On the mechanisms of ATP-induced and succinate-induced redistribution of cations in isolated rat liver cells

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.     

Autoradiographische Untersuchungen über das Wachstum des Reissnerschen Fadens von Cyprinus carpio (L.)

Summary The incorporation of³⁵S-l-cysteine into the hypothalamic-hypophyseal neurosecretory system was studied in normal male albino rats and in similar rats which had received thiouracil or excess thyroxine during 18 days. The study was performed by autoradiography. Labelled cysteine was administered by intraperitoneal injections. The animals were sacrificed 30 min, 3 and 24 hours after injection. From the autoradiographs the grain counts were determined in the supraoptic nucleus, paraventricular nucleus, median eminence and infundibular process. The thyroxine-treated rats in the 30 minute group displayed a statistically almost significantly higher grain count in the supraoptic nucleus than the corresponding controls; the same was observed in the 3 hour group in the median eminence and infundibular process. In the 24 hour group, however, the grain count was clearly lower at all sites examined than in the controls. With the rats that had been under thiouracil treatment statistically significant differences were only established in the 24 hour group. Their grain count was higher at all sites than that of the corresponding controls. From the results the inference is drawn that thyroxine accelerates the protein synthesis in the ganglion cells of the supraoptic nucleus and accelerates the flow of NSM to the infundibular process and its release there into the blood flow. Thiouracil, again, retards the flow and the release.This work was supported by a grant from the Sigrid Jusélius Foundation, Helsinki.     

Dimerization of human 5-lipoxygenase

Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines.     

On the Blood-Brain Barrier to Peptides: [3H]βCasomorphin-5 Uptake by Eighteen Brain Regions In Vivo

After intracarotid injection of [3H]beta-casomorphin-5 (beta CM5) in rats, the accumulation of radioactivity was determined in 18 brain regions and the anterior pituitary. The relative accumulation in all regions significantly exceeded that of [3H]inulin by a factor of 2.5, indicating a low but measurable brain uptake of the peptide. In blood-brain barrier-free areas, the accumulation of radioactivity was 15-fold higher than in blood-brain barrier-protected areas. The relative accumulation was not dependent on the total beta CM5 concentration in the range of 0.3-1.1 microM, and was not depressed by 400 microM L-tyrosine. We conclude that beta CM5, like other peptides, is accumulated in the blood-brain barrier-free areas to a relatively high but differing degree, whereas in the areas with a tight endothelium the accumulation is relatively low and nearly uniform. A binding to endothelial cells may contribute to the low accumulation of beta CM5, especially in blood-brain barrier-protected areas.     

"I" becomes "L": modification of vertical mammaplasty

The problems of the vertical mammaplasty by Lejour (i.e., gathering the skin envelope in one vertical suture, frequent secondary healing problems, and later sagging of the inferior glandular part in the case of large and very large breasts) are well known. A simple modification of the Lejour technique, that is, adding a lateral inframammary scar to shorten the vertical scar length, is presented. The modified L technique was used in 45 patients (90 breasts) between October of 1999 and August of 2001. With an average follow-up of 13 months, the jugular notch-to-nipple distance was 21 cm, the vertical scar length was 8.4 cm, the lateral inframammary scar length was 11 cm, and the average resection weight was 625 g per breast (range, 200 g to 2080 g). Even among patients who had very large glandular bases and resection weights it was possible to achieve a breast base reduction, modeling the glandular corpus to a harmonic, well-projecting, and youthful shape. Slight wound-healing problems with spontaneous cicatrization within 2 weeks occurred in six patients. In two patients who exhibited gigantomastia up to 2080 g per breast, partial mamilla necrosis occurred on one side. Ninety-one percent of the patients reported being "very satisfied" with the outcome, and 9 percent reporting being "satisfied." The authors' modification of the vertical mammaplasty to an L-shaped scar technique enables the surgeon to apply the principles of the Lejour technique for higher resection weights and diminishes wound-healing problems, and it is still a scar-minimizing technique that results in a scar-free cleavage. It is easy to learn and an ideal standard technique for a teaching hospital.     

Saturable Retention of Vasopressin by Hippocampus Vessels In Vivo, Associated with Inhibition of Blood-Brain Transfer of Large Neutral Amino Acids

Vasopressin receptors have been reported in the endothelium of brain capillaries. The function of these receptors is not known. To test the prediction that vasopressin receptors in brain capillary endothelium affect amino acid transport across the blood-brain barrier and to assess the role of vasopressin transport across the cerebral vascular endothelium, we measured (a) the endothelial permeability to the large neutral amino acid leucine in the absence and presence of arginine vasopressin (AVP) and (b) the permeability of the blood-brain barrier to AVP relative to manitol. In brain regions protected by the blood-brain barrier, after circulation for 20 s, coinjection of leucine and AVP intravenously led to a decrease of leucine transport unrelated to changes of blood flow. The decrease was most pronounced in hippocampus (42%) and least pronounced in olfactory bulb and colliculi (17 and 19%, respectively). In the latter regions, the endothelial permeability to AVP did not significantly exceed that of mannitol. In hippocampus and in regions with no blood-brain barrier (pituitary and pineal glands), AVP retention in excess of mannitol retention was blocked by unlabeled AVP. The findings do not contradict the hypothesis of a role for AVP in the regulation of large neutral amino acid transfer into brain tissue.     

Indications for a brain uptake of labelled vasopressin and ocytocin and the problem of the blood-brain barrier.

Fifteen seconds after intracarotid injection of [125J]-lysine vasopressin, [3H]-ocytocin, tritiated water or [3H]-inulin, the distribution of radioactivity in 18 regions of the rat brain and in the anterior pituitary was determined. Comparing the concentration of the different tracers (in % of injected radioactivity per g brain tissue), it is assumed that a small amount of the labelled neurohormones is taken up by the brain, indicating a penetration of the blood-brain barrier and/or an accumulation within the structures of the barrier system.     

Studies on the fate of homologous DNA applied to seedlings of Matthiola incana.

Seedlings of Matthiola incana (crucifer) are able to take up exogenous homologous DNA by the roots. DNA homogenously labelled with [3H]adenine and 5-bromodeoxyuridine is incorporated into the plants in a macromolecular form. Intact donor DNA and a fraction with a buoyant density intermediate between that of the donor and the recipient DNA can be recovered. Analysis of this intermediate fraction by ultrasonication and alkali treatment allows the suggestion that homologous DNA is integrated as a double-stranded DNA which becomes covalently linked to the recipient DNA. Control experiments in which seedlings were incubated in a mixture simulating donor DNA degradation products in the presence and absence of unlabelled competitors suggest that these results are not due to the breakdown of donor DNA and reincorporation of the products during DNA synthesis in the recipient plants. When ultrasonicated or thermally denatured DNA is applied to the plants it may be degraded and reused for recipient DNA synthesis but it is not recovered in a macromolecular form. The possibility that the intermediate DNA fraction arises by bacterial contamination of the plants can be excluded by several arguments. Autoradiographic studies show that at least part of the radioactivity of the donor DNA taken up by the plants is associated with the cell nucleus.