“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH₃ as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH₃ passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH₃ transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH₃ from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH₃ was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH₂OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH₃ gate of CTPS to mediate inter-domain NH₃ transport.     

Uptake of deoxynivalenol by earthworms from <Emphasis Type="Italic">Fusarium</Emphasis>-infected wheat straw

Conservation tillage combined with crop-residue mulching is increasingly important to meet soil protection targets. Concurrently, the health risk of soil-borne pathogenic fungi like Fusarium species, which produce deoxynivalenol (DON) as their major mycotoxin, is increasing. The detritivorous earthworm species Lumbricus terrestris takes part in the efficient degradation of Fusarium-infected and DON-contaminated wheat straw. Against this background, a laboratory study was conducted to quantify by means of ELISA technique the uptake of DON and its possible absorption and accumulation in tissue by L. terrestris in the short-term (5 weeks) and long-term (11 weeks). The DON concentrations in L. terrestris of the Fusarium-infected treatment were significantly different in the order of gut tissue > body wall > gut content at both dates with a decline in the long-term. The DON concentrations in the tissues decreased by an order of magnitude of weeks to months.     

Fusarium spp. and storage fungi in suboptimally stored wheat: Mycotoxins and influence on wheat gluten proteins

When wheat is stored under suboptimal conditions, a further mycotoxin increase of deoxynivalenol (DON), but especially of mycotoxins produced by storage fungi, e.g. ochratoxin A, is possible, lowering wheat quality and food safety. Different storage trials were conducted under suboptimal storage conditions.Fusarium survival during suboptimal storage was monitored by cultural technique and multiplex-PCR and set into relation to DON contents. Furthermore, XANES spectroscopy was applied on a selected storage trial in order to characterize sulfur speciation in low molecular weight (LMW) subunits of glutenin isolated from suboptimally stored wheat samples highly infected withFusarium and from wheat infected withAspergillus andPenicillium. Distinct changes in sulfur speciation were observed in grains infected with storage fungi, especially a significant increase of higher oxidation states (sulfoxide state, sulfonate state). Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Studies on accuracy of trichothecene multitoxin analysis using stable isotope dilution assays

Critical parameters in mycotoxin analysis were examined by using stable isotope-labelled tricho-thecenes. Sample weight was downsized to 1 g without loosing precision when sufficiently homogenized samples were taken for analysis. Complete extraction of trichothecenes could be achieved with a solvent mixture of acetonitrile+water (84+16; v+v) even without the use of stable isotope labelled standards. However, in particular for the analysis of deoxynivalenol the absolute amount of water in the solvent volume used for extraction appeared critical. Depending on the matrix a low water amount resulted in too low quantitative values when no stable isotope-labelled standards are applied to correct for incomplete extraction. In this case the used extraction volume had to be at least 10 ml for 1 g sample when acetonitrile + water (84+16; v+v) was used as extraction solvent. Losses during sample preparation using two different clean-up columns were not observed. On the contrary, matrix suppression in the ESI-interface of the LC-MS equipment was found to be a serious problem. Depending on the matrix, the latter effect resulted in considerably lower values for trichothecenes when no stable isotope-labelled standards were used to counterbalance this suppression. Presented at the 29th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007     

Comparative stemness and differentiation of luminal and basal breast cancer stem cell type under glutamine‐deprivation

Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24⁺ epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAM^high population whereas MDAMB-231-bCSCs increased CD44^high population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of β-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of β-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-020-00603-1.     

The effect of Tyrophagus putrescentiae on Fusarium poae transmission and fungal community in stored barley in a laboratory experiment

The effect of Tyrophagus putrescentiae on Fusarium poae transmission and fungal community composition was studied in nonsterile barley grain. The experiments included following treatments： control barley without mites; barley containing l0 or 50 mites without preincubation on E poae （Tpl0 and TpS0）; barley containing 10 or 50 mites after preincubation on E poae （FTp 10 and FTp50）. The number of mites, suc cessful transfer of E poae, and changes in the fungal communities were examined after 21 d of experiment. Increase of deoxynivalenol （DON） content in the barley was chosen as a criterion of successful Epoae transfer. The preincubation of T. putrescentiae on Epoae increased DON level approximately to 800 and 300μg/kg of grain for FTpl0 and FTpS0, respectively. T. putrescentiae population growth in FTpl0 was lower than in Tpl0, while no difference was found between FTp50 and Tp50. Fungal communities were compared by amplification, cloning and sequencing of ITS fragments, and operational taxonomic units （OTU） analysis. The OTU analysis did not support the transfer ofF. poae via mites. From the analyzed clones, only 13 cloned sequences clustered with E poae in an OTU defined at distance level 0.07. The related clones originated from FTpl0, Tpl 0, Tp50 and control treatments, but not from FTp50. However, the presence of E poae in FTp50 was confirmed by PCR amplification with specific primers. The observation may be explained by different effect of mite population density, that is, in the high density, （FTp50 treatment） the fungus was overgrazed, while the lower population density （FTp 10） supported E poae transfer.