Restructuring of a Peat in Interaction with Multivalent Cations: Effect of Cation Type and Aging Time

It is assumed to be common knowledge that multivalent cations cross-link soil organic matter (SOM) molecules via cation bridges (CaB). The concept has not been explicitly demonstrated in solid SOM by targeted experiments, yet. Therefore, the requirements for and characteristics of CaB remain unidentified. In this study, a combined experimental and molecular modeling approach was adopted to investigate the interaction of cations on a peat OM from physicochemical perspective. Before treatment with salt solutions of Al³⁺, Ca²⁺ or Na⁺, respectively, the original exchangeable cations were removed using cation exchange resin. Cation treatment was conducted at two different values of pH prior to adjusting pH to 4.1. Cation sorption is slower (>>2 h) than deprotonation of functional groups (<2 h) and was described by a Langmuir model. The maximum uptake increased with pH of cation addition and decreased with increasing cation valency. Sorption coefficients were similar for all cations and at both pH. This contradicts the general expectations for electrostatic interactions, suggesting that not only the interaction chemistry but also spatial distribution of functional groups in OM determines binding of cations in this peat. The reaction of contact angle, matrix rigidity due to water molecule bridges (WaMB) and molecular mobility of water (NMR analysis) suggested that cross-linking via CaB has low relevance in this peat. This unexpected finding is probably due to the low cation exchange capacity, resulting in low abundance of charged functionalities. Molecular modeling demonstrates that large average distances between functionalities (∼3 nm in this peat) cannot be bridged by CaB-WaMB associations. However, aging strongly increased matrix rigidity, suggesting successive increase of WaMB size to connect functionalities and thus increasing degree of cross-linking by CaB-WaMB associations. Results thus demonstrated that the physicochemical structure of OM is decisive for CaB and aging-induced structural reorganisation can enhance cross-link formation.     

Simultaneous degradation of 3-chlorobenzoate and phenolic compounds by a defined mixed culture ofPseudomonas spp.

A mixed culture of a chlorobenzoate-(3-CBA)-degradingPseudomonas aeruginosa, strain 3mT, and a phenol/cresols-degradingPseudomonas sp., strain CP4, simultaneously and efficiently degraded mixtures of 3-CBA and phenol/cresols. However, strains 3mT and CP4 usedortho- andmeta-ring cleavage pathways, respectively. Degradation of 3-CBA was complete when the 3-CBA was equal in amount to or less than that of phenol. CP4/3mT inoculum ratios (w/w) of 1:1 or 1:2 gave the most effective degradation of both the substrates in the mixture. The mixed culture degraded equimolar mixtures of 3-CBA/phenol up to 10mm. Equimolar mixtures of 3-CBA ando-, m- orp-cresol were also degraded by the mixed culture.The authors are with the Microbiology and Bioengineering Department, Central Food Technological Research Institute, Mysore-570013, India;     

Degradation of high concentrations of cresols by Pseudomonas sp. CP4

Pseudomonas sp. CP₄, a potent phenol-degrading laboratory isolate could mineralize all three isomers of cresol. This strain readily utilized up to 1.4, 1.1 and 2.2 g/l of o- m- and p-cresol, respectively as the sole sources of carbon and energy. These are the highest concentrations of cresols reported to be degraded by a bacterial strain. The rates of degradation of the three isomers were in the order: o- > p- > m-cresol. All the isomers of cresol were catabolized through a meta-cleavage pathway. Fairly high catechol 2,3-dioxygenase (C230) activity against catechol was observed in the cell-free extracts of the culture grown on these compounds and were in the order: m- > o- > p-cresol.     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

Secretion to the growth medium of an α-amylase by Escherichia coli clones carrying a Bacillus laterosporus gene

-Amylase gene from Bacillus laterosporus P₃ was cloned and expressed in Escherichia coli HB101 and DH5. Up to 92% of the cloned gene product was secreted into the medium by the recombinant E. coli. The recombinant crude enzyme showed improved functionality in terms of activity at a wider pH range and at higher temperature, as compared to the crude enzyme from the donor strain. The improved functionality of the cloned enzyme was due to the absence of a contaminating protease which was co-produced in the donor strain. Sub-cloning of the -amylase gene using the promoter-probe vector, pKT240 in E. coli DH5 indicated the presence of a promoter of B. laterosporus P₃ in the cloned fragment.     

New metabolic pathway for o-cresol degradation by Pseudomonas sp. CP4 as evidenced by 1H NMR spectroscopic studies

Degradation intermediates of o-, m- and p-cresols extracted from resting cells of Pseudomonas sp. CP4, a potent cresol- and phenol-degrading laboratory isolate, were analysed by using ¹H NMR spectroscopy at 270 MHz. Ortho-, meta- and para-cresols were found to be degraded to 2-methyl-4-oxalocrotonate. 3-Methylcatechol from o-cresol was degraded further to 2-ketohex-cis-4-enoate, 4-methylcatechol from m- and p-cresol was degraded to 2-ketohex-cis-4-enoate. Also 2-ketopent-4-enoate was found to be formed from p-cresol. Formation of 2-methyl-4-oxalocrotonate was envisaged as taking place from 5-hydroxy-2-methylmuconic semialdehyde, the ring-cleavage product of 4-methylresorcinol, a possible product by hydroxylation of o-cresol along with 3-methylcatechol. This is a deviation from the hitherto known pathways of o-cresol degradation. Based on these observations, pathways for the degradation of all three isomers of cresol are proposed.     

Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: an indication of cell cycle defects

In the present study, both post-irradiation DNA synthesis and G₁ phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by ³H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G₂ phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G₂ phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G₂ accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cyle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G₂ phase accumulation developed by Lavin et al. (1992) in characterizing AT heterozygote-like cell cycle anomally in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Standardization of assay procedure and some properties of ribonuclease fromaspergillus candidus

Screening of several fungal cultures resulted in the selection of an isolate of Aspergillus candidus which produced a considerable around of RNa-degrading enzyme in both surface and submerged methods of cultivation. The conditions for the assay of the RNAase were standardized at pH 4.5, 55 degrees C and using 0.25% yeast RNA as substrate. The enzyme was stable at pH 5.2. EDTA was found to activate the enzyme slightly. at temperatures 50-60 degrees C there was considerable loss in enzyme activity which was traced to the presence of a contaminating protease which presumably degraded the RNAase optimally at this temperature. The protease could be preferentially inactivated at or above 75 degrees C. The crude enzyme, in addition to RNAase was found to possess DNAase, nonspecific phosphodiesterase and 3'- and 5'-phosphomonoesterase activities.     

Degradation of phenol through ortho-cleavage pathway by Pseudomonas stutzeri strain SPC2

P.Y. ANEEZ AHAMAD AND A.A.M. KUNHI. 1996. Generally pseudomonads degrade phenol through the meta -pathway, but Pseudomonas stutzeri strain SPC2 isolated by flask enrichment of municipal sewage degraded phenol through the ortho -pathway. The strain utilized up to 1200 ppm of phenol as a sole source of carbon and energy. The strain also degraded benzoate and 4-hydroxy and 3,4-dihydroxybenzoates via the ortho -pathway. Cell-free extracts of the strain grown on these substrates showed fairly good catechol 1,2-dioxygenase (C1,2-D) and protocatechuate 3,4-dioxyenase (PCA 3,4-D) activities, the induction of both activities being increased by benzoate. No meta -cleavage activities were detected.