Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.     

Glucose infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

After a pulse of [3-14C]pyruvate, 24 hr starved rats were infused through the portal vein with two different doses of glucose (7.8 or 20.8 mg/min) or the medium, and blood was collected from the inferior cava vein at the level of the suprahepatic veins. The highest dose of glucose enhanced the appearance of [14C]glucose in blood from the 2nd to the 20th min after tracer delivery. It also enhanced production of [14C]glycogen and concentration of glycogen in the liver after 5 and 20 min. At 20 min of glucose infusion the appearance of [14C]glyceride glycerol in liver as well as liver lactate concentration and lactate/pyruvate ratio were increased. The low dose of glucose used enhanced liver values of [14C]glycogen, [14C]glycogen specific activity and glycogen concentration. Our results support the hypothesis that in the starved rat glucose is converted into C3 units prior to being deposited as liver glycogen and based on the liver zonation model (Jungermann et al., 1983) it is proposed that glucose stimulated gluconeogenesis by shifting the liver to the cytosolic redox state as a secondary consequence of increased glycolytic activity.     

Mechanism of inhibition by lithium of sodium transport in the toad bladder

Lithium transport across the urinary bladder of Bufo marinus has been studied by means of the short-circuit current technique, as well as unidirectional ion flux measurements. Exposure to lithium of the epithelial (mucosal) surface of this preparation led to a slow, progressive decrease of ion transport, with increasing discrepancy between short-circuit current and lithium influx; in fact there was still an appreciable lithium influx across bladder exposed to amiloride even though short-circuit current was suppressed. Ohmic conductance and sodium efflux barely increased under these circumstances. Upon replacement of lithium by sodium on the epithelial side, the preparations recovered slowly indeed, and residual lithium could be detected in bladder tissue for more than 2 hr while the rate of sodium extrusion at the basal-lateral cell border was slowed down. Recovery from exposure to lithium was accelerated by vasopressin and amphotericin, both of which facilitate sodium entry at the apical border of the epithelium. Thus the lasting deleterious influence of lithium on sodium transport might result from the fact that this ion, once trapped in the cytoplasm, closes the sodium channels.     

Rapid thyroid-hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.     

Ultrastructural evidence for an osmoregulatory effect of nikkomycin in the mite Tetranychus urticae

Histological effects of the microbial metabolite and chitin synthesis inhibitor complex Nikkomycin (AMS 0896 Bayer Leverkusen) on osmoregulatory organs of all developmental stages of Tetranychus urticae are described. The metabolite, in a concentration of 100 ppm, was applied via the nutritive plant. Mites fed for 2 to 14 days, and then were collected and immediately fixed. Two osmoregulatory organs occur in T. urticae. The Malpighian complex, differentiated only in females, shows an increased number of apical microvilli in the epithelium of the distal regions after metabolite application, thus resulting in an enlargement of the surface area. Changes in the second osmoregulatory organ, the coxal organ, after Nikkomycin application include depositions of membranous bodies in the lumen as well as in cytoplasmic vacuoles of the proximal tubule. Additionally, an increase in the luminal diameter occurs. Numerous vacuoles of different contents are observed in the cytoplasm of the distal tubule. Consequences of histological alterations in osmoregulatory organs after Nikkomycin application are discussed with special reference to the composition of salivary secretions.     

Root growth and litter decomposition in a coffee plantation under shade trees

In a non-fertilized coffee plantation under shade trees the root biomass was excavated to estimate its distribution in the soil profile. One third of total fine (less than 1 mm) roots was found in the first 10 cm of soil; the cumulative total to 30 cm reached 73%. A highly variable and transient amount of fine roots colonized the litter layer. Root production both in the litter and in the first 7.5 cm of mineral soil was estimated from sequential samplings and was 10 g m^–2 yr^–1 and 660 g m^–2 yr^–1 respectively. The decomposition rate of weighed averages of litter fractions in the coffee plantation, calculated as the ratio of litter fall rate to the amount found in the soil was k=4.8. Shade tree leaves, the major component of litter descomposed slower than coffee leaves and these slower than flowers and fruits. Litter bag experiments showed considerable slower rates when mesh was 0.03 mm than 0.5 mm. Nitrogen and phosphorous showed increases in concentrations as decomposition progressed while potassium, calcium and magnesium followed a decrease in concentration that paralleled that of dry weight loss. In comparing the decomposition rate for litter with or without coffee roots growing in the bags, a tendency to show faster decomposition rates was found for the treatment with roots. These differences were however, only significant for one month for shade tree leaves litter. Nitrogen amounts remaining in shade tree leaves litter was lower in the treatment with roots that without roots. Potassium concentration in roots was positively correlated with potassium concentration in decomposing leaf litter where roots were growing. These results suggest that while roots growing attached to decomposing litter had little or no effect in speeding the decomposition process, the superficial roots seem to play an important role in absorbing very efficiently the mineralized nutrients from litter. The anatomical study of roots showed that the plantation is intensely infected with V-A mycorrhiza. External mycorrhizal hyphae did not to play a role in attachment of roots to decomposing litter while root hairs were found to grow in profusion on root surfaces oriented toward litter.     

Physical properties and function of phallolysin

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations.

Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme.     

Role of molybdenum in nitrate reduction by chlorella

Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH₂-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.