In silico feasibility of novel biodegradation pathways for 1,2,4-trichlorobenzene

Background  

Bioremediation offers a promising pollution treatment method in the reduction and elimination of man-made compounds in the environment. Computational tools to predict novel biodegradation pathways for pollutants allow one to explore the capabilities of microorganisms in cleaning up the environment. However, given the wealth of novel pathways obtained using these prediction methods, it is necessary to evaluate their relative feasibility, particularly within the context of the cellular environment.     

Metabolic engineering under uncertainty--II: analysis of yeast metabolism

Yeast metabolism has been used extensively in scientific investigations and industrial applications. Understanding the properties of the yeast metabolic network is crucial, yet unaccomplished due to its high complexity, the different culture conditions, and the uncertainty associated with kinetic parameters. We recently developed a computational and mathematical framework using Monte Carlo method in which parameter uncertainty can be addressed through large-scale sampling procedure. This framework was applied on the compartmentalized central carbon pathways of Saccharomyces cerevisiae metabolism considering the growth environment of batch and chemostat reactor and integrating information from metabolic flux analysis. Statistical analysis of the results indicates that yeast cells growing in batch culture condition exhibit dramatically different control schemes from those growing in a chemostat. The difference is mainly due to the feedback introduced by the constraints of the chemostat. The control of the enzymes on the rates of the substrate uptake, product excretion, and cell growth and its practical implication are discussed. Clustering of the reaction steps according to the similarity of their responses to enzyme activity perturbations reveals functional coupling of metabolic reactions.     

A comparative analysis of the impact on biota and man in the 30 km zone of the Chernobyl NPP

A methodological approach is described for a comparative assessment of ionizing radiation effects on man and biota, based on the use of indices of radiation impact--ratios of actual exposure doses to environmental objects (including humans) and critical ones. As such doses, standards limiting radiation exposure and doses at which phenotypical effects were absent after the Chernobyl accident were employed, respectively for man and biota. For the test site chosen within the 30 km ChNPP zone (region of the Borshchovka settlement), dose burdens to reference biota species and the population (with and without evacuation) and the corresponding radiation impact indices were calculated. For the long term period after the accident radiation safety standards for man are shown to ensure radiation safety for biota as well. At the same time in the early period after the accident the emergency regulations do not guarantee adequate protection of nature, some species of which can be subject to irradiation more than man, even if countermeasures like evacuation are not applied. A conclusion has been made on the necessity of a more detailed and comprehensive analysis of situations when the anthropocentric principle "if radiation standards protect man then biota are also adequately protected" is violated.     

Phosphate determination during hypokinesia and ambulation in establishing phosphate changes in trained and untrained subjects

Hypokinesia (diminished movement) induces phosphate (P) changes; however, it is not known if P change is greater in trained than untrained subjects. Measuring P balance and P retention during hypokinesia (HK) and P load, we studied if changes in P retention and P depletion were significantly (p<0.05) greater in trained than untrained subjects. Studies were done during a 30-d pre-HK period and a 364-d HK period. Forty male trained and untrained healthy individuals aged 24.5±5.4 yr were chosen as subjects. All volunteers were equally divided into four groups: trained ambulatory control subjects (TACS), trained hypokinetic subjects (THKS), untrained ambulatory control subjects (UACS), and untrained hypokinetic subjects (UHKS). All THKS and UHKS were limited to an average walking distance of 0.3 km/d, and TACS and UACS were on an average running distance of 9.8 and 1.8 km/d, respectively. Subjects took daily 12.7-mmol dicalcium-phosphate/kg body weight in the form of supplementation. Negative P balance, fecal P loss, urinary P and calcium (Ca) excretion, serum P, and total Ca (Ca_t) levels increased significantly (p<0.05), whereas P retention, serum 1,25-dihydroxyvitamin D [1,25 (OH)₂D₃] and intact parathyroid hormone (iPTH) level decreased significantly (p<0.05) in THKS and UHKS when compared with their pre-HK values and their respective ambulatory controls (TACS and UACS). However, P retention, P balance, serum, urinary, and fecal P, and serum hormone level changed significantly (p<0.05) more in THKS than UHKS. Retention of P, fecal P, urinary P and Ca loss, serum P and Ca_t level, P balance, 1,25(OH)₂D₃, and iPTH level change insignificantly (p>0.05) in TACS and UACS when compared with their pre-HK control values. It was concluded that significant negative P balance may indicate P depletion, whereas significant P loss in spite of negative P balance and P load may suggest P retention incapacity; however, P depletion was greater in THKS than UHKS. Clearly, P is wasted much more in THKS than UHKS.     

Measurements in potassium-supplemented athletes during and after hypokinetic and ambulatory conditions

Hypokinesia (diminished movement) induces significant potassium (K) changes; however, little is known about K deposition and deficiency during hypokinesia (HK). Using K supplements during and after HK, the aim was to establish body K deposition and K deficiency during HK. Studies were done during the pre-HK period of 30 d, HK period of 364 d, and post-HK period of 30 d. Forty male trained athletes aged 24.9 ± 8.0 y were chosen as subjects. They were equally divided into four groups: unsupplemented active control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented active control subjects (SACS), and supplemented hypokinetic subjects (SHKS). Hypokinetic subjects were limited to an average walking distance of 0.7 km/d. Control subjects ran an average distance of 11.6 km/d. The SHKS and SACS groups took 95.0 mg elemental K/kg body weight daily. Fecal K excretion, urinary sodium (Na) and K excretion, plasma K and Na levels, plasma renin activity (PRA), plasma aldosterone (PA), food and fluid intake, and physical characteristics were measured. During HK, fecal K loss, urinary K and Na loss, and plasma K, Na, PRA, and PA levels increased significantly (p ≤ 0.05), whereas during the initial days of post-HK, the levels of the measured parameters decreased significantly (p ≤ 0.05) in the SHKS and UHKS groups as compared with the SACS and UACS groups, respectively. During HK, body weight, body fat, peak oxygen uptake, food and fluid intake decreased significantly (p ≤ 0.05), whereas during the initial days of post-HK period remained significantly (p ≤ 0.05) depressed and fluid intake increased in SHKS and UHKS groups when compared with the SACS and UACS groups, respectively. However, during HK and post-HK plasma, urinary, and fecal K changed significantly (p ≤ 0.05) more in the SHKS group than in the UHKS group. The deposition of K was significantly (p ≤ 0.05) lower and K deficiency much higher in the SHKS group than in the UHKS group. Fecal K loss, urinary K and Na loss, plasma K, Na, PRA, and PA levels, body weight, body fat, peak oxygen uptake, and food and fluid intake did not change significantly in the SACS and UACS when compared with their baseline control values. It was shown that plasma K concentration and urinary and fecal K excretion increased during HK and decreased significantly (p ≤ 0.05) during post-HK. post-HK. Oral K supplements did not influence plasma or fecal and urinary K either during HK or post-HK. It was concluded that the low plasma K level and fecal and urinary K loss during post-HK may indicate the presence of K deficiency, and increased K in plasma, urine, and feces during HK and in the presence of K deficiency may suggest the body’s inability to retain K during HK.     

Purifying and directional selection in overlapping prokaryotic genes

In overlapping genes, the same DNA sequence codes for two proteins using different reading frames. Analysis of overlapping genes can help in understanding the mode of evolution of a coding region from noncoding DNA. We identified 71 pairs of convergent genes, with overlapping 3' ends longer than 15 nucleotides, that are conserved in at least two prokaryotic genomes. Among the overlap regions, we observed a statistically significant bias towards the 123:132 phase (i.e. the second codon base in one gene facing the degenerate third position in the second gene). This phase ensures the least mutual constraint on nonconservative amino acid replacements in both overlapping coding sequences. The excess of this phase is compatible with directional (positive) selection acting on the overlapping coding regions. This could be a general evolutionary mode for genes emerging from noncoding sequences, in which the protein sequence has not been subject to selection.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

Isolation and purification of proteins from the symbiosome membrane of yellow lupine root nodules

A unique feature of the symbiotic association between legume plants and rhizobia is the plant-derived membrane which separates the symbionts within root nodule; this membrane is termed the peribacteroid membrane (PBM). Although this membrane plays a vital role in facilitating transport and other processes in nodules, little is known about the proteins that are associated with and are an integral part of it. The objective of this work was to apply modern methods of protein purification to the characterisation of proteins of peribacteroid membrane from nodules of yellow lupine (Lupines luteus). The 17-kDa protein was isolated from purified peribacteroid membrane using size exclusion and ion exchange chromatography (FPLC). The N-terminal amino acid sequence of this protein was determined; the sequence does not match any of the previously reported lupine and other legume sequences. Following detergent solubilisation of purified peribacteroid membrane, integral proteins of 15 to 20 kDa were purified by size exclusion chromatography.