) for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Recent evolutionary origin within the primate lineage of two pseudogenes with similarity to members of the transforming growth factor-β superfamily

Using a search engine called Motifer, we searched the public database of the human genome for genes matching a consensus pattern of cysteine residues derived from members of the transforming growth factor-beta (TGF-) superfamily. We identified two genes (named MDF451 and MDF628) that display sequence similarity to members of the TGF- superfamily in the arrangement of six conserved cysteine residues. Phylogenetic analyses revealed that MDF451 and MDF628 constitute a distinct subgroup within the TGF- superfamily, distantly related to the GDNF subfamily of ligands. Both genes could be identified in several primate species in addition to human, including chimpanzee, gorilla, guereza, and green and gray monkey, but not in rodents or other non-primate mammals, and appear not to be present in the genomes of mouse, rat or zebrafish. RNAs for MDF451 and MDF628 were expressed at low levels within distinct regions of the human central nervous system, including adult cerebellum, adult spinal cord and fetal brain. Despite expression at the RNA level, both genes presented a transcribed upstream stop codon that would prevent translation of the TGF--like reading frame. The coding potential of alternative reading frames was not immediately apparent. The two genes may represent TGF--like pseudogenes that have recently appeared in evolution in a common ancestor of the primate lineage by duplication from a GDNF/TGF--like ancestral gene.Received 9 October 2003; received after revision 13 November 2003; accepted 2 December 2003     

Bovine parathyroid catecholamines: A chemical and histochemical study

Summary Bovine parathyroid glands contain large amounts of dopamine (3.4–13.9 pg/g), but very little norepinephrine. Fluorescent histochemistry demonstrates only rare adrenergic nerve terminals on vasculature. Single dopamine-containing cells, most likely mast cells, are scattered in large numbers throughout the connective tissue stroma.     

DDT: The degradation of ring-labeled14C-DDT to14CO2 in the rat

Summary Ring fission ofp, p-DDT was studied in the rat following a single oral dose of 0.74 mg/kg (1.04 Ci) of uniformly ring-labeled¹⁴C-DDT. Expired air was passed through a solution of ethanolamine-ethylene glycol monomethyl ether (12) to trap¹⁴CO₂. A total of 1.6% of the radioactivity administered was recovered in the expired air collected continually for 10 days, indicating that while degradation of the phenyl moiety is not a major route pfp, p-DDT metabolism in the rat, it is equal to the urinary excretion. Nevertheless, these results represent the most radical change accomplished in vivo of a residual insecticide yet reported in mammals.Acknowledgment. We acknowledge the secretarial work of Ms.Elaine Smolko. This study was supported by NIH fellowship No. 1 F22 ES01723-01 and No. HL16264.     

The influence of peripheral or central administration of ondansetron on stress-induced gastric ulceration in rats

Ondansetron (0.08, 0.15 or 0.3 mg/kg) injected s.c., every 12h with the fourth dose given 0.5 h before experiments, dose-dependently lessened gastric glandular mucosal ulceration produced by cold-restraint stress for 2h. When given intracerebrally (i.c.) (0.1, 0.5 or 1g), using the same treatment regimen, infusion of ondansetron 1 g into the nucleus amygdaloideus centralis decreased stress-evoked ulcers; in contrast, injection of the same dose into the nucleus accumbens intensified these lesions. The associated stress-induced stomach wall mast cell degranulation was unaffected by all s.c. or i.c. doses of ondansetron. Pretreatment with disodium cromoglycate i.p. alone, or concurrently with ondansetron s.c., prevented not only ulceration but also mast cell degranulation. 5-Hydroxytryptamine₃ receptor antagonism appears to inhibit stress-evoked ulcers mainly by blocking the peripheral effects of the amine after its release from the gastric mucosal mast cells.     

Thrombospondin co-localises with TGFβ and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17β estradiol

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor-I (TGF), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10^–10 M–10^–11 M of added estradiol. Treatment with 17 estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF and IGF-I, in terms of their distribution and regulation by 17 estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.     

Unterschiedliche Empfindlichkeit verschiedener Angiotensinderivate gegen Abbau durch Plasma oder Nierenhomogenat

Summary -Asp¹-Angiotensin II had a more pronounced action on the blood pressure of nephrectomized rats than the corresponding-compound.-Asp¹-Angiotensins were more slowly destroyed by rat serum, rat kidney homogenate, and human plasma than-compounds, especially-Asp¹-Angiotensin II amide.     

Oxidative stress in the moderately halophilic bacteriumDeleya halophila: Effect of NaCl concentration

The sensitivity ofDeleya halophila to oxidative stress caused by hydrogen peroxide (H₂O₂) was found to vary, depending on the NaCl concentration of the growth medium. Pretreatment of the bacteria at a low concentration of H₂O₂ (50 M) protected the cells against the lethal effects of higher levels (1–2 mM) of H₂O₂. Exposure ofD. halophila cells to 50 M H₂O₂ resulted in the induction of several proteins (hydrogen peroxide-inducible proteins, hips). However, the kinetics of induction, the extent of induction and the number of hips appear to be influenced by the salt concentration of the growth medium. Five of the hips exhibited apparent molecular masses identical to those of five heat shock proteins (hsps).     

H-Y antigen expression in heterogametic males (XY) and females (ZW): a factor in reproductive strategy?

Summary The cells of heterogametic females with ZW sex chromosomes express H-Y or H-W antigen. A hypothesis is formulated to explain why these animals are capable of practicingamphigonia retardata, i.e. delay in actual fertilization of eggs by retaining viable sperm within the oviduct for a considerable time (several months).     

Evidence for a role of IFN γ in control ofListeria monocytogenes in T cell deficient mice

Summary The role of interferon (IFN) in controlling chronic infections ofListeria monocytogenes (Listeria) was studied in athymic C57BL/6nu/nu mice, and by treating thymectomized C57BL/6 +/+ mice with monoclonal rat CD4 and CD8-specific monoclonal antibodies (Mab). Mice treated with a combination of the two T cell subset antibodies were similar to athymic, nude mice in being able to contol Listeria infection, keeping the titers below 3–5 log₁₀ bacteria per organ, but they could not eliminate them completely. Treatment with antibodies to IFN of nude or CD4⁺ + CD8⁺-T cell-depleted mice suffering from chronic Listeria infection caused a marked increase of Listeria titers, in liver and spleen. This result implies a role of IFN in maintaining anti-Listeria resistance in mice lacking mature T cells.     

Chromatography of mast cell sensitizing antibodies in guinea-pigs

Résumé L'inoculation intraveineuse d'albumine humaine (HA), provoque chez le cobaye l'apparition d'un anticorps (-1) sensibilisant les cellules mast (CM) homologues. L'inoculation dans le coussinet plantaire (HA et adjuvant de Freund complet) fait apparaître deux anticorps: l'un (-2) sensibilisant les CM hétérologues, l'autre (dans le-1 ainsi que dans le-2) sensibilisant les CM homologues.

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We thank Mrs.Nava Raz, Mrs.Tamar Minai and Mr.I. Ofek for technical assistance.     

Extensives Grössenwachstum larvaler Speicheldrüsenchromosomen vonDrosophila melanogaster im Adultmilieu

Summary Larval salivary glands of the early third instar have been transplanted into the abdomen of adult females. Under such conditions, the giant chromosomes reach not only the normal and maximal polytaenic size but in several cases, and after prolonged culturein vivo, cells are found which contain supergiant chromosomes.     

Inorganic phosphate promotes relaxation of chemically skinned smooth muscle of guinea-pigTaenia coli

Summary In the presence of calmodulin and phosphate and an ATP-regenerating system, Triton-treated skinned fibers of theTaenia coli could be made to contract and relax by step changes of Ca⁺⁺ within about 30 sec. In the absence of phosphate, relaxation was slower, and during this slow relaxation tension was not maintained actively. The passive tension could be abolished by phosphate (3–6 mM). Phosphate had little effect on contractile tension but decreased the speed of contraction.Supported by the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Miss Claudia Zeugner is acknowledged.     

Isoenzymes de la lactate deshydrogenase au niveau des organes de l'appareil génital du rat mâle greffé avec une tumeur de la glande interstitielle du testicule

Summary The LDH isoenzyme pattern was determined at the level of the genital apparatus organs in the male rat grafted with a testicular interstitial gland tumor. The seminal vesicles exhibit a modified pattern in the grafted animals after 2 months in comparison with the non-grafted rats. The pattern of the tumoral extract, in fact a true leydig cells extract, does not exhibit the specific testicular band-X.

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Isoenzymes de la lactate deshydrogenase au niveau des organes de l'appareil génital du rat mâle greffé avec une tumeur de la glande interstitielle du testicule

     

Migration of lymphoid cells to the bone marrow of rat following eradication of cells in DNA synthesis and in mitosis

Summary Eradication of replicating bone marrow cells of rat by means of combined administration of single doses of hydroxyurea and vinblastin is followed within 9–10 h by an inflow of lymphoid cells of extramedullary origin in the range of 13,200,000/femur. The rat bone marrow with a high content of lymphoid cells was previously shown to be concentrated in stem cells. The factor(s) which convey the information of decrease of replicating marrow cells to extramedullary sites is at present unknown.Acknowledgment. This study was supported by a grant from the Chief Scientist Office, Ministry of Health, Israel.Hydroxyurea for this investigation was given as a gift by the Squibb Institute of Medical Research, Princeton, N.J. USA, to which the authors are indebted.