Free amino acids from pollens

Pollens from Pinus canariensis, P. nigra, P. pinaster, P. pinea, Castanea sativa, Magnolia grandiflora, Olea sativa cv frantoio, cv itrana, cv pisciottana were examined for their free amino acid composition. A large amount of proline was found in all species; pollens of Olea also contain a large amount of serine.     

Gonadal steroid modulation of brain opioid systems

It is becoming increasingly clear that the effects of the opioids and their synthetic analogs on anterior pituitary function largely depend on the steroid milieu present in the animal at time of drug administration. However, it is still unclear whether gonadal steroids regulate the opioid-modulated mechanisms by affecting the number of opiate receptors in the brain. To further investigate these issues, the effects of opiate agonists and antagonists on LH, FSH and prolactin (Prl) secretion have been studied in: (a) normal and castrated male rats, and (b) normally cycling female rats. The binding characteristics of the brain subclass of mu opiate receptors have been analyzed in the same group of experimental animals; this type of receptors seems to be particularly involved in the control of gonadotropin and Prl release. When injected intraventricularly into normal male rats, morphine (200 micrograms/rat) induced in a significant elevation of serum LH levels at 10 and 20 min. In long-term castrated animals the administration of the drug significantly reduced LH secretion at 40 and 60 min after the injection, the inhibition lasted up to 180 min. Morphine, when given intraventricularly to normal males, induced a conspicuous and significant elevation of serum Prl levels at 10, 20, 40 and 60 min after treatment. However, when the drug was administered to castrated rats, it did not significantly affect Prl release at any time interval considered. Morphine intraventricular injections did not modify serum FSH levels either in normal or in castrated male rats. The concentration of mu opiate receptors was found to be similar when measured in the whole brain of normal and orchidectomized rats. In adult cycling female rats, s.c. injections of naloxone (2.5 mg/kg) stimulated LH release in every phase of the estrous cycle; the magnitude of the responses was highly variable, being particularly elevated at 16.00 h of the day of proestrous and at 10.00, 12.00 and 14.00 h of the day of estrous. Conversely, LH response to naloxone was totally obliterated at 18.00 and 20.00 h of the day of proestrous, when the preovulatory LH surge was found to occur. The concentration of brain opiate receptors of the mu type showed significant variations during the different phases of the estrous cycle, with higher levels at 12.00 h of the day of proestrous and at 18.00 h of the day of estrous.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

Summary When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.     

Are highly phosphorylated 40-S subunits preferentially utilized during protein synthesis in a cell-free system from HeLa cells?

It has been concluded from circumstantial evidence obtained with HeLa cells in vivo that the phosphorylation of ribosomal protein S6 increases the affinity of 40S particles for mRNP [Duncan, R. and McConkey, E. H. (1982) Eur. J. Biochem. 123, 535-538; Thomas, G., Martin-Pérez, J., Siegmann, M. and Otto, A.M. (1982) Cell 30, 235-242]. This conclusion needs to be tested in vitro in a reinitiating cell-free translation system from growth-competent cells. We have prepared such a system from HeLa cells and have compared the capacity of homologous 40S subunits of various degrees of phosphorylation to enter the existing polysome pool. The 40S subunits' degree of phosphorylation was manipulated by exposing aliquots of growth-stimulated HeLa cells to hyperthermia (see accompanying paper). 40S subunits from heat-shocked and control cells, despite differences in S6 phosphorylation level as verified by two-dimensional electrophoresis, did not differ with respect to their recruitment into the existing polysome fraction. Owing to the reinitiation activity of the translation system, assay times could be kept sufficiently short, to avoid any serious interference by the S6 phosphatase activities of the system. Our results suggest that increased S6 phosphorylation by itself is not sufficient to accelerate the participation of 40S subunits in protein synthesis.     

Microcythemia from anemic hypoxia and normal erythropoiesis in the newt

Specimens of the newt, Triturus cristatus carnifex (Laurenti), rendered totally anemic, restore erythron by cyclic waves of erythropoietic activity that alternate with intervals of stasis. Hemolysis is obtained by administering 25 mg/liter of acetylphenylhydrazine in the breeding water for 36 h. The first cycle of erythropoietic activity produces microcytes, which have completely differentiated by 8 weeks after treatment. However, if the animals are raised in a hyperbaric chamber at a pressure of 1.5 atmospheres, in order to compensate for hypoxia, normocytes are produced. In both cases the hematocrit and hematic concentration of hemoglobin reach analogous values, so microcythemia appears to be the only effect of hypoxia. The hemoglobin, hematocrit values, and normocyte counts in hyperbaric animals are about one-half those of the controls newts. These data, together with those on the life span of red blood cells (RBC) and time span between two successive erythropoietic cycles (2 months and 1 month, respectively), indicate that the newts normally keep only two sets (one new, one old) of RBC in circulation, whose approximate parameters can be defined as RBC count: 60,000/mm3, hematocrit: 17%, and hemoglobin: 5.4 g/100 ml.     

High extracellular concentrations of potassium and rubidium modulate the synaptic transmission in the frog labyrinth

High extracellular K or Rb levels (20 mM) produce an increase in the resting EPSP and spike frequencies recorded intra cellularly from single fibres of the posterior nerve in the isolated frog labyrinth. The afferent discharge facilitation proved to be inversely related to the fibre's initial resting activity. The K effect is systematically larger than the Rb effect. High sensitive and scarcely sensitive units may be identified with respect to K and Rb action. The present findings suggest that, according to previous models of hair cell functioning, the K and Rb effects are mediated by a raise in intracellular Ca concentration which sustains an increased transmitter release at the cyto-neural junction.     

Mitogen-responsive S6 kinase

Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post-DEAE chromatography fractions of S49 kin- cell extracts, we could detect a highly active effector-independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40-S or 80-S (reassociated from subunits) ribosomal particles being substrate. The optimal K+ concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent Mr of the enzyme, as estimated by gel filtration on Sephadex G-100, is 56,000 (determination in the presence of 200 mM KCl in 25 mM phosphate buffer). Tighter binding to DEAE-Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6-phosphorylating protein kinases: protein kinase C, protease-activated kinase II, histone-4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow-up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogen-responsive S6 kinase(s) [Martini, O. H. W. and Lawen, A. (1985) in Hormones and cell regulation (Dumont, J. E., Hamprecht, B. and Nunez, J., eds) vol. 9, pp. 411-412, Elsevier Company, North-Holland, Amsterdam; Lawen, A. and Martini, O. H. W. (1985) FEBS Lett. 185, 272-276].     

Insulin-induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress

Insulin treatment of HeLa S3 cells activates an S6-phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease-activated kinase II and histone-4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin-induced increase in S6-kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin-induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6-kinase activities from serum- and from insulin-stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitro transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphorylate the (principal) S6 site recognized by the cAMP-dependent protein kinase.