Hormonal Regulation of Cell Elongation in the Hypocotyl of Rootless Soybean: An Evaluation of the Role of DNA Synthesis

A method was developed where soybean seedlings were grown without roots to study the influence of hormones of root origin on shoot growth. Excision of the root resulted in inhibition of apical section growth and DNA synthesis and inhibited elongating section growth. A synthetic cytokinin restored DNA synthesis in the apical section, but did not influence growth in either the apical or elongating sections. Low concentrations of gibberellin with the cytokinin restored growth in the apical section. Gibberellin alone was sufficient to restore growth in the elongating section.An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, inhibited the increase in apical section DNA without inhibiting control or gibberellin-induced growth in the elongating section. Experiments with (14)C-thymidine resulted in no DNA labeling differences in the elongating section under conditions where gibberellin-induced elongation varied from 50% to 73% above controls. It was concluded that gibberellin-induced elongation in soybean hypocotyl occurred in the absence of DNA synthesis. Gibberellin does stimulate DNA synthesis in the apical tissue apart from its effect on cell elongation.Excised soybean hypocotyl elongated maximally at 10(-6)m auxin. At higher auxin concentrations, fresh weight and ethylene production increased, but elongation was reduced. Addition of GA to the higher auxin concentrations resulted in a 50% inhibition in auxin-induced ethylene production and resumption in maximal elongation. Added ethylene inhibited elongation 30% at 2 mul/l. Addition of up to 100 mul/l ethylene did not inhibit elongation with GA present in the incubation medium. Thus GA may counteract ehtylene inhibition of cell elongation in addition to inhibiting ethylene production in auxin-treated tissues.     

The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration

1. The effects of ATP, PP(i) and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ;magnesium' refers to total Mg(2+), both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the K(m) values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6.8mm in systems buffered with either tris-hydrochloric acid or glycylglycine-sodium hydroxide, but the K(m) values were different in these systems. The K(m) for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris-hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine-sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the K(m) values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris-hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10-20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25-50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg(2+) ion. 5. In the presence of 6.8mm-ATP no reaction occurred below 4-6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10-25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6.8mm-PP(i) or 6.8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6-8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (K(i) 2.1x10(-2)m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.     

Biochemical Changes in Serum of Pure and Mixed Streptococcus faecalis and Bacillus subtilis Infections in Rats

Rats inoculated with Streptococcus faecalis developed endocarditis and demonstrated a 6- to 30-fold increase in aldolase, isocitric dehydrogenase, phosphohexose isomerase, and lactic dehydrogenase. The animals infected with Bacillus subtilis did not develop overt disease nor significant increases in enzyme activities, but viable organisms were recovered at 2 weeks. Rats inoculated with mixed culture of these organisms showed a 2- to 10-fold increase of enzyme activities without evidence of pathological anatomic changes. Both organisms were recovered at necropsy. The total protein and glycoproteins followed the patterns of enzyme activities. There were major changes in alpha(1), alpha(2), and beta globulins and glycoglobuulins at the early stages of infection. The protein-bound hexose changes coincided with the severity of S. faecalis infection, but were at normal levels after 72 hr of infection of B. subtilis and S. faecalis mixed infections. The results indicate that B. subtilis infection modified the pathogenicity of S. faecalis and by an unknown mechanism affected protein and glycoprotein production in serum of experimental rats.     

Cerebral O2 metabolism and cerebral blood flow in humans during deep and rapid-eye-movement sleep

It could be expected that the various stages of sleep were reflected in variation of the overall level of cerebral activity and thereby in the magnitude of cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF). The elusive nature of sleep imposes major methodological restrictions on examination of this question. We have now measured CBF and CMRO2 in young healthy volunteers using the Kety-Schmidt technique with 133Xe as the inert gas. Measurements were performed during wakefulness, deep sleep (stage 3/4), and rapid-eye-movement (REM) sleep as verified by standard polysomnography. Contrary to the only previous study in humans, which reported an insignificant 3% reduction in CMRO2 during sleep, we found a deep-sleep-associated statistically highly significant 25% decrease in CMRO2, a magnitude of depression according with studies of glucose uptake and reaching levels otherwise associated with light anesthesia. During REM sleep (dream sleep) CMRO2 was practically the same as in the awake state. Changes in CBF paralleled changes in CMRO2 during both deep and REM sleep.     

Cyanide and methylisocyanide binding to the isolated iron-molybdenum cofactor of nitrogenase

19F NMR and x-ray absorption experiments have been performed with both the isolated FeMo cofactor and the MoFe protein of nitrogenase in search of direct evidence for substrate or inhibitor binding. Using 19F NMR as a probe and p-CF3C6H4S- as the receptor ligand, the data show that the nitrogenase inhibitors CN- and CH3NC bind to the isolated FeMo cofactor-RFS- complex in N-methylformamide with a finite formation constant. Their binding increases the electronic relaxation time of the complex and increases the life-time of the FeMo cofactor-p-CF3C6H4S- bond, Parallel molybdenum K edge and extended x-ray absorption fine structure experiments show that CH3NC does not bind to molybdenum. Although CO and N3- both relieve CN- and CH3NC inhibition of electron flow through nitrogenase, unlike the latter, they do not appear to bind to isolated FeMo cofactor. In experiments with the dithionite-reduced MoFe protein, we did not detect any changes in the molybdenum K edge or extended x-ray absorption fine structure spectra upon addition of CO, N2, C2H2, NaCN, CH3NC, or azide demonstrating that either these substrates and inhibitors do not bind to molybdenum or that the FeMo cofactor site of nitrogenase is inaccessible to substrate binding except under turnover conditions.     

Primary structure of the brain alpha-spectrin [published erratum appears in J Cell Biol 1989 Mar;108(3):following 1175]

We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.     

Preovulatory plasma estradiol-17beta concentrations and ovulation rates in PMSG/anti-PMSG treated heifers

Possibilities for early characterization of the superovulatory response were studied in 41 PMSG/PG-treated dairy heifers, of which 21 received an additional treatment of PMSG-antiserum. Plasma was obtained at 33, 36, 41, 47 and 51 h after PG for hormone analyses. After slaughter at 6 or 7 d after insemination, the number of follicles and corpora lutea (CL) were recorded, and ova were recovered for morphological evaluation. Significant correlations were demonstrated between plasma concentrations of estradiol-17beta (E2) at 33, 36 and 41 h after PG and the ovulation rate (number of CL). Each of these correlations was equal to the one found by using the peak concentration of E2 achieved during the preovulatory E2 surge. In heifers with preovulatory E2 surges, as determined with the blood sampling scheme used, both the ovarian response (number of CL and follicles) and the quality of ova recovered (number of transferable embryos) was clearly better compared to heifers without this surge. None of the parameters studied was affected significantly by treatment with PMSG-antiserum. It is concluded that plasma E2 determinations at fixed times in relation to prostaglandin treatment can be used to characterize the superovulatory response in donor cattle in terms of the ovulation rate and the quality of ova recovered. No evidence was found in favor of using PMSG-antiserum for improving either the superovulatory response or such characterization.     

Failure of IGF-1 to affect protein turnover in muscle from growth-retarded neonatal rats

To investigate the response of the growth retarded neonatal rat to insulin-like growth factor-I (IGF-I) we have measured the effect of IGF-I on in vitro muscle protein synthesis and degradation rates in growth retarded and control neonatal rat pups. The growth retarded pups were growth retarded in utero by ligation of the uterine blood supply at day 17 of gestation. Basal levels of muscle protein synthesis in vitro were significantly lower in growth retarded pups compared with controls. Protein degradation rate were not different in muscles taken from the two groups. IGF-I stimulated protein synthesis in muscle from control pups by 12% and 15% at 20 ng/ml and 200ng/ml respectively. Net protein degradation was inhibited by 20% in the presence of 20ng/ml IGF-I. IGF-I had no effect on net protein synthesis or degradation in muscle from growth retarded pups. Neither Multiplication Stimulating Activity (at 20ng/ml or 200ng/ml) nor insulin (at 40ng/ml or 800ng/ml) was able to increase synthesis or decrease degradation of protein. Specific receptors for IGF-I are present on muscle membranes from both groups. Unlabelled IGF-I was more effective than MSA or insulin in competing with 125I-IGF-I for binding to the receptor. The relative affinities are consistent with type I IGF receptors. The affinity of these receptors for IGF-I was similar (Kd approximately 5nM) in both groups and the receptor concentration in both cases was approximately 250 fmol/mg protein. The refractility of tissue from growth retarded pups to IGF-I may be partially responsible for the lack of catch up growth in growth retarded neonates.