Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

Background  

Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.     

Effects of Xylitol on Xylitol-Sensitive Versus Xylitol-Resistant Streptococcus mutans Strains in a Three-Species in Vitro Biofilm

We studied the effects of xylitol on biofilms containing xylitol-resistant (Xr) and xylitol-sensitive (Xs) Streptococcus mutans, Actinomyces naeslundii and S. sanguinis. The biofilms were grown for 8 and 24 h on hydroxyapatite discs. The viable microorganisms were determined by plate culturing techniques and fluorescence in situ hybridization (FISH) was performed using a S. mutans-specific probe. Extracellular cell-bound polysaccharides (EPS) were determined by spectrofluorometry from single-species S. mutans biofilms. In the presence of 5 % xylitol, the counts of the Xs S. mutans decreased tenfold in the young (8 h) biofilm (p < 0.05) but no effect was seen in the mature (24 h) biofilm. No decrease was observed for the Xr strains, and FISH confirmed these results. No differences were detected in the EPS production of the Xs S. mutans grown with or without xylitol, nor between Xr and Xs S. mutans strains. Thus, it seems that xylitol did not affect the EPS synthesis of the S. mutans strains. Since the Xr S. mutans strains, not inhibited by xylitol, showed no xylitol-induced decrease in the biofilms, we conclude that growth inhibition could be responsible for the decrease of the counts of the Xs S. mutans strains in the clinically relevant young biofilms.     

Effect of dimethyl sulfoxide on enlarged hearts of copper-deficient rats

The purpose of this study was to examine, by transmission electron microscopy (TEM), the nature of the protective effect of dimethyl sulfoxide (DMSO) on hearts of copper-deficient (CuD) rats. Male, weanling Sprague-Dawley rats were fed, in a two-way design, CuD (0.45 micrograms/g) or copper-sufficient (CuS, 5.4 micrograms/g) diets with or without 5% DMSO in their drinking water. After 28 d, CuD rats showed typical signs of copper deficiency, including reduced liver and heart Cu, enlarged hearts, and anemia. DMSO-treated, CuD rats had lower heart weights and higher hematocrits than CuD rats. DMSO enhanced organ Cu concentrations in CuS, but not in CuD rats. TEM of CuD hearts showed myofibrillar distortion and enlarged, vacuolated mitochondria with fragmented cristae; morphometric measurements indicated an enhanced mitochondrial/myofibrillar ratio (mito/myo), but an increase of both mitochondrial and myofibrillar mass relative to CuS hearts. Compared to CuD hearts, DMSO-treated CuD hearts showed better mitochondrial morphology and myofibrillar organization, as well as a greater mito/myo, but lower mitochondrial and myofibrillar masses. Its function as a hydroxyl radical scavenger indicates that DMSO could protect CuD hearts, in particular their mitochondria, against oxidative damage. However, because measurements of thiobarbituric acid reactive substances were not consistent with this theory, other metabolic mechanisms, direct and indirect, must be examined.     

Kinetics and thermodynamics of oxygen, CO, and azide binding by the subcomponents of soybean leghemoglobin

Leghemoglobin shows extreme high affinity behavior in the binding of both oxygen and CO. We have determined the temperature dependence of the rate constants for ligation of oxygen and CO and from these data the thermodynamics (delta G0, delta H0, delta S0) of ligation for the purified components of soybean leghemoglobin. X-ray crystallography has shown that the heme cavity can easily accommodate ligands the size of nicotinate, and analysis of extended x-ray absorption fine structure data has shown that the Fe atom is in the mean plane of the heme in the leghemoglobin-CO complex. Ligation of oxygen and CO are in accord with this picture in that the Ea for oxygen binding is that expected for a diffusion controlled reaction and delta S0 for the ligation of both CO and oxygen is consistent with the simple immobilization of the ligand at the Fe, with no evidence for significant conformational changes in the protein or changes in solvation. At 20 degrees C the rate constants for oxygen and CO binding vary by 26-44% among the eight leghemoglobin components. For azide binding the variation is a factor of 2. These variations appear to arise from amino acid substitutions outside either the heme cavity or the two major paths for ligand entry to the heme. The distribution of leghemoglobin components varies with the age of the soybean nodule during the growing season. The changes in composition alone, however, would only allow the concentration of free oxygen to vary by about 3%. This finding calls into question models that ascribe a significant functional role to changes in the distribution of leghemoglobin components in regulating oxygen concentration in the nodule.     

Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum

Summary Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.     

Ex vivo measurement of lumbar intervertebral disc pressure using fibre-Bragg gratings

Methods were developed to measure intervertebral disc pressure using optical fibre-Bragg gratings (FBGs). The FBG sensor was calibrated for hydrostatic pressure in a purpose-built apparatus and the average sensitivity was determined to be -5.7 +/- 0.085 pm/MPa (mean +/- SD). The average coefficient of determination (r(2)) for the calibration data was 0.99, and the average hysteresis of the sensor was 2.13% of full scale. The FBG was used to measure intradiscal pressure response to compressive load in five lumbar functional spine units. The pressure measured by the FBG sensor varied linearly with applied compressive load with coefficients of determination ranging from 0.84 to 0.97. The FBG sensor's sensitivity to compressive load ranged from 0.702 +/- 0.043 kPa/N (mean +/- SD) in a L1-L2 specimen, to 1.07 +/- 0.069 kPa/N in a L4-L5 specimen. These measurements agree with those of previous studies in lumbar spines. Two strain gauge pressure sensors were also used to measure intradiscal pressure response to compressive load. The measured pressure sensitivity to load ranged from 0.251 kPa/N (L4-L5) to 0.850 kPa/N (L2-L3). The average difference in pressure sensitivity to load between Sensors 1 and 2 was 12.9% of the value for Sensor 1, with a range from 1.1% to 20.4%, which suggests that disc pressure was not purely hydrostatic. This may have contributed to the difference between the responses of the FBG and strain gauge sensors.     

Expression of <Emphasis Type="Italic">Ciona intestinalis</Emphasis> AOX causes male reproductive defects in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question.

Methods

Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the α-tubulin promoter, and wild-type males of the same genetic background.

Results

In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation.

Conclusions

AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.

     

Multiple phosphorylation of rhodopsin and the in vivo chemistry underlying rod photoreceptor dark adaptation

Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.