Depolymerisation and biodegradation of a synthetic tanning agent by activated sludges, the bacteria Arthrobacter globiformis and Comamonas testosteroni, and the fungus Cunninghamella polymorpha

Degradation of a synthetic tanning agent CNSF (a condensation product of 2-naphthatenesulfonic acid (2-NSA) and formaldehyde) by four activated sludges, two previously characterised bacterial strains, Arthrobacter sp. 2AC and Comamonas sp. 4BC, and the fungus Cunninghamella polymorpha, was studied in batch culture at 25 degrees C by determining the changes in the concentrations of CNSF and its component monomers and oligomers (n2-n11). The loss of individual oligomers was correlated with the length of the NSA-CH2 chain. Approximately 25% of the total CNSF was degraded (i.e. mineralised) by the microbes contained in the four activated sludges and by the two bacterial isolates but with different lag phases and at different overall rates. The decline in CNSF concentration was due almost entirely to the biodegradation of the monomers (34.3% of CNSF) and, in particular, 2-NSA (27% of CNSF). There was no change in the n2-n 11 components. The growth of C. polymorpha, on the other hand, arose from extracellular depolymerisation of CNSF oligomers and the biodegradation of the lower molecular mass products. Between 38% and 42% of total CNSF was degraded by C. polymorpha at 25 degrees C. The order of oligomer degradation was inversely related to degree of polymerisation. Eighty percent and 90% of the n4 and n5 and 100% oligomers n6-n11 were degraded after 120 h. At a higher temperature (37 degrees C) oligomers n4-n11 were degraded completely after 120 h. A combination of biodegradation (75%) and sorption to fungal biomass (25%) accounted for the measured loss of all oligomers from the solution phase. The CNSF degradation rates and the volume of fungal biomass produced (and therefore the extent of biosorption) were dependent on the presence of a second carbon source (both optimum at glucose 5 g/l). This is the first report that identifies and distinguishes between depolymerisation, sorption and biodegradation processes in the removal of CNSF and its component oligomers. The use of combinations of the depolymerising fungus C. polymorpha, and the monomer-degrading bacteria, Arthrobacter sp. 2AC and Comamonas sp. 4BC, have potential for wastewater treatment.

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 mu M), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility. (c) 2006 Elsevier Inc. All rights reserved.

The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity

Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim-/- cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton. (c) 2006 Elsevier Ltd. All rights reserved.