Use of a renal tubule cell line (LLC-PK1) to study the nephrotoxic potential of a kappa-type bence-jones protein

Summary The cytotoxicity of a Bence-Jones protein was assessed using a porcine renal tubule cell line (LLC-PK1), with the aim of developing a model for studying the potential nephrotoxicity of these proteins. The effects of a kappa Bence-Jones protein on cell viability were studied by means of biochemical methods (supravital dye uptake and measurement of cellular enzyme activities) and morphological electron microscopy. After a 24-h-treatment with Bence-Jones protein, a moderate cytotoxicity (about 15%) was noted but only a minor difference compared to treatment with bovine albumin in the same conditions. The morphological study showed a few cells in the process of lysis, but their numbers were insufficient for the demonstration of a clear cytotoxic effect. Immunocytochemical studies showed Bence-Jones protein fixation on some cells, especially on the outer membrane. Labeling of the hyaloplasm and basal pole of a few cells pointed to internalization of protein by LLC-PK1 cells. Although the cytotoxicity of the Bence-Jones protein tested here was only moderate, the use of this model enabled its cytotoxic effect to be distinguished from that ofβ-lactoglobulin. This isolate could serve as a “moderate control” for a later study with a BJP having caused acute renal failure.     

Oxidation of phenols by laccase and laccase-mediator systems.

To investigate how solubility and steric issues affect the laccase-catalysed oxidation of phenols, a series of oligomeric polyphenol compounds, having increasing size and decreasing solubility in water, was incubated with laccase. The extent of substrate conversion, and the nature of the products formed in buffered aqueous solutions, were compared to those obtained in the presence of an organic cosolvent, and also in the presence of two mediating species, i.e. N-hydroxyphthalimide (HPI) and 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO). This approach showed not only an obvious role of solubility, but also a significant role of the dimension of the substrate upon the enzymatic reactivity. In fact, reactivity decreases as substrate size increases even when solubility is enhanced by a cosolvent. This effect may be ascribed to limited accessibility of encumbered substrates to the enzyme active site, and can be compensated through the use of the appropriate mediator. While TEMPO was highly efficient at enhancing the reactivity of large, less soluble substrates, HPI proved less effective. In addition, whereas the laccase/HPI system afforded the same products as laccase alone, the use of TEMPO provided a different product with high specificity. These results offer the first evidence of the role of 'oxidation shuttles' that the mediators of laccase may have, but also suggest two promising routes towards an environmentally friendly process for kraft pulp bleaching: (a) the identification of mediators which, once oxidized by laccase, are able to target strategic functional groups present in lignin, and (b) the introduction of those strategic functional groups in an appropriate pretreatment.     

Cell cycle kinetic analysis with flow cytometry in pea root meristem synchronized with aphidicolin

Meristematic cells in pea root were synchronized after treatment with aphidicolin. Flow cytometric analysis of DNA content variations revealed a variety of information: ca 50% of the cells were synchronized in the first part of the S phase; kinetic studies of the S-phase traverse revealed an increase of the DNA synthesis rate from early to late S; some of the cells in the meristem were non-cycling, arrested in Gl (19%) and G2 (12%). The possibility of using such a synchronized system in cytogenetic and biomolecular studies is discussed.     

Fertilizers from composting of olive-mill wastewaters

The aerobic bioremediation of olive-mill wastewaters produces a high quality compost, characterized by a considerable presence of nutrients, mainly organically-bound nitrogen (1.5-3%), a good level of humification (degree of HUMIFICATION=78%; humification INDEX=0.28), and by the absence of phytotoxicity. The agronomic value of a compost thus obtained was assayed both by the ‘crop test’ and following the plant-soil system as influenced by compost supply. Field experiments performed on maize showed that compost, when supplied before sowing in amounts of 60–90 Mg ha⁻¹ (equivalent to a manuring on the basis of organic matter) is able to reduce the need for chemical fertilization. The same quantity supported the nutritional need of rye grass and horticultural plants. A good rest-effect was also recorded. Compost supply enhanced both soil oxygen consumption and nitrogen fixation in the open field. An improvement of activities in the plant-soil system was made evident by pot trials.