Subcutaneous fat necrosis of the newborn: CT findings of head and neck involvement.

We present the CT findings in two newborns with subcutaneous fat necrosis. This is an uncommon disease that occurs in neonates who have had difficult deliveries. The CT findings varied from discrete subcutaneous nodules to a diffuse subcutaneous fullness. Although the subcutaneous disease tends to spontaneously resolve within weeks to months, an associated hypercalcemia may have a fatal outcome.

Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system.

The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice. The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5. Aspergillus niger and Byssochlamys fulva were kept on malt extract agar. Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80 and checked microscopically. The antifungal activity of the combined enzyme system was tested with initial counts of approx. 10(5) cfu.ml-1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg.l-1 thiocyanate and 20 g.l-1 glucose or in apple juice supplemented with the same amount of thiocyanate. The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 degrees C under aerobic conditions. Inactivation was achieved for all test organisms in both media. The yeast strains were found to be least stable while B. fulva was most resistant. A combination of 5 U.ml-1 LPO with 0.5 to 1 U.ml-1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h. The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced. In this medium, B. fulva was inactivated by 20 U.ml-1 LPD and 1 U.ml-1 GOD within 4 h. R. rubra and S. cerevisfiae were unable to survive in apple juice at 5 U.ml-1 LPO combined with 1 U.ml-1 GOD. For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M. rouxii and R. rubra have been affected within the concentration range tested (maximum 3 U.ml-1).

Muscarinic acetylcholine and histamine-receptor mediated calcium mobilization and cell-growth in human ovarian-cancer cells.

The effects of carbachol and histamine on changes in cytosolic-free calcium ([Ca2+]i) and cell proliferation have been characterized in human ovarian cancer cells (OVCAR-3) and non-tumourigenic Chinese hamster ovary cells (CHO). The muscarinic agonist carbachol increased [Ca2+]i significantly with a rapid biphasic response due to both influx of extracellular calcium and release of calcium from intracellular stores. None of these effects were however seen in CHO cells. The increase in cellular calcium by carbachol was also confirmed by calcium uptake experiments using Ca-45. Carbachol increased Ca-45 uptake by 25% in OVCAR-3 cells but had no effect in CHO cells. Histamine also stimulated calcium mobilization in OVCAR-3 cells but had no effect in CHO cells. The response to histamine was also biphasic although the calcium increase was smaller than with carbachol. Data obtained with selective histamine antagonists showed that the response to histamine was mediated by H-1 histaminergic receptors. Both carbachol and histamine also stimulated cell growth of OVCAR-3 cells but were without effect on CHO cells. The cell proliferating effect of carbachol and of histamine on OVCAR-3 cells as well as the increase in [Ca2+], was totally blocked by atropine and selective H-1 histaminergic receptor antagonist pyrilamine, respectively. Fetal calf serum (FCS) which increased [Ca2+]i in both cell lines also caused a substantial increase in cell growth in the two cell lines. Verapamil partially and TMB-8 totally blocked carbachol stimulated release of calcium from intracellular stores, whereas prenylamine had only a minor inhibitory effect on calcium influx. The effect of verapamil and TMB-8 were most likely resulted from their inhibition of cholinergic receptors rather than a direct inhibition of intracellular calcium release. The carbachol induced effects on calcium transients were also partially inhibited by pertussis toxin and the phorbol ester PMA. Our data suggest that the mitogenic action of carbachol occurs through an increase in [Ca2+]i which promote DNA synthesis and cell growth. These data also indicate the involvement of both a pertussis toxin sensitive and insensitive G-protein as well as protein kinase C in the signal transduction pathway induced by carbachol.

Release of intracellular calcium by prenylamine in human ovarian tumour cells.

The effects of the calcium antagonist prenylamine on intracellular calcium concentration were studied in a human ovarian carcinoma cell line, OVCAR-3. Exposure of cells to 100 microM prenylamine resulted in nearly a 10-fold increase in cytosolic free calcium concentration ([Ca2+]i) as measured by Fura-2 fluorescence. In calcium-free medium, although the increase in [Ca2+]i caused by prenylamine was smaller, it was still substantial compared with the basal level. Efflux experiments with 45Ca showed that 100 microM prenylamine increased calcium efflux by 70% compared with control, indicating active extrusion of the elevated [Ca2+]i. The sluggish nature of calcium release and its independence from the pool activated by ionomycin suggest that the calcium was probably not released from endoplasmic reticulum. These results, although paradoxical, provide a new insight into the possible mechanism of action of prenylamine in causing cancer cell death.

Calcium mobilization and cell proliferation activated by extracellular ATP in human ovarian tumour cells.

The effects of ATP on cell proliferation and intracellular calcium concentration ([Ca2+]i) were examined in a human ovarian cancer cell line (OVCAR-3). Micromolar concentrations of ATP promoted a biphasic rise in [Ca2+]i representing a phase with a rapid peak followed by a phase in which the rise was slower and sustained. When the influx of extracellular calcium was blocked by calcium chelation to EGTA, the ATP stimulated rise in [Ca2+]i was rapid and monophasic. Voltage-sensitive calcium channel blockers like nifedipine and verapamil had no effect on the action of ATP while prenylamine totally blocked calcium influx. ATP inclusion in the medium significantly stimulated growth of OVCAR-3 cells. Fetal calf serum (FCS) increased [Ca2+]i with similar biphasic kinetics representing both the entry of extracellular calcium and release of calcium from intracellular stores. FCS also caused a substantial increase in cell growth. From these experiments it was concluded that an increase in [Ca2+]i is obligatory for stimulation of cell growth in OVCAR-3 cells and that this increase probably requires a contribution from the entry of extracellular calcium. The involvement of both pertussis toxin sensitive G-protein and protein kinase C in ATP induced responses was indicated by the data showing interference of the response by pertussis toxin and phorbol-myristate acetate.

Characterisation of muscarinic cholinergic receptors in human ovaries, ovarian tumours and tumour cell lines.

Using [3H]quinuclidinyl benzilate (QNB) as radioligand, muscarinic cholinergic receptor sites in isolated plasma membrane fractions from human ovarian tumours, cultured tumour cells, and normal ovarian tissue were characterised. QNB binding to all preparations, except from poorly differentiated tumour, was specific, saturable, and of high affinity. In contrast to normal ovaries, benign tumours, well differentiated adenocarcinoma and OVCAR-3 cells, the poorly differentiated adenocarcinoma and SKOV-3 cells completely lacked specific QNB binding. The muscarinic receptor densities and the Kd values in preparation from ovaries, receptor-positive tumours and OVCAR-3 cells were similar. QNB binding was strongly inhibited by the classical muscarinic receptor antagonist atropine, but poorly by the agonist carbachol. In contrast to atropine, inhibition by pirenzepine and AF-DX 116 was relatively low. These data suggest that muscarinic receptors in ovaries and ovarian tumours are of m3 type.

Effect of calcium and calcium antagonists on 45Ca influx and cellular growth of human prostatic tumor cells.

Calcium and calmodulin play significant roles in DNA synthesis and cell proliferation. In this work the effects of verapamil, trifluoperazine, and tamoxifen on 45Ca uptake and cell growth in human prostatic tumor cells (DU 145) and human fibroblast cells (1 BR) were studied. Although the maximum proliferation was achieved at a concentration of around 2 mM CaCl2 in both DU 145 and 1 BR, growth of DU 145 cells was considerably greater than 1 BR at all calcium concentrations (0.1-4 mM). Calcium uptake experiments, using 45Ca, revealed that the unstimulated 45Ca uptake in 1 BR fibroblasts was 4-5 times higher than in DU 145 cancer cells. Depolarization with high extracellular K caused a 2-3-fold increase in 45Ca influx in 1 BR but only 25-55% increase in DU 145 cells. Verapamil caused a significant inhibition of cell growth with an IC50 value of 55 microM. Verapamil paradoxically increased 45Ca uptake in both unstimulated and K-stimulated DU 145 cells. Whereas unstimulated 45Ca uptake could be blocked by very low concentrations of lathanum (10 microM), much higher concentrations (1-10 mM) were required to completely block uptake in K-depolarized cells. Both trifluoperazine and tamoxifen also inhibited cell proliferation with an IC50 concentration of approximately 5 microM. These drugs, had, however, no effect on 45Ca uptake either in unstimulated or depolarized cells. The results suggest that voltage-gated calcium channels exist in both DU 145 cancer cells and fibroblasts. However, verapamil, in contrast to 1 BR, failed to block these channels in DU 145 cells. The mechanism of antiproliferative action of verapamil may be related to the observed, although paradoxical, increase in cellular calcium. The effect of trifluoperazine and tamoxifen does not involve changes in transmembrane calcium movements but could be mediated by their inhibition of calmodulin-mediated reactions within the cell.

The effect of magnesium on calcium uptake and contractility in the human myometrium.

High concentrations of magnesium (12 and 24 mM) in the extracellular medium markedly inhibited both spontaneous activity and K+-induced contracture in strips of nonpregnant human myometrium. Net calcium influx measured by the uptake of 45Ca2+ in the myometrium was considerably decreased by high concentrations of magnesium. This was true for both resting (unstimulated) and K+-stimulated uptake of 45Ca2. While calcium uptake in K+-stimulated tissues exposed to 24 mM magnesium was significantly lower than in those exposed to 12 mM, no difference was found in the unstimulated tissues. These data indicate that the tocolytic action of magnesium most probably results from the inhibition of calcium entry into myometrial cells.

Characterization of membrane calcium channels in nonpregnant and pregnant human uterus.

The binding properties of the calcium channel inhibitor [3H]-nitrendipine to membrane fragments prepared from nonpregnant and pregnant human myometrium were investigated. Specific binding sites with high affinity and low capacity were identified in both pregnant and nonpregnant myometrium. Whereas there was no significant difference in the concentration of binding sites in the two types of myometria, the Kd value for binding in pregnant myometrium was lower than that in nonpregnant myometrium (p less than 0.05). Although dihydropyridines effectively competed for [3H]-nitrendipine-binding sites, verapamil was a poor competitor. This was true for both the pregnant and nonpregnant myometrium. The inhibition of nitrendipine binding by dihydropyridines and also verapamil was significantly greater in pregnant than in nonpregnant myometrium. These data suggest that dihydropyridine-type calcium channel inhibitors might provide a useful alternative for treating premature labor.

Possible reflection of intracellular calcium binding in the divergent pattern of relaxation in rat and rabbit uterus.

The mechanical responses to potassium of the rabbit and rat uterus were different. The force of a potassium-induced contracture showed a rapid decline in the rabbit uterus, whereas it was fully maintained for a long period in the rat uterus. The increase in 45Ca influx following exposure to high KCl was substantial and similar (0.3 mmol kg-1) in both rat and rabbit uterus. Calcium uptake in post nuclear supernatant (PNS) from the rat myometrium was significantly lower than that from the rabbit myometrium in both the rate and capacity of uptake. This uptake was reduced to about 10% in the presence of sodium azide in PNS from both rat and rabbit myometria. Both the rate and capacity of calcium uptake by mitochondria isolated from PNS of rat myometrium were considerably lower than that by rabbit myometrial mitochondria. Microsomes isolated from rat and rabbit PNS had very low capacity for calcium uptake compared with mitochondria. No difference was found in the rate or the capacity of uptake between the rat and the rabbit myometrial microsomes. The data support the argument for a role of mitochondria in myometrial relaxation and further provide evidence for a considerable species difference in calcium uptake by myometrial mitochondria.