[Purging of hemopoietic progenitor cells in autologous transplantation]. / Cistení hemopoetických progenitorových bunek pro autologní transplantace.

The authors describe the results of purification of bone marrow and peripheral progenitor cells (PBPC) for clinical transplantations. Vepeside was used to purify in 1990-1995 a total of 41 bone marrows of adults and children. Of these 23 were transplanted. Maphosphamide was used bone marrow purging in two patients; transplantation was performed in one case. By a combination of Vepeside with methylprednisolone haematopoietic cells of 24 patients were purged, transplantations were performed in 10. Three-day cultivation of haematopoietic cells in the presence of Desferal was used for purging cells of 22 patients with neuroblastoma; transplantations were performed in 10 patients. The authors give the values of nucleated cells, haematopoietic colonies of CFU-GM and CD34 positive cells obtained after purification calculated per kg body weight of the patient and the percentage yields.

Alpha 2-adrenergic receptors in brown adipose tissue of infant rats--II. Studies on function and regulation.

1. Clonidine inhibited the forskolin- and MIX-induced rate of lipolysis in brown fat adipocytes isolated from interscapular brown fat of 7-day-old rats. Its effect could be prevented by the alpha 2-antagonist yohimbine. 2. Pertussis toxin prevented the above effect of clonidine, thus indicating that alpha 2-adrenoceptors are linked with adenylate cyclase via the Ni regulatory subunit. 3. Chemical sympathectomy of 5-day-old rats by 6-hydroxydopamine increased the number of low-affinity alpha 2 sites in brown fat. 4. Chronic administration of yohimbine to 2-3-week-old rats also increased the density of alpha 2-adrenoceptors in brown fat. 5. It is suggested that brown fat of infant rats possesses functional alpha 2-adrenoceptors.

Alpha 2-adrenergic receptors in brown adipose tissue of infant rats--I. Identification and characteristics of binding sites in isolated plasma membranes.

1. Alpha 2-Adrenoceptor antagonists [3H]yohimbine and [3H]RX 781094 and the partial alpha 2-agonist [3H]clonidine exhibited specific binding to plasma membrane fragments isolated from interscapular brown fat of 7-day-old rats. 2. Competition studies with epinephrine, yohimbine and practolol revealed that [3H]norepinephrine, the principal in vivo agonist acting upon brown adipocytes, can readily bind to alpha 2-adrenoceptors in brown fat of infant rats. 3. The presence of alpha 2-adrenoceptor subclass in brown fat of infant rats may play a role in the sympathetic regulation of this rapidly proliferating tissue.

Effect of age and diet on cyclic nucleotide concentrations in the intestinal mucosa of developing rats.

Mucosa isolated from the proximal third of the small intestine of infant rats had much lower cyclic nucleotide concentrations (expressed both per unit wet weight and per unit DNA content) than those determined in the intestinal wall. The steady-state concentrations of both cyclic AMP and cyclic GMP in jejunum showed dramatic increases during the first 5 d post partum. Another increase in cyclic nucleotide concentrations was observed in the isolated mucosa between d 15 and 21. Starvation for 24 h always resulted in lower intestinal cyclic nucleotide concentrations than those of the fed littermates. This effect was more pronounced in younger animals and more evident for cyclic AMP values. Three-week-old rats fed a high carbohydrate diet for 24-48 h exhibited more pronounced elevations in the concentrations of cyclic nucleotides from the jejunal mucosa than did rats fed a high fat diet.

Mechanisms of hormonal regulations in brown adipose tissue of developing rats.

The need for nonshivering heat production, a principal function of brown adipose tissue, is accentuated in neonates. Accordingly, brown fat in the rat exhibits a very pronounced process of morphological and functional maturation perinatally, reaches a peak in its differentiation and heat-generating capacity within 1-2 weeks after birth, and undergoes involutive changes later in life. The later process of dedifferentiation can be either prevented or reversed by exposing the animals to cold ambient temperature for a prolonged period of time (cold acclimatization). The regulation of both the tissue maturation processes and the superimposed acute heat production are hormone mediated. Thus, the hormone receptor system within the adipocyte membrane and the sequence of molecular events interconnecting the initial hormonal stimulus with its final intracellular effect(s) are of considerable importance. The brown adipocytes of developing rats possess adrenoreceptors that can be pharmacologically classified as beta 1 (linked to adenylate cyclase) and alpha 2 (possibly linked to guanylate cyclase), multiple forms of cyclic nucleotide dependent and independent protein kinases, a protein kinase inhibitor, and at least two distinct phosphoprotein phosphatases associated with three phosphoprotein phosphatase modulators. The characteristics and developmental alterations of these regulatory components were studied in considerable detail by our group during the past decade. The results uncovered several target systems for ontogenic modifications of hormonal responses. Strong support was obtained for the hypothesis that protein phosphorylation and dephosphorylation is a major molecular mechanism involved in the regulation of both the brown adipocyte function and its proliferative activity during ontogenic development.

The separation and properties of phosphoprotein phosphatase modulators from brown adipose tissue of young rats and the changes in their concentration during development.

The heat-stable modulators of phosphoprotein phosphatase activity have been partically purified from brown adipose tissue. A nonphosphorylatable inhibitor of phosphorylase phosphatase (inhibitor 2) and an activator of phosphohistone phosphatase were similar to the corresponding modulators from muscle and liver in both their physical properties and in their relative effects upon three different preparations of phosphatase. An inhibitor of phosphorylase phosphatase that was only active when phosphorylated was eluted from DEAE-cellulose by 80 mM NaCl (inhibitor 1'). Only a small amount of inhibitor was eluted at 20 mM NaCl (inhibitor 1), which is the concentration that eluted the bulk of the phosphorylatable inhibitor in muscle and liver. Inhibitor 1 and inhibitor 1' had similar physical properties but differed in their activities towards the different phosphatases. It is suggested that they are different forms of the same protein and that inhibitor 1' more closely resembles the native inhibitor. The modulators did not compete with each other and were probably not subunits of a phosphatase complex. However, the direction and timing of the changes in their concentration in brown fat during the developmental period indicate that the inhibitors, at least, perform some useful physiological function in the tissue. The concentration of inhibitor 2 was high before birth and for 10 days after birth, when the tissue was proliferating. The concentration of the phosphorylatable inhibitor was highest immediately after birth and for the next 16 days, which is the period of greatest thermogenic activity of brown fat.

The effect of starvation on obese mice.

Plasma level of total and acylcarnitine and the activities of carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (PCT) in liver and CAT in brown fat were determined in young obese (ob/ob) mice and their littermates during starvation. Plasma levels of acylcarnitine and beta-hydroxybutyrate rose equally in both groups. Total carnitine levels, however, decreased in lean and rose in obese animals. Hepatic PCT and phosphoenolpyruvate carboxykinase activities rose more in lean than obese mice and brown fat CAT activity decreased in the obese group. Fatty acid synthetase activity decreased equally in the liver in obese mice and their lean littermates.

Cyclic nucleotides in breast milk.

Adenosine 3':5' monophosphate3 (cAMP) and guanosine 3':5' monophosphate (cGMP) are known to participate in the regulation of proliferation and differentiation, the processes intimately associated with maturation of the neonate. We have therefore examined their content in the physiological nutrient of the mammalian neonate, the mother's milk. Widely fluctuating concentrations between 0.1 and 0.7, and between 0.01 and 0.15 nmol/ml, were found for cyclic AMP and cyclic GMP, respectively. Concentrations in human breast milk changed during the 5-to 15-min period of one nursing, during any 24-h period, and also during the total lactation period. Levels of cyclic GMP were generally less fluctuating and were lower during afternoon and evening; they were relatively high at the start of lactation and levelled off during the postpartum period. The ratio of the two cyclic nucleotides also fluctuated widely and was significantly different from the ratio determined on blood plasma collected at the same time. Oxytocin injection had no effect on cyclic AMP content of rat milk. The stomach content of the nucleotide in rat pups remained high for at least 1 h after suckling indicating that cyclic nucleotides remain available for intestinal absorption; whether they have any physiological function in the neonate will have to emerge from further studies.

Effect of insulin and prednisolone on cyclic nucleotides and phosphoenolpyruvate carboxykinase activity in brown fat and liver of developing rats.

The concentrations of cyclic AMP and cyclic GMP in brown fat and liver of both suckling and adult rats at fixed times after injection of insulin (2.5 U/100 g body weight) or prednisolone (2.5 mg/100 g body weight) were compared with the activity of phosphoenolpyruvate carboxykinase assayed 24 h after the injections. A stimulus that produced an increase in cyclic AMP content also produced an increase in the enzyme activity. If the content of cyclic GMP was also increased there was no rise in phosphoenolpyruvate carboxykinase activity. A rise in the content of cyclic GMP alone was associated with a reduction in the activity of the enzyme. These preliminary results indicate that cyclic AMP could be involved in the induction of phosphoenolpyruvate carboxykinase and that cyclic GMP may somehow be related to its repression. The known differences in the response of phosphoenolpyruvate carboxykinase activity to insulin and prednisolone in different tissues and at different stages of ontogenic development may thus be linked to differences in the responsiveness of enzymes concerned with the metabolism of cyclic nucleotides.

The response of cyclic nucleotide content in liver and brown fat of rats weaned to different diets.

Rats were weaned on the 18th or 30th postnatal day to a high-fat, high-carbohydrate or atherogenic diet. Twenty-four hours later, hepatic levels of cyclic guanosine 3',5'-monophosphate (cyclic GMP) and adenosine, 3',5'-cyclic monophosphate (cyclic AMP) were found to be higher in male animals aged 31 days fed the high-fat than those fed the high-carbohydrate diet. Prematurely weaned rats (day 18) reacted in the same way. However, feeding either diet resulted in higher hepatic cyclic nucleotide levels than found in rats kept with the dam. The atherogenic diet was least effective in raising these levels. After a 24-hour fast, cyclic nucleotide levels in liver and brown fat were elevated and hepatic levels could not be lowered by 6 hours of feeding the high-fat or atherogenic diet. In male 40-day-old rats, however, feeding for 2 hours was sufficient to lower these levels, more so with a high-carbohydrate than a high-fat diet. Since blood levels of glucagon are high, and those of insulin are low in 18-day-old rats that are not weaned and since their hepatic cyclic nucleotide levels are low, it is suggested that other factors, in addition to blood hormone levels, play a role in regulating cyclic nucleotide levels.

The "second messenger" system in brown adipose tissue of developing rats. Its molecular composition and mechanism of function.

Our studies of the hormonal receptor system and of the sequence of enzymatic events interconnecting the initial hormonal stimulus to the brown adipocyte with its final subcellular effect are summarized here. The hormone-mediated regulatory pathway consists of the adenyl cyclase and the protein kinase systems; the former is composed of the receptor and catalytic sites, the latter of regulatory and catalytic subunits. Emphasis is given currently to the diversity and characteristics of the individual components of the protein kinase system, since it seems to carry out the ultimate unifying mechanism involved in a variety of hormone-mediated functions, i.e. the phosphorylation of a protein molecule.

Fructose-1,6-diphosphatase activity in brown adipose tissue of the developing rat.

An enzyme activity capable of converting fructose-1,6-diphosphate to fructose-6-phosphate was demonstrated to present in crude tissue extracts from brown adipose tissue of the rat. Mg2+ was essential for the expression of activity. EDTA (0.5 mM) increased the activity by 30%. Fructose-1,6-diphosphate in concentrations of 1 and 10 mM inhibits activity by 30% and 60% respectively. A 65% inhibition was observed in the presence of 0.2 micrometer 5' AMP. The activity of the enzyme was measured in rat brown adipose tissue at different stages of development. It rises sharply between day 2 and day 6 and continues to increase reaching a maximum between 6 and 11 days. Thereafter the activity gradually declines to values observed prenatally. The normal developmental rise in activity could be prevented by chemical sympathectomy on day 2. This procedure had no effect when carried out on day 9. There was a significant increase in enzyme activity after cold adaptation. The possible physiological significance of this enzyme in brown adipose tissue is discussed.