Activation of ADP-ribosyltransferase in polyamine-depleted mammalian cells.

Mammalian fibroblasts were cultured in the presence of alpha-methylornithine and/or methylglyoxal bis(guanylhydrazone), which inhibit the synthesis of polyamines. This led to a decrease in the cellular content of the polyamines spermine and spermidine by up to 60% when the cells were grown in the presence of both drugs together. The activity of the chromatin-associated enzyme ADP-ribosyltransferase was enhanced 2-3-fold in the drug-treated cells when measured in cells subsequently rendered permeable to exogenous NAD+, the substrate for the transferase. This is a novel and surprising observation, since the transferase is invariably activated by the addition of polyamines to a suitable incubation system such as permeabilized cells, isolated nuclei or the purified enzyme. We found no evidence that the activation was due to the appearance of DNA strand breaks, by using a variety of procedures including both neutral [the 'nucleoid' technique of Cook & Brazell [(1975) J. Cell Sci. 19, 261-279; (1976) J. Cell Sci. 22, 287-302]] and alkaline sucrose-gradient centrifugation and gel electrophoresis, suggesting that this therefore may not be the only means of regulating the activity of ADP-ribosyltransferase and that polyamines may have a role to play in this regard in vivo.     

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. ³¹P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Blockade of renin or angiotensin for understanding human hypertension: a comparison of propranolol, saralasin and converting enzyme blockade.

To understand the role of the renin-angiotensin-aldosterone system in the pathogenesis of human hypertension, in serial studies we have blocked the system using three different pharmacologic probes: 1) reduction of renin secretion by administration of the beta receptor blocker, propranolol; 2) blockade of the action of angiotensin II by infusion of saralasin, a competitive antagonist of angiotensin II; and 3) blockade of the enzymatic conversion of angiotensin I to angiotensin II by infusing a nonapeptide competitive inhibitor. The depressor responses induced by either propranolol or the nonapeptide expose a significant to major involvement of excess renin--angiotensin in maintaining the hypertension of some 50 to 70% of common forms of hypertension including "essential" hypertension. This subgroup includes nearly all patients with high or "normal" renin--sodium profiles. The considerably lower estimates for a renin factor in essential hypertension suggested by saralasin testing now appear due to the partial agonism of this drug. Further studies are required to determine whether this relative or absolute excess of renin secretion is primarily involved in the hypertension and if not why it fails to shut itself off. Similar studies of normal subjects are also needed to determine whether renin support of blood pressure is proportionately greater or less than in hypertensive subjects. Meanwhile the validation provided by these three different pharmacologic probes portends a burgeoning clinical role for renin--sodium profiling not only in screening for renal and adrenal cortical hypertensions but also for characterizing the vasoconstrictor and volume elements involved in various individual patients and thus enabling more specific treatments of the various subtypes of essential hypertension.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Gingival eruption sequences of permanent teeth in early hominids.

Gingival eruption of the permanent teeth of Australopithecus, Paranthropus, and early Homo, diagnosed from enamel scratches and facets, is similar save for two sequences: eruption of the canines relative to the premolars which may be sexually dimorphic; and agenesis of M3 with delayed eruption of M2, first seen in Homo at two million years. Gingival eruption sequences are similar also for early and modern Homo, except that in some individuals today M3, M2, M1 and I2 take longer to form and emerge through the gingiva as functioning teeth. Probably, from two million years to the present in the evolutionary history of Homo dental development slowed-down. More and more of ontogeny has been taken over for eruption.     

Aerobic respiration in mutants of Escherichia coli accumulating quinone analogues of ubiquinone.

The ability of three naturally occurring analogues of ubiquinone to function in aerobic respiration in Escherichia coli has been studied. The compounds, which differ from ubiquinone in terms of the substituents on the quinone ring, accumulate in the cytoplasmic membranes of ubiE-, ubiF- and ubiG- mutants. One of the analogues (2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone, NMQ), which lacks the 5-methoxyl group of the benzoquinone ring of ubiquinone promoted the oxidation of NADH, D-lactate and alpha-glycerophosphate but not succinate. Electron transport supported by MMQ was found to be coupled to phosphorylation. In contrast, 2-octaprenyl-6-methoxy-1,4-benzoquinone, which lacks both the 3-methyl and 5-methoxyl groups of ubiquinone, and 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, in which the 5-methoxyl group of ubiquinone is replaced by an hydroxyl group, were virtually inactive in the oxidases tested. The ability of MMQ to function in respiration in isolated membranes is consistent with the findings that the growth rate and yield of a ubiF- strain, unlike other ubi- strains, were only slightly lower than those of a ubiF+ strain. The fact that MMQ is active in some but not all oxidases provides further support for the concept that the quinones link the individual dehydrogenases to the respiratory chain and that each dehydrogenase has specific structural requirements for quinone acceptors.     

C-glycosylflavones of the gnetopsida

C-Glycosylflavones have been identified in Ephedra antisyphilitica, Gnetum gnemon and Welwitschia mirabilis. The C-glycosidic moieties of apigenin and luteolin derivatives have been identified as glucose and/or xylose for these species.     

The effects of clipping and fertilization on nitrogen nutrition and allocation by mycorrhizal and nonmycorrhizal Panicum coloratum L., a C4 grass

Summary Mycorrhizal and nonmycorrhizal plants of Panicum coloratum L. were grown in a factorial treatment design under two nitrogen levels and two clipping heights with an unclipped control. The nitrogen concentration in different plant components was determined following 9 weeks of growth under experimental conditions. Mycorrhizal infection increased green leaf and sheath nitrogen concentration by a relatively small, but significant percentage and had no effect on nitrogen allocation to the various plant components. Clipping increased leaf nitrogen concentration but inhibited growth to the extent that, when compared with the unclipped controls, less nitrogen remained in residual plant biomass with up to half of the total nitrogen allocated to offtake (the material removed by clipping). Plants receiving the higher nitrogen fertilization had higher tissue concentration of N and more N allocated to above-ground living tissues. Mycorrhizal infection interacted with clipping height and also with N availability significantly. Infection was unable to ameliorate the negative effects of the most severe clipping regime and of the low nitrogen availability on leaf and sheath N content. This is possibly due to mycorrhizal demand for carbohydrates competing with the carbohydrate requirement of roots for nitrogen uptake.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicol-resistant (CAPR) human x mouse hybrids and one CAPR cybrid derived from CAPR HeLa cells and CAPS mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with 32P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [32P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited "reverse" chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human-mouse hybrid is to retain human mitochondrial DNA.