Detection of nucleic acids in the attomole range using polybiotinylated oligonucleotide probes.

This article describes the optimization of the hybridization signal obtained with biotinylated oligonucleotides. Optimal number and positions of biotin moieties on a 33-base oligonucleotide probe were determined. The quality of avidin-peroxidase conjugate and the choice of chromogenic substrate influenced detection sensitivity. A signal amplification method was also developed for avidin enzymatic conjugates. These improvements allowed the detection of less than 0.02 fmol of target DNA.

Global ischaemia induces a biphasic response of the mitochondrial respiratory chain. Anoxic pre-perfusion protects against ischaemic damage.

Studies of Langendorff-perfused rat hearts have revealed a biphasic response of the mitochondrial respiratory chain to global ischaemia. The initial effect is a 30-40% increase in the rate of glutamate/malate oxidation after 10 min of ischaemia, owing to an increase in the capacity for NADH oxidation. This effect is followed by a progressive decrease in these oxidative activities as the ischaemia is prolonged, apparently owing to damage to Complex I at a site subsequent to the NADH dehydrogenase component. This damage is exacerbated by reperfusion, which causes a further decrease in Complex I activity and also decreases the activities of the other complexes, most notably of Complex III. Perfusion for up to 1 h with anoxic buffer produced only the increase in NADH oxidase activity, and neither anoxia alone, nor anoxia and reperfusion, caused loss of Complex I activity. Perfusing for 3-10 min with anoxic buffer before 1 h of global ischaemia had a significant protective effect against the ischaemia-induced damage to Complex I.

Thyroid hormone receptors in brain and liver during ageing.

The binding properties--binding capacity (MBC) and affinity (Ka)--of T3 nuclear receptors were analyzed in cortex, cerebellum and liver of rats aged 3, 6, 12 and 24 months. A slight but not significant decrease of Ka was observed in different tissues of normal rats. In hypothyroid animals the Ka in cortex at 24 months was significantly lower than at 3 months. During ageing the MBC of brain receptors decreased whereas hepatic receptors were not altered. Hypothyroidism did not further affect the MBC of the receptors. The data indicate that during ageing the T3 nuclear receptors behave differently in brain and liver. The difference in MBC suggests selectivity in organ sensitivity to thyroid hormones.

Effects of alinidine on metabolic response to high-demand myocardial ischemia.

Alinidine is a new bradycardic agent that interferes with ion channels and the if pacemaker current. To determine if alinidine had antiischemic effects unrelated to its bradycardic action, myocardial metabolism was studied during a pacing-stress test in 20 patients with coronary artery disease and angina pectoris, before and after intravenous infusion of alinidine (10 mg, n = 10; 50 mg, n = 10). When compared to the control pacing-stress test, the low dose of alinidine had no significant effect on aortic pressure, coronary sinus flow (-3%, NS), myocardial oxygen extraction, or myocardial lactate uptake. After the high dose of alinidine, aortic pressure and coronary sinus flow remained unchanged but the arteriocoronary sinus difference in oxygen content increased (12.2 +/- 1.3 to 12.7 +/- 1.4 ml/100 ml; p less than 0.0002) above the values observed during the control pacing-stress test, while both the chemical lactate extraction fraction (-19 +/- 30 to 15 +/- 21%; p less than 0.025) and the L-[1-14C]lactate extraction fraction increased. Accordingly, the net myocardial lactate uptake (corrected for production) had increased from 14 +/- 32 during the control pacing-stress test to 29 +/- 24 mumol/min during the pacing repeated after the high dose of alinidine (p less than 0.05). After the high dose of alinidine, the free fatty acid uptake also rose slightly (+23%; NS) and the alanine production was reduced in 7 of 10 patients (-3.6 +/- 1.7 to -1.4 +/- 0.6 mumol/min; NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intracoronary infusion of nicardipine during silent ischaemia on myocardial metabolism and function.

The effects of an intracoronary infusion of nicardipine (0.2 mg over 10 min) on myocardial substrate uptake and function were studied in 16 patients with coronary artery disease and angina pectoris. Silent ischaemia, demonstrated by myocardial lactate production, was induced twice by pacing below anginal threshold. Nicardipine or saline was randomly infused during the first or second pacing. During pacing with nicardipine, no systemic effect was noted but coronary sinus flow increased (+ 18%; P less than 0.015) and myocardial oxygen uptake decreased by 12% (P less than 0.025). Transcardiac lactate production did not improve (-8 to -10 mumol min-1; NS) but net lactate uptake, estimated from radiolabelled lactate uptake, tended to rise and the glutamine uptake increased from 1.8 to 5.5 mumol min-1 (P less than 0.04). During recovery after pacing, lactate production decreased faster and LV peak (+) dP/dt and relaxation rate were significantly better after nicardipine infusion than after saline. Thus, during silent ischaemia induced by an increased oxygen demand, intracoronary nicardipine did not prevent lactate release but allowed a faster metabolic and functional recovery. These beneficial effects of nicardipine could be explained by an improved myocardial perfusion or by an effect on intracellular calcium homeostasis.

Changes in coronary blood flow and myocardial metabolism during aortic balloon valvuloplasty.

The effects of balloon inflation on myocardial perfusion and metabolism were studied during aortic valvuloplasty in 17 patients with aortic stenosis, including 6 with associated coronary artery disease. Coronary sinus flow and blood samples were obtained before and during the first inflation, and 5 to 10 minutes after the last inflation. During inflation, coronary blood flow decreased (272 +/- 111 standard deviation to 166 +/- 92 ml/min; p less than 0.05), myocardial oxygen uptake fell and transcardiac lactate handling shifted from extraction to production (35 +/- 54 to -41 +/- 48 mumol/min; p less than 0.01). At the end of the procedure, aortic valve area had increased from 0.51 +/- 0.22 to 0.81 +/- 0.48 cm2 (p less than 0.002). Coronary sinus flow increased slightly above control values (+6%; difference not significant) and myocardial oxygen and lactate uptakes were back to control values. However, myocardial alanine production had increased from -3.6 to -6.6 mumol/min (p less than 0.05) and glutamine production was reduced or replaced by extraction (-3.3 +/- 2.1 to 3.5 +/- 3.8 mumol/min; p less than 0.05). Recovery of coronary flow, oxygen and lactate uptakes was not significantly different in patients with or without coronary artery disease, although the former patients tended to have less glutamine extraction and less improvement in their ejection fraction at the end of the procedure. Thus, aortic balloon valvuloplasty produces brief episodes of low-flow ischemia. Recovery of oxidative metabolism is almost immediate after deflation and no detrimental effect seems to persist at the end of the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in lipolytic activity of isolated adipocytes from rats throughout life span.

The lipolytic response of isolated adipocytes from 1 1/2-, 6-, 24- and 32-month-old rats to various doses of epinephrine was studied. Both the basal and the maximal stimulated glycerol release were largest in the mature rats (6 months old) which had the largest adipocytes. Expressing glycerol release per unit cell surface area (which was drastically reduced with age) eliminated the difference between mature and senescent rats both in absence of epinephrine and at high doses of the hormone. However, at low epinephrine doses the adipocytes of the very young and the very old rats showed an enhanced response per unit surface area. A simple pharmacodynamic analysis based on the occupancy theory of drug-receptor interaction suggests that the sensitivity of rat adipocyte receptors is increased during senescence; this increase may be related to the decreased surface of old adipocytes. On the other hand, the decreased maximal lipolytic response during senescence may be due in part to a reduced number of receptors and to a reduced sensitivity of the cellular enzymatic system underlying lipolysis.

In vitro effect of thyroid hormones on lipolysis of rat fat cells during aging.

The in vitro effect of triiodothyronine (T3) 10(-5) M upon lipolysis was studied on white fat cells isolated from 1.5-6- and 30-month-old rats. We couldn't see any consistent effect of this hormone upon the basal lipolysis. We observed a T3 effect on epinephrine-stimulated lipolysis on the three groups of animals. After 1 h of incubation the increase of glycerol release varies with the dose of epinephrine; after 3 h the T3 effect persisted only in the 6-month and 30-month groups.

Morphological and metabolic alterations in adipose tissue of very old rats.

Body weight, epididymal adipose tissue weight, fat cell size, and metabolic activity of fat depot were studied in 30-month-old Wistar rats and compared with rats aged 6 and 24 months old. In very old age a significant reduction of body weight was observed and was paralleled with a decline of adipose mass and a decrease in cell volume. In vitro metabolic studies, with and without insulin, showed a greater oxidation of D-U-14C glucose to 14CO2 and a greater incorporation into triglycerides than in the 24-month-old animals when the data were expressed per cell or per cm2 surface area. The resistance to insulin was elevated in spite of a significant decrease of adipocyte size in very old rats.

[Prolonged effects of training on adipose tissue glucose metabolism and insulin responsiveness in adult rats (author's transl)]. / Effets prolongés de l'entrainement physique sur le métabolisme du glucose du tissu adipeux et sa sensibilité a l'insuline chez le rat adulte.

Physical training (forced swimming for 6 weeks) led in resting adult rats modifications of circulating hormones, fuels and substrates, of epididymal adipose tissue composition and of its glucose metabolism. Plasma concentrations of free fatty acids, cholesterol and insulin were decreased by training. The weight of the epididymal fat pad, its triglyceride content and the mean cell size of adipocytes were significantly diminished. Twenty four hours after the end of the final exercise period, incorporation of D-U-14C-glucose into triglycerides was reduced (P less than 0.01) but the response to insulin was greatly enhanced for swimming rats both for oxidation of labelled glucose (P less than 0.001) and its incorporation into lipids (P less than 0.001) (respectively 215 and 225%). This enhancement of insulin sensitivity by training persisted and became more marked one week after the end of the exercise period (respectively 258 and 363%).

[Effects of TSH on thyroid ribonucleic acids (author's transl)]. / Effets de la TSH sur les acides ribonucléiques thyroidiens.

Bovine thyroid slices were incubated with TSH (20 mU/ml) in the presence of 0.04 microgram/ml actinomycin D. The incorporation of 3H Adenosine or 3H Uridine in the microsomal RNA fraction was studied. TSH enhanced the labeling of this RNA. After gel electrophoresis in 100% formamide, the distribution of the radioactivity showed a relatively larger incorporation of the precursors in the region corresponding to heavy molecular weight RNA (3 X 10(6) daltons). These data suggest that TSH could exert a specific effect on transcription in the thyroid gland.