Carbon and energy balances of glucose fermentation with hydrogenproducing bacterium Citrobacter amalonaticus Y19

For the newly isolated H2-producing chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism was studied in batch cultivation at varying initial glucose concentrations (3.5- 9.5 g/l). The carbon-mass and energy balances were determined and utilized to analyze the carbon metabolic-pathways network. The analyses revealed (a) variable production of major metabolites (H2, ethanol, acetate, lactate, CO2, and cell mass) depending on initial glucose levels; (b) influence of NADH regeneration on the production of acetate, lactate, and ethanol; and (c) influence of the molar production of ATP on the production of biomass. The results reported in this paper suggest how the carbon metabolic pathway(s) should be designed for optimal H2 production, especially at high glucose concentrations, such as by blocking the carbon flux via lactate dehydrogenase from the pyruvate node.     

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

Magnetic field dependence of radical-pair decay kinetics and molecular triplet quantum yield in quinone-depleted reaction centers

Radical-pair decay kinetics and molecular triplet quantum yields at various magnetic fields are reported for quinone-depleted reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26. The radical-pair decay is observed by picosecond absorption spectroscopy to be a single exponential to within the experimental uncertainty at all fields. The decay time increases from 13 ns at zero field to 17 ns at 1 kG, and decreases to 9 ns at 50 kG. The orientation averaged quantum yield of formation of the molecular triplet of the primary electron donor, ³P, drops to 47% of its zero-field value at 1 kG and rises to 126% at 50 kG. Combined analysis of these data gives a singlet radical-pair decay rate constant of 5 · 10⁷s^?1, a lower limit for the triplet radical-pair decay rate constant of 1 · 10⁸s^?1 and a lower limit for the quantum yield of radical-pair decay by the triplet channel of 38% at zero field. The upper limit of the quantum yield of ³P formation at zero field is measured to be 32%. In order to explain this apparent discrepancy, decay of the radical pair by the triplet channel must lead to some rapid ground state formation as well as some ³P formation. It is proposed that the triplet radical pair decays to a triplet charge-transfer state which is strongly coupled to the ground state by spin-orbit interactions. Several possibilities for this charge-transfer state are discussed.     

Effect of hyperthermia on intracellular pH in Chinese hamster ovary cells

Chinese hamster ovary cells were heated at 45.5 or 43.0 degrees C at acidic pH (6.7) or normal physiological pH (7.4) to have a survival of 10(-3). The weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C), was used to measure the intracellular pH (pHi) both during and following hyperthermia. Tritiated water and a Particle Data machine were used to measure cellular volume as well. With 99.9% of the cell population destined to die clonogenically, the physiologically alive cells, as determined by the exclusion of trypan blue dye, maintained their pH differential between pHe and pHi as well as unheated cells. Furthermore, the cell's ability to regulate its pHi in response to changes in pHe was not affected by the same hyperthermic treatment. However, cellular volume decreased by 15-30% by 5 h after the onset of heat treatment. We conclude that heat does not perturb the cellular regulation of intracellular H+ concentration. Therefore, there is no thermal damage to the pHi-regulatory mechanism that could be responsible for either heat-induced reproductive cell death or low pH sensitization of heat killing.     

Overexpression of extracellular superoxide dismutase has a protective role against hyperoxia-induced brain injury in neonatal mice

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P< 0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P < 0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.     

Seasonal changes in the activation of crossbridge motions of isolated thick filament from Limulus striated muscle

Summary In dynamic light scattering, measurements of the intensity-intensity time correlation function from a suspension of rod-like particles of length L could reveal dynamical information related to translational and internal motions of those particles. For a suspension of thick filaments isolated from the myosin-regulated, striated muscles of Limulus at KL>1 (where K is the scattering vector), the average characteristic linewidth ( ) increased with the addition of Ca²⁺ or with the depletion of ATP. The increase in the with the addition of Ca²⁺ could be due to the presence of energy-requiring, high-frequency motions of the crossbridges activated by Ca²⁺. The increase in which occurred with the depletion of ATP was assumed to be mainly due to the thermal motions of the crossbridges after they had moved radially away from the filament backbone. The percentage increase in following the addition of Ca²⁺ was found to be seasonal, i.e., values of obtained from thick filaments isolated between the middle of June and the middle of September were smaller than those obtained during the rest of the year. The effect of temperature on the percentage increase in was also different. The increase showed a maximum at about 35°C during the summer and at about 25°C at other times. However, the percentage increase in developed under ATP-depleted conditions showed no temperature-related maximum. The number of bound Ca²⁺ per myosin molecule was 1 during the summer and 2 at other times.Abbreviations DLS dynamic light scattering - L length - K scattering vector - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - average characteristic line width Deceased     

Genetic analysis of the structure and function of transfer messenger RNA pseudoknot 1

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.