A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Aerobic,phenol-induced TCE degradation in completely mixed,continuous-culture reactors

BothPseudomonas putida F1 and a mixed culture were used to study TCE degradation in continuous culture under aerobic, non-methanotrophic conditions. TCE mass balance studies were performed with continuous culture reactors to determine the total percent removed in the reactors, and to quantify the percent removed by air stripping and biodegradation. Adsorption of TCE to biomass was assumed to be negligible. This research demonstrated the feasibility of treating TCE-contaminated water under aerobic, non-methanotrophic conditions with a mixed-culture, continuous-flow system.Initially glucose and acetate were fed as primary substrates. Pnenol, which has been shown to induce TCE-degrading enzymes, was fed at a much lower concentration (20mg/L). Little degradation of TCE was observed when acetate and glucose were the primary substrates. After omitting glucose and acetate from the feed and increasing the phenol concentration to 50mg/L, TCE biotransformation was observed at a significant level (46%). When the phenol concentration in the feed was increased to 420mg/L, 85% of the incoming TCE was estimated to have been biodegraded. Under the same conditions, phenol utilization by the mixed culture was greater than that ofP. putida F1, and TCE degradation by the mixed culture (85%) exceeded that ofP. putida F1 (55%). The estimated percent-of-TCE biodegraded by the mixed culture was consistently greater than 80% when phenol was fed at 420mg/L. Biodegradation of TCE was also observed in mixed-culture, batch experiments.     

Effects of temperature and photoperiod on flowering in Hyptis brevipes

Few tropical species have been tested for their flowering response under controlled conditions. Hyptis brevipes Poit, is an annual herb, commonly found in wet margins of streams and ponds, being considered a weed for some perennial plantations in Brazil. Under experimental glasshouse conditions, this species proved to be an obligate short-day plant. Flowering was delayed when photoperiods longer than 8 h were given, the critical photoperiod being between 12 and 13 h. When both temperature and photoperiod were controlled, at 20°C a longer protoperiod (by almost 1 h) is still inductive compared to 25 and 30°C. The number of short-day cycles required for full induction is relatively high and dependent upon temperature; at 20°C or above, 10 cycles are adequate, but at 15°C, more short-day cycles are needed. The number of inflorescences formed as well as the floral index vary according to daylength × temperature × inductive cycle number, allowing flowering to be assessed quantitatively. Long days are inhibitory to flowering, either suppressing it completely (when symmetrically intercalated among 24 inductive cycles) or preventing the floral index from increasing.     

EFFECTS OF LONG TERM EXPOSURE TO LOW TEMPERATURE ON THE PHOTOSYNTHETIC APPARATUS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The effects of long term exposure to suboptimal growth temperature on the photosynthetic apparatus of Dunaliella tertiolecta Butcher were investigated using carbon fixation rate versus irradiance curves and the variable fluorescence induction method. Carbon fixation rates per unite chlorophyll a at saturating (p^B_m) and subsaturating (α^B) irradiances were 55% and 39% lower, respectively, at 12° C than at 20° C. Chlorophyll a quotas and the spectrally averaged in vivo absorption cross section normalized to chlorophyll a (a^*) were not significantly different at these two temperatures. Analysis of the fluorescence kinetics revealed 1) no significant variations of the amount of PSII photoactive reaction centers per unit chlorophyll a, 2) a 14% decrease of the PSII quantum yield(+) and 3) a 29% decrease of the energy transfer efficiency between the light harvesting chlorophyll a pigment bed and the PSII reaction centers. The decrease in energy transfer efficiency between the antennae and the PSII reaction centers at 12° C was interpreted as a mechanism to avoid photoinhibition.     

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

In contrast to the degradation of penta-and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.Abbreviations PCB polychlorinated biphenyls - CBA chlorobenzoate - D di - Tr tri - Te tetra - Pe penta- - H hexa     

Multiple xenobiotic-inducible P450s are involved in alkoxycoumarin and alkoxyresorufin metabolism in higher plants

O-Dealkylation of two series of fluorescent 7-alkoxy-coumarins and 7-alkoxyphenoxazones by plant cytochrome P450s was investigated in Helianthus tuberosus tuber tissues treated with prototype P450 inducers, environmental pollutants or agrochemicals. Methoxy-, ethoxy-, propoxy-and butoxycoumarins and methoxy- and ethoxyresorufins were metabolized by fplant microsomes. Dealkylation of pentoxy- and benzyloxyresorufins was not detected. All dealkylating activities were enhanced by aging plant tissues in the presence of xenobiotics, in some cases up to 20-fold relative to the activities detected in control tissues. Increases in total P450 in the same tissues never exceeded 3-fold. The isozymes induced by prototype P450 inducers clearly differed from those in mammalian liver. That multiple P450s with overlapping substrate specificities were involved in the metabolism of both alkoxycoumarins and alkoxyresorufins was demonstrated by (1) the differential induction of the activities in response to exposure to xenobiotics, (2) the differential inhibition of the activities by clotrimazole, paclobutrazole and tetcyclacis in aminopyrine and benzo(a)pyrene-treated tissues, and(3) the selective inhibition observed with antibodies raised against purified ethoxycoumarin deethylase fractions. Our results suggest that the measurement of the dealkylation of such fluorescent substrates in plants might be useful to monitor environmental pollution.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

Effects of dehydration on the electron transport of Chlorella. An in vivo fluorescence study

Chlorella was used to study the effects of dehydration on photosynthetic activities. The use of unicellular green algae assured that the extent of dehydration was uniform throughout the whole cell population during the course of desiccation. Changes in the activities of the cells were monitored by measurements of fluorescence induction kinetics. It was found that inhibition of most of the photosynthetic activities started at a similar level of cellular water content. They included CO₂ fixation, photochemical activity of Photosystem II and electron transport through Photosystem I. The blockage of electron flow through Photosystem I was complete and the whole transition occurred within a relative short time of dehydration. On the other hand, the suppression of Photosystem II activity was incomplete and the transition took a longer time of dehydration. Upon rehydration, the inhibition of Photosystem II activity was fully reversible when samples were in the middle of the transition, but was not thereafter. The electron transport through Photosystem I was also reversible during the transition, but was only partially afterward.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F_m maximum fluorescence yield - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_v variable part of fluorescence - PS photosystem - Q_A primary quinone acceptor of Photosystem II