Initial mutations direct alternative pathways of protein evolution

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.     

Modulation of human time processing by subthalamic deep brain stimulation

Timing in the range of seconds referred to as interval timing is crucial for cognitive operations and conscious time processing. According to recent models of interval timing basal ganglia (BG) oscillatory loops are involved in time interval recognition. Parkinsońs disease (PD) is a typical disease of the basal ganglia that shows distortions in interval timing. Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a powerful treatment of PD which modulates motor and cognitive functions depending on stimulation frequency by affecting subcortical-cortical oscillatory loops. Thus, for the understanding of BG-involvement in interval timing it is of interest whether STN-DBS can modulate timing in a frequency dependent manner by interference with oscillatory time recognition processes. We examined production and reproduction of 5 and 15 second intervals and millisecond timing in a double blind, randomised, within-subject repeated-measures design of 12 PD-patients applying no, 10-Hz- and ≥ 130-Hz-STN-DBS compared to healthy controls. We found under(re-)production of the 15-second interval and a significant enhancement of this under(re-)production by 10-Hz-stimulation compared to no stimulation, ≥ 130-Hz-STN-DBS and controls. Milliseconds timing was not affected. We provide first evidence for a frequency-specific modulatory effect of STN-DBS on interval timing. Our results corroborate the involvement of BG in general and of the STN in particular in the cognitive representation of time intervals in the range of multiple seconds.     

Full-length L1CAM and not its Δ2Δ27 splice variant promotes metastasis through induction of gelatinase expression

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.     

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.     

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.     

Repeated high doses of avermectins cause prolonged sterilisation, but do not kill, Onchocerca ochengi adult worms in African cattle

Background

Ivermectin (Mectizan?, Merck and CO. Inc.) is being widely used in the control of human onchocerciasis (Onchoverca volvulus) because of its potent effect on microfilariae. Human studies have suggested that, at the standard dose of 150 μg/kg an annual treatment schedule of ivermectin reversibly interferes with female worm fertility but is not macrofilaricidal. Because of the importance of determining whether ivermectin could be macrofilaricidal, the efficacy of high and prolonged doses of ivermectin and a related avermectin, doramectin, were investigated in cattle infected with O. ochengi.

Methods

Drugs with potential macrofilaricidal activity, were screened for the treatment of human onchocerciasis, using natural infections of O. ochengi in African cattle. Three groups of 3 cows were either treated at monthly intervals (7 treatments) with ivermectin (Ivomec^®, Merck and Co. Inc.) at 500 μg/kg or doramectin (Dectamax^®, Pfizer) at 500 μg/kg or not treated as controls. Intradermal nodules were removed at 6 monthly intervals and adult worms were examined for signs of drug activity.

Results

There was no significant decline in nodule diameter, the motility of male and female worms, nor in male and female viability as determined by the ability to reduce tetrazolium, compared with controls, at any time up to 24 months from the start of treatments (mpt). Embryogenesis, however, was abrogated by treatment, which was seen as an accumulation of dead and dying intra-uterine microfilariae (mf) persisting for up to 18 mpt. Skin mf densities in treated animals had fallen to zero by <3 mpt, but by 18 mpt small numbers of mf were found in the skin of some treated animals and a few female worms were starting to produce multi-cellular embryonic stages. Follow-up of the doramectin treated group at 36 mpt showed that mf densities had still only regained a small proportion of their pre-treatment levels.

Conclusion

These results have important implications for onchocerciasis control in the field. They suggest that ivermectin given at repeated high does may sterilise O. volvulus female worms for prolonged periods but is unlikely to kill them. This supports the view that control programmes may need to continue treatments with ivermectin for a period of decades and highlights the need to urgently identify new marcofiliaricidal compounds.     

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4⁺ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.     

Defective CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis,counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4⁺CD25⁺ T_reg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T_reg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4⁺CD25⁺ T cells in the central and peripheral lymphoid tissues. In addition, CD4⁺CD25⁺ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4⁺CD25⁺ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T_reg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T_reg cells and accessory cells. Our results demonstrate that the decrease in T_reg cell activity in CIA is counter-regulated by endogenous IFN-γ.     

Peroxisome proliferator-activated receptor gamma overexpression inhibits pro-fibrogenic activities of immortalised rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. In response to pro-fibrogenic mediators, PSCs undergo an activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts. Ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma), such as thiazolidinediones, are potent inhibitors of stellate cell activation and fibrogenesis in pancreas and liver. The effects of PPARgamma ligands, however, are at least in part mediated through PPARgamma-independent pathways. Here, we have chosen a different approach to study regulatory functions of PPARgamma in PSCs. Using immortalised rat PSCs, we have established a model of tetracycline (tet)-regulated PPARgamma overexpression. Induction of PPARgamma expression strongly inhibited proliferation and enhanced the rate of apoptotic cell death. Furthermore, PPARgamma-overexpressing cells synthesised less collagen than controls. To monitor effects of PPARgamma on PSC gene expression, we employed Affymetrix microarray technology. Using stringent selection criteria, we identified 21 up- and 19 down-regulated genes in PPARgamma-overexpressing cells. Most of the corresponding gene products are either involved in lipid metabolism, play a role in signal transduction, or are secreted molecules that regulate cell growth and differentiation. In conclusion, our data suggest an active role of PPARgamma in the induction of a quiescent PSC phenotype. PPARgamma-regulated genes in PSCs may serve as novel targets for the development of antifibrotic therapies.     

Azo dye reduction by mesophilic and thermophilic anaerobic consortia

The reduction of the azo dye model compounds Reactive Red 2 (RR2) and Reactive Orange 14 (RO14) by mesophilic (30 degrees C) and thermophilic (55 degrees C) anaerobic consortia was studied in batch assays. The contribution of fermentative and methanogenic microorganisms in both temperatures was evaluated in the presence of the fermentative substrate glucose and the methanogenic substrates acetate, H2/CO2, methanol, and formate. Additionally, the effect of the redox mediator riboflavin on electron shuttling was assessed. We concluded that the application of thermophilic anaerobic treatment is an interesting option for the reductive decolorization of azo dyes compared to mesophilic conditions. The use of high temperature may decrease or even take the place of the need for continuous redox mediator dosage in bioreactors, contrarily to the evident effect of those compounds on dye reduction under mesophilic conditions. Both fermenters and methanogens may play an important role during reductive decolorization of dyes, in which mediators are important not only for allowing the different microbes to participate more effectively in this complex reductive biochemistry but also for assisting in the competition for electrons between dyes and other organic and inorganic electron acceptors.