Biodegradation Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge

This study focuses on the processes influencing hydrocarbon residue persistence in soil, following land treatment of refinery oily sludge. Treating sludge applied to soil resulted in 70% to 90% degradation of total petroleum hydrocarbon (TPH) during 2 months, regardless of their initial concentrations (9 to 60 g/kg soil). Kinetic analyses performed on TPH degradation, in laboratory and field systems, revealed a degradation pattern characterized by two consecutive first-order kinetics reactions in all experimental settings. The first stage lasted about 3 weeks and was characterized by a temperature dependent rate constant of 0.047 day^-1 at 24°C. That value was comparable to the rate constant obtained when combining the individual rate constants of the saturated, aromatic, asphaltene and polar fractions. The subsequent slower stage rate constant was 0.012 day^-1, insensitive to temperature and to hydrocarbon composition. The transition between the two stages (about 21 days) was independent of the experimental temperature and the biodegradation extent during the first stage. It was concluded that the extent of residual accumulation in the soil was determined by the biodegradation efficiency during the first three weeks of treatment when biological processes dominated. During the following period, abiotic processes leading to reduced bioavailability of the TPH were limiting the degradation rate. Practically, as the first few weeks of treatment determine its efficiency, efforts to enhance the biological activity should be directed to that period.     

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.     

Effects of estrone and 17 β-estradiol on vegetative growth of Medicago sativa

Alfalfa ( Medicago sativa L.) plants were grown in the absence or presence of the steroidal estrogens, estrone and 17β-estradiol, under varying conditions. Plants were analysed for the following parameters: plant weight, estrogen content, phytoestrogen content, degree of nodulation and nitrogen fixation activity. It was found that under controlled greenhouse conditions: (1) Treatment with estrogens in the range of 0.005 to 0.5 μg 1⁻¹ increases both shoot and root dry weitht. In contrast, estrogen in concentrations of 50 to 500 μg 1⁻¹ decreases plant growth. (2) The effect of estrogen of growth is most marked in the absence of nitrogen. (3) Estrone is more effective in increasing growth than 17 β-estradiol. (4) In the plants where estrogen induced growth there was no significant increase in nitrogen fixation activity and nodule number. (5) Endogenous estrogen content of the plant did not increase at concentrations (0.005-0.5 μg 1⁻¹) which increased vegetative growth. (6) Endogenous estrogen content of the plant did increase at concentrations of estrogen (50-500 μg 1⁻¹ which inhibited vegetative growth and nodule weight. It can be concluded that estrogen in concentrations found in sewage water (0.3 μg estrogen 1⁻¹) can affect the vegetative growth of alfalfa plants.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

Microbial preparation of L-[15N]tyrosine and [15N]tyramine and their gas chromatographic-mass spectrometric analyses

The preparation of L-[15N]tyrosine and [15N]tyramine by microbial synthesis is described. Immobilized Erwinia herbicola cells were added to a reaction mixture containing phenol, pyruvic acid, and 15NH4Cl. The reaction was driven by excess nonlabeled pyruvate and phenol. Under these denaturing concentrations of phenol, immobilized cells were more effective than free ones. Gram quantities of L-[15N]tyrosine were obtained without label dilution. The conversion of this L-[15N]tyrosine into [15N]tyramine by Streptococcus faecalis was performed at maximal efficiency. Gas chromatographic-mass spectrometric studies and 1H and 15N NMR analyses of the labeled compounds are reported.     

Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.     

Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions

Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.     

Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase

The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo fast collapse to an ensemble of compact structures where the local environment of surface probes seems to be native-like but the two labeled secondary structure elements remain unfolded.     

Complete penetrance of Creutzfeldt-Jakob disease in Libyan Jews carrying the E200K mutation in the prion protein gene.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a prion disease which is manifest as a sporadic, inherited, and transmissible neurodegenerative disorder. The mean age at onset of CJD is approximately 60 years, and as such, many people destined to succumb undoubtedly die of other illnesses first. The delayed onset of CJD has complicated the analysis of inherited forms of the illness and led to the suggestion that mutations in the prion protein (PrP) gene are necessary but not sufficient for prion disease despite genetic linkage; indeed, an environmental factor such as a ubiquitous virus has been proposed as a second necessary factor. MATERIALS AND METHODS: To examine what appeared to be incomplete penetrance, we applied a life-table analysis to clinical and pedigree data from a cluster population of Libyan Jews in which the E200K mutation is prevalent. The study population included 42 affected and 44 unaffected members of 13 Libyan Jewish families, all of whom possessed the E200K mutation. RESULTS: The calculated value using life table analysis is 0.77 at age 70 which increases to 0.89 if a mutation carrier survives to age 80 and 0.96 if age 80 is surpassed. CONCLUSIONS: These data argue that the E200K mutation alone is sufficient to cause prion disease and does so in an age-dependent manner.     

Studies on the mode of action of the antibacterial agent diethyltin dichloride onEscherichia coli

The mode of action of diethyltin dichloride as a representative of the antibacterial dialkyl-substituted tin compounds was studied forEscherichia coli. The oxygen uptake by resting cells ofEicoli with glucose, lactate, pyruvate or glutamate as substrates is strongly inhibited by diethyltin dichloride at a concentration of 0.08mm which is still below the minimum inhibitory concentration for growth on a rich medium. Inhibition of oxygen uptake with glucose or lactate as substrates is accompanied by an increase in the amount of pyruvic acid accumulating. A slight rise in the concentration of -ketoglutaric acid results from the inhibition of succinate and malate oxidation by diethyltin dichloride, while inhibition of -keto acid production is obtained with glutamate as a substrate. Anaerobically, with glucose as a substrate, ethanol formation as well as the production of carbon dioxide and hydrogen are inhibited by diethyltin dichloride. In many respects the effects of diethyltin dichloride on metabolism resemble those of arsenite.The inhibition of pyruvate metabolism probably results in a lack of acetyl-CoA, and this is suggested to be a possible cause of growth inhibition.