Expression of high- and low-affinity receptors for C3a on the human mast cell line,HMC-1

The proteolytic cleavage product of complement component 3, (C3a), like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC-1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC-1 cells. EGTA did not diminish this response, indicating that mobilizable Ca²⁺ was from intracellular stores. Receptors for C3a in HMC-1 cells couple in part to Bordetella pertussis toxin-sensitive G-proteins and, therefore, appear to belong to the family of serpentine receptors that require G-proteins for signal transduction. HMC-1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind ¹²⁵I-C3a with high-(K_d1 = 2.1–4.8 nM) or low-affinity (K_d2 = 30–150 nM), and both receptors are expressed at high level: 3 × 10⁵–6 × 10⁵ C3aR1/cell and 5 × 10⁵–2.3 × 10⁶ C3aR2/cell. Results from cross-linking experiments with ¹²⁵I-C3a fully agree with the presence of two different classes of C3a receptors in HMC-1 cells. Two membrane proteins with apparent molecular masses of 54–61 kDa (p57) and 86–107 kDa (p97) could be covalently modified with ¹²⁵I-C3a, and this cross-linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL-8, NAP-2, GROα, ENA-78, IP10, PF4, MCP-1, 2 and 3, RANTES, MIP-1α, MIP-1β and 1309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC-1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC-1 cell line an excellent choice for the cloning of the receptor genes.     

Serotoninbestimmungen an Thrombocyten und an Fraktionen von Thrombocyten

Zusammenfassung Aus Suspensionen zerstörter Thrombocyten des Menschen wurden reine Fraktionen von Hyalomer und Granulomer hergestellt. Von den Fraktionen wurde jeweils ein Teil für die elektronenmikroskopische Untersuchung, ein anderer Teil für die Bestimmung des Serotonins verwandt. Durch die elektronenmikroskopische Untersuchung jeder einzelnen Fraktion wurde die Reinheit der Fraktionen überprüft. Die Bestimmung des Serotoningehaltes erfolgte am isolierten Rattenmagen. In Kontrollversuchen wurde ferner der Serotoningehalt intakter Thrombocyten bestimmt.Intakte Thrombocyten besitzen im Mittel einen Serotonin-Gehalt von 52 ng/10⁸ Thrombocyten. In den Fraktionierungsversuchen fanden wir 95% des Serotonins in den Hyalomer-Fraktionen und nur 5% des Serotonins in den Granulomer-Fraktionen. In vivo findet sich also der hohe Serotoningehalt fast ausschließlich im Hyalomer der Thrombocyten.

---

Summary Pure fractions of hyalomer and granulomer were prepared from suspensions of destroyed human thrombocytes. One portion of the fractions was used for the electron microscopic studies, and the other part for the determination of serotonin (5-hydroxytryptamine). The electron microscopic investigation allowed to check the purity of each single fraction. On the isolated rat stomach the determination of the concentration of serotonin was performed. In control preparations we studied the serotonin concentration of intact thrombocytes. These thrombocytes have a mean serotonin concentration of 52 ng/10⁸ platelets. In fractioning experiments we found 95% of the serotonin in the hyalomer fractions and only 5% of the serotonin in the granulomer fractions. In vivo therefore, the high concentration of the serotonin is found almost exclusively in the hyalomer of the thrombocytes.

---

---

Die Ergebniss wurden vor der Medizinischen Gesellschaft Düsseldorf am 23. 1. 1963 mitgeteilt. Herrn Prof. Dr.Meessen danken wir für die Anregung und Förderung der Arbeit, Herrn Prof. Dr.Greeff für die Hilfe bei den Serotoninbestimmungen.     

A comparison of the behaviour to ions of the P3 component of the pigeon cone and rat rod electroretinogram

1. Isolated pigeon and rat retinas were incubated in various media and the effect of changing ions on the P III component of the electroretinogram was studied, with particular regard to the influence of calcium on other ions.2. In pigeon, without calcium, reduction of sodium and chloride both reduce P III. This behaviour continues in the presence of calcium but only if the retina has been treated with ouabain. Under these conditions, the effect of calcium is to prevent the appearance of inverted responses.3. Under other conditions, calcium renders the pigeon P III insensitive to change in chloride. Sensitivity is rapidly restored by the use of ouabain, 2,4-DNP, or by cooling.4. Calcium does not alter the linear relationship between sodium and response amplitude.5. In pigeon, increasing potassium increases P III amplitude, though the exact relationship is altered by calcium.6. In the rat retina treated with ouabain, increasing sodium increases P III, and increasing potassium decreases P III. This result is similar to that of Sillman, Ito & Tomita (1969b). If calcium is absent, inverted P IIIs can be obtained.7. If calcium is present in the medium, reducing sodium also reduces rat P III.8. If calcium is reduced below 10(-7)M, then reducing sodium increases P III, and large responses are obtainable from solutions which contain almost no ions. These responses vanish very rapidly if the retina is cooled or treated with ouabain.     

Fine structure of microgametogenesis of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus

Summary The microgamogony of Eimeria ferrisi from experimentally infected mice was investigated with the electron microscope. Microgamonts were recognizable by the presence of peripherally arranged nuclei and the presence of single or paired centrioles between each nucleus and the limiting membrane of the parasite. Often an intranuclear centrocone directed toward the centriole was present. Differentiation of the microgamete began when elevations of the limiting membrane, which indicated the commencement of flagellar development, appeared above the centrioles. This event was accompanied by the segregation of nuclear content into a dense osmiophilic portion and an electron-pale portion. Then followed a gradual protrusion of the dense portion of the nucleus and developing flagella into the parasitophorous vacuole. A dense ring developed at the base of the differentiating microgamete, resulting in the formation of a stalk which was occupied by the residual portion of the nucleus. Fully developed microgametes became detached and occupied the parasitophorous vacuole along with the residual cytoplasm. Microgametes had an anterior perforatorium, a dense elongate nucleus, with an anteriorly positioned mitochondrion in a small groove of the nucleus. Usually two flagella were present but one microgamete appeared to have three. Polysaccharide first appeared when differentiation was in progress and increased until large numbers of granules were present in the microgamont cytoplasm.Abbreviations AM Amylopectin - B Basal Body of Flagellum - CC Centrocone - CE Centriole - DR Dense Ring - ER Endoplasmic Reticulum - F Flagellum - HC Host Cell - HN Host Cell Nucleus - MI Mitochondrion - MN Microneme - MP Micropore - MT Microtubule - N Nucleus - P Perforatorium - PL Osmiophilic Plate - PV Parasitophorous Vacuole - RN Residual Nucleus Supported in part by the Deutsche Forschungsgemeinschaft¹, the Alexander von Humboldt Foundation² and a Faculty Development Grant to Andrews University by the Merck Foundation, Rahway, New Jersey, USA     

Localization of corticotropin-releasing activity in the rat hypothalamus

Hypothalamic nuclei were removed from frozen sections of rat brain and examined for their corticotropin-releasing activity. The highest concentration was measured in the median eminence. In addition there was significantly more activity detected in the nuclei paraventricularis, supraopticus, suprachiasmaticus and arcuatus than in the other nuclei.     

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis.     

Compartmental aspects of Na+ saturation kinetics in prog skina

Epithelial membranes are multicompartment structures of microscopic and submicroscopic dimensions. Therefore, interpretations of kinetic data on solute fluxes, based on the standard three compartment model are open to criticism. We have obtained an integrated view of the kinetics of Na⁺ transport in frog skin epidermis by application of the computer simulation method. Epidermis and whole skin models were designed which resemble photomicrographs of these tissues. Justification is given for the way in which internal and external chamber compartments are connected (topology). The epidermis model has eight passive, and two active transfer sites. Our primary aims were 1) simulation of the transepidermal Na⁺ influx and the concomitant Na⁺ backflux saturation kinetics, and 2) localization of the so-called “outer” and “inner” Na⁺ responsive borders in epidermis. The analysis, based on methodical variations of transfer coefficients, suggests involvement of the “composite desmosomes” and the transepithelial Na⁺-pump leak pathway. These are located in the outer and the inner region of the epidermis, respectively. Reasonable functional agreement between epidermis and model was also seen in 1) Na⁺ saturation kinetics in ouabain, poisoned system, 2) relative independence of the two borders to the “trans” [Na⁺] in the external solutions, and 3) equal energy requirement, for the transmembrane Na⁺ pump, Na⁺/O₂∼-20. This work was supported by NIH Grant GM 03545-23     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.