Eukaryotic DNA topoisomerase I reaction is topology dependent.

The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.     

The combined effects of dopamine (DA) and the DA antagonists EGYT-2509, chlorpromazine and haloperidol on the kidney function

In anaesthetized dogs renal function was investigated in four successive 20-min periods in four experimental series. (1) In the first series following the first period (serving as control) 2.5 micrograms/kg/min of dopamine (DA) dissolved in 0.5 ml/min of Ringer's solution was infused into the left renal artery (period 2), than during periods 3 and 4. It was found that first (period 2) and second (period 3) doses of DA induced a significant decrease of about 20-30% in renal vascular resistance, and an increase of about 15-25% in renal blood flow. At the same time, systemic arterial blood pressure fell by 10%. The other investigated parameters of the left kidney (Cinulin, CPAH, sodium, potassium and water excretion) did not differ from the respective parameters of the intact right kidney. (2) In the second experimental series following the first period (prior to period 2) 1.0 mg/kg of the DA antagonist EGYT 2509 was administered intravenously. Prior to the period 3 again 1.0 mg/kg of EGYT 2509 and prior to period 4 2.0 mg/kg of EGYT 2509 was given intravenously. During periods 2 through 4 2.5 micrograms/kg/min of DA was infused into the left renal artery. It could be ascertained that EGYT 2509 abolished the renal effects of DA while not inducing any decrease in arterial blood pressure. (3) In the third experimental series, following the control period, prior to periods 2,3 and 4, 1.0 mg/kg, 1.0 mg/kg and 2.0 mg/kg chlorpromazine respectively, was administered i.v. followed by the infusion of DA into the left renal artery. After the administration of chlorpromazine arterial blood pressure and renal vascular resistance fell concomitantly and DA failed to induce any further changes in these parameters. According to our experiments chlorpromazine abolishes the effect of DA on kidney function. (4) In the fourth series, prior to DA infusion the dogs were given 0.5 mg/kg (period 2) then again 0.5 mg/kg and finally 1.0 mg/kg of haloperidol intravenously. Haloperidol decreased arterial blood pressure as well as renal vascular resistance, thus renal blood flow did not change. Renal blood flow could then be increased by DA infused into the left renal artery. It seems that haloperidol could not abolish the vascular effects of DA in the kidney. Our experiments indicate that substance EGYT 2509 possesses the most marked dopaminergic antagonistic effect, chlorpromazine had also been effective, while haloperidol had proved to be practically ineffective.     

Inhibition of mitochondrial respiration by mepivacaine

In the last years our researches on neurotropic drugs follow our hypothesis that the strong effects on nervous system have always hidden more widespread effects on all tissues and cells. It is often required to employ local anesthetics in practising dentistry and orthodontics, particularly when children have to be treated. We have assayed in vitro one of these dental anesthetics, mepivacaine, on liver rat mitochondria: it depresses the respiration coupled to phosphorylation in mitochondria having a good respiratory control; so respiratory control too is depressed, but P/O ratio is unaffected; also respiration uncoupled by 2.4-dinitrophenol is depressed. Depressing respiration cooperates with anesthesia; unchanging P/O is good for the health of the cells and tissues treated by the mepivacaine.     

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.     

NCI-H716 cells as a model for endocrine differentiation in colorectal cancer

In colonic neoplasms, endocrine differentiation is encountered not only in carcinoid tumors but also in adenocarcinomas, where endocrine cells may represent a distinct line of differentiation in the tumor. The significance of endocrine differentiation in colorectal cancer is not well established, partly because of the paucity of tumor cell lines which can serve as a model for studying endocrine differentiation. In this report we describe the properties of NCI-H716 cells, a cell line derived from a poorly differentiated adenocarcinoma of the caecum, under various in vitro conditions and as xenografts in athymic mice. Phenotypical properties were immunohistochemically assessed using a panel of differentiation related antibodies, and also by Northern blot analysis and by electron microscopy. Receptors for biogenic amines and peptide hormones were analyzed by ligand binding assay. These studies show that:

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara)

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA3 gibberellic acid - NAA -naphthaleneacetic acid - PAS periodic acid Schiff - PCM protoplast culture medium - PCV packed cell volume     

The primary structure and oxygen-binding properties of the single haemoglobin of the high-Antarctic fish Aethotaxis mitopteryx DeWitt

Summary The complete amino acid sequence of the single haemoglobin of the Antarctic fish Aethotaxis mitopteryx DeWitt has been established by automated repetitive Edman degradation on the intact and cleaved (enzymatically and chemically) and chains. A very high sequence identity with other Antarctic fish haemoglobins has been detected. The haemoglobin has a moderate Bohr effect and no Root effect. Organic phosphates and chloride also regulate oxygen binding only to a moderate extent. The lack of Root effect is consistent with the substitution His — Val at the HC3 C-terminal position of the chain. The low overall heat of oxygenation suggests that in this species oxygen transport is an energy-saving process, presumably related to cold adaptation. The comparative analysis of the haemoglobins of Antarctic fishes emphasises some unique features of the oxygen-transport system of A. mitopteryx, which are likely to be related to its also rather unique mode of life.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Role of vasopressin in the modifications of renal function during aging]

In the course of aging, the renal concentrating ability is markedly reduced. This defect may result from an inappropriate synthesis of antidiuretic hormone in the central nervous system or may be due to an impaired renal response to vasopressin. The two hypotheses have been studied in vivo in rats and in vitro in mice. The results of these studies indicated that: 1) dehydration induces a comparable release of vasopressin along the hypothalamo-hypophysial axis in 10, 20 and 30 month-old rats; 2) there is no change with age of the number of nephrons, single nephron filtration rate or transport capacity of Henle's loop of cortical nephrons which could account for an impaired renal response to vasopressin; 3) the reduced concentrating ability of the kidney appears to be linked to a decreased response of the medullary thick ascending limb of Henle's loop which in part is responsible for the cortico-papillary gradient of solutes within the kidney.     

Effects of order of presentation of exercise intensities and of sauna baths on perceived exertion during treadmill running

Thirteen male subjects performed a running test on the treadmill consisting of four standard exercise intensities [65%, 75%, 85%, 95% maximal O2 uptake (VO2 max)] presented in ascending, descending or random order. At the end of each exercise intensity, O2 consumption, heart rate (fc), venous blood lactate concentration [( Ia]b) and perceived exertion were assessed. This last variable was determined according to the Borg nonlinear CR-20 scale. The same variables were also determined during exercise at a standard intensity (65% or 95% VO2 max) performed before and after a Finnish sauna bath. Ratings of perceived exertion showed a good test-retest reliability (r = 0.77); they were the same when the exercise intensity was expressed in relative (%VO2 max) or absolute (speed) terms, and were independent of the order of presentation of the exercise. The latter had no effect on fc either but it did, however, influence [Ia]b, which was significantly higher in the descending, as compared to the ascending or random modes of presentation. The sauna bath increased fc at a given exercise intensity, but left perceived exertion and [Ia]b unchanged. It was concluded that at least under the present experimental conditions, fc and venous [Ia]b do not play a major role as determinants of perceived exertion.     

Observations on egg production rates and seasonal changes in the internal morphology of Mediterranean populations of Acartia clausi and Centropages typicus

Egg production rates in wild populations of Acartia clausi and Centropages typicus, sampled biweekly in the Gulf of Naples from October 1985 to July 1987, showed marked seasonal fluctuations with maximum values in early spring that proceeded the annual maxima for adult female densities in summer. A positive correlation between chlorophyll a concentrations and egg production was evident only during the early spring phytoplankton bloom. A strong diminution in egg deposition occurred later in spring and continued throughout the summer notwithstanding high chlorophyll concentrations. In winter, when population abundances for adult females were lowest, egg production rates were always higher than in summer. Differences in egg production rates coincided with pronounced morphological changes between summer and winter populations of both species. The most striking of these changes consisted, in winter, in the presence of a dark brown fluid-like mass of granular material that seemed to freely bathe the gonads. The presence of this substance only during periods of elevated egg production suggests that it may enhance egg production rates when the adult population reaches minimum annual levels. Such a mechanism of self-regulation may operate to dampen the effects of environmental variability thereby contributing to maintain a conservative structure in coastal copepod communities.